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ImageJ User Guide - RSB Home Page

1. Process gt Binary gt commands 29 8 4 Dilate Adds pixels to the edges of objects in a binary image Use Filters gt Maximum to perform grayscale dilation on non thresholded images SEE ALSO Binary Options XXII Interpreting Binary Images 29 8 5 Open Performs an erosion operation followed by dilation This smoothes objects and removes isolated pixels SEE ALSO Binarye Options XXII Interpreting Binary Images 29 8 6 Close Performs a dilation operation followed by erosion This smoothes objects and fills in small holes The command has a tailing hyphen to differentiate it from File Close w SEE ALSO Binaryb Options XXII Interpreting Binary Images 29 8 7 Outline Generates a one pixel wide outline of foreground objects in a binary image The line is drawn inside the object i e on previous foreground pixels 29 8 8 Fill Holes This command fills holes 4 connected background elements in objects by filling the background 34 29 8 9 Skeletonize IMPROVED IN IJ 1 46R Repeatably remove pixels from the edges of objects in a binary image until they are reduced to single pixel wide shapes topological skeletons As explained in X XI Skeletonize vs Skeletonize 3D there are several skeletonization algorithms ImageJ implements a thinning algorithm from Zhang and Suen A fast parallel algorithm for thinning digital patterns CACM 27 3 236 239 114 Last u
2. Certain plugins however provide self updating mechanisms e g ObjectJ and the OME Bio Formats 2 Last updated 2012 10 02 Installing and Maintaining ImageJ Related Software Fiji compares to ImageJ as Ubuntu compares to Linux The main focus of Fiji is to assist research in life sciences targeting image registration stitching segmentation feature extraction and 3D visualization among others It also supports many scripting languages BeanScript Clojure Jython Python Ruby see Scripting in Other Languages Importantly Fiji ships with a convenient updater that knows whether your files are up to date obsolete or locally modified Comprehensive documentation is available for most of its plugins The Fiji project was presented publicly for the first time at the ImageJ User and Developer Conference in November 2008 MBF ImageJ The MBF ImageJ bundle or ImageJ for Microscopy formerly WCIF ImageJ features a collection of plugins and macros collated and organized by Tony Collins at the MacBiophotonics facility McMaster University It is accompanied by a comprehensive manual describing how to use the bundle with light microscopy image data It is a great resource for microscopists but is not maintained actively lagging behind the development of core ImageJ Note that you can add plugins from MBF ImageJ to Fiji combining the best of both programs Actually you can use multiple ImageJ distributions simultaneously assemble
3. ImageJ _ User Guide IMAGEJ F1J1 1 46 ImageJ User Guide IJ 1 46r Tiago Ferreira Wayne Rasband Tuesday 2 October 2012 FOREWORD The ImageJ User Guide provides a detailed overview of ImageJ and inherently Fiji the standard in scientific image analysis see XX VI Focus on Bioimage Informatics It was thought as a comprehensive fully searchable self contained annotatable manual see Conventions Used in this Guide A HTML version is also available as well as printer friendly booklets see Guide Formats Its latest version can always be obtained from http imagej nih gov ij docs guide The source files are available through a Git version control repository at http fiji sc guide git Given ImageJ s heavy development this guide will always remain incomplete All Im ageJ users and developers are encouraged to contribute to the ImageJ documentation resources see Getting Involved Contents Release Notes for ImageJ 1 46r Noteworthy Macro Listings Conventions hu Getting Started 1 Introduction 2 Installing and Maintaining ImageJ 2 1 ImageJDistributions 22 Related Sonware 5 446 656 amp Ba bbe eae Me owe BE e Zo WNC 6 we OHS ERASE ROS ee eee eR AA A 3 Getting Help 3 1 Help on Image Analysis sk gta ew wh es So OS di GB we we ds Sie HE OrOM ADO a E A ne es dea Ba A A S II Working with ImageJ 4 Using Keyboard Shortcuts 5 Finding Commands 6 Undo and Redo 7 I
4. AA Fast Hartley Transform TIFF PUE ei Fiji Is Just ImageJ UEPs PITS stos Flexible Image Transport System URL HOS A Frames Per Second voxel CIP ewe fos hee Graphics Interchange Format WYSIWYG Le risas Graphical User Interface HDR ciate aed High Dynamic Range HEX AP Hexadecimal AA Hypermedia Image Processing Reference HSB AA Hue Saturation Brightness HTML HyperText Markup Language POR rin Integrated Development Environment Elisa ImageJ IPR tias Insight Segmentation and Registration JAR sla cache Java ARchive PEG orir ac os Joint Photographic Experts Group Knime Konstanz Information Miner KNIP enesis Knime Image Processing 181 Lookup table Lempel Ziv Welch Maximum Intensity Projection Motion JPEG Not a Number Open Microsopy Environment Operating System Pixels per cycle Portable BitMap Portable Gray Map Picture element Portable Network Graphics Pixels per inch Portable PixMap Random Access Memory Regular expression Red Green Blue Region Of Interest Shift key Tagged Image File Format Ultimate Eroded Points Uniform Resource Locator Volumetric pixel What You See Is What You Get Index 3D Viewer 89 3D Filters 20 124 3D Gaussian blur 124 3D Object Counter 20 3D Projection 89 3D ROIs 20 3D Viewer 20 89 Acceptable manipulation 5 ActionBar 25 Add see Math 117 Add images see Image
5. Cancel SEE ALSO Image Adjust gt Size Image gt Transform gt Bin Image gt Stacks gt Tools gt Grouped Z Project 28 12 Transform gt This submenu contains commands that perform geometrical image transformation on the active image or stack 28 12 1 Flip Horizontally Replaces the image or selection with a x mirror image of the original 28 12 2 Flip Vertically Turns the image or selection upside down y mirror 28 12 3 FlipZ Reverses the order of the slices in a stack z mirror 28 12 4 Rotate 90 Degrees Right IN IJ 1 46R Rotates the entire image or stack clockwise 90 98 Last updated 2012 10 02 Image gt Transform gt 28 12 5 Rotate 90 Degrees Left IN IJ 1 46R Rotates the entire image or stack counter clockwise 90 28 12 6 Rotate Angle degrees 8 0 Grid Lines 3 Interpolation Bicubic Fill with Background Color wi Enlarge Image to Fit Result M Preview OO RGB of Color48 x 162 pixels RGB age or selection clockwise the specified Use this dialog to rotate the active im number of degrees Set Grid Lines to a value greater than zero to superimpose a grid on the im age in Preview mode Two resam pling methods are possible Bilinear and Bicubic interpolation cf Image Size With 8 bit and RGB images check Fill with Background Color to fill with the background color instead of zero cf Color Picker K Check E
6. Horizontal ee Vertical M Autoscale After Filtering reduce edge artifacts before the Fourier trans The Bandpass Filter uses a special algorithm to M Saturate Image when Autoscaling O Display Filter form the image is extended in size by attaching mirrored copies of image parts outside the orig O gt Cancel OK T O ut inal image thus no jumps occur at the edges Filter Large Structures Down to Smooth variations of the image with typical sizes of bright or dark patches larger than this value are suppressed background Filter Large Structures Up to Determines the amount of smoothing Objects in the image smaller than this size are strongly attenuated Note that these values are both half the spatial frequencies of the actual cutoff The cutoff is very soft so the bandpass will noticeably attenuate even spatial frequencies in the center of the bandpass unless the difference of the two values is large say more than a factor of 5 or so Suppress Stripes Select whether to eliminate Horizontal or Vertical stripes Removal of horizontal stripes is similar to subtracting an image that is only blurred in the horizontal direction from the original Tolerance of Direction This is for Suppress Stripes higher values remove shorter stripes and or stripes that are running under an angle with respect to the horizontal vertical direction 122 Last updated 2012 10 02 Process gt FFT gt Autosca
7. Process gt Enhance Contrast Output is a binary image with foreground 255 and background 0 using an inverted or normal LUT depending on the Black Background option in Process Binary Options see XXII Interpreting Binary Images The number of particles as obtained by Analyze Analyze Particles in the output image does not depend on the selected Output Type Note that Segmented Particles will usually result in particles touching the edge if Exclude Edge Maxima is selected Exclude Edge Maxima applies to the maximum not to the particle Find Maxima does not work on stacks but the FindStackMaxima macro runs it on all the images in a stack and creates a second stack containing the output images The FindMaxi maRoiManager macro demonstrates how to add particles found by Find Maxima to the ROI Manager Points at maxima Multi point selection 29 5 Enhance Contrast IN IJ 1 46R bl Enhances image contrast by using either histogram stretching o or histogram equalization Both methods are described in detail in the Hypermedia Image Processing Reference Contrast Stretching and Histogram Equalization Analyze Particles C Normalize O Equalize histogram M Process all 27 slices O Use stack histogram This command does not alter pixel values as long as Normalize Equalize Histogram or Normalize All n Slices in the case of stacks are not checked Saturated Pixels Deter
8. Function Formula Comments Straight line y a bzx cf Pixel Inverter Analyzeb Calibrate The range of x values should not be too far from 0 especially for higher order polynomials gnd_gth d del y at ba ca ire Ath should fulfill vean O Max TMin a a 5th should fulfill Enean lt Guise Win 2 7h amp 8 should fulfill EMean K Max Min Power y axx Optionally fit can be performed without linear Boonen ETET regression during minimization Most curve fitting programs such as Microsoft Excel use Exponential ber regression Fitting without regression assumes 0X ross ES Te equal weight for all data points and is insensitive to zero or negative data E tial CA y ax 1 0 cf FRAP Profiler plugin recovery 7 Los y axln bx y a bxIn x c Rodbard y aoe 42 see DeLean A Munson PJ Rodbard D i Simultaneous analysis of families of sigmoidal Rodbard died NIH Image T Te curves application to bioassay radioligand assay j and physiological dose response curves Am J Inverse eS Ea 1 0 Physiol 1978 Aug 235 2 E97 102 PMID 686171 Rodbard d x Gaussian y a b a xe End 42 Gamma variate y b x x a xe a SEE ALSO E CurveFittingDemo E RodbardSigmoidFit and E PlotSigmoidDerivatives macros Profile Plot Options 149 Last updated 2012 10 02 Analyze gt Tools gt 30 14 5 ROI Manager Show All RO
9. TrakEM2 As mentioned earlier TrakEM2 features powerful tools for multi dimensional regions of interest 13 SEE ALSO Imager Stacksp 3D Project Orthogonal Views H Analyze gt Surface Plot XXI Skeletonize vs Skeletonize 3D 3D tools in Fiji Three Pane Crop 3D image processing tutorials on the ImageJ wikipage 2l Last updated 2012 10 02 Settings and Preferences 13 Settings and Preferences ImageJ preferences are automatically saved in a preferences file the ElIJ_prefs txt text file This file is stored in G Library Preferences on Mac OS X in G imagej on Linux and Windows with referring to the user s home directory Several macros and plugins also write parameters to this file If the E IJ_prefs txt is erased using Edit gt Optionsp Reset ImageJ will create a new one the next time it is opened resetting all parameters to their default values Sometimes it may be useful to override or restore certain settings that may have been changed during a working session For example the Limit to threshold option Analyze gt Set Measurements will affect most measurements performed on thresholded images Thus it may be wise to check the status of this parameter before each analysis specially when working on multiple computers 3 Ensuring Specific Settings at Launch macro AutoRun setOption DebugMode true setOption Bicubic true setOption Display Label true setUption Limit t
10. 0 Magenta 255 0 255 Red 255 0 0 Orange 255 150 0 HAG 33 19 00 HAC 33 419 400 HAC 33 19 00 HAC 33 419 400 5 4C 33 19 400 HAC 33 419 400 Yellow 255 255 0 99 7F 66 4C 33 19 00 White 255 255 255 FF HOC B2 499 7F 66 4C 433 19 00 FFFFFF Gray 127 127 127 WEG HB 09 7F 466 4C 33 19 00 66 4C 33 19 00 Wier Black 0 0 0 FF E5 CC B2 999 Alpha blending values can be added to the beginning of hex triplets to modulate color transparency e g FFTF FF 0000 defines red at 50 opacity In ImageJ the hash prefix is optional ColorChoosers in the Color Picker window display hex values of RGB colors The built in macro function toHex returns hexadecimal representations of decimal numbers and can also be used to convert RGB color values see RGBtoHEX macro Several other macros e g MakeOverlay exemplify how to annotate images using hex colors 103 Last updated 2012 10 02 Image gt Overlay gt 28 14 3 Hide Overlay Causes ImageJ to stop displaying the overlay displayed by Show Overlay 28 14 4 Show Overlay Displays an overlay that was hidden by Hide Overlay 28 14 5 From ROI Manager Creates an overlay from the selections on the ROI Manager list see Analyze gt Tools gt ROI Manager Note that previously added Overlays will be removed 28 14 6 To ROI Manager Copies the selections and images in the current overlay to the ROI Manager where they can be edited
11. 33 Pointer 45 72 Polar coordinates 119 Polygon selection 31 Polynomial regression see Curve fitting 147 Pop up menu see Contextual menu 41 Power spectrum 120 PPI 59 Preferences see Settings 21 66 Print 59 Processing 106 Progress bar 29 Projection 88 89 93 Proxy server 71 Python 26 Quit 59 R GNU S see Interoperability 3 4 RAM see Memory 28 70 Raw 56 Raw density 137 Reciprocal 118 Record see Macro recorder 155 Rectangular selection 29 Redo 9 Reduce 92 95 Reflex angles 33 Regex 50 51 Renyi see Threshold 77 Resampling see Interpolation 80 Reset 73 160 Results table 42 Revert 59 RGB 14 74 79 RGB 80 ROI see also Selection 17 ROI see Selection 16 ROI Manager 103 Rolling ball 129 Rotate 98 Rounded rectangle 30 Roundness 136 184 Ruby 26 Sagittal see Orthogonal views 88 SalsaJ 4 Sample Images 49 Scale 97 Scale bar 152 Scientific notation 137 Screenshot 159 Scrolling 36 Search 158 Segmented Line selection 32 Selection 16 Composite 18 65 Properties 64 Refine 30 Restore 62 ROI Manager 148 Settings 21 66 69 72 115 121 Shadows 110 Shanbhag see Threshold 77 Shape descriptors 136 Sharpen 106 Shortcuts 8 156 165 Simple Neurite Tracer 21 Simplex see Curve fitting 147 64 bit 70 Skeleton 113 Skewness 137 Sliding paraboloid see Rolling ball 129 Smooth 106 Sobel edge 106 Solidi
12. Converts a stack into a hyperstack RGB stacks are converted into 3 channel hyperstacks Order is the order of the channels c slices z and frames t within the stack ImageJ hyperstacks are always in czt order Stacks not in czt order will be shuffled to be in czt order The channel Display Mode can be Composite Color or Grayscale cf Channels Tool Z SEE ALSO Hyperstack to Stack Converts a hyperstack into a stack in czt order SEE ALSO Stack to Hyperstack 28 7 4 Reduce Dimensionality This command 26 reduces the dimensionality of an hyperstack by creating a new hyperstack with for example all the channels and time points at a given z position or all the z slices for the current channel and time point 96 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Image gt Crop X Uncheck Channels n to delete all but the current channel Slices n Create Image With to delete all but the current z slice and Frames n to delete all but the O Channels 2 current time point Check Keep Source and the original stack will not M Slices 5 be deleted M Frames 51 1x5x51 16MB The expected dimensions and size of the reduced stack are displayed in Eil Keep Source the dialog Cancel SEE ALSO Hyperstack to Stack 28 7 5 Channels Tool Z Opens the Channels widget or brings it to the front if it is already open is the keyboard shortcut for this command This t
13. It can create batch applications as well as 2 06 interactive applications The applications include the topics DNA combing quantification of stained proteins in cells comparison of intensity ratios between nuclei and cytoplasm and counting nuclei stained in different channels ObjectJ ObjectJ the successor of object image supports graphical vector objects that non destructively mark images on a transparent layer Vector objects can be placed manually or by macro commands Composite objects can encapsulate different color coded marker 3 Last updated 2012 10 02 Installing and Maintaining ImageJ Related Software structures in order to bundle features that belong together ObjectJ provides back and forth navigation between results and images The results table supports statistics sorting color coding qualifying and macro access SalsaJ SalsaJ is a student friendly software developed specifically for the EU HOU project It is dedicated to image handling and analysis of astronomical images in the classroom SalsaJ has been translated into several languages TrakEM2 TrakEM2 is a program for morphological data mining three dimensional modeling and image stitching registration editing and annotation 13 TrakEM2 is distributed with Fiji and capable of 3D modeling Objects in 3D defined by sequences of contours or profiles from which a skin or mesh can be constructed and visualized in 3D Relat
14. SEE ALSO Despeckle 29 6 6 Remove NaNs This filter replaces NaN Not a Number pixels in 32 bit float images by the median of the neighbors inside the circular kernel area defined by Radius 31 It does not remove patches of NaNs Help Cancel larger than the kernel size however he a Radius 2 0 pixels M Preview Note that some ImageJ filters such as Process gt Filters Gaussian Blur Mean and Variance destroy the surrounding of NaN pixels by setting it also to NaN Other filters may produce invalid results in the position of NaN pixels SEE ALSO NaNs txt a macro that demonstrates how to create count and remove NaNs 29 7 Shadows Commands in this submenu produce a shadow effect with light appearing to come from a direction corresponding to the command name East North Northeast Northwest South Southeast Southwest and West The commands use Convolve 3 x 3 ImageJ s 3 x 3 convolution function The Shadows Demo command uses all eight kernels to demonstrate the speed of Convolve 3 x 3 The illustration below shows four of the Shadows convolution kernels 111 Last updated 2012 10 02 Process gt Binary gt 1 2 1 1 2 1 1 0 1 1 0 1 Original 0 1 0 0 1 0 it 2 2 1 3 1 2 1 1 2 1 1 0 1 1 0 1 AAA Original tif AAP North tif AAA South tif AA East tif West tif 256x254 pixels 8 bit inverting LUT 64 x pixels x pixels 3 bit inverting p 256x254 pixel
15. Select All A wae ae waitForUser title message Zap Gremlins Copy to Image Info The ImageJ editor version 1 43n The editor is a simple text editor featuring Function Finder F an essential tool when writing Macros The Fiji Script Editor is a more advanced editor featuring syntax highlight and support to all of Fiji s scripting languages 23 Editor Macros Scripts and Plugins can be opened and executed in the ImageJ editor The editor commands are organized in five menus File Edit Font Macros and Debuge File gt Basic file operations Open Save Print etc are listed in this menu The last saving directory is kept in E IJ_prefs txt the IJ preferences file see Settings and Preferences Edit gt Similarly to any other text editor this menu contains commands related to text handling as well as commands for locating text Specially useful are Go to Line I Ctrl L This dialog box enables you to quickly go to a specified line of code Zap Gremlins This command finds and deletes the extraneous non visible non printing characters that sometimes appear when cutting and pasting from other sources such as email messages that may contain extraneous control characters or any non ASCII characters Copy to Image Info This command will copy the selected text or the entire contents of the editor if no selection is present to the image header being available through the Image gt Show Info i c
16. s FFT Filter plugin The Process Binary Fill Holes algorithm was contributed by Gabriel Landini The Skeletonize3D plugin was written by Ignacio Arganda Carreras based on an ITK imple mentation by Hanno Homann It implements a 3D thinning algorithm from Lee et al Building skeleton models via 3 D medial surface axis thinning algorithms CVGIP 56 6 462 478 1994 Multi threading support for all Process Filters gt commands was contributed by Stephan Saalfeld and Michael Schmid in ImageJ 1 45c The faster and more accurate version of Process Filters Gaussian Blur implemented in ImageJ 1 38r was contributed by Michael Schmid The NonBlockingGenericDialog class used by the Process Batch gt Macro command was added by Johannes Schindelin The rolling ball code of Process gt Subtract Background is based on the NIH Image Pascal version by Michael Castle and Janice Keller The sliding paraboloid algorithm was written by Michael Schmid The Analyzeb Distribution command was written by Gabriel Landini The scaled color bar implemented in Analyzep Histogram h was contributed by Bob Dougherty The much improved CurveFitter Analyze gt Tools gt Curve Fitting implemented in IJ 1 46f was contributed by Michael Schmid The Rodboard and Gaussian functions were originally contributed by David Rodbard NIH and Stefan W rz DKFZ respectively The ROI Manager XOR command Analyzeb Tools gt ROI Manager was added by
17. the support for 3D ROIs selections containing contiguous cluster of voxels is somewhat limited in ImageJ This limitation has been addressed by ImageJ2 and several IJ1 plugins The list below summarizes some of the ImageJ plugins that deal effectively with multi dimensional objects Note that a manual installation of these tools as standalone ImageJ plugins is a challenging task given their special dependencies reason why they are all bundled as part of Fiji 3D Filters Specialized 3D filters such as Process Filters gt Gaussian Blur 3D can be installed to perform 3D operations Examples are the 3D processing package by Thomas Boudier 51 and the 3D binary filters by Benjamin Schmid 3D Object Counter 3D Object Counter 3D OC counts and qualifies 3D objects in a stack 8 similarly to the 2D analysis performed by Analyze gt Analyze Particles It is complemented by 3D Roi Manager 51 a companion plugin that adds a 3D ROI Manager to ImageJ 3D Viewer 3D Viewer brings powerful hardware accelerated 3D visualization to ImageJ 101 extending the limited functionality of Imager Stacks gt 3D Project In the ImageJ 3D Viewer stacks can be displayed as texture based volume renderings surfaces or orthoslices It is macro recordable and can be used by other plugins as a high level programming library for 3D visualization Simple Neurite Tracer Simple Neurite Tracer allows semi automated segmentation of tubular structures in 3D 71
18. the title of the image will be printed at the top of the page Selection only If checked current selection will be printed instead of the entire image Rotate 90 If checked the image will be rotated 90 to the left before being printed Print actual size Considers the DPI information in the image header typically 72 cf Jpeg For a higher value use a unit of inch in the Analyzer Set Scale dialog E g setting Distance in Pixels to 300 Known Distance to 1 and Unit of Length to inch will set the DPI to 300 26 13 Print p Prints the active image The size of the printed image will normally be slightly less its size on the screen unzoomed Use the Page Setup dialog to increase of decrease the size of printed images Images larger than the page are scaled to fit Overlays are embedded when printing images 26 14 Quit Prompts you to save all unsaved images and then exits You can also exit ImageJ by clicking on the close button in its window s title bar 60 Last updated 2012 10 02 Edit gt 27 Edit 27 1 Undo z Described in Undo and Redo 27 2 Cut x Copies the contents of the current image selection to the internal clipboard filling the selection with the current background color SEE ALSO Edit Copy to System Paste Control 27 3 Copy c Copies the contents of the current image selection to the internal clipboard If there is no selection copies the entire active image The
19. 02 Analyze gt Set Measurements the Feret s diameter is displayed as Feret Angle as well as the minimum caliper diameter MinFeret The starting coordinates of the Feret diameter FeretX and FeretY are also displayed see also Feret s Diameter macro and Chamfer distances and Geodesic diameters plugin Integrated density The sum of the values of the pixels in the image or selection This is equivalent to the product of Area and Mean Gray Value With IJ 1 44c and later Raw integrated density sum of pixel values is displayed under the heading RawIntDen when Integrated density is enabled The Dot Blot Analysis tutorial demonstrates how to use this option to analyze a dot blot assay Median The median value of the pixels in the image or selection Skewness The third order moment about the mean The documentation for the Moment Calculator plugin explains how to interpret spatial moments Uses the heading Skew Kurtosis The fourth order moment about the mean Uses the heading Kurt Area fraction For thresholded images is the percentage of pixels in the image or selection that have been highlighted in red using Image Adjust gt Threshold T For non thresholded images is the percentage of non zero pixels Uses the heading Area Stack position The position slice channel and frame in the stack or hyperstack of the selection Uses the headings Slice Ch and Frame N B For line selections the heading Length is cre
20. 170 Last updated 2012 10 02 Credits C1 C2 Os C4 C5 C6 C7 Cs C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 The ImageJ installer for Windows is created using the Inno Setup installer generator ImageJ exe the Windows that launches ImageJ Elij jar was contributed by George Silva Support for ZIP compressed TIFFs was contributed by Jason Newton in IJ 1 45g The macro editor s Function Finder Macros gt Find Functions was written by J r me Mutterer The Elliptical Selection Tool was contributed by Norbert Vischer The Brush Selection Tool is based on the ROI Brush Tool plugin from Tom Larkworthy and Johannes Schindelin Jean Yves Tinevez and Johannes Schindelin authors of the Fiji Arrow Tool contributed code to the Arrow Tool Michael Schmid added 4 connected and 8 connected tracing with tolerance to the Wand Tool Macro Toolsets distributed with ImageJ have been contributed by Gilles Carpentier J r me Mutterer and Tiago Ferreira The Pixel Inspector is a plugin tool conversion of Michael Schmid s Pixel Inspector plugin In IJ 1 431 and earlier the File gt Import gt Results command was based on J r me Mutterer s Import Results Table macro Marcel van Herk added URLs support to the File Import Stack From List command in IJ 1 45f Michael Schmid contributed improvements to the
21. 2 2 gt gt Segmented Line Selection Tool Works exactly as described for the Polygon Selection Tool Create a segmented line selection by repeatedly clicking with the mouse Each click will define a new line segment Double click when finished or click in the small box at the starting point The points that define a segmented line selection can be moved or deleted and new points can be added Length and line angle are displayed in the status bar during drawing see Toggling Calibrated Units Modifier keys Shift clicking on an existing vertex adds a new one adding a new segment to the segmented line Alt Alt clicking on an existing vertex of the segmented line removes it SEE ALSO Polygon Selection Tool Freehand Selection Tool V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 2 3 Freehand Line Selection Tool Select this tool and drag with the mouse to create a freehand line selection SEE ALSO Freehand Selection Tool Overlay Brush V Toggling Calibrated Units XVII Embed ding Color Annotations in Grayscale Images Toolbar Shortcuts 19 3 Arrow Tool This tool shares the same toolbar slot with the Line Selection Tools and can also be installed on a dedicated toolbar slot using the More Tools Menu menu see Arrow Double clicking on the tool icon opens its Options prompt 6 32 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R
22. 2012 10 02 Edit gt Options gt 27 13 Options Use commands in this submenu to change various ImageJ user preference settings 27 13 1 Line Width Displays a dialog box that allows to change the line width in pixels of line selections see Line Selection Tools and concomitantly the lines gen eee erated by the Edit Draw d command This legacy command has been superseded by the Image Adjust gt Line Width widget but required since the latter is not recordable see Plugins gt Macros Record 27 13 2 Input Output IN IJ 1 46R 1 0 Options JPEG quality 0 100 Specifies the compression JPEG quality 0 100 75 level used by File gt Save Asp Jpeg Requesting a GIF and PNG transparent index 1 higher degree of compression a lower value will Al atencion boctabless te result in smaller files but poorer image quality Note that lossy JPEG compression creates serious L Use JFileChooser to open save artifacts see II Image Types Lossy Compression O Save TIFF and raw in Intel byte order and Metadata Results Table Options M Copy column headers GIF and PNG transparent index Specifies the Y Copy row numbers transparent color used for images saved in GIF Sawe column headers Save row numbers and PNG formats Use 1 for none Note that PNG and GIF transparency only works with 8 bit Cancel ok images File extension for tables Sets the default extension to be used
23. 24 bit RGB images allow 16 7 million of colors see Image Types and Formats and concomitantly smaller file sizes Reduction of true color values to a 256 color palette is performed by color quantization algorithms ImageJ uses the Heckbert s median cut color quantization algorithm see Image Type gt menu which in most cases allows indexed color images to look nearly identical to their 24 bit originals SEE ALSO Image Lookup Tables gt and LUT Menu True Color Images As described in Image Types and Formats true color images such as RGB images reflect genuine colors i e the green in an RGB image reflects green color in the specimen Color images are typically produced by color CCD cameras in which color filter arrays Bayer masks are placed over the image sensor Color Spaces and Color Separation Color spaces describe the gamut of colors that image handling devices deal with Because human vision is trichromatic most color models represent colors by three values Mathematically these values color components form a three dimensional space such as the RGB HSB CIE Lab or YUV color space RGB Red Green Blue is the most commonly used color space However other alternatives such as HSB Hue Saturation Brightness provide significant advantages when processing color information In the HSB color space Hue describes the attribute of pure color and therefore distinguishes between colors Saturation sometimes called p
24. 44d an later ImageJ no longer automatically installs at startup plugins in JAR file directories that start with a lower case letter Developing ImageJ Plugins More information on how to develop ImageJ plugins can be obtained on the following documents 1 Developer Resources Page on the ImageJ website Help Dev Resources http imagej nih gov 1ij developer index html 2 Dedicated tutorials on Fiji s webpage http fiji sc wiki index php Introduction_into_ Developing Plugins 3 Dedicated tutorials on the ImageJ Documentation Portal http imagejdocu tudor lu 4 Dedicated tutorials on the ImageJDev webpage http developer imagej net ides SEE ALSO Macros Scripts Editor Fiji Script Editor 20 Last updated 2012 10 02 Running ImageJ From the Command Line 17 Scripting in Other Languages Support for other languages is possible in ImageJ using Fiji and its powerful editor Fiji adds extra support for BeanShell Clojure Python and Ruby The following documents will introduce you to the advanced scripting capabilities of Fiji 1 The extensive tutorial on scripting Fiji with Jython by Albert Cardona http www ini uzh ch acardona fiji tutorial 2 Dedicated tutorials on the Fiji webpage http fiji sc wiki index php Scripting comparisons Fiji Script Editor Fiji features a more powerful script editor than ImageJ s built in Editor The Fiji editor is an invaluable help when writ
25. 5 ImageJ for Microscopy see MBF ImageJ 3 ImageJ2 5 10 ImageROI 20 66 100 Imaris 4 Immunohistochemistry see Histochemical staining 15 Import 50 Installation 164 Integrated density 137 Interleave 93 Interoperability 4 Interpolation 68 80 IsoData see Threshold 77 Java 23 24 67 70 159 JavaScript 23 24 JFileChooser 67 JPEG 11 13 55 Quality 67 JPEG2000 12 Jython see Python 26 Keyboard see Shortcuts and Modifier keys 8 Knime 4 KNIP see Knime 4 Kurtosis 137 Label ROIs 149 152 Lanes see 1 D Gel analysis 143 183 Layers see ImageROI Overlays 101 Levels 76 Levels see Brightness amp Contrast 75 Li see Threshold 77 Linear regression see Curve fitting 147 Linux 2 LOCI Bio Formats see Bio Formats 11 54 Log 118 Logical operations Images 128 ROIs 150 Lossless compression 10 Lossy compression 12 56 LUT 14 53 58 104 LUT Menu 38 LZW compression 10 Mac OS X 2 Macro functions 44 163 Macro recorder 155 Macro tools 40 Macros 23 119 126 155 Install 155 Magenta Green Blue MGB 16 Magnifying Glass Tool 35 Mailing lists see Help resources 6 Mask 64 111 112 125 Math 117 MATLAB see Interoperability 4 Max 118 MaxEntropy see Threshold at Maxima 106 MBF ImageJ 3 6 13 Measure 126 131 Measurements 135 Memory 28 51 52 70 159 Metadata 12 81 pManager 3 MIJ see Interoperability 4
26. 82 83 84 85 86 87 88 89 F Leymarie and M D Levine Fast raster scan distance propagation on the discrete rectangular lattice CVGIP Image Understanding 55 1 84 94 1992 doi DOI 10 1016 1049 9660 92 90008 Q Richard M Lindley Daniel B Hawcutt M Gwen Connell David H Edgar and Simon E Kenny Properties of secondary and tertiary human enteric nervous system neurospheres J Pediatr Surg 44 6 1249 55 discussion 1255 6 Jun 2009 doi 10 1016 j jpedsurg 2009 02 048 Mark H Longair Dean A Baker and J Douglas Armstrong Simple neurite tracer open source software for reconstruction visualization and analysis of neuronal processes Bioinformatics 27 17 2453 4 Sep 2011 doi 10 1093 bioinformatics btr390 H M Macdonald D M L Cooper and H A McKay Anterior posterior bending strength at the tibial shaft increases with physical activity in boys evidence for non uniform geometric adaptation Osteoporos Int 20 1 61 70 Jan 2009 doi 10 1007 s00198 008 0636 9 Philippe Mailly Suzanne N Haber Henk J Groenewegen and Jean Michel Deniau A 3D multi modal and multi dimensional digital brain model as a framework for data sharing J Neurosci Methods Dec 2009 doi 10 1016 j jneumeth 2009 12 014 Peter A McAtee Ian C Hallett Jason W Johnston and Robert J Schaffer A rapid method of fruit cell isolation for cell size and shape measurements Plant Methods 5 5 Jan 2009 doi 10 1186 1746 4811 5 5 R
27. AVI reader and AVI writer plugins Karen Collins contributed improvements to the FITS Writer File gt Save Asp FITS command The Edit Selection gt Fit Circle command based on a MATLAB script by Nikolai Chernov was contributed by Michael Doube and Ved Sharma The Edit Selection gt Create Selection command is based on the Threshold _ To Selection plugin written by Johannes Schindelin IJ 1 46f adopted Johannes Schindelin s RMI based OtherInstance class from Fiji which works on multi user machines and is more secure The Color Picker Image Colort Color Picker K was written by Gali Baler a 2003 2004 intern from Bethesda Chevy Chase High School The 16 different thresholding methods available in the Image Adjust gt Threshold T tool were implemented by Gabriel Landini Michael Schmid contributed improvements to the downsizing kernel used by Image Adjust gt Size and Imagep Scale E as well as undo support for Imagep Scale E The Image Adjust gt Color Threshold command implements Gabriel Landini s Threshold Colour plugin The Reslice and the ZProject plugin Image gt Stacks Reslice and Z Project commands were contributed by Patrick Kelly and Harvey Karten of the University of California San Diego The Imagep Stacks Orthogonal Views H command is based on Dimiter Prodanov s StackSlicer plugin and Albert Cardona s Updater class Michael Doube added support for XZ and YZ view con
28. Apply Brightness slider Increases or decreases image brightness by moving the display range Hold ing down will simultaneously adjust all channels of a composite image Contrast slider Increases or decreases contrast by varying the width of the display range The narrower the display range the higher the contrast Holding down will simultaneously adjust all channels of a composite image Auto ImageJ will automatically optimize brightness and contrast based on an analysis of the image s histogram Create a selection and the entire image will be optimized based on an analysis of the selection The optimization is done by allowing a small percentage of pixels in the image to become saturated displayed as black or white Each additional click on Auto increases the number of saturated pixels and thus the amount of optimization A run Enhance Contrast saturated 0 35 macro call is generated if the command recorder Plugin Macro Record is running Reset Restores the original brightness and contrast settings The display range is set to the full pixel value range of the image A resetMinAndMax macro call is generated if the command recorder is running Holding down restores original settings in all channels of a composite image Set Allows to enter the minimum and maximum display range values in a dialog box A setMinAndMax macro call is generated if the command recorder is running 76 Last updated 2012 10 02 IMPROV
29. Be Ge ates Be oes ae a E eee oe ea 39 ES AR cre 2 eo tat a sheet tue de A 39 TOS Overlay DTS em dE ve OS ADA A we eS ee a 39 OPO o ss he tay ic weds eae ee nh ae Eek eee eRe ete O aces Gop ae che G1 he ode oe 40 1920 PIIN pCO gt Es EDS AREA E A Sx 40 A A E cat sat Vk cee E AR 41 MR a e AA II RE 41 20 Custom Tools 41 21 Contextual Menu 42 22 Results Table 43 23 Editor 44 24 Log Window 46 25 Customizing the ImageJ Interface 46 25 Floating Behavior of Malm Window 4 284 4 iea8 445 sea 46 A POMC E ye SH Ee es Be A7 V Menu Commands 26 File gt 26 1 49 NA II oe EY 49 111 202 Ope ss Ola ox ewe ae a 26 3 Open Next O 26 4 Open Samples gt 26 5 Open Recentb 26 0 IMPOFED gt ara 26 7 Close w exc 26 8 ClosesAll cesa os ss 209 AA 26 10 Save Sb 26 11 Revert r e si s e me Ze 26 12 Page Setups 2 x aca ai DOM or Ns se WO ee end ee aks De NOME a Se alas oes eho 27 Edit gt Zeal AUNGON Z sorna 2D CUE ea oe atte Di COPY lOs od dee 27 4 Copy to System ao Pastel sue nia 27 6 Paste Control Pi O as Hews oats eet Ee a 27 8 Clear Outside lb a ecw a 2 EME mh acide anh ooh eS ce ZIM Draw fd reos ro A A 27 12 Selection ARES D aa a oe HR 28 Image DON TYDER ers od Bead 28 2 Adjust iio et 28 3 Show Info i 28 4 Properties P 285 5 COMORES e cai wh 23 0 SEACKSE ei a a 28 7 Hyperstacksb DSS CODA
30. Character Classes Example Meaning Delimit the set of characters to match aA Either lower or upper case A Character ranges 0 9 Any digit from 0 through 9 l Any character 0 9 A digit plus any other character t Zero or more of the preceding item Any character sequence Zero or one of the preceding item 0 9 An optional digit One or more of the preceding item O 9 At least a digit Negation 0 9 Any character that is not a digit amp amp AND Intersection 0 98z82 3 A digit that is not 3 OR Alternation 0 9 l a zA Z A digit or lower or upper case letter 92 Last updated 2012 10 02 IMPROVED IN IJ 1 46R File gt Import gt IX REDUCING MEMORY REQUIREMENTS WHEN IMPORTING IMAGES Since ImageJ 1 44d the File Import gt gt Image Sequence command no longer features the Convert to 8 bit Grayscale checkbox This option was used to reduce memory requirements but used different scaling for each imported image As a replacement use the Use virtual stack option and then convert to 8 bit using File Type 8 bit Memory requirements can also be reduced by using the Scale Images option The amount of memory allocated to ImageJ can be adjusted in Edit gt Options Memory amp Threads Use this command to import images that are not in a file format directly supported by ImageJ You will need to know certain in R16 bit Signed formation about the layout of the image
31. Commands in this submenu such as Inverse FFT operate on the 32 bit FHT not on the 8 bit power spectrum All other ImageJ commands only see the power spectrum Two FFT dedicated tutorials are available on the ImageJ website FFT Measurements and FFT Filtering 29 10 1 FFT Computes the Fourier transform and displays the power spectrum Polar coordinates of measured point selections are recorded by Analyze gt Measure m If the mouse is over an active frequency domain FFT window its location is displayed in polar coordinates The angle is expressed in degrees while the radius is expressed in pixels per cycle p c The radius is expressed in units per cycle e g mm c if the spatial scale of the image was defined using Image Properties P or Analyze Set Scale Although outdated the ImageFFT documentation summarizes important frequency domain methodologies 120 Last updated 2012 10 02 Process gt FFT gt 29 10 2 Inverse FFT Computes the inverse Fourier transform You can filter or mask spots on the transformed frequency domain image and do an inverse transform to produce an image which only contains the frequencies selected or which suppresses the frequencies selected Use ImageJ s selection tools and fill clear commands to draw black or white areas that mask portions of the transformed image Black areas pixel value 0 cause the corresponding frequencies to be filtered removed and white a
32. Composite Selections The brush diameter can be adjusted by double clicking on the tool icon Modifier keys Holding Shift forces the Brush Selection Tool to add pixels to the selection Alt Holding Alt forces the Brush Selection Tool to subtract pixels from the selection SEE ALSO XIII Converting Composite Selections Toolbar Shortcuts 19 1 6 Polygon Selection Tool Creates irregularly shaped selections defined by a series of line segments Segment length and angle are displayed in the status bar during drawing see V Toggling Calibrated Units To create a polygon selection click repeatedly with the mouse to create line segments When finished click in the small box at the starting point or double click and ImageJ will automatically draw the last segment The vertex points that define a polygon selection can be moved and modifier keys can be used to delete or add new vertexes to the polygon Modifier keys Shift clicking on an existing vertex of the polygon adds a new corner point smoothing the polygon edge Alt Alt clicking on an existing vertex of the polygon removes it SEE ALSO Segmented Line Selection Tool Enlarge V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 1 7 Freehand Selection Tool As with the polygon selection tool ImageJ automatically draws the last segment Location and intensity of starting pixel are displayed in the status bar during dr
33. Copies the screen to an RGB image and displays that image in a new window Holding will capture the screen while a modal dialog box is active if the dialog is based on ImageJ s GenericDialog class SEE ALSO Capture Image Flatten F 31 3 7 Capture Image Copies a WYSIWYG version of active image to an RGB image and displays that image a new window SEE ALSO Flatten F Capture Screen g 31 3 8 ImageJ Properties This command displays various ImageJ properties Java version OS name and version path separator location of directories screen size etc in a text window Holding lists all Java properties SEE ALSO Status bar Help gt About ImageJ 31 3 9 Threads This command lists in a text window the currently running threads and their priorities 31 3 10 Benchmark Runs 62 image processing operations on the current image and displays the elapsed time in the ImageJ status bar Additional benchmarks test results and source code are available in the Benchmarks package of plugins SEE ALSO FAQs on the ImageJ Wikipage 160 Last updated 2012 10 02 IJ 1 46R Plugins gt New gt 31 3 11 Reset Use this command to unlock a locked Image or to reclaim memory used by the clipboard and undo buffers cf Undo and Redo K Clipboard Undo Buffer SEE ALSO Edit gt Options Reset 31 4 New This submenu contains commands opening editor windows that can be used to edit and run macros scri
34. Edit gt Options gt Conversions Copy to clipboard Specifies which data is copied to the clipboard Choose Data only to copy the table without headers x y and Data to copy the current position x y values followed by remaining data or Header and Data to copy the table with headers Tables are copied as tab delimited values SEE ALSO Text Images Imagep gt Transform gt Image to Results Results to Image File gt Save Asp Text Image Import gt Text Image Toolbar Shortcuts 40 Last updated 2012 10 02 IJ 1 46R IJ 1 46R IJ 1 46R IJ 1 46R IJ 1 46R Custom Tools Spray Can 19 21 Spray Can The Spray Can Airbrush tool draws random pixels in the current foreground color paint see Color Picker K and XVII Embedding Color Annotations in Grayscale Images It behaves as a traditional airbrush or spray paint Holding the main mouse button without moving the cursor will build up paint as if pressing the nozzle of an aerosol paint can Spray width Dot size and Flow rate can be specified by double clicking on the tool icon This tool is useful to generate random spot noise Use it to e g assess the effectiveness of median filtering Load the Spray Can tool apply it over an image and toggle the Preview option in the Process Filters gt Median prompt SEE ALSO Process Noise gt Add Noise Salt and Pepper Toolbar Shortcuts 19 22 gt gt s Stacks Menu A drop down menu collecting several comma
35. J M Fischer Sae Uchida and Karl Messlinger Measurement of meningeal blood vessel diameter in vivo with a plug in for ImageJ Microvasc Res 80 2 258 66 Sep 2010 doi 10 1016 j mvr 2010 04 004 Manuel G Forero Jenny A Pennack Anabel R Learte and Alicia Hidalgo DeadEasy Caspase Automatic Counting of Apoptotic Cells in Drosophila Plos One 4 5 e5441 Jan 2009 doi 10 1371 journal pone 0005441 Seth T Gammon W Matthew Leevy Shimon Gross George W Gokel and David Piwnica Worms Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources Anal Chem 78 5 1520 7 Mar 2006 doi 10 1021 ac051999h Max Gassmann Beat Grenacher Bianca Rohde and Johannes Vogel Quantifying Western blots pitfalls of densitometry Electrophoresis 30 11 1845 55 Jun 2009 doi 10 1002 elps 200800720 Eben Gering and Carter T Atkinson A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis J Parasitol 90 4 879 81 Aug 2004 Nicola Goodall Lilian Kisiswa Ankush Prashar Stuart Faulkner Pawel Tokarczuk Krish Singh Jonathan T Erichsen Jez Guggenheim Willi Halfter and Michael A Wride 3 Dimensional modelling of chick embryo eye development and growth using high resolution magnetic resonance imaging Exp Eye Res 89 4 511 21 Oct 2009 doi 10 1016 j exer 2009 05 014 John D Gottsch Olof H Sundin Erik V Rencs David G Emmert Walter J Stark Clement J Cheng and Gr
36. Johannes Schindelin The ROI Manager Multi Measure command Analyze gt Tools gt ROI Manager is based on Bob Dougherty s Multi Measure plugin The ROI Manager Multi Plot command Analyze gt Tools gt RO Manager was contributed by Philippe Gendre The Analyze gt Tools gt Synchronize Windows command an improved version of the SyncWindows _ plugin by Patrick Kelly was contributed by Joachim Walter The Control Panel Plugins Utilities gt Control Panel U was written by Cezar M Tigare The Command Finder Plugins Utilities gt Find Commands was written by Mark Longair The E PlugInTool class was inspired by Johannes Schindelin s Abstract Tool class in Fiji Other additions improvements and reproducible bug reports have been contributed by Adrian Daerr Airen Peraza Ajay Gopal Albert Cardona Alberto Duina Alden Dima Andreas Maier Andrew French Andrii Savchenko Arttu Miettinen Aryeh Weiss Balazs Nyiri Barry DeZonia Bill Mohler Bob Hamilton Bob Loushin Bruno Vellutini Burri Olivier Carlos Becker Carne Draug Charles Anderson Cheryl McCreary Christian Moll Christophe Leterrier Christopher Harrison Damon Poburko Daniel Hornung Daniel Kalthoff Daniel Senff David Gauntt David McDonald Denny Hugg Dimiter Prodanov Divakar Ramachandran Dorai Iyer Duncan Mak 1172 Eik Schumann Emmanuel Levy Erik Meijering Fabian Svara Francis Burton Frank Sprenger Franklin Shaffer Frederic
37. Log window In the Log Window double click on a file path 1 Image samples mitosis test data zip to have it open by ImageJ 25 Customizing the ImageJ Interface Most settings determining the look and feel of ImageJ are listed in Edit Options gt namely Edito Options Appearance and Edit Options Misc see also Settings and Preferences However other aspects of the ImageJ interface can also be personalized 25 1 Floating Behavior of Main Window It is possible to place the Main ImageJ window above all other windows at all time using a simple JavaScript instruction 1J getInstance setAlwaysOnTop true To test it copy this one line script to the clipboard or download ElAlways_on_Top js from the online scripts repertoire switch to ImageJ type File gt Newb System Clipboard V then type Macros Evaluate JavaScript j To create an Always on Top command save this script in the plugins folder as E Always_on_Top js and run Helpe Refresh Menus to start using the new command Macro 5 Customizing the Float Behavior of IJ s Main Window exemplifies how to set this option at launch SEE ALSO I I Frontmost Window and Window Activation 46 Last updated 2012 10 02 J 1 46R Customizing the ImageJ Interface Pointer Examples of modified crosshair pointers more visible on grayscale images The default crosshair cursor can be replaced by any image saved as E crosshair cursor gif in GImageJ images
38. Min 118 MinError see Threshold 77 Minima 107 MIP see Projection 89 MJPG 11 54 Modeling see TrakEM2 and Bio 4 Modifier keys 8 166 Moments see Threshold 77 Montage see Stacks Montage 87 More Tools Menu 36 Morphological operators 111 Multi Plot 151 Multi point tool 34 Multiplicative inverse see Reciprocal 118 Multiply see Math 117 Multiply images see Image calculator 127 NaN 110 119 Navigator palette 153 Nelder Mead see Curve fitting 147 NetBeans 25 Noise 40 106 107 109 124 128 Non background pixels 145 Non destructive annotations see Overlay 19 100 OME Bio Formats see Bio Formats 11 54 Opacity see Blend 102 Optical density see Calibration Optical density 138 Options see Settings 21 66 OR see Logical operations 128 150 Orthogonal views 88 Otsu see Threshold 77 Outliers 110 Outline 113 Oval selection 30 Overlay 19 51 100 101 150 Labels 103 Options 104 ROI Manager 103 150 Overlay Brush 38 PackBits compression 10 Paint Bucket Tool see Flood Filler 38 Paintbrush see Brush and Overlay Brush 37 Panel figures see Stacks Montage 87 Paste 60 Pencil 39 Perimeter 136 PGM 11 Pixel Inspector 39 Pixel size 82 Planar views see Orthogonal views 88 Plot profile 68 91 141 142 151 Plugin Tools 40 Plugin tools 40 Plugins 23 24 163 plugins config 47 PNG 11 58 Transparency 67 Point tool
39. Opens a stack or virtual stack from a text file or URL containing a list of image file paths 11 The images can be in different folders but they must all be the same size and type The Virtual Stack From List macro demonstrates how to generate a list of images and then use that list to open the images as a virtual stack The OpenStackUsingURLs macro demonstrates how to how to open an image series from a remote server 26 6 9 TIFF Virtual Stack Opens a TIFF file as virtual stack see Virtual Stacks and III Opening Virtual Stacks by Drag amp Drop 26 6 10 AVI Uses a built in version of the AVI reader plugin to open an AVI file JPEG or PNG compressed or uncompressed as a stack or virtual stack one slice per video frame 12 Animation speed is retrieved from image frame rate AVI files can also be opened using File gt Open o or drag and drop but macros must use this command to O Use Virtual Stack gain access to the dialog box options C Convert to Grayscale O Flip Vertical ImageJ supports a restricted number of AVI formats including MJPG Cancel motion JPEG and various YUV 4 2 2 4 2 0 compressed formats cf plugin source code The OME Bio Formats library see Non native Formats extends support to MSRLE and MSV1 encoded formats First Frame 1 Last Frame 27 The dialog promt allows you to choose if frames should be converted to 8 bit grayscale or flipped vertically For large files an option to open the
40. Outlines 0 Background 255 If In situ Show is checked the original image will be replaced by this image Masks 8 bit binary image containing filled outlines of the measured particles gray levels Masks 0 Background 255 If In situ Show is checked the original image will be replaced by this image Ellipses 8 bit binary image containing the best fit ellipse cf Edit gt Selection gt Fit Ellipse of each measured particle gray levels Ellipses 0 Background 255 If In situ Show is checked the original image will be replaced by this image 133 Last updated 2012 10 02 Analyze gt Analyze Particles Count Masks 16 bit image containing filled outlines of the measured particles painted with a grayscale value corresponding to the particle number If In situ Show is checked the original image will be replaced by this image Overlay Outlines Displays outlines of the measured particles in the image overlay removing previously added Overlays Overlay Masks Displays filled outlines of the measured particles in the image overlay removing previously added Overlays Original thresholded Outlines Bare Outlines Masks ABLA blobs gif Masks of blobs gif 256x254 pixels 8 bit inverting LUT 64 Ellipses Count Masks Ellipses of blobs gif Count Masks of blobs gif 256x254 pixels 8 bit 64K x pixels 16 bit K p Do Od D
41. ROI Manager in which selections are being added allowing ROIs to be on hold This concept of multiple distinct selections has been dramatically improved in ImageJ2 so we urge you to download IJ2 if multiple ROIs are important in your workflows Importantly overlay selections are vector graphics composed of mathematically defined paths as opposed to raster graphics in which objects are defined by pixels and are not affected by scaling i e do not become pixelated Most of overlay related commands are listed in the Image gt Overlay gt and in the ROI Manager window Analyzeb Tools gt ROI Manager Appearance of overlay selections can be adjusted using Image Overlay gt Overlay Options Labels As mentioned in II Image Types Lossy Compression and Metadata overlays are saved in the header of tif images and do not need to be saved externally when using TIFF the default file format of ImageJ The major advantages of overlays are summarized below Storage of ROIs In ImageJ it is only possible to have a single ROI at a time However it is possible to add selections to the image overlay using Image gt Overlay gt Add Selection b Once added to the image overlay ROIs can be re activated by Alt clicking Control clicking or long pressing 1 4 second or longer Activated ROIs can be deleted by pressing the key Selections can also be added and recovered in bulk using the Image Overlay gt From ROI Manager To ROI M
42. ROIs to create a composite selection Combines all ROIs if none is selected XOR Uses the exclusive or operator on the selected ROIs to create a composite selection 43 All ROIs are considered if none is selected Split Splits the current selection it must be a composite selection into its component parts and adds them to the ROI Manager Add Particles Adds objects segmented by the particle analyzer to the ROI Manager Requires that Record Starts be checked in the Analyze gt Analyze Particles dialog box Particle analyzer objects can also be added to the ROI Manager by checking Add to Manager in the Analyze Particles dialog box 151 Last updated 2012 10 02 Analyze gt Tools gt Multi Measure F Measure All 27 Slices Multi Measure Measures all the ROIs on all slices in the One Row Per Slice stack creating a Results Table with either one row per Enabling both options will result slice if One Row Per Slice is checked in the dialog or one row per measurement 44 in a table with 90 columns Cancel ok MM Logical operations using the ROI Manager Analyze gt Tools ROI Manager Multi Plot Runs Analyze gt Plot Profile k on the selected ROIs on a single graph 45 All selections are plotted if none is selected When plotting less than seven selections colored lines are drawn blue ROI 1 green ROI2 magenta ROI3 red ROI 4 cyan ROI 5 and yellow ROI 6 Profiles with m
43. SEE ALSO Noise Add Noise Accurate Gaussian Blur plugin AnimatedGaussianBlur macro 29 11 38 Gaussian Blur 3D This command calculates a three dimensional 3D gaussian lowpass filter using a 3 D Gaussian It works with Stacks and Hyperstacks but not single slice Color Composite Images Refer to Gaussian Blur for more information on sigma values SEE ALSO Gaussian Blur 3D source code 29 11 4 Median Reduces noise in the active image by replacing each pixel with the median of the neighboring pixel values 29 11 5 Mean Smooths the current image by replacing each pixel with the neighborhood mean 29 11 6 Minimum This filter does grayscale erosion by replacing each pixel in the image with the smallest pixel value in that pixel s neighborhood SEE ALSO Binary Erode 29 11 7 Maximum This filter does grayscale dilation by replacing each pixel in the image with the largest pixel value in that pixel s neighborhood SEE ALSO Binary Dilate 125 Last updated 2012 10 02 Process gt Batch gt 29 11 8 Unsharp Mask Sharpens and enhances edges by subtracting a blurred version of the image the unsharp mask from the original Radius Sigma 1 0 pixels Unsharp masking subtracts a blurred copy of the image Mask Weight 0 1 0 9 0 60 and rescales the image to obtain the same contrast of large low frequency structures as in the input image This is equivalent to adding a high pass filtered image
44. Stack histogram If checked ImageJ will first compute the histogram of the whole stack or hyperstack and then compute the threshold based on that histogram As such all slices are binarized using the single computed value If unchecked the threshold of each slice is computed separately Auto Uses the currently selected thresholding method to automatically set the threshold levels based on an analysis of the histogram of the current image or selection Apply Sets thresholded pixels to black and all other pixels to white For 32 bit float images Apply will also run Process gt Math gt NaN Background Reset Disables thresholding and updates the histogram Set New threshold levels can be entered into this dialog box SEE ALSO XX Creating Binary Masks Color Threshold Wand Tool Analyzer Analyze Parti cles 28 2 5 Color Threshold Thresholds 24 bit RGB images based on Hue Saturation and Brightness HSB Red Green and Blue RGB CIE Lab or YUV components Ranges of the filters can be set manually or based on the pixel value components of a user defined ROI This command implemented in version 1 431 is an experimental built in version of the Threshold Colour plugin 20 and is not yet fully integrated into ImageJ AAA FluorescentCells tif E O FluorescentCells tif Threshold Color experimental RGB IME ME 4 Thresholding method Default H Threshold color Black 34 Color space RGB
45. Starting Image Increment Scale Images Information width x height xdepth size of the stack to be created is displayed at the bottom of the dialog File Name Contains or Enter Pattern png Number of Images Specifies how many images to open O Convert to RGB Sort Names Numerically O Use Virtual Stack 198 x 255 x 28 5 4MB a Increment If set to 2 every other image will be opened if set to 3 to every third image will be opened etc Starting Image If set to n import will start with the n image in the folder File Name Contains Enter a string into this field and ImageJ will only open files whose name contains that string ol Last updated 2012 10 02 File gt Import gt Enter Pattern Regular expressions regex can be typed here for advanced filtering see Basic syntax of regular expressions Scale Images Setting a value less than 100 will reduce memory requirements E g entering 50 reduces the amount of memory needed to open a stack by 25 two dimensional images 0 5 x 0 5 0 25 of the original data This value is ignored if Use Virtual Stack is checked Convert to RGB Allows a mixture of RGB and grayscale images to be opened by converting all the sequence to RGB Note that if this option is unchecked and the first imported image is 8 bit then all the remaining images in the sequence will be converted to 8 bit Checking this option circumvent
46. The second group controls measurement settings The eighteen checkboxes of the first group are Area Area of selection in square pixels or in cali brated square units e g mm pm etc if M Area M Mean gray value Analyze gt Set Scale was used to spatially M Standard deviation O Modal gray value calibrate the image O Min max gray value M Centroid Center of mass M Perimeter C Bounding rectangle J Fit ellipse Mean gray value Average gray value within the M Shape descriptors O Feret s diameter selection This is the sum of the gray val D Integrated density L Median ues of all the pixels in the selection divided O Skewness O Kurtosis l ER G sack positon by the number of pixels Reported in cali ad S brated units e g optical density if Analyze O Invert Y coordinates O Scientific notation Calibrate was used to calibrate the im Y Add to overlay age For RGB images the mean is calculated Redirect to None b by converting each pixel to grayscale using Decimal places 0 9 3 the formula gray red green blue 3 Or gray 0 299 xred 0 587 x green 0 114 x blue Help Cancel ok _ if Weighted RGB Conversions is checked in Edit Options gt Conversions 136 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Analyze gt Set Measurements Standard deviation Standard deviation of the gray values used to generate the mean gray value Uses the Results Table heading StdDev Modal g
47. Tools Arrow 19 13 gt 4 Arrow Installs a copy of the Arrow Tool on the first available toolbar slot or the last if no free slots are available so that it can be accessed without the need of selecting it on the Line Selection Tools dropdown menu Refer to the original Arrow Tool for details and modifier keys SEE ALSO Color Picker window XVII Embedding Color Annotations in Grayscale Images Brush Overlay Brush Pencil Text Tool Toolbar Shortcuts 19 14 gt 4 Brush A freehand paintbrush tool that draws invasively as opposed to the Overlay Brush that draws on a non destructive image over Color white 4 lay see Overlays and Image blobs gif 54 pixels 8 bit inverting Brush width a Shift drag to change width Overlay A commands Alt drag to draw in background color i tg Color Picker shift k changes color Double clicking on the tool icon opens its Options dialog box Undo in which is possible to specify the Brush width in pixels and Color Being an annotation tool the paintbrush paints in foreground color as reflected its icon see XVII Embedding Color Annotations in Grayscale Images when working with non RGB images The Color dropdown menu provides a convenient way to reset the foreground color to one of the default options bypassing the need of opening the Color Picker window evoked using As previously described see Undo and Redo undo is restricted to last drawing step The Brush
48. Wand Tool icons Draw ing colors are displayed in the Color Picker Tool foreground and background colors and drawing tools such as the Arrow Brush Cancel OK Flood Filler and Pencil foreground color only Background white Selection magenta SEE ALSO Imager Color Color Picker K Using a Keyboard Shortcut to Change Selection Color 7 Using a Keyboard Shortcut to Change Selection Color This macro loops through the all the possible Selection colors using q as a keyboard shortcut Var cIdx macro Change Selection Color q color newArray red green blue magenta cyan yellow orange black white run Colors selection color lcldz 10 gt if cIdx color length cldx 0 i 27 13 10 Appearance IN IJ 1 46R This dialog contains options that control how images are dis Bosse eee ee played an option to display better looking toolbar icons and J Open images at 100 an option to set the menu font size _ Black canvas OU No image border Interpolate zoomed images Uses interpolation instead of O Use inverting lookup table E Antialiased tool icons pixel replication when displaying zoomed images Menu font size lo points Open Images at 100 Newly open images are displayed Help Cancel OK using 100 magnification 1 image pixel 1 screen pixel a 70 Last updated 2012 10 02 Edit gt Options gt Black Canvas Causes the
49. What is inside the selection will be outside and vice versa 64 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IJ 1 46R IMPROVED IN IJ 1 46R Edit gt Selection gt 27 12 10 Create Mask Creates a new 8 bit image called Mask whose pixels have a value of 255 inside the selection and 0 outside see ROI manipulations By default this image has an inverting LUT so black is 255 and white is O unless Black Background in Process Binary gt Options is checked SEE ALSO Process Binary Convert to Mask XX Creating Binary Masks XXII Interpreting Binary Images 27 12 11 Create Selection Creates a selection from a thresholded image or a binary mask 15 27 12 12 Properties y Opens a dialog box that allows the user to assign a contour color Stroke color and a contour width Width to the active Name MySelection selection or a filling color Note that selections can be either Stroke color fics00_ d filled or contoured but not both The default selection colors Width 3 black blue cyan green magenta orange red white yellow can be typed textually Any other color must be typed using hex notation see XIX Hexadecimal Color Values ALO Properties Fill color none List coordinates 31 Set Stroke width to 0 to have selections drawn using a width _ of one pixel regardless of the image magnification see XVIII pra Working with Zoomed Canvases With Text Too
50. _ OK Image macros examples SetOverUnderThresholdColors txt Plugins gt Utilities gt Search related not only to a image processing routine e g background or co localization but also to a practical context such as radiogram cell or histology Indeed ImageJ source files contain detailed annotations useful to both developers and regular users that want to know more about ImageJ routines and algorithms Search and Find Commands I are described in detail in Plugins Utilities gt SEE ALSO Control Panel U Keyboard Shortcuts and E SourceCodeRetriever a macro that searches for a menu entry and retrieves the source file of the respective command 6 Undo and Redo Probably the first thing you will notice is that ImageJ does not have a large undo redo buffer Undo Edit gt Undo z is currently limited to the most recent image editing filtering operation With time you will appreciate that this is necessary to minimize memory overhead Nevertheless with IJ 1 45 and later Undo z is in most cases undoable and can be applied to multiple images if Keep multiple undo buffers is checked in Edit gt Options gt Memory amp Threads If you cannot recover from a mistake you can always use Filet gt Revert r to reset the image lo its last saved state For selections Edit gt Selection gt Restore Selection E can be used to recover any misdealt selection 9 Last updated 2012 10 02 Image Types an
51. amount of image data copied is shown in the status bar SEE ALSO File Internal Clipboard Paste Control 27 4 Copy to System Copies the contents of the current image selection to the system clipboard SEE ALSO File Newb System Clipboard V Copy c Paste Control 27 5 Paste v Inserts the contents of the internal clipboard or from the system clipboard if the internal clipboard is empty into the active image The pasted image is automatically selected allowing it to be dragged with the mouse Click outside the selection to terminate the paste Select Edit gt Undo z to abort the paste operation SEE ALSO Paste Control 27 6 Paste Control AAN Paste Control Y Copy Blend Difference Transparent white Transparent zero AND OR After pasting use the Paste Control pop up menu to control how the image currently being pasted is transferred to the destination image Transfer Mode Except for Blend and Transparent the Paste Control transfer modes are the same as those listed in the de scription of Process gt Image Calculator The Blend mode is the same as the Image Calculator Average mode In Transparent mode white black pixels are transparent and all other pixels are copied unchanged Subtract Multiply Divide Min Max SEE ALSO Image Overlay gt Add Image 61 Last updated 2012 10 02 Edit gt Clear 27 7 Clear Erases the contents of the selection to
52. and Pencil tools are in all similar differing only on brush stroke size Modifier keys Shift dragging on the canvas will adjust the brush size Alt Holding Alt makes the brush paint in background color SEE ALSO Overlay Brush Pencil Freehand Line Selection Tool Color Picker window XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 15 gt gt D Developer Menu A drop down menu collecting several online resources and commands that are useful when writing Macros Plugins or troubleshooting ImageJ operations Debug mode activates ImageJ s debugging mode Edit gt Options Misc SEE ALSO Extending ImageJ Editor Help Plugins Macros Utilities gt Newb Common Commands Menu Tool Stacks Menu LUT Menu 38 Last updated 2012 10 02 IJ 1 46R IJ 1 46R IJ 1 46R IJ 1 46R IJ 1 46R IJ 1 46R Tools Flood Filler 19 16 gt amp Flood Filler A paint bucket tool that fills with the current foreground color adjacent pixels that have the same value as the clicked pixel Double click on the tool icon to specify the flood type in terms of pixel connectivity 4 connected or S connected To spread the fill to contiguous pixels within an intensity range use the Wand Tool instead Double click on the Wand Tool icon to set a Tolerance value then press Edit gt Fill f to fill with foreground color highlighted in the Flood Filler icon or Backspace Edit gt Clea
53. and right to left reverse cycle 10 Cycling Through the Toolbar Using Keyboard Shortcuts These two macros loop through the tools listed in an array using F1 gt and F2 as keyboard shortcuts forward and reverse cycling var index var tools newArray rectangle roundrect oval ellipse brush polygon freehand line itreeline polyline lt Marrow wand dropper angle point multipoint text macro Cycle Tools Fwd Fi setTool tools Lindex if index tools length index 0 macro Cycle Tools Rwd F2 if index lt 0 index tools length 1 setTool tools Lindex A tool can be defined either by its name or by its position in the toolbar using setTool id which allows assigning keyboard shortcuts to Custom Tools and items loaded by the More Tools Menu e g setToo1 21 activates whatever tool has been installed on the last slot of the toolbar It is also possible to temporarily activate a tool The macro below taken from the Rename and Save ROI Sets toolset activates the Color Picker Tool when is pressed but restores the previously active tool as soon as the mouse is released 11 Temporary Activation of a Tool macro Pick Color Once F3 tool IJ getToolName setTool dropper while true getCursorLoc z y Z flags if flags amp 16 0 setTool tool exit
54. available memory are displayed at the bottom of the O Avoid interpolation use 1 pixel spacing dialog Increase Output spacing to reduce the size of the Voxel size 3x3x2 5 um output stack Output Size 1MB 3075MB free A dialog allows you to specify the spacing of the recon structed slices Help Cancel ok Output spacing Determines the number of orthogonal slices that will be reconstructed In creasing Output spacing reduces the size of the output stack Start at Determines the image edge top left bottom or right from which reconstruction starts Start at is replaced by Slice count if there is a line selection With lines selections a stack is created by shifting by Output spacing the line down and to the left to generate additional slices for the output stack In this case the size of the output stack in determined by Slice count Flip vertically If checked each slice in the output stack will be flipped vertically Rotate 90 degrees If checked each slice in the output stack will be rotated 90 Avoid interpolation If checked no interpolation will be done Help Opens http imagej nih gov ij docs menus image html reslice SEE ALSO Dynamic Reslice and Radial Reslice plugins 28 6 10 Orthogonal Views H OO t1 head tif ALBO Yz 109 bd x216 pixels 16 b SUb pixels 16 Provides an orthogonal view display of the current stack or hyperstack 22 E g if a stack displays sagittal sections
55. bioinformatics btq013 C Igathinathane LO Pordesimo and WD Batchelor Major orthogonal dimensions measurement of food grains by machine vision using ImageJ Food Res Int 42 1 76 84 Jan 2009 doi 10 1016 j foodres 2008 08 013 C Igathinathane LO Pordesimo EP Columbus WD Batchelor and SR Methuku Shape identification and particles size distribution from basic shape parameters using ImageJ Comput Electron Agr 63 2 168 182 Jan 2008 doi 10 1016 j compag 2008 02 007 Brian A Irving Judy Y Weltman David W Brock Christopher K Davis Glenn A Gaesser and Arthur Weltman NIH ImageJ and Slice O Matic computed tomography imaging software to quantify soft tissue Obesity Silver Spring 15 2 370 6 Feb 2007 doi 10 1038 oby 2007 573 Noel Jabbour Priya D Krishna James Osborne and Clark A Rosen A new approach to geometrical measurements in an animal model of vocal fold scar J Voice 23 1 88 94 Jan 2009 doi 10 1016 j jvoice 2007 07 002 Yoonseok Kam Audrey Karperien Brandy Weidow Lourdes Estrada Alexander R Anderson and Vito Quaranta Nest expansion assay a cancer systems biology approach to in vitro invasion measurements BMC Res Notes 2 130 Jan 2009 doi 10 1186 1756 0500 2 130 Pasi Kankaanp Lassi Paavolainen Silja Tiitta Mikko Karjalainen Joacim P iv rinne Jonna Nieminen Varpu Marjom ki Jyrki Heino and Daniel J White BiolmageXD an open general purpose and high throughput image processing platform
56. calculator 127 Algorithm Gift wrap 63 Heckbert quantization 14 74 Newton based Pratt fit 63 Alpha blending value see Blend 102 Analyze 131 AND see Logical operations 128 150 Angle tool 33 Animation 55 94 Annotation Layers see ImageROI Overlay 66 101 Annotations 32 35 37 38 84 Grayscale images 85 Non destructive image overlay 19 100 Appearance 69 Area 135 Area under the curve see Definite integral 143 Arrows 32 36 Aspect ratio 29 30 80 136 AutoRun 21 49 AVI 11 54 58 Background see Subtract Background 128 Background pixels 145 Batch processing 125 155 BeanShell 26 Bee Lambert law 143 Benchmark 159 Bicubic interpolation see Interpolation 80 Bilinear interpolation 68 Bilinear interpolation see Interpolation 80 Binary 111 112 Bio Formats 2 4 59 Bio formats see LOCI 11 54 Bio 3 BiolmageXD 4 Bitwise operations 117 Black background 78 112 116 Blend 20 100 102 BoneJ 3 Bounding box 65 Brightness Contrast 75 76 78 108 Brush 37 Brush selection tool 30 Bug reporting see also Debug 7 Calibration 140 Optical density 138 152 Spatial 82 138 152 Calibration bar 152 Caliper length see Feret 136 Canvas 81 Capture image 159 Catmull Rom see Interpolation 80 CCD 14 110 Cell Counter plugin 33 34 CellProfiler see Interoperability 4 Center of mass 136 Centroid 136 Channels 82 96 CIE 80 CIE Lab 14 79 Circularit
57. center of the line drawn is displaced from the selection edge by 1 2 pixel to the bottom right Thus the line center the line in case of line width 1 is inside the selection at the top and left borders but outside at the bottom and right borders Rectangular selections but not polygonal selections or traced selections that happen to be rectangular are an exception to this rule For rectangular selections one pixel wide outlines are always drawn inside the rectangle Thicker lines are drawn as for the other selection types 62 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Edit gt Invert I 27 11 Invert I Creates a reversed image similar to a photographic negative of the entire image or selection For 8 bit and RGB images see Image Types and Formats Invert always uses min 0 and max 255 regardless of the data values For 16 bit and 32 bit images the actual minimum and maximum are used rather than the full range of the pixel type SEE ALSO Image gt Lookup Tablesp Invert LUT 27 12 Selection gt Osana Fit Fit Fit Convex Make Create Area to Make MRS 8 Spline Circle Ellipse Hull Inverse Mask Line Band Bax 8 SOO CMTE ROI manipulations using Edit gt Selection gt commands General handling of ROIs and Overlays is described in Selections 27 12 1 Select All a Creates a rectangular selection that is the same size as the image 27 12 2 Select None A Deactivates the se
58. commands in a hierarchical tree structure Click on a leaf node to launch the corresponding ImageJ command or plugin Double click on a tree branch node folder to expand or collapse it Click and drag on a tree branch node folder to display its descendants in a separate RGB Stack 7 Window Level HSE Stack Color Balance Threshold child window In a child window click on Show Parent to re open the parent window SEE ALSO Find Commands I 31 3 2 Find Commands I Command Finder Adaptive 3 DThreshold Colocalisation Threshold Color Threshold DynamicThreshold 1d Entropy Threshold The quickest way to find a command without having to navigate through all the menus 48 Evoke the prompt by pressing as in com mand Launcher or Locator If you type part of a command name the list will only show com mands that match that substring If only a single command matches then that command can be run by pressing Enter If multiple commands match click with the mouse to select a command to run Alternatively pressing the up or down keys will move keyboard focus to the list and the selected command can be run by pressing Enter Pressing switches focus back to the prompt Double clicking on a command will run that command Pressing closes the window Show full information EJ Close when running a TT IA Run Export Close 158 Last update
59. coronal YZ projection image and transverse XZ projection image will be dis played through the data set The two extra planar views are displayed in sticky panels next to original image and can be toggled using H the command shortcut The intersection point of the three views follows the location of the mouse click and can be controlled by clicking and dragging in either the XY XZ or YZ view XY and XZ coordinates are displayed in the title of the projection panels The mouse wheel changes the screen plane in all three views Voxel dimensions can be adjusted in Image Properties P 89 Last updated 2012 10 02 Image Stacks gt SEE ALSO 3D Project and 3D Viewer 101 Volume Viewer Stack Slicer Display3_ TP plugins Edit gt Options gt DICOM 28 6 11 Z Project Projects an image stack along the axis perpendicular to image plane the so called z axis 21 With hyperstacks the projection is performed on the active time frame or for all time points if All Time Frames is checked Five different projection types are supported The preferred projection method is stored in the preferences file Start slice 1 Average Intensity projection outputs an image wherein each pixel stores average intensity over all images in stack at corresponding Stop slice 5 _Average v Average Intensity Max Intensity Min Intensity Sum Slices Standard Deviation Median pixel locati
60. from the edges of the image This Fill Holes setting also affects Binary Close which erodes from the edges unless this checkbox is selected Skeletonize EDM output Determines the output type for the Binarye Distance Map Ultimate Points and Voronoi commands Set it to Overwrite for 8 bit output that overwrites the input image 8 bit 16 bit or 32 bit for separate output images 32 bit output has floating point subpixel distance resolution Do This drop down menu allows one to test the chosen settings by previewing each binary operation Erode Dilate Open Close Outline Fill Holes Skeletonize on the active image This option is only available when the active image is binary Help Opens http imagej nih gov ij docs menus process html Foptions 29 9 Math The commands in this submenu add subtract multiply etc a constant to each pixel in the active image or selection A Preview option is available for most operations pa Taia process Stace With stacks the dialog depicted on the left is displayed Process all 27 images There is Choose Yes to process entire stack or No to process only no Undo if you select Yes the active slice The dialog is not displayed if Hide Process LA eS NN Qu An a i No Cancel Yes Stack dialog is checked in Edit gt Options Misc SEE ALSO Memory Threads 29 9 1 Add Adds a constant to the i
61. if New overlay is Cancel OK checked Also if no selection exists and the command is run a warning message is displayed in which is possible to remove the existing overlay by running Remove Overlay o id Note that measured selections Analyze gt Measure m can J Remove existing overlay be added automatically to the image overlay by selecting the Co Add to overlay checkbox in Analyze gt Set Measurements SEE ALSO Overlay Options Labels Selection gt Properties y XIX Hexadecimal Color Values ROI Manager 28 14 2 Add Image Blends two open images by adding an image to the overlay of frontmost image The image to be blended can be of any type see Image Types and Formats but cannot be larger than the host image A blending alpha value can be specified in the Opacity 0 100 field The initial X Y location is based on the existing rectangular selection if any 102 Last updated 2012 10 02 IMPROVED IN IJ 1 46R CHANGED IN IJ 1 46R Image Overlay gt By default the created overlay image cannot be moved around the canvas i e is not a image selection ImageROI but are OoOO gt stored in the TIFF header and can be saved and restored Image to add blobs gif H when saving images in TIFF format On the other hand ee 104 image selections can be created using Edit gt Selection gt Image to o 129 Selection or by running Image Overlay gt To ROI Manager Opacity 0
62. image canvas white by default to be rendered in black This is useful when looking at X ray images in order to avoid high contrasting intensities at the image edges No image border Displays images without the default one pixel wide black border Use inverting lookup table Causes newly opened 8 bit images to have inverted pixel values where white 0 and black 255 This is done by both inverting the pixel values and inverting the LUT Use the Image Lookup Tables gt Invert LUT command to invert an image without changing the pixel values Double Buffer Selections Reduces flicker when working with complex selections but it also increases memory usage and slows screen updates It is not needed on Mac OS X which has built in double buffering Antialiased tool icons Smooths and darken the tool icons in the Main ImageJ window This option is enabled by default on all operating systems On Windows XP enable Clear Type sub pixel anti aliasing to improve the quality of text in menus Menu font size Specifies the size of the ImageJ window menu font Use a size of 0 zero to use Java s default menu font size Changing the font size requires the restarting of ImageJ This option is ignored on Mac OSX Help Opens http imagej nih gov ij docs menus edit html appearance SEE ALSO Customizing the ImageJ Interface 27 13 11 Conversions Conversion Options M Scale When Converting Use this dialog to set options that control how images
63. images including geometric transformations Process gt Image processing including point operations filters and arithmetic operations Analyze gt Statistical measurements profile and histogram plotting and other operations related to image analysis Plugins gt Commands for creating editing and managing add ons see Extending ImageJ listing all the user installed Macros Scripts and Plugins installed in the E ImageJ plugins directory Windowb Selection and management of open windows Help gt Updates documentation resources and version information VII ORGANIZING COMMANDS IN THE MENU BAR The Plugins gt menu can become easily cluttered after the installation of several plugins Since Plugins gt reflects the hierarchy of directories in S ImageJ plugins up to two subfolders sub menus i e subfolders can be created to keep the menu organized preventing it from running off the bottom of the screen E g to move the EPS Writer plugin into a Plugins gt Input Output gt PDF gt submenu one would move EJEPS_Writer class into GImageJ plugins Input Output PDF In addition checking the Move isolated plugins to Misc menu checkbox in Edit gt Options gt Misc will compact the menu list by moving to Plugins Miscellaneous all the plugins with only one command that try to install themselves in submenus Note that external plugins can be installed in any of the ImageJ menus This is the case of plugins packaged in JAR fi
64. language SEE ALSO Macros Function Finder F 164 Last updated 2012 10 02 Help gt 33 10 Update ImageJ Image Updater Upgrade To daily build 5 You are currently running v1 430 If you click OK Image will quit and you will be running the upgraded version after you restart Image i 4 e Cancel OK 33 11 Refresh Menus Update ImageJ Upgrades ImageJ to the latest Elij jar at http imagej nih gov ij upgrade or downgrades to one of the earlier versions at http imagej nih gov ij download jars Select daily build from the drop down menu and ImageJ will be upgraded to the latest daily build at http imagej nih gov ij ij jar SEE ALSO Installing and Maintaining ImageJ Use this command to update ImageJ s menus after adding or removing plugins or macros to the plugins folder Prior to ImageJ 1 44b this command was named Update Menus 33 12 About Pluginse This submenu displays information about some of the plugins in the ImageJ plugins folder To be included in this submenu a plugin must be packaged as a JAR file There is an example at rsb info nih gov ij plugins jar demo html 33 13 About ImageJ About Image Opens an image containing information about the ImageJ version the author the website Java version and memory available Note that clicking in the Status bar is a quicker way to show this information SEE ALSO Plugins Utilities
65. moved resized or re colored see ROI Manager Note that previous items in the ROI Manager list will be deleted 28 14 7 Remove Overlay Permanently clears the overlay so that it cannot be restored using Show Overlay 28 14 8 Flatten F Creates a new RGB image that has the overlay rendered as pixel data The RGB image is the same size as the active image unlike Plugins Utilities gt Capture Image which creates a WYSIWYG What You See Is What You Get image that is the same size as its window Stacks must first be converted to RGB Image Type gt submenu see also Image Types and Formats when flattening all slices in the stack NEw IN 28 14 9 Labels This prompt defines if and how Overlays should be labelled Color magenta 4 dde Font size 112 It sets the behavior of Image ARES Overlay gt Add Selection b An O Use names as labels alyze gt Measure m after acti M Draw backgrounds vating the Add to overlay option O Bold ne in Analyzeb Set Measurements Cancel O Analyze gt Analyze Particles and Q the ROI Manager Show All display Color The color of the label as one of the default selection colors ay Labels Font size Specifies the font size of the label 12 72 pt 104 Last updated 2012 10 02 Image Lookup Tables gt Show labels If overlays should be decorated with a text label This option is inactive by default Use names as labe
66. movie as a virtual stack is also available see Virtual Stacks It is also possible to specify the starting and ending frame Enter 0 zero to specify the last frame 1 to specify the second last frame etc SEE ALSO File gt Save Asp AVI 26 6 11 XY Coordinates Imports a two column text file such as those created by File Save Asp XY Coordinates as a polygon selection The selection is displayed in the current image or if the current image is too small in a new blank image Coordinates of active selection at evenly spaced one pixel intervals can be retrieved using the List coordinates options in Edit gt Selection gt Properties y 26 7 Close w Closes the active image 26 8 Close All ALU Close All There are 2 images with unsaved changes If you Closes all open images An alert is displayed if click OK they will be closed without being saved there are unsaved changes Cancel C OK gt C 0K 59 Last updated 2012 10 02 Filep Save s 26 9 Save s Saves the active image in TIFF format the default format of ImageJ cf II Image Types Lossy Compression and Metadata To save only a selected area create a rectangular selection and use the Image Duplicate D command Note that Save s and File Save Asp Tiff are redundant commands 26 10 Save As Use this submenu to save the active image in TIFF GIF JPEG or raw format Can also be used to save measurement
67. nih gov ij macros js Thread safe JavaScript code can be generated using the Recorder Plugins gt Macros Record Scripts can be opened in the editor as any other macro Scripts with the extension El js can be run using Macros Run Macro otherwise Macros gt Evaluate JavaScript Ctrl J must be used JavaScript Programming Resources on ImageJ JavaScript scripting include 1 The ImageJ web site with growing documentation http imagej nih gov ij developer javascript html 2 Tutorials on the Fiji webpage http fiji sc wiki index php Javascript Scripting 3 Online scripts repository http imagej nih gov ij macros js SEE ALSO Macros Plugins Editor Fiji Script Editor 16 Plugins Plugins are a much more powerful concept than Macros and Scripts and most of ImageJ s built in menu commands are in fact implemented as plugins Quoting Werner Bailer 3 24 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Plugins Advantages and disadvantages of JavaScript in ImageJ A thorough comparison between different scripting languages is available on the Fiji webpage JavaScript Advantages Full access to ImageJ and Java APIs Standardized Richer language objects operator break continue etc Extensive documentation JavaScript Disadvantages Slower especially starting up No equivalent of macro sets Cannot use most of ImageJ s 360 built in macro functions Requires knowledge
68. not read correctly Attempts to open AVIs in other formats will fail SEE ALSO Non native Formats II Image Types Lossy Compression and Metadata X Warning on JPEG Compression Non native Formats When opening a file ImageJ first checks whether it can natively handle the format If ImageJ does not recognize the type of file it calls for the appropriate reader plugin using HandleExtraFileTypes a plugin bundled with ImageJ If that fails it tries to open the file using the OME Bio Formats library if present a remarkable plugin that supports more than one hundred of the most common file formats used in microscopy If nevertheless the file cannot be opened an error message is displayed Because both these plugins are under active development it is important that you keep them updated The OME Bio Formats library can be updated using its self updating plugin Plugins gt LOCI gt Update LOCI Plugin or Fijit s built in updater Help Update Fiji The following websites provide more information on the OME Bio Formats http loci wisc edu bio formats image http fiji sc Bio Formats http loci wisc edu bio formats using bio formats In addition the ImageJ web site lists more than sixty plugins that recognize more exotic file formats The ImageJ Documentation Portal also maintains a somewhat outdated list of file formats that are supported by ImageJ SEE ALSO Native Formats Filer gt Import gt II I
69. number with lead aa ing zero s 00 00 Converts the label into a min a Se utes seconds timestamp 00 00 00 Converts the label Preview into a hours minutes seconds timestamp Text Stamps Help Cancel n contents of the Text field Label Displays slice abels Starting value and Interval Specify the first value and the numeric steps to be applied Note that with timestamps metric time values must be used e g an Interval of 3600 will create 1 hour increments Text The string to be drawn after each number when the Format chosen is either 0 or Text label without numeric sequence Use overlay If checked labels will be created as non destructive image Overlays Note that previously added overlays will be removed Also note that Virtual Stacks and Hyperstacks can only have overlay labels Use text tool font If checked labels will be created using the typeface and style specified in the Fonts widget If unchecked labels are typeset using ImageJ s default font sans serif typeface Help Opens http imagej nih gov ij docs menus image html label SEE ALSO Make Montage Stacks Tools Insert 92 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Image gt 28 6 15 Tools 28 6 15 1 Combine AAA Combiner Stackl mri stack 1 tif H Stack2 mri stack 2 tif O Combine vertically A Help Cancel Stacks gt Combines two stacks Width x Height x De
70. on the ImageJ website Toolsets Toolsets are macro files E txt and El ijm files containing up to eight macro tools along with any number of ordinary macros Toolsets are listed on the More Tools Menu menu if installed in the GImageJ macros toolsets directory Choosing a toolset e g Lookup Tables replaces all previously installed tools As mentioned E ImageJ macros StartupMacros txt contains the tools loaded at startup This file can be customized using Plugins Macros Startup Macros or by holding when choosing Startup Macros from the More Tools Menu menu ImageJ feature several pre installed toolsets 8 and many others are available on the ImageJ website Toolsets can also be created by choosing Toolset Creator a convenient way to create groups of Menu Tools listing Plugins gt commands 41 Last updated 2012 10 02 Contextual Menu SEE ALSO More Tools Menu Tools documentation Plugins gt Newer Macro Tool Plugin Tool 21 Contextual Menu As mentioned earlier macros and macro tools in the ElStartupMacros txt are automatically installed in the Plugins Macros gt submenu and in the toolbar when Im ageJ starts up Rename Duplicate Original Scale Paste Control In addition the ElStartupMacros txt file also installs the contextual popup menu displayed when right clicking on an image Other macros and toolsets e g Magic Montage may also replace the default menu with specialized ones In this
71. overlay is preserved when an image is saved in TIFF format cf II Image Types Lossy Compression and Metadata 101 Last updated 2012 10 02 Image Overlay gt 90 80 70 60 50 40 30 20 10 BE AM PA A MO PLC 18 lt A PET las ll LT INE bau b a CA O OE ar a tal PF F N T 1 J a E A a baer wes ITI da al O IO i _ AI ene Via DAA Al a B r CIA da TT al TOT Aare E Ji T ha i M P Ba F aa de ll F ae Non destructive image Overlays Outputs from E Grid_Overlay E MakeOverlay and ROI Color Coder that exemplify the usage of most Image gt Overlay gt submenu commands The Overlay Brush tool can also be used to create freehand annotations 28 14 1 Add Selection b Selections are immediately added to the current overlay when Stroke color red pressing B Pressing will display a dialog box in Font size 18 which Stroke color and width and Fill color can be set Except for text selections and as explained in Editp Selection gt Properties ly Stroke contour color and Width Stroke color red are ignored if a Fill color is specified Colors are specified Width 1 using the name of one of the default selection colors black blue cyan green magenta orange red white and yellow or Fill color none using hex notation see XIX Hexadecimal Color Values O New overlay a Previously added Overlays are removed
72. results lookup tables selections and selection XY coordinates 26 10 1 Tiff Saves the active image or stack in TIFF format in redundancy with Filep Save s TIFF is the only format other than raw that supports all ImageJ data types 8 bit 16 bit 32 bit float and RGB and the only format that saves spatial and density calibration In addition Selections and Overlays are also saved in the TIFF header By default 16 bit and 32 bit images are saved using big endian byte order Check Save TIFF and Raw in Intel Byte Order in the Edit gt Options Input Output dialog box to save using little endian byte order SEE ALSO Native Formats I Image Types Lossy Compression and Metadata X Warning on JPEG Compression 26 10 2 Gif Saves the active image in GIF format RGB images must first be converted to 8 bit color using using Image Typer 8 bit Color The value to be used as the transparent index 0 255 can be set in the Edit gt Options Input Output dialog box Stacks are saved as animated GIFs Use Imageb Stacks gt Toolst gt Animation Options Alt or right click on the on the play pause icon that precedes the stack slider to set the frame rate 26 10 3 Jpeg Saves the active image in JPEG format Edit JPEG Quality Edit gt Options Input Output dialog box to specify the JPEG compression level 0 100 This value is shown on the title of the save dialog prompt Lower values produce smaller f
73. supporting BeanShell Jython JRuby and other scripting languages It does not support Function Finder F but selecting a built in macro function and running Tools gt Open Help on Macro Functions retrieves the documentation for the selected function SEE ALSO Scripting in Other Languages Running ImageJ From the Command Line IJ_ ED a plugin by J r me Mutterer that binds jEdit to ImageJ 18 Running ImageJ from the Command Line ImageJ was devised as a desktop application It can however run without a graphics environment headless mode by adding a special library headless jar to the ij jar classpath that overrides 26 Last updated 2012 10 02 Running ImageJ From the Command Line key ImageJ classes to work better headlessly As described on the Fiji website this strategy is implemented in Fiji through the headless command line flag see also Running ImageJ in headless mode and Using Cluster for Image Processing with IJ Headless operations are simplified in ImageJ2 ImageJ recognizes the following command line options file name ijpath path port macro path arg batch path arg eval macro code run command debug Opens a file Examples DLODS LE Users wayne images blobs tif eslx ti1f Specifies the path to the directory containing the plugins directory Example ijpath Applications ImageJ Specifies the port ImageJ uses to determine if another instance is running Ex
74. surfaces To enable this feature set Opacity to a value greater than zero and select either Mean Value or Brightest Point projection Surface Interior Depth Cueing Depth cues can contribute to the three dimensional quality of projection images by giving perspective to projected structures The depth cueing parameters determine whether projected points originating near the viewer appear brighter while points further away are dimmed linearly with distance The trade off for this increased realism is that data points shown in a depth cued image no longer possess accurate densitometric values Two kinds of depth cueing are available Surface Depth Cueing and Interior Depth Cueing Surface Depth Cueing works only on nearest point projections and the nearest point component of other projections with opacity turned on Interior Depth Cueing works only on brightest point projections For both kinds depth cueing is turned off when set to zero i e 100 of intensity in back to 100 of intensity in front and is on when set at 0 lt n 100 i e 100 n of intensity in back to 100 intensity in front Having independent depth cueing for surface nearest point and interior brightest point allows for more visualization possibilities Interpolate Check Interpolate to generate a temporary z scaled stack that is used to generate the projections Z scaling eliminates the gaps seen in projections of volumes with slice spacing greater than 1 0 pixels Th
75. the Java version of the class files created by Plugins Compile and Run Plugins compiled with a Target pinta Target of 1 6 will not run on earlier versions of Java A Target of 1 4 should be used to create plugins capable of running on all versions ImageJ Target cannot be set higher than the version of Java ImageJ is currently running on Compile and Run C Generate debugging info javac g TETE E TIETA Cam l Help Cancel J OK Generate Debugging Info javac g If checked information needed by Java debuggers will be included in the class files Help Opens http imagej nih gov ij docs menus edit html compiler 27 13 15 DICOM This dialog sets options related to the handling of DICOM images Namely if ImageJ should open DICOM images as 32 bit float if voxel ne eet T depth should be calculated based on the distance between the first j and last slice and if coronal transverse sections should be mirrored when using the Image Stacks gt Orthogonal Views H command With IJ 1 45 the DICOM reader applies the Rescale Slope value when Open as 32 bit float is enabled and tag 0028 1053 is not 1 0 Mm DICOM Options Orthogonal Views O Rotate YZ O Flip XZ Cancel ok gt SEE ALSO Image Types and Formats 27 13 16 Misc Displays a dialog box for configuring several advanced A i in Se settings that do not fit elsewhere in the ImageJ interface Divide by zero va
76. the current background color and keys are shortcuts to this command With stacks a dialog is displayed offering the option to clear the selection in all stack images Clear by pressing to avoid this dialog SEE ALSO Clear Outside Fill f Color Picker Tool 27 8 Clear Outside Erases the area outside the current image selection to the background color SEE ALSO Clear Fill f Color Picker Tool 27 9 Fill f Fills the current selection with the current foreground color With stacks a dialog is displayed offering the option to fill the selection in all stack images Fill the selection by pressing to avoid this dialog SEE ALSO Clear Draw d Color Picker Tool 27 10 Draw d Outlines the current selection using the current foreground color and line width The foreground and background colors can also be set using the Edit gt Options Colors command Use the Edit gt Optionse Line Width command or double click on the line tool to change the line width With stacks a dialog is displayed offering the option to draw the selection in all stack images Draw the selection by pressing D to avoid this dialog SEE ALSO Analyze gt Label Color Picker Tool XVII Embedding Color Annotations in Grayscale Images XI Drawing Lines Wider Than One Pixel XI DRAWING LINES WIDER THAN ONE PIXEL If the line width is an even number the selection boundary is at the center of the line If the line width is odd 1 3 the
77. the currently displayed slice Holding will skip ten slices backward SEE ALSO Arrow Keys 28 6 5 Set Slice AANA Set Slice Slice Number 1 27 1 e 0 Cancel OK 28 6 6 Images To Stack Displays a specified slice The user must enter a slice number greater than or equal to one and less than or equal to the number of slices in the stack Creates a new stack from images currently displayed in separate windows Method If images differ in size a drop down menu allows to choose a conversion method Images to Stack The 2 images differ in size smallest 117x157 largest 256x254 They will be converted to a stack using the specified method Method Copy center Y Copy center Copy top left Scale smallest Scale largest Images to Stack Name Stack Title Contains C Bicubic Interpo M Use Titles as L Name Stack U Keep Source Ir Title Contains M Use Titles as Labels Keep Source Images gt Cancel OK Copy center and Copy top left Stack will have the width of the widest open image and the height of the highest open image Smaller images will then be copied either to the center or to the upper left corner of the slice Borders are filled with pixels that have a value of zero Scale smallest and Scale largest Stack will have the dimensions of the smallest largest open image Other images are scaled to the new slice d
78. the left most lane if the lanes are vertical or the top lane if the lanes are horizontal 145 Last updated 2012 10 02 Analyze gt Tools gt 2 Select Gels gt Select First Lane 1 and the lane will be outlined and Lane 1 selected displayed in the status bar 3 Move the rectangular selection right to the next lane or down if the lanes are horizontal and select Gels Select Next Lane 2 The selected lane is outlined and labelled and Lane n selected is displayed in the status bar 4 Repeat the previous step for each remaining lane 5 Select Gels gt Plot Lanes 3 to generate the lane profile plots 6 Use the Straight Line Selection Tool to draw base lines and or drop lines so that each peak of interest defines a closed area ImageJ will automatically switch to the Straight Line tool Note that you can hold to constrain lines to be either horizontal or vertical To access to all the lanes it may be necessary to scroll the image vertically using the Scrolling Tool Hold down the space bar to temporarily switch to this tool 7 For each peak measure the size by clicking inside the peak with the Wand Tool If necessary scroll the image vertically by holding down the space bar and dragging 8 Select Gels gt Label Peaks to label each measured peak with its size as a percent of the total size of the measured peaks SEE ALSO Calibrate SinglePanelGelAnalyzer macro Video tutorial on ImageJ
79. the range of Channels c Slices 2 and Frames t to be Channels c 1 2 duplicated dos c EE With single images hold to skip the dialog box Title mitosis 1 tif Frames t 1 51 Cancel OK SEE ALSO Rename ancel y 97 Last updated 2012 10 02 Image gt Rename 28 10 Rename Renames the active image mri stack tif _ SEE ALSO Duplicate D q Cancel HL OK 28 11 Scale E Resizes the image or current area selection by scale factors entered into a dialog box As with Image Size two resam pling methods are possible Bilinear and Bicubic interpolation X Scale Y Scale Z Scale For the best looking results particularly with graphics and text use integer scale factors 2 3 5 etc and check Average when downsizing with scale factors less than 1 0 19 Also when downsizing smoothing the source image prior to scaling Width pixels Height pixels 226 Depth images 27 Interpolation Bicubic may produce better looking results O Fill with background color Scaled image selection are copied to a new image named Title M Average when downsizing if Create new window is checked If scaling a selection that A cue eae will not be copied to a new image check Fill with Background Title mri stack 1 tif Color to fill with the background color instead of zero Entire stacks or hyperstacks in the Z Dimension will be scaled if Process entire stack is checked
80. the reference point 0 0 always in pixels of the image coordinate system see also Invert Y coordinates in Analyze gt Set Measurements As explained in XXIII Global Calibrations check Global to make the current settings global 1 e applied to all images opened during the current session SEE ALSO Analyzep Set Scale ImageShow Info i 28 5 Colore This submenu contains commands that deal with color images 28 5 1 Split Channels Splits an RGB image or stack into three 8 bit grayscale images containing the red green and blue components of the original The window names have an appended red green and blue With composite images and or hyperstacks e g File Open Samples Organ of Corti 2 8M 4D stack this command splits the stack into separate channels SEE ALSO Merge Channels IMPROVED 28 5 2 Merge Channels IN IJ 1 46R Merges 2 7 images into an RGB image or multi channel composite image Select the channel order color using the C1 C7 dropdown menus Select None to skip a channel 83 Last updated 2012 10 02 Image Color gt gt Merge Channels Create composite If checked a multi channel composite image C1 red None ie see Color Composite Images will be created If unchecked C2 green Cai an RGB image is created instead When creating composite E images original LU Ts and display ranges are preserved C3 blue C 3 tif e unless Ignore source LUTs is che
81. the simplicity of macro language and feel less integrated in ImageJ 14 Macros A macro is a simple program that automates a series of ImageJ commands The easiest way to create a macro is to record a sequence of commands using the command recorder Plugins gt Macros Record A macro is saved as a text file E txt or El ijm extension and once installed executed by selecting the macro name in the Plugins gt Macros submenu by pressing a key or in the case of Macro tools by clicking on an icon in the ImageJ toolbar In addition any macro file placed in GImageJ plugins with an El ijm extension will be installed in the Plugins gt menu like any other plugin before version 1 41 only files with an underscore in the name would be listed There are more than 300 example macros on the ImageJ Web site To try one open it in a browser window and drag it directly to the Main ImageJ window or copy it to the clipboard Ctrl A switch to IJ and run File gt New System Clipboard V Ctrl v pasting the macro into a new Editor window Run it using the editor s Macros gt Run Macro command Ctrl R Most of the example macros are also available in the macros folder inside the ImageJ folder Macro Programming The ImageJ community has created excellent tutorials on macro programming These resources are indispensable guides to the ImageJ macro language 1 The ImageJ Macro Language Programmer
82. to remove points Similarly when using Edito Draw d marks are painted with the diameter of Mark Width SEE ALSO Point Tool Cell Counter plugin Toolbar Shortcuts 19 7 Wand Tool Creates a selection by tracing objects of uniform color or thresholded objects To trace an object either click inside near the right edge or outside to the left of the object To automatically outline and measure objects have a look e g at the WandAutoMeasureTool macro To visualize what happens imagine a turtle that starts moving to the right from where you click looking for an edge Once it finds the edge it follows it until it returns to the starting point Note that the wand tool may not reliably trace some objects especially one pixel wide lines unless they are thresholded highlighted in red using Image Adjust gt Threshold T Double clicking on the wand tool icon or running Edit gt Options Wand Tool opens the configuration dialog box in which three modes 4 connected 8 connected or Legacy plus a tolerance value can be set 7 34 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Tools Text Tool O 4 Connected 2400 _ __ AAA 8 Comnected 2400 Mode Legacy 0 0 y fe A Cancel OK The Wand Tool 4 8 connected particles can be traced within an intensity range Tolerance The wand takes the pixel value where you click as an initial value It
83. unless the width of the initial selection is at least twice its height 144 Last updated 2012 10 02 Analyze gt Gels gt AAA Lane rd 1 of gel gif AOA Plot of Lane 1 of gel gif b Ll K 5 gel gif Uncalibrated OD 1 0 1 0 Vertical scale factor Horizontal scale factor W Uncalibrated OD M Label with percentages C Invert peaks CP i Cancel ok APA Results of Lane 1 Area Percent 1 4820 175 36 631 8338 731 63 369 Select Next Lane To be used after the first rectangular ROI is moved over the adjacent lanes Note that all selections must have the same dimensions Plot Lanes Generates the lane profile plots ImageJ assumes that only one plot is created per analysis As a consequence re running this command more than once within the same analysis will cause an error message You must first use the Select First Lane command To recreate plotted profiles use the Re plot Lanes command Re plot Lanes Recreates the lane profile plots If Plot Lanes has not yet been run an error message is displayed The data needed to re plot the lanes 1s not available Reset Resets the analysis Label Peaks Uses the area measurements obtained with the Wand lool to label lane peaks see Gel Analyzer Options Gel Analyzer Options Use this dialog to control the behavior of the gel analyzer Vertical Horizontal scale factor Specifies th
84. useful tool for biological image processing and analysis Microsc Microanal 13 200 201 Jan 2007 doi 10 1017 S1431927607076611 Fathima Shihana Dhammika Menike Dissanayake Nicholas Allan Buckley and Andrew Hamilton Dawson A Simple Quantitative Bedside Test to Determine Methemoglobin Ann Emerg Med Oct 2009 doi 10 1016 j annemergmed 2009 07 022 Tal Shprung and Illana Gozes A novel method for analyzing mitochondrial movement inhibition by paclitaxel in a pheochromocytoma cell model J Mol Neurosci 37 3 254 62 Mar 2009 doi 10 1007 s12031 008 9129 8 S Sieuwerts FAM de Bok E Mols WM de Vos and JETvan Hylckama Vlieg A simple and fast method for determining colony forming units Lett Appl Microbiol 47 4 275 278 Jan 2008 doi 10 1111 3 1472 765X 2008 02417 x A Sol J Mas and I Esteve A new method based on image analysis for determining cyanobacterial biomass by CLSM in stratified benthic sediments Ultramicroscopy 107 8 669 73 Aug 2007 doi 10 1016 3 u1tramic 2007 01 007 179 1109 1110 111 112 113 114 115 116 1117 118 David Stepensky FRETcalc plugin for calculation of FRET in non continuous intracellular compartments Biochem Biophys Res Commun 359 3 752 8 Aug 2007 doi 10 1016 j bbrc 2007 05 180 Jason R Swedlow and Kevin W Eliceiri Open source bioimage informatics for cell biology Trends in Cell Biology 19 11 656 60 Nov 2009 doi 10 1016 j tcb 2009 08
85. when ImageJ quits resetting all parameters to their default values when ImageJ is restarted As mentioned in Settings and Preferences ElIJ_prefs txt holds all the settings and preferences of ImageJ and is stored in G Library Preferences on Mac OSX in G imagej on Linux and Windows with referring to the user s home directory Several macros and plugins also write parameters to this file SEE ALSO 3 Ensuring Specific Settings at Launch Pluginsp Utilities gt Reset 74 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IJ 1 46R Image gt 28 Imager 28 1 Type Use this submenu to determine the type of the active image or to convert it to another type An attempt to perform an unsupported conversion causes a dialog box to be displayed that lists the possible conversions Supported conversions in ImageJ Image gt Type gt submenu Note that ImageJ2 supports many more types of image data VFrom Too 8 bit 16 bit sabe SP RGB RGB BSB color color stack stack 8 bit IS I S IS 16 bit I S E I S I S 32 bit IS IS IS 8 bit color I S e I RGB color I S IS on IS LS RGB stack I HSB stack I I Single images only S Stacks 8 bit Converts to 8 bit grayscale ImageJ converts 16 bit and 32 bit images to 8 bit by linearly scaling from min max to 0 255 where min and maz are the two values displayed in the Image gt Adjust gt Brightness Contrast C lmage gt Show Info i displa
86. 007 Philippe Th venaz and Michael Unser User friendly semiautomated assembly of accurate image mosaics in microscopy Microsc Res Tech 70 2 135 46 Feb 2007 doi 10 1002 jemt 20393 Zhuang Tian Li Qiang Liu Chifang Peng Zhenxing Chen and Chuanlai Xu A new development of measurement of 19 Nortestosterone by combining immunochromatographic strip assay and ImageJ software Food Agr Immunol 20 1 1 10 Jan 2009 doi 10 1080 09540100802621017 Edmund Y Tong Geoffrey C Collins April E Greene Colozzi Julia L Chen Philip D Manos Kyle M Judkins Joseph A Lee Michael J Ophir Farrah M Laliberte and Timothy J Levesque Motion based angiogenesis analysis a simple method to quantify blood vessel growth Zebrafish 6 3 239 43 Sep 2009 doi 10 1089 zeb 2008 0554 T Vrekoussis V Chaniotis I Navrozoglou V Dousias K Pavlakis E N Stathopoulos and O Zoras Image analysis of breast cancer immunohistochemistry stained sections using ImageJ an RGB based model Anticancer Res 29 12 4995 8 Dec 2009 Thomas Walter David W Shattuck Richard Baldock Mark E Bastin Anne E Carpenter Suzanne Duce Jan Ellenberg Adam Fraser Nicholas Hamilton Steve Pieper Mark A Ragan Jurgen E Schneider Pavel Tomancak and Jean Karim H rich Visualization of image data from cells to organisms Nature Methods 7 3 Suppl S26 41 Mar 2010 doi 10 1038 nmeth 1431 Jennifer L West and Ian D Cameron Using the medical image processing package Image
87. 1 46R Analyze gt Summarize Record Starts This option allows plugins and macros to recreate particle outlines using the doWand x y macro function The CircularParticles macro demonstrates how to use this feature In situ Show If checked the original image will be replaced by the binary mask specified in the Show drop down menu Note that this option does not apply to Overlay Outlines and Overlay Masks that are always displayed as non destructive image Overlays on the measured image Help Opens http imagej nih gov 1j docs menus analyze htmlFap SEE ALSO Particle Remover plugin 4 8 Connected Particle Analysis 30 3 Summarize For each column in the Results Table calculates and displays the mean standard deviation minimum and maximum of the values in that column This command is also available by right clicking on the Results Table label Area Mean Mode Min Max Perim Circ Feret mtDen Median 63 blobs gif 5 3001 1 884E2 1 360E2 1 280E2 2 480E2 3 5561 5 268E 1 1 603E1 9 984E3 1 840E2 1 000E2 64 blobs gif 4 9001 1 729E2 1 7602 1 280E2 2 2402 3 673E1 4 565E 1 1 703E1 8 472E3 1 760E2 1 000E2 Mean 347 547 187 727 203 375 128 230 375 67 902 0 834 24 875 67786 250193 500 100 SD 217 766 19 042 32 933 0 26 096 26 562 0 144 9 516 44012 25 3 111 Min 1 128 128 128 2 828 0 406 1 414 128 128 Max 902 248 128 248 132 225 1 52 202 178720 240 eee a PF SEE ALSO Analyze Particles Di
88. 100 50 after adding the image to the overlay De 3 Cancel OK a SEE ALSO Paste Control Blend transfer mode Image Stacks gt Tools gt Insert XIX HEXADECIMAL COLOR VALUES Hexadecimal hex notation is frequently used in computing to summarize binary code in a human friendly manner Here are some decimal hexadecimal equivalents Dec 0123456789 10 11 12 13 14 15 16 17 18 19 20 30 40 50 60 70 80 90 100 110 255 Hex 0123456789 A BCOD E F 1011 12 13 14 1E 28 32 3C 46 50 54 64 GE FF Decimal RGB color values that typically range from 0 to 255 are succinctly represented by two hexadecimal digits ranging from 00 through FF A hex color value is a 6 digit three byte hexadecimal number hex triplet in the form RRGGBB in which RR specifies the red GG the green and BB the blue value Opacity of hex triplets can be modified by an optional transparency alpha channel giving rise to a 8 digit four byte hex number in the form AARRGGBB with AA specifying the alpha blending value In ImageJ the alpha channel codes for opacity and ranges from 00 fully transparent to FF solid color Alpha values can be omitted for fully opaque colors The table below provides some hexadecimal color values in 10 transparency increments and can be used to create semi transparent Overlays Color RGB triplet Dey wave Hex triplet 100 90 80 70 60 50 40 30 20 10 0 Blue 00 259 Cyan 0 255 255 Green 0 255
89. 1365 2818 2009 03144 x David Hecker Joachim Kappler Alexander Glassmann Karl Schilling and Wolfgang Alt Image analysis of time lapse movies a precision control guided approach to correct motion artefacts J Neurosci Methods 172 1 67 73 Jul 2008 doi 10 1016 j jneumeth 2008 04 010 Stephan Hegge Mikhail Kudryashev Ashley Smith and Friedrich Frischknecht Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection Biotechnol J 4 6 903 13 Jun 2009 doi 10 1002 biot 200900007 J Hegyi V Hegyi G Messer P Arenberger T Ruzicka and C Berking Confocal laser scanning capillaroscopy a novel approach to the analysis of skin capillaries in vivo Skin Res Technol 15 4 476 81 Nov 2009 doi 10 1111 j 1600 0846 2009 00393 x 176 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Ricardo Henriques Mickael Lelek Eugenio F Fornasiero Flavia Valtorta Christophe Zimmer and Musa M Mhlanga QuickPALM 3D real time photoactivation nanoscopy image processing in ImageJ Nat Methods 7 5 339 40 May 2010 doi 10 1038 nmeth0510 339 E lannuccelli F Mompart J Gellin Y Lahbib Mansais M Yerle and T Boudier NEMO a tool for analyzing gene and chromosome territory distributions from 3D FISH experiments Bioinformatics 26 5 696 7 Jan 2010 doi 10 1093
90. 16 bit images Calibrate Function Straight Line 2nd Degree Polynomial 3rd Degree Polynomial 1 5 13 518576 4th Degree Polynomial b 0 956813 fee c 0 839117 Exponential d 365 424344 Power R12 0 981385 Log Rodbard Gamma Variate y a b In x c Rodbard NIH Image Exponential with Offset pixel value _ lt a Gaussian Open Save Exponential Recovery EAN EA AE aoa E j H fi 1 i 1 A A List Save Copy X 187 6 Y 0 41 Global calibration g M Show plot Pixel Inverter Uncalibrated OD Help Cance rie Calibration Function T T De m ps 0 a 5 SEE ALSO Optical Density Calibration tutorial Global Calibrations Calibration Bar Curve Fitting Image Types and Formats 30 10 Histogram h Calculates and displays a histogram of the distribution of gray values in the active image or selection The X axis represents the possible gray values and the Y axis shows the number of pixels found for each gray value The horizontal LUT bar below the X axis is scaled to reflect the display 9 This shortcut is shown on Windows and Linux but not on Mac OSX as it conflicts with the system wide Hide shortcut However the shortcut without holding down Cmd does work on OS X 140 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Analyze gt Histogram h XXIII GLOBAL CALIBRATIONS Calibration settings related to spatial pixel width height and v
91. 2012 10 02 Getting Help Help on ImageJ imagingbook com Web site of Digital Image Processing An Algorithmic Introduction using Java by Wilhelm Burger and Mark Burge 11 This technical book provides a modern self contained intro duction to digital image processing techniques Numerous complete Java implementations are provided all of which work within ImageJ Hypermedia Image Processing Reference HIPR2 Developed at the Department of Artificial Intelligence in the University of Edinburgh provides on line reference and tutorial information on a wide range of image processing operations IFN wikipage The Imaging Facility Network IFN in Biopolis Dresden provides access to advanced microscopy systems and image processing The website hosts high quality teaching material and useful links to external resources stereology info Stereology Information for the Biological Sciences designed to introduce both basic and advanced concepts in the field of stereology SEE ALSO ImageJ Related Publications on page 174 3 2 Help on ImageJ Below is a list of the ImageJ help resources that complement this guide see Guide Formats Specific documentation on advanced uses of ImageJ macro programming plugin development etc is discussed in Extending ImageJ l The ImageJ online documentation pages Can be accessed via the Help gt Documentation command The Fiji webpage http fiji sc The ImageJ Informatio
92. 2012 10 02 Help gt 33 Help Many of the commands in this menu use ImageJ s BrowserLauncher to open a Web page using the user s default browser On Linux BrowserLauncher looks for netscape firefox konqueror mozilla opera epiphany or lynx and uses the first one it finds 33 1 ImageJ Website Opens the ImageJ home page 33 2 ImageJ News Opens the News section of the ImageJ website 33 3 Documentation Opens the Documentation section of the ImageJ website 33 4 Installation Opens the Installation section of the ImageJ website specifically dedicated to the OS in which ImageJ is running i e either Linux Mac OS 9 Mac OS X or Windows Worth reading are the sections Known Problems and Troubleshooting 33 5 Mailing List Opens the ImageJ Interest Group page on the NIH LISTSERV facility Here you can search the mailing list archives post a message to the list join or leave the list or update options 33 6 Dev Resources Opens the Developer Resources section of the ImageJ website 33 7 Plugins Opens the Plugins page on the ImageJ website which lists more than 500 ImageJ plugins 33 8 Macros Opens the macros directory on the ImageJ website which contains more than 400 ImageJ macros 33 9 Macro Functions Opens the Macro Functions reference page an indispensable guide to the built in functions that can be called from the ImageJ macro
93. 4 M Dark background Original Filtered Select Segmentation of DAPI stained nuclei using Image gt Adjust gt Color Threshold 80 Last updated 2012 10 02 Image gt Adjust gt Pass If checked values within range are thresholded and displayed band pass filter otherwise values outside the selected range are thresholded band reject filter Thresholding Method Allows any of the 16 different automatic thresholding methods to be selected see Threshold T Threshold Color Selects the threshold color either Red Black White or Black amp White see Threshold T Color space Selects the color space HSB RGB CIE Lab or YUV see Color Spaces and Color Separation Dark background To be checked when features are lighter than the background see XXII Interpreting Binary Images The state of the checkbox is remembered across restarts Original Restores the original image and updates the buffer when switching to another image Filtered Shows the filtered image Note that the final thresholded image type is RGB not 8 bit gray see Image Types and Formats Select Creates a ROI selection based on the current settings The selection is made according to the settings defined in the Process Binary Options dialog Sample Experimental Sets the ranges of the filters based on the pixel value components in a user defined ROI Stack Processes the remaining slices of the stack if any using t
94. 46r has been intentionally omitted The guide was typeset with T XLive 2012 on Mac OS 10 6 8 All illustrations were created with ImageJ Fiji loaded with G Landini s IJ Robot and J Schindelin s Tutorial Maker plugins Screenshots were produced by the screencapture shell utility controlled by the following IJ macro exec screencapture ciWo gt dev null 2 gt 1 amp run System Clipboard setLineWidth 1 setForegroundColor 111 121 132 drawRect 0 0 getWidth getHeight The HTML version was produced with eLyXer 1 2 and formatted using CSS code from Alex Fern ndez and Michael Hiineburg JavaScript code from Ciar n O Kelly Stuart Langridge and Tiago Ferreira It uses SyntaxHighlighter and icons from the Tango Desktop Project Getting Involved Your help is needed to improve ImageJ Even if you are not a programmer your participation is important Are you a skilled ImageJ user You might want to help out with the documentation effort Write a FAQ How To Tutorial or Video Tutorial on the ImageJ Documentation Portal Help us updating the ImageJ User Guide Share the add ons you may have created with the community Subscribe the mailing list and help answering the questions raised by other users Are you know knowledgeable in image processing Please join the mailing list and participate in the discussions You could also write a Tutorial on the ImageJ Documentation Portal Do you have a st
95. 9 Plugin Tools 40 Point 33 Scrolling 36 Spray Can 40 Stacks Menu 40 Text 35 Toolset Switcher see More Tools Menu 36 Toolsets 36 40 Topological Skeleton see Skeleton 113 Tracing see Wand tool 34 185 TrakEM2 4 Transform 64 97 Translate 98 Transparency see Blend 102 Tutorials 6 Undo 9 59 71 Unsharp mask 125 Updates 2 164 Variance 125 Virtual stacks see Stacks Virtual 12 Voronoi 115 Voxel depth 82 Wand tool 34 Watershed 107 115 Web browser 163 Window management 162 Window Synchronization 153 Windows OS 2 XOR see Logical operations 128 150 Yen see Threshold 77 YUV 11 14 79 YUV 80 Z Profile 91 Z Projection see Projection 89 93 ZIP 49 56 150 Archived TIFF files 10 Compressed TIFF 56 Lossless compression 10 Zoom 35 99 Fit to Screen 100 Colophon The initial contents of this guide have been retrieved in 2009 from the ImageJ website using html2text Since then it has been complemented and updated using informations posted on the ImageJ mailing list ImageJ Documentation Portal Fiji ImageJ and ImageJDev websites and Tony Collins ImageJ for Microscopy manual Nevertheless because there has never been accompanying documentation for some of the 350 described commands several sections have been written from scratch based on the relevant ImageJ source code and authors own experience Legacy nomenclature that became obsolete with version 1
96. Adjust gt Brightness Contrast C Adjust gt Threshold T Show Info i Properties P command Shortcut BOER ACA AG ale la BOBE WU rf Q A space oe UN UN gt gt m gt E BEE gt BEE r 3 AB 102 3 166 Description Create new image or stack Create new text window Create image from system clipboard Open file any format recognized by ImageJ Open next image in folder Opens the Blobs gif example image Close the active window Save active image in Tiff format Revert to saved version of image Print active image Undo last operation Copy selection to internal clipboard and clear Copy selection to internal clipboard Paste contents of internal clipboard Erase selection to background color Fill selection in foreground color Draw selection Invert image or selection Select entire image Remove selection Restore previous selection Defines selection properties Add selection to ROI Manager Adjust brightness and contrast Adjust threshold levels Display information about active image Display image properties Key Modifiers List of ImageJ 1 46r built in shortcuts This table can be obtained within ImageJ using the Plugins gt Shortcuts List Shortcuts command Command Operation Shortcut Color gt Color Picker K Stacks Next Slice gt Stacks p gt Previous Slice lt Stacks Reslice Stacks Orthogonal Views H S
97. Adjust gt Scale E Image gt Stacks Tools gt Grouped Z Project 99 Last updated 2012 10 02 Image gt Zoom gt 28 12 9 Image to Results Prints the active area selection to the Results Table clearing previous results The entire image is processed Ue CUTI when no area ROI exists XY coordinates are detailed in column and row headers Calibrated and floating point images are listed with the precision specified by Decimal places in Analyze gt Set Measurements blobs gif 800 For RGB images each pixel is converted to grayscale using the formula gray red green blue 3 or the formula gray 0 299 x red 0 587 x green 0 114 x blue if Weighted RGB to Grayscale Conversion is checked in Edit Options Conversions SEE ALSO Text Images Pixel Inspector Results to Image File gt Save Asp Text Image Import gt Text Image E Stack_to_Results js a script that displays the contents of a stack using one image per column 28 12 10 Results to Image The reverse of Image to Results converting the tabular data in the Results Table into a 32 bit image named Results Table Column and row headers are ignored SEE ALSO Text Images Pixel Inspector File gt Save Asp Text Image Import Text Image 28 13 Zoom This submenu contains commands that control how the current image is displayed The and or and keys are the preferred way to use the In and Out commands W
98. Area Selection Tools as an eraser press to erase in order to isolate the single graph curve to be measured Alternatively use one of the drawing tools Pencil or Brush to paint directly in background color 3 Open the Threshold T tool Shift T and adjust the threshold levels so that the curve is highlighted in red 4 Select the curve with the Wand Tool and run Edit Clear Outside to erase everything on the canvas but the curve 5 With the line still highlighted by the threshold widget run Analyze gt Tools Analyze Line Graph to get the XY coordinates of the traced line you can hold down while selecting the command to reveal the actual image that is processed 6 On the newly obtained plot select List Copy or Save these commands are described in Plot Profile k to export the curve coordinates into a spreadsheet application The exported values are tabulated in pixel coordinates unless the digitzed graph has been spatially calibrated using Analyzeb Set Scale or Image Properties P 147 Last updated 2012 10 02 Analyze gt Tools gt SEE ALSO Figure Calibration a simple plugin to retrieve data from other types of graphs such as scatter plots and histograms 30 14 4 Curve Fitting IN R AOO Curve Fitter AOO y a b x c x x User defined 1 Fit Open Apply M Show settings a Straight Line ot y soe re xx mp ps 2nd Degree Polynomial a 0 21071 2 00 4 5 3rd
99. Blur 29 6 3 Salt and Pepper Adds salt and pepper noise to the image or selection by randomly replacing 2 5 of the pixels with black pixels and 2 5 with white pixels This command only works with 8 bit images SEE ALSO Spray Can Filters Gaussian Blur 110 Last updated 2012 10 02 Process gt Shadows gt 29 6 4 Despeckle This is a median filter It replaces each pixel with the median value in its 3x3 neighborhood This is a time consuming operation because for each pixel in the selection the nine pixels in the 3x3 neighborhood must be sorted and the center pixel replaced with the median value the fifth Median filters are good at removing salt and pepper noise 29 6 5 Remove Outliers SLU Rec O Replaces a pixel by the median of the pixels in the surrounding D if it deviates from the median by more than a certain value the threshold Useful for correcting e g hot pixels or dead pixels of a CCD camera Radius 2 0 pixels Threshold 50 Which Outliers Bright P Radius Determines the area uncalibrated i e in pixels used ES Preview for calculating the median Run Process Filters gt Show Caned o Circular Masks to see how radius translates into an h e i area Threshold Determines by how much the pixel must deviate from the median to get replaced in raw uncalibrated units Which Outliers Determines whether pixels brighter or darker than the surrounding the median should be replaced
100. C tool Image gt Adjust gt Brightness Contrast C is to use the overall stack histogram 28 2 1 Brightness Contrast C Use this tool to interactively alter the brightness and contrast of the active image With 8 bit images brightness and contrast are changed by updating the image s lookup table LUT so pixel values are unchanged With 16 bit and 32 bit images the display is updated by changing the mapping from pixel values to 8 bit display values so pixel pixel values are also unchanged Brightness and contrast of RGB images are changed by modifying the pixel values Histogram The line graph at the top of the window which is superim posed on the image s histogram shows how pixel values are mapped to 8 bit 0 255 display values The two numbers under the plot are the minimum and maximum displayed pixel values These two values i a define the display range or window ImageJ displays images by nimam linearly mapping pixel values in the display range to display values Pp in the range 0 255 Pixels with a value less than the minimum are diia displayed as black and those with a value greater than the maximum Brightness are displayed as white a GA lt gt Contrast Minimum and Maximum sliders Control the lower and upper limits Cao Che of the display range Holding down will simultaneously adjust all channels of a composite image e g File Open Samples HeLa Cells 1 3M 48 bit RGB EA PT Set
101. CONVERTING COMPOSITE SELECTIONS Enter zero in the Edito Selection gt Enlarge dialog box to convert Composite Selections into polygon selections Note however that the conversion may fail if the composite ROI is composed of more than one piece and or contains internal holes 27 12 15 Make Band Takes an area selection and creates a band with a thickness of the Make Band specified number of pixels see ROI manipulations If you imagine the band as a doughnut shape then the original selection corresponds to ES as the hole i e the band is made by growing out the original selection Cancel OK AA SEE ALSO MakeSelectionBand the macro that implemented this command in previous IJ versions 27 12 16 Specify Opens a dialog that allows the user to define a rectangular or elliptical selection Width and Height are the dimensions of the selection X Coordinate and Y Coordinate define the position of the selection Check Oval to create an elliptical selection If Centered is checked the selection is positioned so X Coordinate and Y Coordinate define the center of the selection otherwise they define the upper left corner Width Height X Coordinate Y Coordinate O Oval _ Centered AN This command is also available through the ROI Manager More gt Cancel Ok _ drop down menu see Analyze gt Tools gt RO Manager 27 12 17 Straighten This command straightens a curved obje
102. Color Picker window XVII Embedding Color Annotations in Grayscale Images Brush Overlay Brush Pencil Text Tool Toolbar Shortcuts 19 4 A Angle Tool This tool allows you to measure an angle defined by three points Double click on the angle tool icon to enable the measurement of reflex angles The angle is displayed in the status bar while the selection is being created or adjusted Press Analyze gt Measure m to record the angle in the Results Table SEE ALSO Toolbar Shortcuts 19 5 Point Tool Use this tool to create a point selection to count objects or to record pixel coordinates Modifier keys Shift clicking adds more points creating a multi point selection see Multi point Tool Point count is displayed on the Status bar Alt Alt clicking on a point deletes it Alt clicking and dragging with the Rectangular Selection Tool or Oval Selection Tool deletes multiple points 33 Last updated 2012 10 02 Tools Multi point Tool Point Tool Double clicking on the point tool icon or running Editt gt eae els Options Point Tool displays its configuration dialog box E ET eee Mark Width If greater than zero a mark of the specified O Auto Next Slice diameter will be permanently drawn in the current fore Add to ROI Manager ground color cf Color Picker K Note that marks M Label Points Selection Color magenta modify the image it may be wise to work with a copy and co
103. Degree Polynomial ci par 3 00 8 0 4th Degree Polynomial R42 0 9874 ig cee 5th Degree Polyn A A Formula i 6th Degree Polyn 7th Degree Polyn Formula y a b x c x AM E re z 8th Degree Polyn e Simplex Fitting Options Power Power linear reg Function name User defined Formula y a b x c x x Exponential A 00 Formula y a b x c x x Exponential linear regression a C List Save Copy Status Success Exponential with Offset Initial b 1 00 Number of completed minimizations 2 Exponential Recovery Initial c 1 00 Number of iterations 821 max 9000 Log Time 99 ms Maximum iterations 4500 y a b In x c Sum of residuals squared 5 94693 Rodbard Number of restarts 2 Standard deviation 1 09059 Rodbard NIH Image Error tolerance 1 10A x 10 RA2 0 98744 Inverse Rodbard Parameters Gaussian es r y a 0 21071 Gamma Variate Cancel b 0 43178 y NUser defined gt ImageJ s CurveFitter Analyze gt Tools gt Curve Fitting ImageJ 1 46g ImageJ s CurveFitter provides a simple tool for fitting various functions to X and Y data using an improved multithreaded simplex algorithm 42 This strategy an iterative method is a kind of trial and error procedure in which the parameters of the fitting model are adjusted in a systematic way until the equation fits the data as close a
104. ED IN IJ 1 46R Image gt Adjust gt A 16 bit image consists of 65536 possible gray levels Most of times however the relevant image information is contained only within a narrow range of the grayscale This is the case e g in low light microscopy in which signal is restricted to the lower end of the grayscale The Set Display Range dialog allows you to choose how to scale the range of gray levels of 16 bit images ADOS Minimum displayed value Maximum displayed value Unsigned 16 bit range O Propagate to all open images LU Propagate to all other 4 channel images O Propagate to the other 3 channels of this image _ Set Display Range 0 255 Automatic Y Automatic Y 8 bit 0 255 10 bit 0 1023 12 bit 0 4095 15 bit 0 32767 16 bit 0 65535 Cancel O OK FD Automatic Automatically selects the best range given the intensity values of the image based on the percentage of the total number of pixel values from the lowest to highest pixel value 8 bit 0 255 Gray level range of 0 255 10 bit 0 1023 Gray level range of 0 1023 12 bit 0 4095 Gray level range of 0 4095 15 bit 0 3276 7 Gray level range of 0 32767 16 bit 0 65535 Gray level range of 0 65535 Check Propagate to all open images to apply these values to the rest of the images currently open With multi channel images the option to propagate the specified range to the remaining channels is also
105. Edit gt Selection gt Convert to Mask XX Creating Binary Masks XX CREATING BINARY MASKS Four ImageJ commands can be used to create binary masks 1 Edit Selection gt Create Mask 2 Process Binary Make Binary 3 Process Binary gt Convert to Mask 4 Imager Adjust gt Threshold T Apply By default these commands will produce binary images with inverted LUT so that black is 255 and white is 0 see Invert LUT This behavior can be reversed by checking Black Background in Process gt Binary Options before running the above commands i e an inverting LUT will not be used black will be 0 and white 255 This choice can be confirmed when running Make Binary and Convert to Mask on thresholded images It can also be imposed at startup see Settings and Preferences SEE ALSO XXII Interpreting Binary Images 29 8 3 Erode Removes pixels from the edges of objects in a binary image Use Filters gt Minimum to perform grayscale erosion on non thresholded images SEE ALSO Binarye Options XXII Interpreting Binary Images 113 Last updated 2012 10 02 Process gt Binary gt Original Voronoi Original Distance Map Ultimate Points Watershed Voronoi Ultimate Points AOO Voronoi O a Orig sNoconols UE x pixels It inverting pIx v AOOO Original 200x180 pixels 8 bit 35K OOP EDM OOC UEP Watershed 200x180 pixels 8 bit inverting x pixels o bit inverting x pixels o bit
106. Endrov 4 Eroded points 114 Erosion 112 Ethics see Acceptable manipulation 5 Euclidian distance map 107 114 115 Exponential transformation 118 Eye dropper 36 Fast Fourier transform 119 Fast Hartley Transform 119 Feret 136 Fiji 2 4 6 15 20 24 26 50 Filters 121 123 125 Find see Search 157 Fit Circle 63 Cubic spline 62 65 Curve see Curve fitting 147 Ellipse 63 136 FITS 11 58 Flatten 103 Floating point coordinates see Sub pixel selections 63 Flood Filler 38 Fractal box count 146 Frame interval 82 Freehand area selection 31 Freehand line selection 32 Frequency domain 119 Gamma correction 118 Gaussian Blur 124 1 D Gel analysis 143 GIF 55 Transparency 67 Gift wrap algorithm see Convex hull 63 Global calibration see Calibration 140 Graph 68 142 146 Grayscale morphology 112 Grouped Z Projection 93 HandleExtraFileTypes 50 Hartnack see IJ icon 174 Headless mode 26 Heat maps 101 Heckbert quantization 14 74 Help resources 5 7 163 Hexadecimal Hex colors 85 102 Histogram 140 Histogram measurements 134 Holes 113 133 Hot pixels 110 HSB 14 74 79 HSB 80 Huang see Threshold 77 Hyperstacks 95 Icy 4 IJ icon 174 Image calculator 127 Image formats Native 10 Non native 11 Image processing help 5 Image selection see ImageROI 20 100 Image sequence 50 57 Image SXM 4 Image types 10 13 74 ImageDev see ImageJ2
107. Hessman Gabriel Landini Gilles Carpentier Gregory Reneff Hiroki Hakoshima Ian Lim Ingo Bartholomaeus Jakob Preus Jamie Robinson Jan Eglinger Jan Funke Jarek Sacha Jay Unruh Jean Pierre Clamme Jerome Mutterer Jesper Pedersen Jim Passmore Joachim Wesner Johannes Hermen Johannes Schindelin Johannes Weissmann John Oreopoulos John Pearl Jonathan Silver Jose Wojnacki Juan Grande Julian Cooper Kai Uwe Barthel Karen Collins Kees Straatman Kevin Moll Kris Sheets Mark Krebs Mark Longair Martin Dressler Mat Al Tamimi Matthew Smith Michael Cammer Michael Doube Michael Ellis Michael Schmid Michel Julier Nagananda Gurudev Nico Stuurman Norbert Vischer Olaf Freyer Oliver Bannach Olivier Bardot Paul Jurczak Peter Haub Rainer Engel Raymond Coory Reinhard Mayr Richard Cole Richie Mort Robert Dougherty Shannon Stewman Simon Roussel Stefan Starke Stephan Saalfeld Steven Green Thomas Boudier Thorsten Wagner Tiago Ferreira Tomas Karlsson Tseng Qingzong Ulf Dittmer Uwe Walschus Valerio Mussi Ved Sharma Vytas Bindokas Wilhelm Burger Winfried Wurm Xiao Chen Zeljka Maglica XXVI FOCUS ON BIOIMAGE INFORMATICS In July 2012 Nature Methods issued a focus dedicated to the role of bioimage informatics in microscopy the tools that are available for scientific image processing and the challenges and opportunities in the field The special issue features a large collection of articles discussing ImageJ Fiji
108. I cis se ap ae hee ea 28 9 Duplicate D 28 10 Rename aoaaa 28 11 Scales A 28 12 Transform gt xe x asa aa ZO LO LOON aen Dr wk 26 TA yera y E so He SX 28 15 Lookup Tables gt 29 Process 29T SOOSI sx leas 29 2 Sharpen aoaaa ee a Ee 29 9 Aind Edges c wsos giei 29 4 Find Maxima 29 5 Enhance Contrast 1V 29 6 Noise eee soe erect A at OE et oe Be 110 ZO ShadowS gt ers wwe a amp Old Sok amp Sl IA OE oh ws 111 DOSS DIAS da Do Ae Ae ss EA os Se de 112 20 9 Mathes 4 42 vo oS do ove Se Re eee a EME HREAR SEEDS EE 117 DONO Pie oo oa 8 eee te e Ge ek ke ee eee oe ee eee 120 20 Ml FIE e 8 a Kick BES Sch ek oS oh Ew OS A BD SO ROS nd 3S a 124 DO E des oe ok oe Oe we et OS ES E A 126 DO AS Image Galca giz or dao de lead ee ee Se ee ds A oa a 128 20 VA Subtract BACK Bro ld aad amp o e le eo PS E ES 129 ZO Repeat Command R ect cae a So Sp te ds Be ee A ee Be OE Ad oo 131 30 Analyzer 132 UL Measures citas barriada SEO ib 132 30 2 Analyze Particles 4 4 44 ss a Bk E be RS OO A a 132 UA U E e as Axle co Grid A Syd eS Moko od eed OG ate a 135 SUE DISPO as 2 156 8 heeds e peda as Hoke Bee oe ie eb 135 SU apal eae ok ah ee eee ek ee A A a e 136 30 6 Clear iResullS y aoe xp ae o o eh Ace e es es a amp Bee da 136 30 7 Set Measurement a sofa piru a Balk oe oe ee we RS Ee OO ee ES 136 Gute Se ocalerb Fat an bee a aa ae Eee See a he Ot ee Gc 139 SOLO Cara Gaar aaa ee Oe A e
109. IMPROVED IN IJ 1 46R Tools FO Arrow Tool Width O Size ES Color black Style Notched P O Outline Double head Cancel OK Filled Notched Open Single Ne N YL head Double A Xx NS head Outline kal YW N Angle Tool Being an annotation tool arrows are created using foreground color see Color Picker K and not selection color see Point Tool Width and Size in pixels can be adjusted by dragging the respective sliders or by direct input Apart from the arrow styles listed here a Headless option is also available As for painting tools Brush Flood Filler and Pencil the Color dropdown menu provides a convenient way to reset the foreground color to one of the default options i As with any other selection add arrows to the non destructive overlay by pressing Image gt Overlay gt Add Selection b or D Edit gt Draw d to permanently draw the arrow on the image see XVII Embedding Color Annotations in Grayscale Images when working with non RGB images The same modifier keys described to the Straight Line Selection Tool apply to the arrow tool Forces the line to be either horizontal or vertical Alt Keeps the line length fixed while moving either end of the line Forces the two points that define the line to have integer coordinates when creating a line on a zoomed image While moving either end of the line the line is rotated resized about its center SEE ALSO
110. ImageJ Prop erties 165 Last updated 2012 10 02 Part VI Keyboard Shortcuts The following table summarizes the keyboard shortcuts built into ImageJ You can create additional shortcuts or override built in ones by creating simple macros and adding them to the ElStartupMacros txt You can also assign a function key to a menu command using Plugins gt Shortcuts Create Shortcuts Several of these shortcuts accept key modifiers as described in Key Modifiers Also note that except when using the Text Tool you do not need to hold down the control key to use a keyboard shortcut unless Require control key for shortcuts in Edit Options Misc is checked SEE ALSO Using Keyboard Shortcuts Finding Commands E KeyboardShortcuts txt macro demonstrating how assign shortcuts to custom macros Toolbar Shortcuts List of ImageJ 1 46r built in shortcuts This table can be obtained within ImageJ using the Plugins gt Shortcuts List Shortcuts Command Operation File gt NewbImage n Newer Text Window N New System Clipboard V Open o Open Next O Open Samples Blobs 25K Close w Save s Revert r Print p Edit gt Undo z Cut x Copy c Paste v Clear Fill f Draw d Invert I Selection gt Select All a Selection gt Select None A Selection gt Restore Selection E Selection gt Properties y Selection gt Add to Manager t Image
111. Imager Stacks gt Tools gt Animation Options Alt and Image Properties P as long as the unit of Frame Interval is sec SEE ALSO File gt Importp AVI 26 10 9 PNG Saves the active image in PNG Portable Network Graphics format All image types except RGB are saved as 8 bit PNGs 16 bit images are saved as 16 bit PNGs With 8 bit images the value to be used as the transparent index 0 255 1 for none can be set in the Editp Options Input Output dialog box Overlays are embedded when saving in PNG format 26 10 10 FITS Saves the active image in FITS Flexible Image Transport System format 13 26 10 11 LUT Saves the active image s lookup table to a file The 768 byte file consists of 256red values 256 green values and 256 blue values 26 10 12 Results Exports the contents of the Results window as a tab delimited or comma delimited El csv text file Prior to ImageJ 1 44b this command used to be named Measurements 26 10 13 Selection Saves the current area selection boundary to a file that can be later retrieved using File gt Open o to restore the selection Active Selections and Overlays are saved in the TIFF header by default see File gt Save Asp Tiff 26 10 14 XY Coordinates Exports the XY coordinates of the active ROI as a two column tab delimited text file ROI coordinates can also be retrieved using the List coordinates op
112. J for astronomy arXiv astro ph Jan 2006 arXiv astro ph 0611686v1 Jonas G Wilson Leedy and Rolf L Ingermann Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters Theriogenology 67 3 661 72 Feb 2007 doi 10 1016 j theriogenology 2006 10 003 XMa and MSHutson Quantifying the intercellular forces during drosophila morphogenesis American Physical Society page 29003 Mar 2006 URL http meetings aps org link BAPS 2006 MAR H29 3 180 List of Abbreviations and Acronyms Alt cas cw ae ds Alt Option or Meta key EUT stiee ese API ie Meee Application Programming AAA pende MIP ASCIL a eadoss American Standard Code for MJPCG Information Interchange NaN res Diseno Binary digit i i OME resar BMP Sipisnada Bitmap Image File Device Independent Bitmap DIB OP rs DDD Issac Bits per pixel A CODY sioda Charge Coupled Device PENG tios CUP ads eu Color Picker PEM tes CON cms Comma Separated Values PIMC 53 5558 CUE vee Computed Tomography PING Sxapawens CUT baie aaa Control key In this guide also the Ppl 6 Command key in Apple PPM keyboards Prsrssdcizs DICOM Digital Imaging and Communications in Medicine RAM esas DET a cos Dots Per Inch TEQeX EDM AAA Euclidian Distance Map RGB FAQ susatnenda Frequently Asked Questions ROI AA Fast Fourier Transform Shift
113. L Manager BOO ROI Manager O O O mri stack 1 tif 0013 0057 0051 Addit 186x226 pixels 8 bit 0026 0175 0067 gt d aoaia Triang i R Show All with Labels 0026 0112 0054 Update lor ES eg OO Omisa 0928 0185 0141 O Delete 186X 6 pixels 3 bIt 0001 0092 0095 i ERA f Some Annotation 7 0024 0098 0156 Rename 0026 0018 0141 Stroke Color white s 5 t Measure oO o i alll Font Size 16 X coordinate __ 4 Deselect Y coordinate More Fill Color 77000088 Slice number Options SOLO Options More y M Associate Show All ROIs with slices Deselect Select All m Al O Restore ROIs centered ow O Use ROI names as labels W Labels Cancel OK Misc operations via drop down menu also available when right clicking on list area The ROI Manager Analyze gt Tools gt ROI Manager ImageJ 1 46p Show All with without labels The ROI Region of Interest Manager is a tool for working with multiple selections Selections can be from different locations on an image from different slices of a stack or from different images All selection types including points lines and text are supported Add Click Add to add the current selection to the list or press E the keyboard shortcut for the Edit gt Selection gt Add to Manager t command The ROI manager creates a three part label The first part stacks only is the slice number the seco
114. Maxima Output Type Single Points Output Type Maxima Wit Output Type Segmented P O Results O Results 234 253 J 1 65 176 253 234 229 167 223 103 214 256x254 pixels 83 bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 X pixels amp bit 233 184 84 177 10 173 WOON DU hWN a 212 162 10 252 151 i 11 169 142 12 220 125 12 2 Q1 Process gt Find Maxima ImageJ 1 43s Six outputs are possible Single Points Maxima Within Tolerance Segmented Particles Point Selection List and Count Noise Tolerance Maxima are ig nored if they do not stand out from the surroundings by more than this value calibrated units for calibrated images In other words a threshold is set at the maximum value minus noise Y Single Points tolerance and the contiguous O Exclude Edge Maxima aima Within Tolerance area around the maximum above M Above Lower Threshold segmented Particles Bi iaht Background hiu Selection the threshold is analyzed For ei accepting a maximum this area must not contain any point with a value higher than the maximum Noise Tolerance 50 Output type Single Point M Preview Point Selection 65 Maxima Fa KE k Cancel OK o o T Only one maximum within this area is accepted Output Type Can be see Process gt Find Maxima ou
115. Nature Methods 9 7 683 9 Jun 2012 doi 10 1038 nmeth 2047 Christof Karmonik Michele York Robert Grossman Ekta Kakkar Krutina Patel Hani Haykal and David King An image analysis pipeline for the semi automated analysis of clinical MRI images based on freely available software Computers in biology and medicine Jan 2010 doi 10 1016 j compbiomed 2009 12 003 V Kaynig T Fuchs and J M Buhmann Neuron geometry extraction by perceptual grouping in sstem images In Computer Vision and Pattern Recognition CVPR 2010 IEEE Conference on pages 2902 2909 june 2010 doi 10 1109 CVPR 2010 5540029 St phane Kerner Daniel Etienne Jacques Malet Francis Mora Virginie Monnet Corti and Philippe Bouchard Root coverage assessment validity and reproducibility of an image analysis system J Clin Periodontol 34 11 969 76 Nov 2007 doi 10 1111 j 1600 051X 2007 01137 x German Kilimnik Abraham Kim Junghyo Jo Kevin Miller and Manami Hara Quantification of pancreatic islet distribution in situ in mice Am J Physiol Endocrinol Metab 297 6 E1331 8 Dec 2009 doi 10 1152 ajpendo 00479 2009 Y J Kim B F M Romeike J Uszkoreit and W Feiden Automated nuclear segmentation in the determination of the Ki 67 labeling index in meningiomas Clin Neuropathol 25 2 67 73 Jan 2006 Anna Kirilova Gina Lockwood M Math Perry Choi Neelufer Bana Masoom A Haider Kristy K Brock Cynthia Eccles and Laura A Dawson Three dimensional mot
116. Platelets 21 1 60 6 Feb 2010 doi 10 3109 09537100903410609 Kevin M Schartz Kevin S Berbaum Robert T Caldwell and Mark T Madsen WorkstationJ workstation emulation software for medical image perception and technology evaluation research Medical Imaging 2007 Image Perception 6515 49 Mar 2007 doi 10 1117 12 708482 Johannes Schindelin Ignacio Arganda Carreras Erwin Frise Verena Kaynig Mark Longair Tobias Pietzsch Stephan Preibisch Curtis Rueden Stephan Saalfeld Benjamin Schmid Jean Yves Tinevez Daniel James White Volker Hartenstein Kevin Eliceiri Pavel Tomancak and Albert Cardona Fiji an open source platform for biological image analysis Nature Methods 9 7 676 82 Jun 2012 doi 10 1038 nmeth 2019 Benjamin Schmid Johannes Schindelin Albert Cardona Mark Longair and Martin Heisenberg A high level 3D visualization API for Java and ImageJ BMC Bioinformatics 2010 11 274 11 274 Jan 2010 doi 10 1186 1471 2105 11 274 Caroline A Schneider Wayne S Rasband and Kevin W Eliceiri NIH Image to ImageJ 25 years of image analysis Nature Methods 9 7 671 Jun 2012 URL http www nature com nmeth journal v9 n7 full nmeth 2089 html doi doi 10 1038 nmeth 2089 Ruggero Scorcioni Susan N Wright JPatrick Card Giorgio A Ascoli and German Barrionuevo Point analysis in Java applied to histological images of the perforant pathway A user s account Jan 2008 doi 10 1007 s12021 008 9011 4 Joel B Sheffield ImageJ a
117. Plugin command More information on image filters can be obtained by looking up related keywords convolution Gaussian median mean erode dilate unsharp etc on the Hypermedia Image Processing Reference index SEE ALSO Memory amp Threads 29 11 1 Convolve Does spatial convolution using a kernel entered into a text area A kernel is a matrix whose center corresponds to the source pixel and the other elements correspond to neigh boring pixels The destination pixel is calculated by multiplying each source pixel by its corresponding ker nel coefficient and adding the results If needed the input image is effectively extended by duplicating edge pixels outward There is no arbitrary limit to the size M Normalize Kernel of the kernel but it must be square and have an odd Preview gt e Cancel width mum do de a om do de 6 6 3 3 3 3 olivo i i Ll Dto A Open Save Rows in the text area must all have the same number of coefficients the rows must be terminated with a carriage return and the coefficients must be separated by one or more spaces Kernels can be pasted into the text area using Checking Normalize Kernel causes each coefficient to be divided by the sum of the coefficients preserving image brightness The kernel shown is a 9 x 9 Mexican hat which does both smoothing and edge detection in one operation Note that kernels can b
118. This macro replaces Red with Magenta in RGB images using Process gt Image Calculator command if bitDepth 24 exit This macro requires an RGB image setBatchMode true title getTitle r title red g title preen b title blue run Split Channels imageCalculator Add b r run Merge Channels red amp r green amp blue kb rename title MGB setBatchMode false 1 Each channel is kept separate from the others and can be turned on and off using the Channels tool Image gt Color gt Channels Tool Z This feature allows e g to perform measurements on a specific channel while visualizing multiple 2 Channels can be 8 16 or 32 bit and can be displayed with any lookup table 3 More than 3channels can be merged or kept separate 2 Replace Red with Magenta ijm Using Image Color gt Channels This macro replaces Red with Magenta in RGB images using the Image gt Color gt Channels tool if bitDepth 24 Ignore non RGB images exit This macro requires an RGB image setBatchMode true Enter Batch mode title getTitle Retrieve the image title run Make Composite Run Image gt Color gt Make Composite run Magenta Run Image gt Lookup Tables gt Magenta on channel 1 runs RGB Color Run Image gt Type gt RGB Color rename title MGB Rename the image setBatchMode false Restore GUI mode 10 Selections Selections
119. X Maz values can be specified to fix the X axis range Y Maz Fixes the Y axis range Type Auto to have the range determined by the largest bin count The getHistogram and Plot getValues macro functions can be used to get the Value and Count data displayed when you click the List button cf 8 Obtaining Histogram Lists 8 Obtaining Histogram Lists 1 Single images r n Bile bolo 1 getHistogram values counts 256 for i 0 i lt values length i print values i countsli Pf 2 Entire stacks run fl Head 2 4M 16 bits FUNC Histogram stack Plot getValues values counts for i 0 i lt values length i print values i countsli 30 11 Plot Profile k Displays a two dimensional graph of the intensities of pixels along a line or rect angular selection The X axis represents distance along the line and the Y axis is the pixel intensity NaN values in 32 bit images see Image Types and Formats are ignored 100 For rectangular selections or line se lections wider than one pixel displays 7 0 a column average plot where the X Distance pixels axis represents the horizontal distance through the selection and the Y axis the vertically averaged pixel intensity To average horizontally hold or check Vertical Profile in Edito Options Profile Plot Options For real time examinations activate Live mode to continuously update the profile as the
120. a compressed ZIP archive 57 Last updated 2012 10 02 File gt Save Asp OA Text Image txt Jer fee fes fea cs fce Jer cs co cio eri cia cis cia cis ere Bs It UR o o oS o a So oS o 0 O 0 O 0 O 0 O 0 O 0 O OOOO ODO O O 8 O amp oO oO eo oOo 8 DOC ooo OOOO ODO OOO oOo O O ODO oaoqcdoo a amp oO oOo oOo ogo 8 8 ao geo eo amp Coo coo ree eo DO cea amp O OOO ODO DO 0 8 O amp Text Images File gt Import gt Text Image and File gt Save Asp Text Image 26 10 6 Raw Data Saves the active image or stack as raw pixel data without a header 8 bit images are saved as unsigned bytes unsigned 16 bit images are saved as unsigned shorts and signed 16 bit images e g File gt Open Samples CT 420K 16 bit DICOM are saved as signed shorts 32 bit images are saved as floats and RGB images are saved in three bytes per pixel 24 bit interleaved format 16 bit and 32 bit float images are saved using big endian byte order unless Export Raw in Intel Byte Order is checked in the Edit gt Options Input Output dialog box 26 10 7 Image Sequence Save Image Sequence Saves a Stack or a hyperstack as an image se quence Format TIFF Save Image Sequence Format Specifies the output format that can be set to either BMP FITS GIF JPEG PGM PNG Raw Text Image TIFF or Zip cf Image Types and Formats In IJ 1 44 and later Overlays are embedded whe
121. acks gt Reduce Dimensionality 28 6 15 4 Reverse Alias for the Image Transform gt Flip Z command 93 Last updated 2012 10 02 Image Stacks gt 28 6 15 5 Insert IN IJ 1 46R Inserts a Source image into a Destination image at the spec DO Stack Inserter ified X and Y Location pixel coordinates Source and TOREN Destination can be single images or stacks but must be of the same type see Image Types and Formats The Destination Destination tl head tif 19 image will be permanently modified once Source has been X Location 0 inserted Note that when Source is a single image Edit gt Y Location 0 Selection gt Image to Selection can be used together with Image Overlay gt Add Image to create image selections Cancel WOK ImageROIs a more convenient way of blending two open images SEE ALSO Image Type Imageb Stacks gt Label 28 6 15 6 Montage to Stack CUP i ea Stack Maker cc Converts a montage image to an image stack based on the specified Images per row 2 number of rows and columns taking into account a Border width Images per column 2 This is the opposite of what the Image Stacks gt Make Montage Border width 0 command does Cancel SEE ALSO Demontager plugin 28 6 15 7 Make Substack IN IJ 1 46R Extracts selected images from the active stack copy or ing them to a new stack in the order of listing or Enter a range e g 2 14 a range with increme
122. ad a fresh copy the next time it is launched Evaluate JavaScript j Ctrl J Runs JavaScript code in the editor window Note that Run Macro runs JavaScript code if the title of the file ends with js Debug This menu contains seven commands related to the macro debugging You can debug a macro using the commands in the Debug menu You start a debugging session initiating Debug Macro You can then single step through the macro code by repeatedly running Step Debug Macro d Ctrl D Starts running the macro in debug mode and opens the Debug window which initially displays the memory usage number of open images and the active image s title The macro stops running at the first executable line of code which is highlighted Use one of the following commands to continue execution Step e Ctrl E Executes the highlighted statement and advances to the next The variable names and values in the Debug window are updated Trace t Ctrl T Runs the macro displaying variable names and values in the Debug window as they are encountered Fast Trace T Ctrl Shift T Same as above but faster Run Runs the macro to completion at normal speed similarly to Macros Run Macro Run to Insertion Point E Runs the macro to a statement that was previously defined by clicking the mouse on an executable line of code Abort Exits debug mode SEE ALSO Extending ImageJ Macros and Scripts Th
123. age gt Zoom To Selection SEE ALSO Manipulating ROIs Tools 168 Last updated 2012 10 02 Toolbar Shortcuts Ctrl or Cmd Key Modifications 34 3 Ctrl or Cmd Key Modifications Rectangular Selection Tool and Oval Selection Tool Selection is resized around its center Straight Line Selection Tool Line is rotated resized around its center SEE ALSO Manipulating ROIs Tools 34 4 Space Bar Any Tool Switch to the Scrolling Tool 34 5 Arrow Keys Moving Selections The four arrow keys move selection outlines one pixel at a time Resizing Selections Rectangular and oval selections are resized by holding while using the arrow keys Stacks Navigation The and keys substitute for and for moving through a stack If there is a selection you must also hold Hyperstacks Navigation The and keys change the channel Hold to move through the slices and to move through the frames Zooming The and keys zoom the image in and out If there is a selection you must also hold either or SEE ALSO Manipulating ROIs Zoom gt 35 Toolbar Shortcuts Keyboard shortcuts cannot be used directly to activate tools in the ImageJ Toolbar with the exception of the Magnifying Glass and the Scrolling Tool However shortcuts can be assigned to macros that use the setTool macro function The set of macros listed below taken from E ToolShortcuts exemplify how to assign the function keys through to some of the most commonly used Tools Once copie
124. ages In the case of non native formats batch operations will be influenced by the behavior of the reader plugin or library cf Non native Formats 126 Last updated 2012 10 02 Process gt Batch gt 29 12 1 Measure This command measures all the images in a user specified folder by running the Analyzer Measure m command in all images of the chosen directory Note that measurements are performed on non thresholded images In the case of TIFF images saved with active selections measurements are performed on the ROI and not the whole image SEE ALSO Analyze gt Set Measurements Batch gt Macro 29 12 2 Convert Batch Convert CEE Desktop Rat me pue i or resizes multiple images rom a specified folder Out put y Desktop Converted PA Gane mm dile Selects ia source a containing e images to be processed Interpolation Bilinear 4 A ra Output Selects the destination folder where cale Factor 1 00 the processed images will be stored Cancel Convert Me Output Format Specifies the output format that can be set to TIFF 8 bit TIFF JPEG GIF PNG PGM BMP FITS Text Image ZIP or Raw cf Image Types and Formats and File gt Save As gt submenu Interpolation The resampling method to be used in case Scale Factor is not 1 00 see Im agep Size and Imagep Scale E For better results Average when downsizing is automatically selected when scaling d
125. al intensity optical tablet or step wedge see optical density calibration tutorial Autogain must be disabled during digitization to ensure generally linear intensity responses In addition images should also be scanned with sufficiently high spatial and dynamic bit depth resolutions the optical not interpolated resolution of a scanner can also be measured e g high contrast patterns 84 6 um 1 300inch apart should be distinguishable on images scanned at 300 dpi Note that in general a posteriori background corrections on scanned images should also be avoided All these issues have been discussed in detail in Gassmann et al Quantifying Western blots pitfalls of densitometry 38 N B The paper benchmarks a legacy version of Process gt Subtract Background 30 13 Gelso Use the commands in this submenu to analyze one dimensional electrophoretic gels Elec trophoretic densitometry is discussed in more detail in XXIV Using Scanners in Densitometry These commands use a simple graphical method that involves generating lane profile plots drawing lines to enclose peaks of interest and then measuring peak areas i e definite integrals using the Wand Tool Note that this technique cannot be used to compare bands on different gels unless gels are calibrated to known standards The commands listed in this submenu are Select First Lane Requires a rectangular selection Note that lanes are assumed to be vertical
126. all transparent points along each ray and projects their mean value It produces images with softer edges and lower contrast but can be useful when attempting to visualize objects contained within a structure of greater brightness e g a skull Slice Spacing The interval in pixels between the slices that make up the volume ImageJ projects the volume onto the viewing plane at each Rotation Angle Increment beginning with the volume rotated by Initial Angle and ending once the volume has been rotated by Total Rotation Lower Upper Transparency Bound Determine the transparency of structures in the vol ume Projection calculations disregard points having values less than the lower threshold or greater than the upper threshold Setting these thresholds permits making background points those not belonging to any structure invisible By setting appropriate thresholds you can strip away layers having reasonably uniform and unique intensity values and highlight or make invisible inner structures Note that you can also use Image Adjust gt Threshold T to set the transparency bounds Opacity Can be used to reveal hidden spatial relationships especially on overlapping objects of different colors and dimensions The surface Opacity parameter permits the display of weighted combinations of nearest point projection with either of the other two methods often giving the observer the ability to view inner structures through translucent outer
127. alled in the first available slot or in the last slot if no free slots are available Calibration Menu Custom tools gt Remove Tools 19 12 More Tools Menu The eight Toolbar slots between the Color Picker Tool and the More Tools Menu can be customized using this drop down menu named Toolset Switcher in previous IJ versions Tool configurations are stored in the ImageJ preferences file see Settings and Preferences and retrieved across restarts The More Tools list is populated by E StartupMacros txt in GImageJ macros Toolsets installed in G ImageJ macros toolsets built in tools loaded from Hlij jar Arrow Brush Developer Menu Flood Filler LUT Menu Overlay Brush Pencil Spray Can and Stacks Menu and Single Tools installed in GImageJ plugins Tools At startup the default set of tools is typically loaded from ElStartupMacros txt Later on tools can be appended or replaced Single Tools are installed in the first available slot or in the last slot if no free slots are available Toolsets replace all the eight slots in the toolbar Choose Remove Tools to reset the toolbar The icons for drawing tools installed from this menu reflect the foreground color see Color Picker K and are updated when the foreground color changes Modifier key Shift clicking on the menu icon will open the selected macro El txt and El ijm files SEE ALSO Custom Tools Toolbar Shortcuts 37 Last updated 2012 10 02
128. alues and can have ANY intensity value including NaN Not a Number RGB Color Color Images that can display 256 values in the Red Green and Blue channel These are 24 bit 23X8 images RGB color images can also be 32 bit color images 24 bit color images with additional eight bits coding alpha blending values i e transparency Native Formats Natively i e without the need of third party plugins ImageJ opens the following formats TIFF GIF JPEG PNG DICOM BMP PGM and FITS Many more formats are supported with the aid of plugins These are discussed in Non native Formats TIFF Tagged Image File Format is the default format of ImageJ cf File Save s Images can be 1 bit 8 bit 16 bit unsigned 32 bit real or RGB color TIFF files with multiple images of the same type and size open as Stacks or Hyperstacks ImageJ opens lossless compressed TIFF files see II Image Types Lossy Compression and Metadata by the LZW PackBits and ZIP Deflate Inflate 2 compression schemes In addition TIFF files can be opened and saved as ZIP archives Tiff tags and information needed to import the file number of images offset to first images gap between images are printed to the Log Window when ImageJ is running in Debug Mode Edit gt Options Misc see Settings and Preferences A numeric variable is signed if it can represent both positive and negative numbers and unsigned if it can only represent positive num
129. ambert law so that you are aware that the optical density absorbance of the staining to be measured must be proportional to the concentration of the probed material An obvious corollary of the Beer Lambert law is that saturated stains or overexposed films cannot be quantified Generally speaking office scanners are not suitable for densitometric analysis That being said and in the absence of better alternatives they can be used to digitize gels and X ray films if the following guidelines are respected Samples must be scanned in transmission mode Most flatbed scanners have the light source and detector located on the same side of the instrument reflection mode Under these conditions light interacts with semi transparent objects in a complex manner with reflected light re passing through the object on its way to the detector In addition thick samples such as Coomassie stained gels are imaged from the surface under reflection light Scanners can only serve as densitometers if the light source and detector are on opposite sides of the instrument transmission mode This is critical for quantitation of absorbances and requires an adapter for transparencies or negatives to be mounted or built in on the scanner The scanner must offer a linear intensity response The response characteristics of most scanner detectors is unknown therefore the film gel should always be scanned next to a photographic gray scale image of known optic
130. amples port1 use default port address 1 port2 use default port address 2 port0 do not check for another instance OtherInstance Runs a macro or script passing it an optional argument which can be retrieved using getArgument Examples macro analyze ijm macro analyze Users wayne images stackl Runs a macro or script in batch mode no GUI passing it an optional argument ImageJ exits when the macro finishes Evaluates macro code Examples eval print Hello world eval return getVersion Runs an ImageJ menu command Example run About ImageJ Runs ImageJ in debug mode SEE ALSO Linux installation ImageJ Documentation Portal Command line Zi Last updated 2012 10 02 Part IV ImageJ User Interface Unlike most image processing programs ImageJ does not have a main work area ImageJ s main window is actually quite parsimonious containing only a menu bar at the top of the screen on the Mac containing all the Menu Commands a Toolbar a Status bar and a Progress bar Images histograms profiles widgets etc are displayed in additional windows Measurement results are displayed in the Results Table Most windows can be dragged around the screen and resized The window version 1 46j 10 11 12 A B C D E F G H 133 Toloa lu elslolalr gt ii Status bar bar 1 Rectangular Selection Tool and Rounded 8 Wand Tool Rectangular Selection Tool 9 Text Tool 2 Oval Selec
131. anager commands Non destructive annotations Overlays are the best way of annotating images in ImageJ examples As vector graphics overlays do not change pixel values can be scaled without loss of quality even at high zoom levels see XVIII Working with Zoomed Canvases and can be displayed at different opacity values see XIX Hexadecimal Color Values RGB snapshots of the image with embedded overlays can be created by holding F the shortcut for Image Overlay gt Flatten F Flattened images with the overlay rendered as pixel data are also created when saving the image as PNG or JPEG File gt Save Asp or when printing the image canvas Filep Print p The Flatten command is also listed in the ROI Manager Image ROIs An imageROI image selection is a ROI that displays an image as an overlay As described in Edit gt Selection gt Image to Selection and Imager Overlay gt Add Image this allows multiple images to be blended on a single image canvas 20 Last updated 2012 10 02 IJ 1 46R 3D Volumes AA ImageJ 3D Viewer Eja am File Edit View Add Landmarks Help ya y f E j AOO ImageJ 3D Viewer File Edit View Add Landmarks Help 3D Viewer Fiji 1 460 bringing hardware accelerated 3D visualization to ImageJ As ex plained in 3D Volumes most of plugins that truly extend ImageJ functionally to multi dimentional data are bundled as part of Fiji 12 3D Volumes Currently
132. and Related Software including Cardona A Tomancak P Current challenges in open source bioimage informatics 14 Carpenter AE et al A call for bioimaging software usability 18 Eliceiri KW Biological imaging software tools 30 Schindelin J et al Fiji an open source platform for biological image analysis 100 Schneider CA et al NIH Image to ImageJ 25 years of image analysis 102 A full announcement can be found on the Fiji news channel 173 ImageJ Related Publications The following references are a small sample particularly biased towards the life sciences of the bibliography directly related to ImageJ the standard in scientific image analysis These publications include 1 technical articles and books describing routines implemented in ImageJ 2 research articles that have made extensive use of ImageJ as a scientific tool or 3 reviews that discuss ImageJ pertinently A similar list is maintained on the Fiji website To cite ImageJ one of the citations is possible see FAQs l Rasband WS ImageJ U S National Institutes of Health Bethesda Maryland USA imagej nih gov ij 1997 2012 Abramoff MD Magalhaes PJ and Ram SJ Image Processing with ImageJ Biophotonics International 11 7 36 42 2004 PDF 1 Schneider CA Rasband WS and Eliceiri KW NIH Image to ImageJ 25 years of image analysis Nature Methods pp 671 2012 102 To cite Fiji S
133. and V Hartenstein Drosophila brain development closing the gap between a macroarchitectural and microarchitectural approach Cold Spring Harbor Symposia on Quantitative Biology 74 235 48 Jan 2009 doi 10 1101 sqb 2009 74 037 Albert Cardona Stephan Saalfeld Stephan Preibisch Benjamin Schmid Anchi Cheng Jim Pulokas Pavel Tomancak and Volker Hartenstein An integrated micro and macroarchitectural analysis of the Drosophila brain by computer assisted serial section electron microscopy PLoS Biol 8 10 Jan 2010 doi 10 1371 journal pbio 1000502 Albert Cardona and Pavel Tomancak Current challenges in open source bioimage informatics Nature Methods 9 7 661 5 Jun 2012 doi 10 1038 nmeth 2082 Thomas Carlier Ludovic Ferrer Jean B Berruchon Regis Cuissard Adeline Martineau Pierre Loonis and Olivier Couturier Quality controls for gamma cameras and PET cameras development of a free open source ImageJ program Medical Imaging 2005 Physics of Medical Imaging Edited by Flynn 5745 1237 Apr 2005 doi 10 1117 12 595539 R Carmona D Mac as J A Guadix V Portillo J M P rez Pomares and R Mu oz Ch puli A simple technique of image analysis for specific nuclear immunolocalization of proteins Journal of microscopy 225 Pt 1 96 9 Jan 2007 doi 10 1111 3j 1365 2818 2007 01719 x Anne E Carpenter Thouis R Jones Michael R Lamprecht Colin Clarke In Han Kang Ola Friman David A Guertin Joo Han Chang Robert A Lindquist Jas
134. and thus sharpens the image C Preview a Cancel OK T Radius The standard deviation blur radius cf Gaussian Blur of the Gaussian blur that is subtracted Increasing the Gaussian blur radius will increase contrast Mask Weight Determines the strength of filtering whereby MaskW eight 1 would be an infinite weight of the high pass filtered image that is added Increasing the Mask Weight value will provide additional edge enhancement 29 11 9 Variance Highlights edges in the image by replacing each pixel with the neighborhood variance 29 11 10 Show Circular Masks Generates a stack containing examples of the circular masks used by the Median Mean Minimum Maximum and Variance filters for various neighborhood sizes 29 12 Batch This submenu allows the execution of commands in a series of images without manual intervention Batch gt commands are non recursive i e they are applied to all the images of the chosen Input folder but not its subfolders Nevertheless a directory hierarchy can be transversed using ImageJ macro language cf BatchProcessFolders macro Three critical aspects to keep in mind when performing batch operations that modify processed images Files can be easily overwritten since the batch processor will silently override existing files with the same name The destination Output folder should have adequate disk space to receive the created im
135. ands are applied to all ROIs Properties Similarly to Edit gt Selection gt Properties y opens a dialog box in which is possible to assign a contour color Stroke color of a certain Width or a Filling color Set 150 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IJ 1 46R CHANGED IN IJ 1 46R IMPROVED IN IJ 1 46R Analyze gt Tools gt Stroke width to O to have selections drawn using a width of one pixel regardless of the image magnification see XVIII Working with Zoomed Canvases As previously mentioned selections can be either filled or contoured but not both The nine default selection colors black blue cyan green magenta orange red white yellow can be typed textually Any other color must be specified using hex notation see XIX Hexadecimal Color Values With Text Tool selections it is also possible to specify the Font size and Justification If multiple ROIs have been selected from the ROI Manager s list see XXV Selecting ROIs in the ROI Manager properties will be applied to the specified Range of selections Flatten F Alias for Image Overlay gt Flatten F Show All Toggles the display of all ROI Manager Overlays If Labels is active ROIs will also be labelled Once Show All is checked ROIs can be re activated by Alt clicking Control clicking or long pressing 1 4 second or longer Re activated sele
136. ann Amit Bar Or and Alyson E Fournier NeuriteTracer a novel ImageJ plugin for automated quantification of neurite outgrowth J Neurosci Methods 168 1 134 9 Feb 2008 doi 10 1016 j jneumeth 2007 08 029 Jonathan Popko Adelaide Fernandes Dora Brites and Lorene M Lanier Automated Analysis of NeuronJ Tracing Data Cytom Part A 75A 4 371 376 Jan 2009 doi 10 1002 cyto a 20660 Stephan Preibisch Stephan Saalfeld Johannes Schindelin and Pavel Tomancak Software for bead based registration of selective plane illumination microscopy data Nature Methods 7 6 418 9 Jun 2010 doi 10 1038 nmeth0610 418 178 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 Stephan Preibisch Stephan Saalfeld and Pavel Tomancak Globally optimal stitching of tiled 3D microscopic image acquisitions Bioinformatics 25 11 1463 5 Jun 2009 doi 10 1093 bioinformatics btp184 Olaf Ronneberger Kun Liu Meta Rath Dominik Ruess Thomas Mueller Henrik Skibbe Benjamin Drayer Thorsten Schmidt Alida Filippi Roland Nitschke Thomas Brox Hans Burkhardt and Wolfgang Driever ViBE Z a framework for 3D virtual colocalization analysis in zebrafish larval brains Nature Methods Jun 2012 doi 10 1038 nmeth 2076 R Ropolo O Rampado P Isoardi A Izzo L Savio T Cammarota O Davini R De Lucchi and G Gandini Automated quality control in comp
137. are converted from one type to another wi Weighted RGB Conversions 0 30 0 59 0 11 EES aS Cancel OK T i Scale When Converting ImageJ will scale from min max to 0 255 when converting from 16 bit or 32 bit to 8 bit or to scale from min max to 0 65535 when converting from 32 bit to 16 bit Note that Scale When Converting is always checked after ImageJ is restarted Weighted RGB Conversions 0 30 0 59 0 11 When checked the formula gray 0 299 x red 0 587 x green 0 114 x blue is used to convert RGB images to grayscale If it is not checked the formula gray red green blue 3 is used The default weighting factors 0 299 0 587 0 114 which are based on human perception are the ones used to convert from RGB to YUV the color encoding system used for analog television The weighting factors can be modified using the setRGBWeights macro function SEE ALSO Image Types and Formats Embedding Color Annotations in Grayscale Images fA Last updated 2012 10 02 Edit gt Options gt 27 13 12 Memory amp Threads Memory 64 bit Use this dialog to specify the maximum amount of mem ae a alado si ory available to ImageJ and the number of threads used ii is E by filters when processing stacks Java applications such wi Keep multiple undo buffers as ImageJ will only use the memory allocated to them M Run garbage collector on status bar click typically 640 MB but this dialog allows the user to Help Fa
138. are created or resized selection properties e g location width etc are displayed on the status bar In addition clicking on ImageJ s status bar forces the Java garbage collector to run which may help to reclaim unused memory see Edit gt Options gt Memory amp Threads You can assess this by running Plugins gt Utilities gt Monitor Memory each click on the Status bar should lead to a spike in the ImageJ s memory utilization AA ear ES PILA LACIE Image 1 4305 Java 1 6 0_17 64 bit 3344K of 1166MB lt 1 hk SEE ALSO Plugins Utilities gt ImageJ Properties Help About ImageJ V TOGGLING CALIBRATED UNITS If a spatial scale has been defined in Image Properties P or Analyzeb Set Scale selection properties are displayed in the Status bar in calibrated units Resizing or moving while holding down forces this information to be displayed in pixels Progress bar The progress bar located to the right of the status bar shows the progress of time consuming operations It will not appear if the operation requires less then approximately one second 19 Tools 19 1 Area Selection Tools These tools share the first four toolbar slots As described in Toolbar use the right click drop down menu to switch a different tool Selection Color can be changed by double clicking on the Point Tool Multi point Tool 19 1 1 Rectangular Selection Tool Location width height and aspect ratio
139. are displayed in the status bar during drawing see V Toggling Calibrated Units Modifier keys Selection is constrained to a square Alt Current aspect ratio is maintained while resizing With arrow keys width and height are changed one pixel at a time Selection is resized around the center SEE ALSO Rounded Rectangular Selection Tool Specify XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 29 Last updated 2012 10 02 Tools Area Selection Tools 19 1 2 Al Rounded Rectangular Selection Tool IN IJ 1 46R eel ane ee This tool creates rectangular shapes with rounded corners It shares the same toolbar slot and the same modifier keys with the Rectangular Selection Tool Double clicking on its icon opens the depicted dialog in which is possible to Stroke color 99ff0000 specify Stroke width 69 ejg Corner diameter 40 EJ CIA Fill color Stroke width The width of the contour Cancel OK Corner diameter The arc size at the vertices Stroke Fill Color The contour stroke color or the filling color of the rounded rectangle As explained in Edit Selection Properties y selections can be either filled or contoured but not both The nine default selection colors black blue cyan green magenta orange red white yellow can be typed as text Any other color must be typed in hex notation see XIX Hexadecimal Color Values SEE ALSO Rectangular Selection Tool XVII Em
140. ated For straight line selections Angle is recorded even if Fit Ellipse is unchecked Also note that measurements that do not apply to certain selection types may be listed as NaN Infinity or Infinity The second part of the dialog controls measurement settings Limit to threshold If checked only thresholded pixels are included in measurement calcula tions Use Image Adjust gt Threshold T to set the threshold limits This setting affects only thresholded images see Settings and Preferences Display label If checked the image name and slice number for stacks are recorded in the first column of the Results Table e g mri stack tif 9 For renamed selections Edit gt Selection gt Properties y or selections measured via ROI Manager s measure com mand see ROI Manager the selection label is appended e g blobs gif 0339 0163 or blobs gif mySelection Invert Y coordinates If checked the XY origin is assumed to be the lower left corner of the image window instead of the upper left corner see also Image Properties P Scientific notation If checked measurements are displayed in scientific notation e g 1 48E2 Add to Overlay If checked measured ROIs are automatically added to the image overlay see Overlays Appearance of overlay selections can be adjusted using Image Overlay gt Overlay Options Labels Redirect to The image selected from this popup menu will be used as the targe
141. atershed result og y Process gt Binary gt Watershed running in Debug mode 29 8 12 Watershed Watershed segmentation is a way of automatically separating or cutting apart particles that touch It first calculates the Euclidian distance map EDM and finds the ultimate eroded points UEPs It then dilates each of the UEPs the peaks or local maxima of the EDM as far as possible either until the edge of the particle is reached or the edge touches a region of another growing UEP Watershed segmentation works best for smooth convex objects that don t overlap too much Enable debugging in Edit gt Options gt Misc and the Watershed command will create an animation that shows how the watershed algorithm works cf online example SEE ALSO Find Maxima Segmented Particles output for watershed segmentation of grayscale images 29 8 13 Voronoi Splits the image by lines of points having equal distance to the borders of the two nearest particles Thus the Voronoi cell of each particle includes all points that are nearer to this particle than any other particle When particles are single points this process is a Voronoi tessellation also known as Dirichlet tessellation The output type Overwrite 8 bit 16 bit or 32 bit of this command can be set in the Process gt Binary gt Options dialog box In the output the value inside the Voronoi cells is zero the pixel va
142. atio to a value other than 1 0 enables support for different horizontal and vertical spatial scales e g 100 pixels cm horizontally and 95 pixels cm vertically To set the Pixel Aspect Ratio 1 Measure the width and height in pixels of a digitized object with a known 1 1 aspect ratio 2 Enter the measured width in pixels in Distance in Pixels Enter the known width in Known Distance 3 Calculate the aspect ratio by dividing the width by the height and enter it in Pixel Aspect Ratio When Global is checked the scale defined in this dialog is used for all opened images during the current session instead of just the active image see XXIII Global Calibrations Click to Remove Scale resets Distance in Pixels field and Known Distance to zero and the Unit of Length to pixel SEE ALSO Tutorials showing how to use this command Examples of Image Analysis Using ImageJ by Larry Reinking Measuring DNA Contour Lengths and Spatial Calibration Fiji The Copy Pixel Size plugin 30 9 Calibrate Use this dialog box to calibrate an image to a set of density standards for example radioactive isotope standards or a calibrated optical density step tablet Note that in general calibrations cannot be applied to 32 bit images the pixel intensity unit of 32 bit images can still be changed nevertheless The calibration procedure is done in three steps 1 Use Analyze gt Clear Results to reset the measurement counter use one of t
143. available Apply Applies the current display range mapping function to the pixel data If there is a selection only pixels within the selection are modified This option currently only works with 8 bit images 8 bit stacks and RGB stacks This is the only B amp C option that alters the pixel data of non RGB images SEE ALSO Window Level Enhance Contrast Color Balance XIV Applying Auto Bright ness Contrast to Entire Stacks XVI Brightness Contrast of High Bit Depth Images XV Display Range of DICOM Images 28 2 2 Window Level Window Center Level 94 Window Width 188 Pp pp Level 94 pp a y Window 188 Cancel U Propagate to all open imac a Ca E Auto jf Reset E This command and Brightness Contrast C B amp C are redundant but Window Level W amp L behaves in a manner closer to that imple mented on medical image terminals by interactively adjusting the Window range of minimum and maximum Contrast and Level position of that range in the grayscale intensity space Brightness If the B amp C window is opened it will be closed and the W amp L window will be opened at the same location T7 Last updated 2012 10 02 Image gt Adjust gt XV DISPLAY RANGE OF DICOM IMAGES With DICOM images ImageJ sets the initial display range based on the Window Center 0028 1050 and Window Width 0028 1051 tags Click Reset on the W amp L Image
144. awing SEE ALSO Freehand Line Selection Tool Polygon Selection Tool Enlarge V Toggling Cal ibrated Units XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 2 Line Selection Tools Use these tools to create line selections The three line selection tools share the same toolbar slot As described in Toolbar use the right click drop down menu to switch between line tools Double click on any line tool to specify the line width by opening the Image Adjust gt Line Width widget on which is also possible to apply a cubic spline fit to a polyline selection Check the Sub pixel resolution checkbox in Edit Options gt Profile Plot Options to create line selections with floating point coordinates see Selections With Sub pixel Coordinates 31 Last updated 2012 10 02 Tools Arrow Tool 19 2 1 Straight Line Selection Tool Length and line angle are displayed in the status bar during drawing see V Toggling Calibrated Units Modifier keys Forces the line to be either horizontal or vertical Alt Keeps the line length fixed while moving either end of the line Forces the two points that define the line to have integer coordinates when creating a line on a zoomed image While moving either end of the line the line is rotated resized about its center SEE ALSO Calibration Bar Set Scale V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19
145. b1420s92 Kevin W Eliceiri Michael R Berthold Ilya G Goldberg Luis Ibanez B S Manjunath Maryann E Martone Robert F Murphy Hanchuan Peng Anne L Plant Badrinath Roysam Nico Stuurmann Jason R Swedlow Pavel Tomancak and Anne E Carpenter Biological imaging software tools Nature Methods 9 7 697 710 Jun 2012 doi 10 1038 nmeth 2084 175 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Fern n Federici Lionel Dupuy Laurent Laplaze Marcus Heisler and Jim Haseloff Integrated genetic and computation methods for in planta cytometry Nature methods Jun 2012 URL http www nature com nmeth journal v9 n5 full nmeth 1940 html doi 10 1038 nmeth 1940 J r me N Feige Daniel Sage Walter Wahli B atrice Desvergne and Laurent Gelman PixFRET an ImageJ plug in for FRET calculation that can accommodate variations in spectral bleed throughs Microsc Res Tech 68 1 51 8 Sep 2005 doi 10 1002 jemt 20215 L Ferrer T Carlier A Lisbona and M Bardies An imageJ plugin to create whole body transmission scan using CT scanner a validation study Eur J Nucl Med Mol I 34 5198 S198 Jan 2007 L Ferrer Y Grealou D Autret S Gaudaire G Brunet G Delpon A Lisbona B Bridji I Resche C Rousseau T Carlier and M BardiAs A new ImageJ plugin to correct for partial effect volume Eur J Nucl Med Mol I 31 5230 5230 Jan 2004 Michael
146. bedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 1 3 O Oval Selection Tool Location width height and aspect ratio are displayed in the status bar during drawing see V Toggling Calibrated Units Modifier keys Selection becomes circular Alt Current aspect ratio is maintained while resizing With arrow keys width and height are changed one pixel at a time Selection is resized around the center SEE ALSO Elliptical Selection Tool Specify V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 1 4 OJ Elliptical Selection Tool IN IJ 1 46R Ellipse properties are adjusted by dragging the four handlers on its antipodal points 4 To rotate or resize drag the handlers on its major axis transverse diameter To adjust eccentricity drag the handlers on its minor axis conjugate diameter SEE ALSO Oval Selection Tool XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 30 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Tools Line Selection Tools 19 1 5 2 Brush Selection Tool Adjusts refines the shape of area selections using a circular brush 5 Clicking inside the area selection and dragging along its boundary will expand the boundary outwards Clicking outside the area selection and dragging along its boundary will shrink the boundary inwards Once the tool has been applied ImageJ will treat the adjusted ROIs as
147. ber of Labels Adjusts the total number of values displayed Decimal Places Adjust the Location At Selection l number of decimal places Fill Color None present in the labels Label Color Black 0 amaa Font Size Adjusts labels font Number of Labels 5 size labels are drawn bold Decimal Places 2 if Bold Text is checked Font Size 14 Zoom Factor 10 Zoom Factor Magnifies the entire calibration bar can vas Enter a value less than 1 to reduce the bar size The Calibration Bar Macros can be used to add a calibration bar to a stack or to all the images and stacks in a folder C Bold Text E 3 Cancel OK SEE ALSO Calibrate XXIII Global Calibrations 30 14 8 Synchronize Windows Synchronizes mouse motion and input between multiple windows so that a ROI drawn in one image is replicated in all other Synchronized windows 46 A synchronization cursor indicates the location of the mouse across the synchronized window set Synchronized window set Images to be synchronized are specified in this list containing all open images To select a consecutive group of images click the first item press and hold down Shift and then click the last item Alternatively click on the first item and drag it across To select non consecutive images press and hold down Ctrl then click each item to be selected Use the Un Synchronize All buttons to de select all listed images Sync cursor If checked th
148. bers 10 Last updated 2012 10 02 Image Types and Formats DICOM Digital Imaging and Communications in Medicine is a standard popular in the medical imaging community Support in ImageJ is limited to uncompressed DICOM files DICOM files containing multiple images open as Stacks Use Imager Show Info i to display the DICOM header information A DICOM sequence can be opened using File gt Import gt Image Sequence or by dragging and dropping the folder on the ImageJ window Imported sequences are sorted by image number instead of filename and the tags are preserved when DICOM images are saved in TIFF format ImageJ supports custom DICOM dictionaries such as the one at http imagej nih gov ij download docs DICOM _ Dictionary txt More information can be found at the Center for Advanced Brain Imaging FITS Flexible Image Transport System image is the format adopted by the astronomical community for data interchange and archival storage Use ImageShow Info i to display the FITS header More information here PGM Portable GrayMap PBM Portable BitMap and PPM Portable PixMap are simple image formats that use an ASCII header More information here AVI Audio Video Interleave is a container format which can contain data encoded in many different ways ImageJ only supports uncompressed AVIs various YUV 4 2 2 compressed formats and PNG or JPEG encoded individual frames Note that most MJPG motion JPEG formats are
149. by clicking on and dragging the vertical lines that separate the column headings Selected rows can be deleted by pressing the backspace key The arrow keys can be used to vertically scroll the window 22 Results Table Most of ImageJ analyses are printed to the Results table Table commands are organized in four menus File gt Edit Font gt and Results gt A contextual menu listing the majority of these commands can be accessed by right clicking anywhere in the Results window File gt Save As Exports the measurements as a tab delimited or comma delimited text file as defined in Results gt Options File gt Rename Renames the table Because ImageJ outputs measurements exclusively to the Results table renaming the table will freeze its contents File gt Duplicate Creates a new table containing a copy of the data Note that ImageJ will not output measurements to duplicated tables Font gt This menu contains commands to adjust font size Results gt Clear Results Alias for the Analyze gt Clear Results command Results gt Summarize Alias for the Analyze gt Summarize command Results gt Distribution Alias for the Analyze gt Distribution command Results gt Set Measurements Alias for the Analyze gt Set Measurements command Results gt Options Opens the Edit gt Options gt Input Output dialog in which is possible to specify if column headers and row numbers should be saved or copied f
150. by three white males which is not unlikely considering the current population in science the probability that at least one of them is colorblind is whopping 22 One practical point defined by the Color Universal Design is the use of magenta in red green overlays see also 66 Magenta is the equal mixture of red and blue Colorblind people that 1 See Wootton R Springall DR Polak JM Image Analysis in Histology Conventional and Confocal Microscopy Cambridge University Press 1995 ISBN 0521434823 This section is partially extracted from Masataka Okabe and Kei Ito Color Universal Design CUD How to make figures and presentations that are friendly to Colorblind people http jfly iam u tokyo ac jp color accessed 2009 01 15 15 Last updated 2012 10 02 Color Images Red green images and partial color blindness Deuteranopia second panel protanopia third panel are the most common types of partial color blindness red green confusion Tritanopia blue orange confusion fourth panel is quite rare Replacing Red with Magenta in RGB Images bottom row is a simple way to compensate for color vision deficiencies have difficulties recognizing the red component can easily recognize the blue hue The region of double positive becomes white which is easily distinguishable for colorblind In ImageJ this is easily accomplished using the Image Color gt Merge Channels or using the ImageJ macro language see IV R
151. case re installing the Startup Macros using the More Tools Menu will revert the contextual menu to its default Record Capture Screen Monitor Memory Startup Macros Search The ImageJ Macro Language Programmer s Reference Guide explains how this menu can be customized Find Maxima Subtract Background The menu that is displayed when a user right clicks or ctrl clicks on an image window can be customized through installation of the Popup Menu macro Any menu has a name and a list of menu items The newMenu name items macro function allows the creation of a new menu This menu passes the chosen item as a simple string to the Popup Menu macro From this point you can decide what to do according to what item was chosen 4 Customizing the Image Popup Menu The Popup Menu macro defines the menu that is displayed when right clicking or ctrl clicking on an image It is part of the startup gt macros StartupMacros txt and several other macro toolsets ae var pmCmds newMenu Popup Menu newArray Help Rename Duplicate Original Scale Baste Control gt Record Capture Screen gt Monitor Memory Startup Macros o st a Search nad Maximov macro Popup Menu cmd getArgument if cmd Help showMessage About Popup Menu To Customize this menu edit the line that starts with n tvar pmCmds in ImageJ macros Start
152. chindelin J et al Fiji an open source platform for biological image analysis Nature Methods pp 676 82 2012 100 To cite this document Ferreira T and Rasband WS ImageJ User Guide IJ 1 46 imagej nih gov ij docs guide 2010 2012 11 2 3 4 5 6 7 8 9 10 11 MD Abramoff PJ Magalhaes and S J Ram Image Processing with ImageJ Biophotonics International available at http webeye ophth uiowa edu dept biograph abramoff imagej pdf 11 7 36 42 2004 Simon Andrews Jonathan Gilley and Michael P Coleman Difference Tracker ImageJ plugins for fully automated analysis of multiple axonal transport parameters J Neurosci Methods 193 2 281 7 Nov 2010 doi 10 1016 j jneumeth 2010 09 007 W Bailer Writing ImageJ Plugins A Tutorial Upper Austria University of Applied Sciences Dept of Media Technology and Design Hagenberg Austria http www gm fh koeln de konen WPF BV tutorial ImageJ_ V1 71 pdf Jan 2006 Daniel P Barboriak Anthony O Padua Gerald E York and James R Macfall Creation of DICOM aware applications using ImageJ J Digit Imaging 18 2 91 9 Jun 2005 doi 10 1007 s10278 004 1879 4 David J Barry Cecilia Chan and Gwilym A Williams Morphological quantification of filamentous fungal development using membrane immobilization and automatic image analysis J Ind Microbiol Biotechnol 36 6 787 800 Jun 2009 doi 10 1007 s10295 009 0552 9 Delphin
153. cked Source LUTs are C4 gray C 4 tif H always ignored when creating RGB images F C5 cyan None e l Keep source Images If checked source images will not be C6 magenta C 2 tif hey disposed C7 yellow None P Ignore source LUTs If checked LUTs of source images are M Create composite ignored In this case merged channels will adopt the Keep source images lookup table mentioned besides the channel choice i e red vi Ignore source LUTs green blue gray cyan magenta yellow As mentioned Cancel this option is assumed when merging into RGB SEE ALSO Channels Tool Z Lookup Tables gt and IV Replacing Red with Magenta in RGB Images 28 5 3 Channels Tool Z Alias for Image gt Hyperstacks gt Channels Tool Z 28 5 4 Stack to RGB Converts a two or three slice stack into an RGB image assuming that the slices are in R G B order The stack must be 8 bit or 16 bit grayscale Also converts composite images e g File gt Open Samples HeLa Cells 1 3M 48 bit RGB into RGB 28 5 5 Make Composite Converts in place an RGB image a 2 7 image stack or a 2 7 channel hyperstack into a composite color image Use the Channels Tool Z tool Shift Z to enable and disable F Composite l the channels of a composite image Use Brightness Contrast Lan Grayscale C Shift C to adjust the brightness and contrast of the current channel Make Compo
154. cks The first line of a slice label up to 60 characters is displayed in parentheses in the image subtitle the line of information above the image The macro functions setMetadata Label string and getMetadata Label can be use to set and retrieve the current slice label SEE ALSO Stacksb Label 28 6 15 10 Start Animation Animates the active stack by repeatedly displaying its slices frames in sequence It is run more easily by clicking on the play icon preceding stack sliders see Stacks To stop the animation click on the slider pause icon click on the image or use Stop Animation evoked by the same shortcut As such stacks animation can be toggled using LA The frame rate is displayed in the status bar Open the Animation Options Alt dialog box to specify the animation speed pressing or right clicking on the on the slider play pause icon opens the Animation Options dialog Note that more than one stack can be animated at a time 28 6 15 11 Stop Animation 1 Terminates animation of the active stack see Start Animation 28 6 15 12 Animation Options Alt 55 Animation Options Use this dialog to set the animation speed in frames per second Speed 0 1 1000 fps S p i PES e By the starting and ending frame or to enable oscillating animations ic Me Selecting Start Animation animates the stack as soon as the dialog is dismissed Last Frame 27 O Loop Back and Forth M S
155. criptors of complexity _ E a om c The command counts the number of boxes of an in creasing size needed to cover a one pixel binary object boundary and implements the method described in T G Smith Jr G D Lange and W B Marks Frac box size C tal Methods and Results in Cellular Morphology J tist Cave Copy Neurosci Methods 69 1123 126 1996 A plot is generated with the log of size on the X axis and the log of count on the Y axis and the data is fitted with a straight line The slope S of the line is the negative of the fractal dimension i e D slope Size S and count C are printed to the Results Table Refer to the source code for additional information SEE ALSO Fractal Dimension and Lacunarity plugin 30 14 3 Analyze Line Graph This command uses the Particle Analyzer to extract sets of coordinate data from digitized line graphs The following procedure describes how to use it 1 Open the image containing the graph For practice use the File Open Samples Line Graph 21K sample image Make sure your graph is a grayscale image Image gt Type gt 8 bit Analyze Line Graph will assume that the graph is displayed on a white background so images with darker backgrounds must be adjusted beforehand see e g Edito Invert I and Process Binary gt Make Binary 2 Set background color to white using the Color Picker Tool or the Color Picker window Use any of the
156. ct in an image see Straightening filamentous objects The curved object must first be outlined using the Segmented Line Selection Tool Double click on the line tool icon to open the Imagep Adjust gt Line Width widget in order to adjust the width of the line selection By default the Straighten command fits a cubic spline curve to the points that define the line so it is not necessary to check the Spline Fit checkbox Note that Straighten also works with straight line selections In this case the object defined by the line selection is rotated to be horizontal SEE ALSO Straighten plugin Image Transform gt 27 12 18 To Bounding Box Converts a non rectangular selection to the smallest rectangle that completely contains it 27 12 19 Line to Area IN IJ 1 46R Converts a line selection to an area traced ROI 66 Last updated 2012 10 02 IJ 1 46R IMPROVED IN IJ 1 46R Edit gt Selection gt pate 155 Spline Fit vs Line Width Edit gt Selection gt Straighten As described in Segmented Line Selection Tool the points of a polyline selection can be repositioned dragged deleted using Alt click or duplicated using Shift click Press Edit gt Selection gt Restore Selection E to restore accidentally deleted lines 27 12 20 Area to Line Converts a non composite area selection to its enclosing outline see ROI manipulations The obtained line will have the width speci
157. ctions that are moved or edited are automatically updated When Show All is active and the ROI Manager window is closed a dialog box is displayed providing the option to save the displayed ROIs by moving them to the image overlay by running Image Overlay gt From ROI Manager When saving the image as tiff Overlays are stored in the TIFF header and can later be retrieved using Image Overlay gt To ROI Manager Save the 5 displayed ROIs as an overlay Save as Overlay Labels Toggles overlay labels displayed by Show All Labels are customized using More gt Labels a shortcut to Image Overlay gt Labels More gt Displays a drop down menu with several additional commands this menu is also available when right clicking on the ROI Manager s list area Open Opens a El roi file and adds it to the list or opens a ZIP archive zip file and adds all the selections contained in it to Save Fill the list ree Save Saves the selected ROI as aE roi file If no ROIs are selected OR Combine saves all the ROI Manager selections in a ZIP archive XOR Split Fill Alias for Edito Fill f Add Particles Multi Measure Draw Alias for Edit gt Draw d Multi Plot ak AND Uses the conjunction operator on the selected ROIs to create a SsPecily Raneve ice nio composite selection All ROIs are considered if none is selected Help Options OR Combine Uses the union operator on the selected
158. d 2012 10 02 Plugins gt Utilities gt Show full information If checked the Command Finder will display the location of the listed menu entries Fuzzy matching Fiji only Activates approximate string matching Useful if you are not sure about the command spelling Close when running If checked the Command Finder will dismiss after choosing Run or pressing I Export Prints the filtered list of commands to an ImageJ table SEE ALSO Control Panel U Search Finding Commands 31 3 3 Search Searches for macros E txt E ijm scripts El js E py E rb E clj El bsh plugins source E java and El html Threshold files containing a particular string M Search contents Search is performed recursively subdirectories are included lgnore case NS and results displayed in the Log Window In the Log window O Search plugins folder double click on a file path to have it open O Search scripts folder J Search source folder Search contents Specifies if the search should be restricted to Cancel OK filenames or extended to file contents If checked the line number where the string was found is displayed Ignore case Specifies if the search should be case insensitive Search macros folder Extends the search scope to E ImageJ macros Search plugins folder Extends the search scope to E ImageJ plugins Search scripts folder Extends the search scope to ElImageJ scripts if present Sea
159. d the undo buffer is reset every time the active frontmost image changes Run garbage collector on status bar click If checked forces the Java garbage collector to run every time the user clicks on the Status bar which may help to reclaim unused memory see also Plugins Utilities gt Monitor Memory Help Opens http imagej nih gov ij docs menus edit html memory SEE ALSO FAQs on the ImageJ wikipage 27 13 13 Proxy Settings oxy sartings Use this dialog to modify the proxy settings of the Java Proxy server II Virtual Machine This may be required for ImageJ to connect to the internet in certain machines running behind HTTP proxies For example proxy settings may be required to update ImageJ using the Help gt Update ImageJ command Help Cancel OK or to open the images in the File gt Open Samples gt submenu Port amp 080 M Or use system proxy settings To use the system proxy settings enable the Or use system proxy settings option this will set the java net useSystemProxies property to true To configure your proxy settings manually i Last updated 2012 10 02 Edit gt Options gt specify the address of the HTTP proxy in Proxy server and the port the proxy listens on normally 8080 in Port Settings will be saved in the ImageJ preferences file E IJ_Prefs txt 27 13 14 Compiler Displays a dialog box with options for the Plugins gt Compile and Run command Target Specifies
160. d Formats In ImageJ the equivalent to Redo is the Process gt Repeat Command R that re runs the previous used command skipping Edit gt Undo z and File gt Open o commands SEE ALSO Plugins Utilitiest gt Reset Multi Undo plugin 7 Image Types and Formats Digital Images are two dimensional grids of pixel intensities values with the width and height of the image being defined by the number of pixels in x rows and y columns direction Thus pixels picture elements are the smallest single components of images holding numeric values pixel intensities that range between black and white The characteristics of this range 1 e the number of unique intensity brightness values that can exist in the image is defined as the bit depth of the image and specifies the level of precision in which intensities are coded e g A 2 bit image has 2 4tones 00 black 01 gray 10 gray and 11 white A 4 bit image has 2 16 tones ranging from 0000 0 to 1111 16 etc In terms of bits per pixel bpp the most frequent types of images Image gt Type that ImageJ deals with are ImageJ2 supports many more types of image data 8 bit Images that can display 256 2 gray levels integers only 16 bit Images that can display 65 536 21 gray levels integers only 32 bit Images that can display 4 294 967 296 232 gray levels real numbers In 32 bit images pixels are described by floating point v
161. d in real time as the mouse is dragged over the image It is useful to examine how a filter changes the pixel data E g load Pixel In spector move the cursor over an im age and run Process Filters gt Gaussian 30 56 72 B8 104 104 31 48 56 72 80 80 32 40 40 48 48 48 33 40 40 40 40 40 32 32 Blur When toggling the Preview checkbox you will be able to monitor in real time how different Sigma radius change pixel values Cancel C ox In the Pizel Values table columns and row headers x amp y positions are expressed in pixel coordinates The y axis direction is determined by the Invert Y coordinates value in Analyze gt Set Measurements The center position current cursor is printed in red x y value When the table is in the foreground the arrow keys can be used to nudge the neighborhood square outlined in red and the table can be copied into the clipboard by pressing c For settings press the Prefs button at the top left of the table Radius Specifies the size of the table 3 x 3 for radius 1 5 x 5 for radius 2 etc Grayscale readout The numeric output for 8 and 16 bit grayscale images Can be Raw the default Calibrated see Analyze Calibrate or Hexadecimal Hex RGB readout The numeric output for RGB images Can be R G B triplets Gray Value or Hexadecimal Hex see XIX Hexadecimal Color Values The mean grayscale value is determined by the weighting factors specified in
162. d spatial calibration bar Width Length of the bar in calibrated units dii E Height Height of the bar in pixels Height in pixels 4 Font Size 16 Font Size Adjusts the font size of the Color White scale bar label Background None Color Adjusts the text color see XVII Location Upper Left Embedding Color Annotations in Grayscale Images M Bold Text O Hide Text O Serif Font O Overlay Background Adjusts the filling color of the label text box e Cancel OK tie Location Adjusts the position of the calibration bar If there is a selection the bar is initially drawn at the selection Bold Text Serif Font Specify if label should be typeset in boldface serif typeface Hide Text If checked the bar is drawn without label Overlay If checked the bar is created as a non destructive image overlay see Overlays and Image Overlay gt submenu If unchecked the scale bar is drawn invasively SEE ALSO Analyzep Set Scale Image Properties P XXIII Global Calibrations 30 14 7 Calibration Bar Creates an RGB copy of the current image and displays a labelled calibration bar on it see Analyze gt Calibrate 153 Last updated 2012 10 02 Analyze gt Tools gt Location Defines the position of the bar If an area selection is active the bar is initially drawn at that selection Fill Color Defines the bar s background color Label Color Adjusts the text color Num
163. d to the El ImageJ macros StartupMacros txt file they will be automatically installed at startup 9 Assigning Keyboard Shortcuts to ImageJ Tools These macros allow tools to be selected by pressing function keys Add them to ImageJ macros StartupMacros txt and they will be automatically installed when ImageJ starts macro Rectangle fi setTool rectangle macro Elliptical 2 setTool elliptical macro Brush f3 setTool brush macro Polygon f4 setTool polygon macro Freehand f5 setTool freehand macro Straight Line f6 setTool line macro Segmented Line f7 setTool polyline macro Arrow f8 setTool arrow macro Angle f9 setTool angle macro Multi point f101 tsetlool multipoint macro Wand f11 setTool wand macro Magnifying Glass f12 setTool zoom 169 Last updated 2012 10 02 Toolbar Shortcuts This approach however requires the user to memorize a large number of shortcuts In addition it may be difficult to assign so many hot keys without conflicting with previously defined ones see Plugins gt Shortcuts An alternative way to control the toolbar using the keyboard is to create macros that progressively activate tools from a predefined sequence The next example demonstrates such strategy It is composed of two macros activated by and that iterate through the toolbar from left to right forward cycle
164. de Rolling Ball Background Subtraction the plugin that implemented this command in versions up to 1 39e 29 15 Repeat Command R Reruns the previous command The Edit gt Undo and File Open commands are skipped SEE ALSO Undo and Redo 131 Last updated 2012 10 02 Analyze gt 30 Analyze gt This menu contains commands related to statistical measurements on image data profile and histogram plotting and plugins related to image analysis 30 1 Measure m Based on the selection type calculates and displays on the Results Table either area statistics line lengths and angles or point coordinates Performed measurements can be specified in the Set Measurements dialog box Area statistics are calculated for the complete image if there is no selection or for a selected subregion defined by one of the Area Selection Tools For linear selections Straight Segmented and Freehand lines see Line Selection Tools length and angle straight lines only are also calculated For Point selections see Point Tool and Multi point Tool the X and Y coordinates are recorded Note that Measure m will paint invasively a mark over the measured point in foreground color unless Mark Width in the Point Tool options dialog box is set to zero With RGB images results are calculated using brightness values RGB pixels are converted to brightness values using the formula value red green blue 3 or value 0 299 x red 0 587 x
165. dless of the image magnification 0 Width To thicken ROI edges at higher zoom levels set Stroke width to a non zero value in Edit Selection gt Properties y Image Overlay gt Overlay Options or ROI Manager s Properties 28 13 4 View 100 5 Displays the image using 100 magnification 1image pixel 1 screen pixel Enable Open Images at 100 in the Edit Options gt Appearance dialog to have images automatically opened at 100 magnification 28 13 5 To Selection Zooms in based on the current selection Note that in the absence of a selection this command zooms the image to a fit to screen level see Magnifying Glass 28 13 6 Set Zoom 102 X center 128 Zooms the active image to an exact value e g 37 4 overcoming the predefined zoom steps described in Image Zoom In The zoomed een 127 canvas will be centered at X Y center pixel coordinates 28 Cancel ok 28 14 Overlay gt As mentioned previously this submenu contains commands for creating and working with non destructive image Overlays An overlay consists of one or more selections arrows lines points shapes and text see ImageJ Toolbar Overlays can also be composed of image selections imageROls that behave partially as regular Selections see Add Image Press Add Selection b to add the current selection to the overlay Press Flatten F to create an RGB image with the overlay embedded in it The
166. downloadable ZIP archive is also available at http imagej nih gov ij docs user guide zip Printable booklets Two sided booklets that can be printed on a duplex unit printer by setting the automatic duplex mode to short edge binding Two formats are available A4 http imagej nih gov ij docs guide user guide A4booklet pdf and letter size paper http imagej nih gov ij docs guide user guide USbooklet pdf Conventions Used in this Guide Throughout the guide internal links are displayed in gray e g Part IV ImageJ User Interface Links to external URLs such as the ImageJ website http imagej nih gov ij are displayed in dark blue ImageJ commands are typed in sans serif typeface with respective shortcut keys flanked by square brackets e g Image Duplicate D As explained in Using Keyboard Shortcuts this notation implies Shift modifiers i e D means pressing DJ d only the D key and assumes that Require control key for shortcuts in Edit Options Misc is unchecked Note that references to the key include the key of Macintosh keyboards Useful tips and reminders are placed in Noteworthy notes numbered with upper case roman numerals e g 1 Frontmost Window and Window Activation The full list of these notes is available on page viii Filenames directories and file extensions are typed in monospaced font marked by a related icon e g file ElStartupMacros txt in folder G Applicatio
167. dragging as long as the cursor is within the selection and has changed to an The status bar displays the coordinates of the upper left corner of the selection or the bounding rectangle for non rectangular selections as it is being moved To move the contents of a selection rather than the selection itself Edit gt Copy c Edito Paste v and then click within the selection and drag Selections can be nudged one pixel at a time in any direction using the arrow keys Note that the up and down keys zoom the image in and out in the absence of selections see Arrow Keys shortcuts The Brush Selection Tool can be used to perform fine adjustments of ROI contours Most ROIs can be resized one pixel at a time by holding while using the arrow keys In general see Area Selection Tools and Line Selection Tools for details selections are resized by dragging one of the selection handlers While dragging holding resizes the selection around its center holding imposes a fixed aspect ratio and holding forces a 1 1 aspect ratio SEE ALSO Key Modifiers 18 Last updated 2012 10 02 IJ 1 46R IMPROVED IN IJ 1 46R Overlays Composite Selections 10 2 Composite Selections Composite selections are non contiguous ROIs containing more than O C2 one cluster of pixels and or ROIs containing internal holes Composite e ES ROIs are typically originated with the Brush Selection Tool but they can be defined with any other selection tool using k
168. e ImageJ Macro Language Programmer s Reference Guide Fiji s Introduction into Macro Programming Plugins gt Macros gt Record Fiji Script Editor IJ_ ED 45 Last updated 2012 10 02 Customizing the ImageJ Interface 24 Log Window The Log window is used to display useful infor mation about ongoing operations It is frequent for plugins and macros to send messages to the mitosis test tif opening 254 NewSubfileType value 0 256 ImageWidth value 256 Log window reporting progress errors or trou 257 ImageLength value 256 bleshooting information 258 BitsPerSample value 8 262 Photolnterp value 1 If you are troubleshooting a problem you can 270 ImageDescription value 194 count 10 check Debug mode in Edit gt Options Misc to 273 StripOffsets value 768 h I J ota to the L 277 SamplesPerPixel value 1 ave mageJ ou pu ing messages to the Log 278 RowsPerStrip value 256 window ImageJ will exit debug mode as soon as 279 StripByteCount value 65536 the Log window is closed 262 XResolution value 297 283 YResolution value 305 E In addition Tiff tags and information needed to 296 ResolutionUnit value 1 MECA import files are printed to the log window when 50838 MetaDataByteCounts val 50839 MetaData value 22022 Metadata 0 6c61626c 336 slice labels ImageJ runs in Debug Mode Most of the general shortcut keys described in Editor apply to the
169. e S A Beeckman Geert Meesen Patrick Van Oostveldt and Daisy Vanrompay Digital titration automated image acquisition and analysis of load and growth of Chlamydophila psittaci Microsc Res Tech 72 5 398 402 May 2009 doi 10 1002 jemt 20694 A M Bell K Parton and E Smith Earth Tutor An Interactive Intelligent Tutoring System for Remote Sensing American Geophysical Union 52 08 Dec 2005 c 2005 American Geophysical Union S Bolte and F P Cordeli res A guided tour into subcellular colocalization analysis in light microscopy Journal of microscopy 224 Pt 3 213 32 Nov 2006 doi 10 1111 3j 1365 2818 2006 01706 x Kerry M Brown Duncan E Donohue Giampaolo D Alessandro and Giorgio A Ascoli A cross platform freeware tool for digital reconstruction of neuronal arborizations from image stacks Neuroinformatics 3 4 343 60 Jan 2005 doi 10 1385 NI 3 4 343 Emel Bulut and Biinyamin Sahin A new method of assessing the size of mandibular cysts on orthopan tomograms projection area fraction J Craniofac Surg 20 6 2020 3 Nov 2009 doi 10 1097 SCS 0b013e3181bd302e Wilhelm Burger and Mark James Burge Digital image processing An algorithmic introduction using Java ISBN 978 1 84628 379 6 Springer Jan 2008 URL http www imagingbook com 174 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A Cardona S Saalfeld P Tomancak
170. e and antialiasing Smooth checkbox of the font used by the Text Tool and Image Stacksp gt Label It is also possible to adjust the horizontal text alignment using the style drop down menu Left the default Right or Centered The widget is more easily opened by double clicking on the Text Tool SansSerif KF 118 amp M Smooth 27 13 4 Profile Plot Options Use this dialog to control how plots generated by ImageJ are displayed Image Stacks Plot Z axis Profile Analyze gt Plot Profile k Analyze gt Calibrate Analyze gt Tools gt Curve Fitting Multi Plot Analyze gt Tools gt ROI Manager etc Plot Width and Plot Height Specify the length in pixels of the X axis Plot Width and Y axis Plot Height Fixed y axis Scale If checked the Y axis range is fixed and gt gt Profile Plot Options the specified Minimum Y and Maximum Y values are used otherwise plots are scaled based on the minimum and maximum gray values Width pixels 300 Height pixels 150 Minimum 0 00 Maximum Y 0 00 Do not Save x values If checked List Save and Copy buttons will appear in profile plot windows L Fixed y axis scale Auto close If checked profile plot windows will be automat O Do not save x values irene ically closed when List Save and Copy are clicked O Vertical profile On O List values O Interpolate line profiles M Draw grid li
171. e bottom of the window It has two modes Foreground and Background To change modes click on the desired selection box Clicking on the Foreground Background Switcher button sets the current foreground to the background and vice versa The Black White Reset button sets the foreground to black and the background to white AAO CP Color Selectors Color Ramp Foreground Color Algorithm Red LOOO Background Color 4B H SP Red 4 gt 4 0 v Yellow Green 4 DE k 255 Green Black White Cyan Ramp Blue Hex values EEA 64 c8Ffc8 Magenta Foreground Color aL Ei Foreground Background Switcher Background Color L Black White Reset Color Picker and Color Choosers IJ 1 46c The CP window can be activated using the keyboard shortcut for Image Colorb Color Picker K or by double clicking on the Color Picker Tool on the ImageJ Toolbar Color Choosers are evoked by double clicking on a color of the CP window and can be used to retrieve Hexadecimal Color Values The eye dropper is drawn in the current foreground color while the frame around it is drawn in background color Foreground color is also reflected in drawing tools such as the Arrow Brush Flood Filler and Pencil tools The color palette is based on HSB Hue Saturation and Brightness color model see Color Spaces and Color Separation Hue increases as you go down the palette while saturation and brightness values are split horizontal
172. e cursor is synchronized across selected images with the mouse pointer changing to a red X shape cursor When unchecked mouse movements become restricted to the active image Sync channels If checked the channel slider c is synchronized across the synchronized window set Image coordinates If checked spatial calibration units will be used instead of pixels coordi nates For proper registration this option should be unchecked when syncing images with different pixel sizes see Image Properties P Sync z slices If checked the depth slider z is synchronized across the synchronized window set 154 Last updated 2012 10 02 IJ 1 46R Analyze gt Tools gt Synchronize Windows E Pee MAX_mitosis tif 150 mitosis tif MAX _mitosis tif M 5ync Cursor _ Sync z Slices M 5ync Channels M Sync t Frames Cal Image Coordinates El Image Scaling Synchronize All Unsynchronize All Analyze gt Tools gt Synchronize Windows IJ 1 46j This command can be used to measure ROIs across images transfer ROIs from a reference image e g a Maximum Intensity Projection MIP to multiple images or to use an un zoomed copy of the active image as a navigator palette Sync t frames If checked the frame slider t is synchronized across the synchronized window set Image scaling If checked positions to different windows are translated via offscreen coordinates providing correct regi
173. e ee la Dae Ee 139 EO MISCO rela Wl i state A eee ai et Se vs Se Sea oe 140 SEL Actress aa A a es Ee OS we Ae eee ee 142 SOM Suace FICE oe oh od ode cb hte ws e E Beds Soe ee eh De e ee I ee e 143 EE O ea ewe ee ET 144 SOMA TOS era a ewok a ah 21 Eh asd bk a e a Baws 146 31 Plugins gt 156 el GROG aao seats E iss Serie ex ee tends take Gems Go oe Ses ho eas Se n 156 Ue SMOC Ga Ge cach task Hever Coe ae i a as Gees te ee ee A te te es Be e 157 Oley WEISS Es ads oe ts aus e ci Se ve ck Bea ee tea ae ah oe ews oe e ee aes Bes wae ae 158 SUZ NaNe p gs tec ha oo ad ss oe oh oid GE mes oe WS wey ees Be Seneca A Gp ae ee 161 ES os A gee a OO bed Sy eC OO Ea Sa ee top Pee 162 32 Window gt 163 2 A EA A RAE 163 O22 PUR IBenine tab e g ara ar ea daa 163 la COSCA trata kde da A is es a a A a E 163 O E A A A Wy a ee 163 33 Help gt 164 ook Wie Be Web ere aeee eea et eh Be A ee tes ee ee a ey Oe N 164 Joe IMALel NEWS i ore bm E wie ee a ee EO Es 164 Boao DOCU ON Sit otto a Jy See este dl a ee a da oe eae a Ge 164 Oa Imsa Ms as este ace dk ce A ee A AA A E 164 D TOU IM A te ve ch tee ab nw oe GG ieee Ao ys ae es Ge Oe ee aan es on 164 330 Dey IRESOUNGCS se derribos Recah a Ta eres gota de Sep er Oe oh Se OE cee Be sip eee Be te 164 Sot RMS Se k e 0 wa di A A He Hoe Ge Gk A Gh Ges 164 Doro MacOS s se ave Heh dd he eet Si es So we a oe oe ee Oh ee Oe ee oe 164 aoc Macro FUNCOMS as sc bd Ae oe oe ew Oe eS a ore aS 164 o AE o
174. e gt Open File Import gt AND DRAG amp DROP While the File Open lo command opens formats natively supported by ImageJ images and non images files the File gt mport gt submenu provides access to plugins for additional file types e g reading raw files images in ASCII format or loading images over the network Most of ImageJ s Input Output plugins are installed on this submenu Note that almost every format known to ImageJ can be opened by dragging and dropping the file into the Main ImageJ window E g in the illustration below a remote macro file is opened by dragging its URL directly from a Web browser 26 3 Open Next O Closes the current image and opens the next image if any in its directory Holding opens the previous image if any in its directory 26 4 Open Samples Opens example images hosted on the ImageJ Web site These sample images are useful for creating testing and debugging macros since routines can be applied to the same image regardless of where the macro is run Among all probably the most used is blobs gif Open Samples gt Blobs 25K B Sample images can be downloaded from http imagej nih gov ij images or in bulk from either http imagej nih gov i download sample images zip or in Fiji by running File Open 50 Last updated 2012 10 02 File gt Open Recent gt Samples gt Cache Sample Images The AutoRun macro in the ElStartupMacros txt file can then b
175. e saved as a text file by clicking on the Save button displayed as an image using File gt Import Text Image scaled to a reasonable size using Image gt Adjust gt Size and plotted using Analyze Surface Plot SEE ALSO ConvolutionDemo macro 124 Last updated 2012 10 02 IJ 1 46R Process gt Filters gt 29 11 2 Gaussian Blur Gaussian Blur This filter uses convolution with a Gaussian function for smoothing 37 Sigma Radius 2 00 O Scaled Units um Sigma is the radius of decay to e7 61 i e the standard devi M Preview ation o of the Gaussian this is the same as in Adobe Photoshop PAS but different from ImageJ versions till 1 38q in which radius was 2 5xo cf GaussianBlur java Like all ImageJ convolution operations it assumes that out of image pixels have a value equal to the nearest edge pixel This gives higher weight to edge pixels than pixels inside the image and higher weight to corner pixels than non corner pixels at the edge Thus when smoothing with very high blur radius the output will be dominated by the edge pixels and especially the corner pixels in the extreme case with a blur radius of e g 10 the image will be replaced by the average of the four corner pixels For increased speed except for small blur radii the lines rows or columns of the image are downscaled before convolution and upscaled to their original length thereafter
176. e scale factor at which the lane profile plots are displayed Uncalibrated OD If checked ImageJ will convert gray values to uncalibrated optical density values As explained in Analyze gt Calibrate ImageJ converts pixel intensities into optical density using the function Unc OD log p 255 pixel value As mentioned earlier the conversion can only be performed on 8 bit images Thus when dealing with higher bit depth images see Image Types and Formats the gel analyzer works on a 8 bit copy of the gel kept hidden from the user when using this option Label With Percentages If checked the Label Peaks command will print to the Results Table table the peak percentage and use it to label the plot The percentage value is obtained by dividing the area of each peak by the sum of all measured peaks from all lanes Invert Peaks If checked peaks will be inverted i e bands darker than background will have positive peaks bands lighter than background will have negative peaks This setting does not change the analysis see Image gt Lookup Tables Invert LUT For practice refer to the video tutorial on the ImageJ wikipage and use the File gt Open Samples Gel sample image 1 D gel to perform the following steps Note that a copy of the gel image with the lane outlines can be created at any point using the Image Overlay gt Flatten F command 1 Use the rectangular selection tool to outline the first lane This should be
177. e simplex fitting options namely Maximum number of iterations The number of maximum iterations in which the CurveFitter will try to improve upon the parameter values to get the best fit Usually the algorithm reaches optimal convergence before reaching the default value 148 Last updated 2012 10 02 Analyze gt Tools gt Number of restarts To ensure that the result is trustworthy i e that it did that it did not get stuck or find a local minimum CurveFitter tries at least twice to find the minimum with different starting points If the two results are not the same Number of restarts determines how often it may start two additional runs until the best two results agree within the error tolerance There is no limit for the number of restarts apart from the maximum number of iterations 4 Click Apply to create a 32 bit copy see Image Types and Formats of the current image transformed with the chosen function CurveFitter s built in functions A more detailed summary of the E CurveFitter s abilities is available online The complete documentation can be accessed through the ImageJ API Most functions use linear regression to determine the first two coefficients directly which generally increases the quality of the result 42 As explained in Curve Fitting simplex fitting options can be adjusted by selecting the Show settings checkbox For polynomials the proposed criteria are known to yield optimal convergence
178. e used to change the default path of sample images allowing a complete off line usage of the File gt Open Samples gt submenu 6 Setting File gt Open Samples for Offline Usage This macro calls the Prefs setImageURL method to change the default path of Sample Images http imagej nih gov ij images to a local gt subfolder of ImageJ s directory named samples Note that Fiji lt gt provides this feature by default 7 macro AutoRun dir getDirectory imagej samples if File exists dir dir replace dir 20 if startsWith getInfo os name Windows dir replace dir File separator call 17 Prefs setlimagesUkRL file dir 26 5 Open Recent gt The submenu shows a list of the 15 recently opened files Click on a filename to open it 26 6 Import gt This submenu lists the installed image reader plugins SEE ALSO Non native Formats Acquisition plugins Input Output plugins VirtualStackFrom List macro VIII Opening Files File gt Open File gt Import gt and Drag amp Drop IMPROVED 26 6 1 Image Sequence IN IJ 1 46R A Sequence Options Opens a series of images in a chosen folder as a stack Im ages may have different dimensions and can be of any format supported by ImageJ see Image Types and Formats and Han dleExtraFileTypes plugin Non image files Scripts E 1lut E roi ElRoiSet zip etc are ignored Number of Images
179. ect that is not part of the background Rolling Ball Radius The radius of curvature of the paraboloid As a rule of thumb for 8 bit or RGB images it should be at least as large as the radius of the largest object in the image that is not part of the background Larger values will also work unless the background of the image is too uneven For 16 bit and 32 bit images with pixel value ranges different from 0 255 the radius should be inversely proportional to the pixel value range e g for 16 bit images pixel values 0 65535 typical values of the radius are around 0 2 to 5 Light Background Allows the processing of images with bright background and dark objects Separate Colors RGB images only If unchecked the operation will only affect the brightness leaving the hue and saturation untouched Create Background Don t Subtract If checked the output is not the image with the background subtracted but rather the background itself This option is useful for examining the background created in conjunction with the Preview option Create Background can be also used for custom background subtraction algorithms where the image is duplicated and filtered e g removing holes in the background before creating the background and finally subtracting it with Process Image Calculator Sliding Paraboloid If checked the rolling ball is replaced by a paraboloid that has the same curvature at the apex as a ball of that radi
180. ection Plugins gt Utilities gt ImageJ Properties List all Java properties Area Selection Tools Subtract current selection from the previous one Rectangular Selection Tool and Oval Selection Tool Current aspect ratio is maintained while resizing Straight Line Selection Tool Keeps the line length fixed while moving either end of the line Forces the two points that define the line to have integer coordinate values when creating a line on a zoomed image Segmented Line Selection Tool and Polygon Selection Tool Alt clicking on a node deletes it Point Tool Alt clicking on a point deletes it Color Picker Tool Alt clicking on an image picks up background color All Tools Show location and size in pixels rather than calibrated units 34 2 Shift Key Modifications Image gt Adjust gt Threshold T Adjusting Min also adjusts Max Image gt Adjust gt Brightness Contrast C Apply adjustments to all channels of a composite image Installed Macros and Scripts Open instead of run Rectangular Selection Tool and Oval Selection Tool Forces 1 1 aspect ratio Area Selection Tools Add selection to previous one Polygon Selection Tool Shift clicking on a node duplicates it Straight Line Selection Tool Forces line to be horizontal or vertical Segmented Line Selection Tool Shift clicking on a node duplicates it Point Tool Shift clicking adds points Multi point Tool behavior Magnifying Glass Shift clicking and dragging runs Im
181. ectional data exchange between Matlab and ImageJ allowing to exchange images between the two imaging software MIJ also allows MATLAB to access all built in functions of ImageJ as well as third party ImageJ plugins The developers provide more information on the MIJ and Matlab File Exchange websites Fiji features Miji m which makes even more convenient to use the libraries and functions provided by Fiji s components from within Matlab SEE ALSO ImageJ related links list of related imaging software on the ImageJ2 website 4 Last updated 2012 10 02 Getting Help ImageJ2 2 3 ImageJ2 ImageJDev is a federally funded multi institution project dedicated to the development of the next generation version of ImageJ ImageJ2 ImageJ2 is a complete rewrite of ImageJ that includes the current stable version ImageJ ImageJ1 with a compatibility layer so that old style plugins and macros can run the same as they currently do in ImageJ1 Below is a summary of the ImageJDev project aims To create the next generation version of ImageJ and improve its core architecture based on the needs of the community To ensure ImageJ remains useful and relevant to the broadest possible community main taining backwards compatibility with ImageJ1 as close to 100 as possible Expand functionality by interfacing ImageJ with existing open source programs To lead ImageJ development with a clear vision avoiding duplicat
182. egory W Schmidt Analysis and documentation of progression of Fuchs corneal dystrophy with retroillumination photography Cornea 25 4 485 9 May 2006 doi 10 1097 01 ico 0000178726 11693 14 Adam S Green Paul R Ohmann Nick E Leininger and James A Kavanaugh Polarization Imaging and Insect Vision The Physics Teacher 48 17 Jan 2010 c 2010 American Institute of Physics doi 10 1119 1 3274352 Dilraj Grewal Rajeev Jain Gagandeep Singh Brar and Satinder Pal Singh Grewal Pentacam tomograms a novel method for quantification of posterior capsule opacification Invest Ophthalmol Vis Sci 49 5 2004 8 May 2008 doi 10 1167 iovs 07 1056 J C Grochowsky L W Alaways R Siskey E Most and S M Kurtz Digital photogrammetry for quantitative wear analysis of retrieved TKA components J Biomed Mater Res Part B Appl Biomater 79 2 263 7 Nov 2006 doi 10 1002 jbm b 30537 Muriel Hachet Haas No l Converset Olivier Marchal Hans Matthes Sophie Gioria Jean Luc Galzi and Sandra Lecat FRET and colocalization analyzer a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug in Microsc Res Tech 69 12 941 56 Dec 2006 doi 10 1002 jemt 20376 A J Hand T Sun D C Barber D R Hose and S MacNeil Automated tracking of migrating cells in phase contrast video microscopy sequences using image registration Journal of microscopy 234 1 62 79 Apr 2009 doi 10 1111 j
183. els in size Edit gt Options gt Profile Plot Options 8 Open the B amp C widget at its last saved screen position Image gt Adjust gt Brightness Contrast C SEE ALSO Customizing the ImageJ Interface FAQs on ImageJ wikipage VII Organizing Com mands in the Menu Bar 22 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Part III Extending ImageJ ImageJ capabilities can be extended by loadable code modules in the form of macros scripts or plugins 300 macros 500 plugins and 20 scripts are available through the ImageJ web site Below is a short description of these three type of ImageJ add ons Macros The easiest way to execute a series of ImageJ commands The ImageJ macro language a Java like language contains a set of control structures operators and built in functions and can be used to call built in commands and other macros Macro code is stored in text files E txt and El ijm extensions Plugins Much more powerful flexible and faster than macros most of ImageJ s built in menu commands are actually plugins but harder to write and debug Plugins are written in the Java programming language El java source files and compiled to El class files Scripts ImageJ uses the Mozilla Rhino interpreter to run JavaScripts Similarly to plugins scripts have full access to all ImageJ and Java APIs but do not need to be compiled scripts and macros run interpretively On the other hand scripts lack
184. en resized Use ROI names as labels If checked ROI names are used as selection labels when in the Labels checkbox is active If unchecked the ROI position in the Manager s list is used Selections can be renamed using either Rename or Properties 152 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IJ 1 46R Analyze gt Tools gt XXV SELECTING ROIS IN THE ROI MANAGER For most ROI Manager operations Deselect works as a Select All button e g to measure all ROIs in manager one would press Deselect then Measure In addition it is possible to select contiguous ROIs in the ROI Manager list with a single Shift click Non contiguous ROIs can be selected by Control click Command click on Mac OS X When Show All is active Overlays that are not stored in the ROI Manager will not be re activated by Alt clicking Control clicking or long pressing 1 4 second or longer This is a reminder that those overlay selections added to the image overlay via Image Overlay gt Add Selection b are not under the control of the ROI Manager You will be able to activate them as soon as Show All is unchecked SEE ALSO ROI Manager Tools E RoiManagerMacros ROI Color Coder Edit gt Selection Im age gt Overlay gt Analyze gt Tools gt Synchronized windows XIX Hexadecimal Color Values XII Transferring Selections Between Images IMPROVED 30 14 6 Scale Bar IN IJ 1 46R ANA MAX _confocal series tif MCB Draws a labelle
185. eplacing Red with Magenta in RGB Images IV REPLACING RED WITH MAGENTA IN RGB IMAGES When building RGB images magenta can be obtained using the Image gt Color gt Merge Channels Previously created RGB images can be converted to MGB using Image Color gt Channels Tool Z Alternatively the Process gt Image Calculator command can be used to add the red channel to the blue channel Both these approaches can be automated using the ImageJ macro language as exemplified by Macros 2 and 1 Once saved in the S ImageJ plugins folder these Macros are treated as regular ImageJ commands In Fiji as expected the procedure of modifying RGB images is simpler one just needs to run Image gt Color gt Replace Red with Magenta For even more convenience Fiji provides an analogous command that replaces the system clipboard s image with a magenta green one It is also possible to simulate color blindness using the Vischeck or Dichromacy plugins or in Fiji using the Image Color Simulate Color Blindness command Color Composite Images In a composite image colors are handled through channels The advantages with this type of image over plain RGB images are One advantage of Dichromacy over the Vischeck plugin is that it can be recorded and called from scripts and macros without user interaction 16 Last updated 2012 10 02 Selections 1 Replace Red with Magenta ijm Using Process gt Image Calculator
186. ey modifiers The following modifier keys can be use to create composite selections Drawing outside current selection while pressing Shift creates new content To add a non square rectangle or ellipse the Shift key must be released after adding the selection Alt Drawing inside current selection while pressing Alt creates a hole removing content from the ROI Note that some operations may not be performed properly on complex ROIs In these cases it may be useful to convert a composite ROI into a polygon using the Edit gt Selection gt Enlarge command as explained in XIII Converting Composite Selections SEE ALSO Wand Tool ROI2PolylineROI macro 10 3 Selections With Sub pixel Coordinates Since ImageJ 1 46 selections can be defined with subpixel accuracy beyond the nominal pixel resolution of the image Floating point selections Line Selections see Line Selection Tools are created with floating point coordinates if the Sub pirel resolution checkbox is active in Edit Options Profile Plot Options Sub pixel coordinates of pre existing selections can be interpolated using the Edit Selection gt Interpolate command Interpolated points are easily noticeable on small selections created on images zoomed 1200 or greater 83 5 112 5 le With sub pixel resolution Without n Interpolated selections ROIs drawn with left or without middle sub pixel accuracy For line selections see Line Selection Tools t
187. fied in the Imager Adjust gt Line Width widget Note that by design Area to Line does not create closed paths E g the converted outline of a rectangular selection will be composed of only three segments with the first and fourth corner points of the rectangle being disconnected SEE ALSO Composite Selections Line to Area 27 12 21 Image to Selection Creates an image selection ImageROI Image selections are BOO Image ROI Overlays that can be moved around the canvas see Image Overlay gt Add Image Once created opacity of the blended image can be re adjusted at any time using Edit gt Selection gt Opacity 0 100 50 Properties y Use Edit gt Selection gt Restore Selec cm tion E to recover the blending image after clicking outside its limits Use Flatten F to finally embed the imageROl Note that image selections behave only partially as regular selections e g can be added to the ROI Manager list can be moved beyond image boundaries but cannot be resized or rotated However they are stored in the TIFF header and can be saved and restored when saving images in TIFF format Image blobs gif SEE ALSO Paste Control Blend transfer mode Image Stacks gt Tools Insert ROI Man ager 27 12 22 Add to Manager t Adds the current selection to the ROI Manager Analyze gt Tools gt RO Manager If there is no selection the ROI Manager is opened 67 Last updated
188. file 16 bit Unsigned Width Height tones A i j 32 bit Signed including the size of the image and the offset Offset to First Image 0 32 bit Unsigned ae 32 bit Ret to the beginning of the image data Number of Images ae ae 4 bit Re Gap Bewese images es as Interleaved RGB images have pixels stored Sie 24 bit RGB Planar contiguously rgbrgbrgb in a single image La te 15 ero i O Little Endian Byte Order ms e 7 plane Planar RGB images have the red green J Open All Files in Folder 32 bit pa and blue image data stored in separate 8 bit Use Virtual Stack 32 bit ABCR sample planes ImageJ saves RGB images p 1 Bitmap both TIFF and raw in interleaved format Help J Cancel Image Type There are fourteen choices depicted above 16 bit signed integer images are converted to unsigned by adding 32 768 1 bit Bitmap images are converted to 8 bit Image Width The number of pixel in each row of image data Image Height The number of rows in the image Offset to First Image The number of bytes in the file before the first byte of image data Number of Images The number of images stored in the file If this value is greater than the actual number of images the resulting stack will get truncated to the actual size Gap Between Images The number of bytes from the end of one image to the beginning of the next Set this value to width x height
189. gold analysis Computers amp Geosciences 35 347 Feb 2009 doi 10 1016 j cageo 2007 11 012 Fabrice de Chaumont St phane Dallongeville Nicolas Chenouard Nicolas Herv Sorin Pop Thomas Provoost Vannary Meas Yedid Praveen Pankajakshan Timoth e Lecomte Yoann Le Montagner Thibault Lagache Alexandre Dufour and Jean Christophe Olivo Marin Icy an open bioimage informatics platform for extended reproducible research Nature Methods 9 7 690 6 Jun 2012 doi 10 1038 nmeth 2075 Simon A W G Dello Ronald M van Dam Jules J G Slangen Marcel C G van de Poll Marc H A Bemelmans Jan Willem W M Greve Regina G H Beets Tan Stephen J Wigmore and Cornelis H C Dejong Liver volumetry plug and play do it yourself with ImageJ World J Surg 31 11 2215 21 Nov 2007 doi 10 1007 500268 007 9197 x M Doube ImageJ and analysis of correlated confocal and BSE SEM imaging Scanning 28 2 93 94 Jan 2006 doi 10 1111 j 1469 7580 2012 01514 x Michael Doube Michal M Klosowski Ignacio Arganda Carreras Fabrice P Cordelieres Robert P Dougherty Jonathan S Jackson Benjamin Schmid John R Hutchinson and Sandra J Shefelbine BoneJ Free and extensible bone image analysis in ImageJ Bone 47 6 1076 9 Dec 2010 doi 10 1016 j bone 2010 08 023 Arthur Edelstein Nenad Amodaj Karl Hoover Ron Vale and Nico Stuurman Computer control of microscopes using pManager Curr Protoc Mol Biol Chapter 14 Unit14 20 Oct 2010 doi 10 1002 0471142727 m
190. green 0 114 x blue if Weighted RGB Conversions is checked in Edit gt Options gt Conversions Intensity statistics Mean Modal Median Min amp Maz Gray Value Standard Deviation and Integrated Density can be performed on area line and multi point selections With lines these parameters are calculated from the values of the pixels along the line see Plot Profile k With area selections the following parameters can measured Area Center of Mass Centroid Perimeter Bounding Rectangle Shape Descriptors Fitted Ellipse Feret s Diameter Skewness Kurtosis and Area Fraction SEE ALSO Results Table Analyze Particles Summarize Distribution Set Measurements Batch gt Measure 30 2 Analyze Particles This command counts and measures objects in binary or thresholded images Image Adjust gt Threshold T or Color Threshold Analy sis is performed on the existing area selection or on the entire image if no selection is present gt Analyze Particles Size umA 2 D Infinity O Pixel units Circularity 0 00 1 00 Show Nothing It works by scanning the image or selection Nothing until it finds the edge of an object It then out Outlines wi Display results M Exclude on Bare Outlin i Clear results O Include hole ala sari lines the object using the Wand Tool measures AW TEs L Record start Macks it using the Measure m command f
191. gt 29 10 4 FFT Options Displays the FFT Options dialog box The first group of check boxes specifies which image s are created by the FFT command Display M FFT Wind O id A Display FFT Window The standard output It consists of J Fast Hartley Transform an 8 bit image of the power spectrum and the actual data o e Ie which remain invisible for the user The power spectrum fo Dg E oa image is displayed with logarithmic scaling enhancing Cancel the visibility of components that are weakly visible The actual data are used for the Inverse FFT command Display Raw Power Spectrum The power spectrum without logarithmic scaling Display Fast Hartley Transform The internal format used by the command which is based on a Hartley transform rather than Fourier transform Display Complex Fourier Transform A stack with two slices for the real and imaginary parts of the FFT Do Forward Transform If checked the current image is transformed immediately when closing the FFT Options dialog 29 10 5 Bandpass Filter FFT Bandpass Filter Removes high spatial frequencies blurring the image and low spatial frequencies similar to Filter Large Structures Downto 40 pixels subtracting a blurred image It can also sup Filter Small Structures Up to 3 pixels press horizontal or vertical stripes that were Suppress Stripes r created by scanning an image line by line 33 Tolerance of Direction 5
192. gt Find Commands I In addition ImageJ features a find function that locates macros scripts and plugins source E java files on your computer the Plugins Utilities gt Search command Because most of IJ source files contain circumstanced comments you can use this utility to retrieve files Undo and Redo I FRONTMOST WINDOW AND WINDOW ACTIVATION In ImageJ all operations are performed on the active frontmost image which has its title bar highlighted If a window is already open it will activate when its opening command is re run e g if the B amp C window is already opened Image gt Adjust gt Brightness Contrast C pressing its keyboard shortcut Shift C will activate it Pressing on any image will bring the Main ImageJ window to the foreground In addition it is also possible to permanently place the main window above all other windows see Floating Behavior of Main Window Plugins gt Utilities gt Find Commands I Command Finder Adaptive 3DThreshold Colocalisation Threshold Color Threshold DynamicThreshold 1d Entropy Threshold Show full information Close when running Threshold PA AA i A if i A i A Run Export Close M Search contents Ignore case M Search macros folder O Search plugins folder O Search scripts folder O Search source folder Image macros AutoThresholdAndSegment txt Image macros examples AutoThresholdingDemo txt y Cancel
193. he Area Selection Tools and Analyze Measure m to record the mean gray value of each of the standards 2 When finished making the measurements select Analyzep Calibrate to display the Calibrate dialog box To calibrate the image enter the known standard values in the 139 Last updated 2012 10 02 Analyze gt Histogram h right column The left column will be already populated with the measured mean gray values Select a curve fitting method from the popup menu enter the unit of measurement and click OK If Show plot is checked ImageJ will then display the calibration function on a separate window Note that both columns must contain the same number of values 3 If the calibration function is not satisfactory bring up the Calibrate dialog box again and select a different curve fitting method In addition to the functions that can be chosen from the drop down menu described in Curve Fitter s built in functions two other functions are available that do not require any measurement of OD standards Uncalibrated OD As mentioned in Gels gt Gel Analyzer Options causes ImageJ to convert gray values from 8 bit images to uncalibrated optical density values using the func tion Unc OD log 255 Pixel value This conversion can only be performed on 8 bit images Pixel Inverter Linear function defined by Inverted pixel Bit depth 1 Pixel value with Bit depth being 255 for 8 bit images or 65535 for
194. he current settings Macro Creates a macro based on the current settings which is sent to the Macro Recorder window Plugins gt Macros Record if open Help Opens the built in help dialog SEE ALSO 3D Color Inspector Color Histogram Threshold T Wand Tool Analyze gt Analyze Particles 28 2 6 Size Scales the active image or selection to a specified Width and Height in pixels Width pixels 186 Check Constrain aspect ratio and ImageJ will adjust either the Height or the Width to maintain the original aspect ratio When applicable other dimensions can also be resized Depth images in stacks Depth slices and Time frames in hyperstacks Height pixels 226 Width pixels 186 ad Height pixels 226 M Constrain aspect ratio M Ave rage when downsizing None Y Bilinear Bicubic Check the Average when downsizing checkbox for better results when scaling down images 19 Two resampling methods are possible Bilinear and Bicubic interpolation The implementation of the bicubic method Catmull Rom interpolation is derived from Burger and Burge 2008 11 Interpolation Cancel SEE ALSO Canvas Size Image gt Scale E Ilmage gt Transform gt Bin 81 Last updated 2012 10 02 Image Show Info i 28 2 7 Canvas Size IMPROVED IN IJ 1 46R Resize Image Canvas Changes the canvas size without scaling the actual image TA
195. he image dimensions in pixels Set Slices to a value greater than one to create a stack Fill With Width Height 40 pixels 1 Slices Cancel OK SEE ALSO Image Hyperstacks gt New Hyperstack Image Types and Formats 26 1 2 Hyperstack Alias for the Image gt Hyperstacks gt New Hyperstack command 26 1 3 Text Window N Creates a new text window with the title Untitled txt SEE ALSO Plugins Newb Text Window Macro Table 26 1 4 Internal Clipboard Opens the contents of the internal ImageJ clipboard SEE ALSO Edit Copy c Cut x Paste Control 26 1 5 System Clipboard V Opens the contents of the operating system clipboard SEE ALSO Editt Copy to System Cut x Paste Control 49 Last updated 2012 10 02 File gt Open o 26 2 Open o Opens an image and displays it in a separate window Image files must be in TIFF GIF JPEG DICOM BMP PGM or FITS format or in a format supported by a reader plugin Also opens ImageJ and NIH Image lookup tables E lut extension Tables in tab delimited text format El x1s or El csv extension see Results Table Selections E roi or El zip extension Text files E txt El ijm E js and El java extensions SEE ALSO File Import Image Types and Formats Virtual Stacks VIII Opening Files File gt Open File gt Import gt and Drag 4 Drop VIII OPENING FILES Fil
196. hen a selection exists zooming with the Arrow Keys requires holding down either or Ctrl 28 13 1 In Zooms in to next higher magnification level and if possible enlarges the window As explained in Magnifying Glass there are 21 possible levels shown in the title bar 3 1 4 2 6 3 8 3 12 5 16 7 25 33 3 50 75 100 150 200 300 400 600 800 1200 1600 2400 and 3200 28 13 2 Out Zooms out to next lower magnification level and if needed shrinks the window 28 13 3 Original Scale 4 Displays the image at the magnification used when the image was first opened As a shortcut double click on the Magnifying Glass tool 100 Last updated 2012 10 02 IJ 1 46R IJ 1 46R IMPROVED IN IJ 1 46R Image Overlay gt XVIIl WORKING WITH ZOOMED CANVASES Images are magnified using and or and if no selection exists Magnification occurs around the cursor or to the center of the image when the cursor lays outside the image canvas The Zoom indicator in the upper left corner of magnified images shows what portion of the image is currently displayed At high magnification levels the pixel grid becomes visible by default unless Interpolate zoomed images is checked in Edit gt Options Appearance To scroll a magnified image hold down the space bar Scrolling Tool shortcut while dragging the cursor By default Overlays and the active selection are displayed with a 1 pixel wide contour regar
197. his option can be enabled in Edit Options Profile Plot Options by activating the Sub pixel resolution checkbox Pixel coordinates of area selections see Area Selection Tools can be interpolated using Edit gt Selection gt Interpolate The image on the right is the output of E SubPixelSelections js a script that demonstrates how to create selections at sub pixel resolution without the need of setting any option in ImageJ SEE ALSO Zoomp Magnifying Glass 11 Overlays Overlays are non active selections displayed over the pixel data on the image overlay and are the core of non destructive image processing in ImageJ In a way you can think of the image 19 Last updated 2012 10 02 Overlays Polygons 8 bit 61K a E 250x250 pixels 256x254 pixels blobs gif 8 bit inverting LUT 641 p Cell Colony pixels 8 bit 38K ImageWithOverlay tif 6 bit 196K me 359x280 pixels 1 BRS C Clown amp Lena x200 pixels RGB 250K y E A sm at c 57 114 121x154 pi The cell outlines were created by the particle analyzer Non destructive operations using the image overlay Overlays can be used to annotate images store ROIs and blend images ImageROls at multiple opacity levels Refer to the Image Overlay gt documentation for further examples You can download the frontmost image to practice overlay editing overlay as an invisible
198. hold level to create the binary image Auto thresholding is displayed in the Status bar cf Threshold T With stacks the Convert to Mask dialog box is displayed Note that for non thresholded images and stacks Make Binary and Convert to Mask behave similarly SEE ALSO Editp gt Selection Convert to Mask XX Creating Binary Masks XXII Interpreting Binary Images 112 Last updated 2012 10 02 Process gt Binary gt 29 8 2 Convert to Mask Converts an image to black and white The mask will have an inverting LUT white is 0 and black is 255 unless Black Background is checked in the Process gt g Calculate Threshold tor Each Image Binary Options dialog box If a threshold has not been ee set automatic threshold levels will be calculated cf Make Binary Note that for non thresholded images and stacks Make Binary and Convert to Mask behave similarly With stacks the depicted dialog is displayed Convert all images in stack to binary TA TA Cancel j OK e i Calculate Threshold for Each Image If checked threshold levels will be calculated for each individual slice otherwise the calculated threshold of the currently displayed slice will be used for all slices Black Background Defines whether the background is black and the foreground is white Note that the value of this checkbox reflects and sets the global Black Background value of Process Binary Options SEE ALSO Make Binary
199. i Process gt submenu Smooth S Sharpen Find Edges and Enhance Contrast 29 1 Smooth S Blurs the active image or selection This filter replaces each pixel with the average of its 3x3 neighborhood 29 2 Sharpen Increases contrast and accentuates detail in the image or selection but may also accentuate noise This filter uses the following weighting factors to replace each pixel with a weighted average of the 3x3 neighborhood L l 12 l l i l 29 3 Find Edges Uses a Sobel edge detector to highlight sharp changes in intensity in the active image or selection Two 3x3 convolution kernels shown below are used to generate vertical and horizontal derivatives The final image is produced by combining the two derivatives using the square root of the sum of the squares 1 2 1 1 0 1 0 0 2 06 2 2 l 1 0 1 29 4 Find Maxima Determines the local maxima in an image and creates a binary mask like image of the same size with the maxima or one segmented particle per maximum marked 29 Analysis is performed on the existing rectangular selection or on the entire image if no selection is present For RGB images maxima of luminance are selected with the luminance defined as weighted or unweighted average of the colors depending on how Weighted RGB to Grayscale Conversion is set in Edit gt Options gt Conversions 107 Last updated 2012 10 02 Process gt Find
200. i Width and H eight may be either expanded or contracted Az Ea If the canvas size is increased the border is filled with the current background color see Color Picker K i or if Zero Fill is checked the border is filled with pixels O Zero Fill bic ey that have a value of zero The position of the old image Top Right er Center Left within the new canvas may also be specified Y Center Position Center anal A Cancel ay SEE ALSO Size ImagebScale E Imagep Bottom Lef i ee Tra nsform gt Bin XVII Embedding Color Bottom Right Annotations in Grayscale Images 28 2 8 Line Width This widget is used to adjust the width of line selec tions in pixels It is opened more easily by double clicking on the Line Selection Tools icon Checking Spline Fit fits a cubic spline curve to the points that define the line O Spline Fit SEE ALSO Edit Options gt Line Width Edit Selection gt Fit Spline 28 3 Show Info i IN A40R AA Info for t1 head tif Opens a text window containing information about These comments were added to the TIFF file h a ld dea ls using this macro the active image including the pixel or voxel size info File openAsString since IJ 1 44k For DICOM and FITS images also l displays file header information Use the popup menu right click in the Info window to save the informa Title tl head tif Cut Width 384 00 mm 256 Cop
201. i ie oe oe N 25 2 Pointer At startup ImageJ will search for a GIF image named Elcrosshair cursor gif in the GiImageJ images directory If present it will be used to replace the default crosshair cursor The pointer can also be changed to an arrow by toggling Use pointer cursor on Edit gt Options gt Misc 5 Customizing the Float Behavior of IJ s Main Window These macros can be added to the ImageJ macros StartupMacros txt file gt in order to set the floating behavior of the Images main window option 1 Run ImageJ plugins Always_on_Top js command at launch by gt adding it to the AutoRun macro macro AutoRun run Always on Top option 2 Execute the script at launch by adding it to AutoRun macro AutoRun eval script IJ getInstance setAlwaysOnTop true option 3 Toggle the setAlwaysOnTop option using a shortcut e g Fl var afloat macro Toggle AlwaysOnTop Fi booleans newArray true false eval script IJ getInstance setAlwaysOnTop booleans afloat afloat afloat 47 Last updated 2012 10 02 Part V Menu Commands As described in ImageJ User Interface the menu bar lists all ImageJ commands It is organized in eight menus File gt Basic file operations opening saving creating new images Most are self explanatory Edit gt Editing and drawing operations as well as global settings Image Conversion and modification of
202. ia Java plugins Custom acquisition analysis and processing plugins can be developed using ImageJ s built in editor and Java compiler User written plugins make it possible to solve almost any image processing or analysis problem Being public domain open source software an ImageJ user has the four essential freedoms defined by the Richard Stallman in 1986 1 The freedom to run the program for any purpose 2 The freedom to study how the program works and change it to make it do what you wish 3 The freedom to redistribute copies so you can help your neighbor 4 The freedom to improve the program and release your improvements to the public so that the whole community benefits ImageJ is being developed on Mac OS X using its built in editor and Java compiler plus the BBEdit editor and the Ant build tool The source code is freely available The author Wayne Rasband wsr nih gov is a Special Volunteer at the National Institute of Mental Health Bethesda Maryland USA SEE ALSO History of ImageJ at imagejdev org A somehow outdated list of ImageJ s features is available at http imagej nih gov ij features html Installing and Maintaining ImageJ 2 Installing and Maintaining ImageJ ImageJ can be downloaded from http imagej nih gov ij download html Details on how to install ImageJ on Linux Mac OS 9 Mac OS X and Windows 1 are available at http imagej nih gov ij docs install Help Installation command Specially
203. icardo B Medeiros Kate J Papenfuss Brian Hoium Kristen Coley Joy Jadrich Saik Kia Goh Anuratha Elayaperumal Julio E Herrera Ernesto Resnik and Hsiao Tzu Ni Novel sequential ChIP and simplified basic ChIP protocols for promoter co occupancy and target gene identification in human embryonic stem cells BMC Biotechnol 9 59 Jan 2009 doi 10 1186 1472 6750 9 59 E Meijering M Jacob J C F Sarria P Steiner H Hirling and M Unser Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images Cytometry Part A the journal of the International Society for Analytical Cytology 58 2 167 76 Apr 2004 doi 10 1002 cyto a 20022 C dric Messaoudi Nicole Garreau de Loubresse Thomas Boudier Pascale Dupuis Williams and Sergio Marco Multiple axis tomography applications to basal bodies from Paramecium tetraurelia Biol Cell 98 7 415 25 Jul 2006 doi 10 1042 BC20050097 C dric Messaoudii Thomas Boudier Carlos Oscar Sanchez Sorzano and Sergio Marco TomoJ tomography software for three dimensional reconstruction in transmission electron microscopy BMC Bioinformatics 8 288 Jan 2007 doi 10 1186 1471 2105 8 288 K Moodley and H Murrell A colour map plugin for the open source Java based image processing package ImageJ Computers amp Geosciences 30 6 609 618 Jan 2004 doi 10 1016 j cageo 2004 03 017 Richard L Mort Quantitative analysis of patch patterns in mosaic tissues with ClonalTools
204. ifying glass icon to revert to the image s original magnification As explained in Image gt Zoom In there are 21 possible magnification levels 3 1 4 2 6 3 8 3 12 5 16 7 25 33 3 50 75 100 150 200 300 400 600 800 1200 1600 2400 and 3200 Modifier keys Clicking and dragging while holding down the Shift key runs Image Zoom To Selection Alt Image zooms out right click behavior SEE ALSO XVIII Working with Zoomed Canvases Zoom commands Toolbar Shortcuts 19 10 amp Scrolling Tool Allows you to scroll through an image that is larger than its window You can temporarily activate this tool except when using the Text Tool by holding down the space bar SEE ALSO XVIII Working with Zoomed Canvases Toolbar Shortcuts 19 11 4 Color Picker Sets the foreground drawing color by picking up colors from any open image Colors can also be picked up from the Color Picker CP window Image Colors Color Picker K using any tool In the icon the eye dropper is drawn in the current foreground color while the frame around it is drawn in the current background color Edit Draw d and Edito Fill f use the foreground color Editt gt Clear Clear Outside and Cut x use the background color Double clicking on the tool icon will display the Color Picker window Modifier key Alt Alt clicking with the Color Picker Tool on the image canvas picks up background color SEE ALSO Color Pic
205. iles but poorer quality Larger values produce larger files but better quality Color sub sampling is disabled when the value is set to 100 reducing the likelihood of color artifacts By default the DPI in the JPEG header is set to 72 For a higher value use a unit of inch in the Analyzep Set Scale dialog E g setting Distance im Pixels to 300 Known Distance to 1 and Unit of Length to inch will set the DPI to 300 Overlays are embedded when saving in Jpeg format see Image gt Overlay gt Flatten F SEE ALSO II Image Types Lossy Compression and Metadata X Warning on JPEG Compression 56 Last updated 2012 10 02 IMPROVED IN IJ 1 46R File gt Save Asp X WARNING ON JPEG COMPRESSION The JPEG format uses Lossy compression that leads to severe artifacts that are not compatible with quantitative analyses As such it should only be used for presentation purposes if file size is an issue but even then a lossless format such as PNG is probably more suitable The illustration below exemplifies the consequences of saving images in a lossy format To replicate it open the Mandrill sample image by drag and drop or alternatively using File gt Import gt URL and typing the image s path http imagej nih gov ij images baboon jpg duplicate it Imagep gt Duplicate D save the duplicate as JPEG File gt Save Asp Jpeg run File gt Revert r so that the saved version is reloaded by ImageJ and calculate
206. ills it to r a y M Add to Manager UL In situ Sho Count Macks Overlay Outlines Overlay Masks make it invisible then resumes scanning until it reaches the end of the image or selection Press to abort this process Help Cancel Size Particles with size area outside the range specified in this field are ignored Values may range between 0 and Infinity For spatial scaled images cf Set Scale values are expressed in physical size square units or in pixels if Pixel Units is checked Enter a single value and particles smaller than that value will be ignored 132 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Analyze gt Analyze Particles Original thresholded Exclude on Edges Include Holes Size 0 50 Circ 0 5 1 0 AOO DemoBlobs gif O Exclude on Edges APA Include Holes A AA S 0 50 C 0 5 1 0 x pixels 6 It x pixels 6 It inverting x pixels o It inverting x pixels 6 It inverting Size 0 20 Circ 0 0 0 5 Size 0 20 Circ 0 3 0 5 Size O0 oo Circ 0 0 0 3 Size 50 20 Circ 0 5 1 0 A A A S 0 0 C 0 0 0 5 S 0 co C 0 3 0 5 S 0 co C 0 0 0 3 A A A S 50 o C 0 5 1 0 Include x pixels 8 bit inverting y x pixels 8 bit inverting x pixels 8 bit inverting x pixels 8 bit inverting j Particle Analyzer Analyze gt Analyze Particles Features of th
207. imensions Bicubic interpolation is used if Bicubic interpolation is checked cf Imageb Size and Imagep Scale E Name Specifies the title of the stack to be created Title Contains Enter a string into this field and ImageJ will only convert to stack images whose name contains that string Last updated 2012 10 02 Image Stacks gt Montage Columns Rows Scale Factor First Slice Last Slice Increment Border Width Font Size M Label Slices O Use Foreground Color MP Cancel OK Bicubic Interpolation If checked bicubic interpolation cf Adjust gt Size will be used if any of the Scale methods was previously chosen Use Title as Labels If checked image titles without extension will be used as stack labels As described in Stacks gt Tools gt Remove Slice Labels these labels up to 60 characters correspond to the image subtitle the line of information above the image Keep Source Images If checked original images are kept 28 6 7 Stack To Images Converts the slices in the current stack to separate image windows SEE ALSO Stacks gt Images To Stack 28 6 8 Make Montage Produces a single grid image containing the individual images that compose Stacks and 4D Hyperstacks This can be useful for visual comparisons of a series of images stored in a stack and to create panel figures for publication and presentations A dialog box allows you to specify the magnificati
208. ing scripts in any of Fiji s supported languages including the ImageJ macro language The editor features full undo support syntax highlighting tabs bookmarks and several other tools that simplify scripting workflows in ImageJ For more information visit Fiji s editor website at http fiji sc wiki index php Script Editor LO StartupMacros txt File Edit Language Templates Run ADOSI Tabs _ Startup Open Help on Macro Functions ___ Extract source jar StartupMacros txt C Je A See file About Startup Macros i reate new p E f Focus on the main Fiji window 5 Malalalalelajololelojolejojolejojojolajojojajojojejojojok AutoRun macro HAHA AAA AA a a a a a a a a a a a a a a a a a a 7 macro AutoRun fldr getDirectory imagej samples Find Next Calibration if File exists fldr fldr getDirectory imagej Sample TReplacewith o if File exists fldr 2 path replace fldr 20 Replace All 13 in windows backslashes give problems Match Case Regex 4 pene P 14 if startswith getInfo os name Wind Gf Mark All Whole Word 1 path tere strings split fldr File separator for i 0 i lt strings length i path path strings il call ij Prefs setImagesURL file path Started StartupMacros ijm at Mon Dec 27 13 06 44 EST 2010 The Fiji Script Editor ImageJA 1 44m The Fiji Editor is an advanced text editor
209. ings and Preferences You can use the Process Binary Options dialog to assess the impact of the Black background option Create a binary image choose an operation from the Do drop down menu activate the preview feature and toggle the Black background checkbox SEE ALSO Settings and Preferences XX Creating Binary Masks 29 9 3 Multiply Multiplies the image or selection by the specified real constant With 8 bit images results greater than 255 are set to 255 With 16 bit signed images results greater than 65 535 are set to 65 535 29 9 4 Divide Divides the image or selection by the specified real constant Except for 32 bit float images attempts to divide by zero are ignored With 32 bit images dividing by zero results in Infinity Infinity or NaN 0 0 pixels when the source pixels are positive negative or zero The divide by zero value can be redefined using Edit gt Options Misc 29 9 5 AND Does a bitwise AND of the image and the specified binary constant 29 9 6 OR Does a bitwise OR of the image and the specified binary constant 29 9 7 XOR Does a bitwise XOR of the image and the specified binary constant 118 Last updated 2012 10 02 Process gt Math gt 29 9 8 Min Pixels in the image with a value less than the specified constant are replaced by the constant 29 9 9 Max Pixels in the image with a value greater than the specified con
210. ins menu preventing it from running off the bottom of the screen When this option is enabled plugins that attempt to install themselves in a submenu with only one command are instead installed in the Plugins Miscellaneous submenu An example of such a plugin is TurboReg which normally creates a Plugins gt TurboReg submenu that contains only one command Run single instance listener If checked ImageJ will use sockets to prevent multiple in stances from being launched 16 On Windows this avoids the problem where another copy of ImageJ starts each time an image is dragged and dropped on the ImageJ icon It also prevents multiple instances when running ImageJ from the command line Note that you may get a security alert the first time ImageJ starts with this option enabled ImageJ does not require external socket access so it is okay to deny it access in the security alert This option is set by default with new Windows installations Debug mode If checked causes ImageJ to display debugging messages in the Log Window In addition some commands e g Process Binary gt Skeletonize and Watershed produce detailed outputs when Debug mode is active Close the Log window to disable display of debugging messages Help Opens http imagej nih gov ij docs menus edit html misc SEE ALSO Customizing the ImageJ Interface 27 13 17 Reset 00 Reset Preferences Causes the IJ preferences file E 1J_prefs txt to be o EL iad deleted
211. intosh It runs either as an online applet or as a downloadable application on any computer with a Java 1 5 or later virtual machine Downloadable distributions are available for Windows Mac OS X and Linux It can display edit analyze process save and print 8 bit 16 bit and 32 bit images It can read many image formats including TIFF GIF JPEG BMP DICOM FITS and raw It supports stacks and hyperstacks a series of images that share a single window It is multithreaded so time consuming operations such as image file reading can be performed in parallel with other operations It can calculate area and pixel value statistics of user defined selections It can measure distances and angles It can create density histograms and line profile plots It supports standard image processing functions such as contrast manipulation sharpening smoothing edge detection and median filtering It does geometric transformations such as scaling rotation and flips Image can be zoomed up to 32 1 and down to 1 32 All analysis and processing functions are available at any magnification factor The program supports any number of windows images simultaneously limited only by available memory Spatial calibration is available to provide real world dimensional measurements in units such as millimeters Density or gray scale calibration is also available ImageJ was designed with an open architecture that provides extensibility v
212. ion of efforts To provide a central online resource for ImageJ program downloads a plugin repository developer resources and more Be sure to follow the ImageJ2 project news and the ImageDev blog for updates on this exciting project 3 Getting Help 3 1 Help on Image Analysis Below is a list of online resources in no particular order related to image processing and scientific image analysis complementing the list of external resources on the IJ web site Ethics in Scientific Image Processing Online learning Tool for Research Integrity and Image Processing This website created by the Office of Research Integrity explains what is appropriate in image processing in science and what is not Digital Imaging Ethics at the Cellular Imaging Facily Core SEHSC This website compiled by Douglas Cromey at the University of Alabama Birmingham discusses thoroughly the topic of digital imaging ethics It is recommended for all scientists The website contains links to several external resources including 1 What s in a picture The temptation of image manipulation 2004 M Rossner and K M Yamada J Cell Biology 166 1 11 15 doi 10 1083 jcb 200406019 2 Not picture perfect 2006 Nature 439 891 892 doi 10 1038 439891b Scientific Image Processing What you need to know about scientific image processing Simple and clear this Fiji webpage explains basic aspects of scientific image processing 9 Last updated
213. ion of liver tumors using cine magnetic resonance imaging Int J Radiat Oncol 71 4 1189 1195 Jan 2008 doi 10 1016 j ijrobp 2007 11 026 Atsuko Kobayashi Takumi Fujigaya Masayuki Itoh Takahisa Taguchi and Hiroshi Takano Technical note a tool for determining rotational tilt axis with or without fiducial markers Ultramicroscopy 110 1 1 6 Dec 2009 doi 10 1016 j ultramic 2009 08 007 Janos Kriston Vizi Ng Wee Thong Cheok Leong Poh Kwo Chia Yee Joan Sim Poh Ling Rachel Kraut and Martin Wasser Gebiss an image plugin for the specification of ground truth and the performance evaluation of 3d segmentation algorithms BMC Bioinformatics 12 232 2011 doi 10 1186 1471 2105 12 232 G Landini and G Perryer Digital enhancement of haematoxylin and eosin stained histological images for red green colour blind observers J Microsc 234 3 293 301 Jun 2009 doi 10 1111 j 1365 2818 2009 03174 x Derick Lau Anthony Seibert David Gandara Luko Laptalo Este Geraghty and Christopher Coulon Computer assisted image analysis of bronchioloalveolar carcinoma Clin Lung Cancer 6 5 281 6 Mar 2005 Charles A Lessman Ravikanth Nathani Rafique Uddin Jamie Walker and Jianxiong Liu Computer aided meiotic maturation assay CAMMA of zebrafish Danio rerio oocytes in vitro Mol Reprod Dev 74 1 97 107 Jan 2007 doi 10 1002 mrd 20530 177 69 70 71 72 73 74 75 76 77 78 79 80 81
214. ional modeling The extraction of the map that describes links between objects For example which neuron contacts which other neurons through how many and which synapses SEE ALSO BiolmageXD Endrov Image SXM 2 2 2 ImageJ Interoperability Several packages exist that allow ImageJ to interact with other applications environments Bitplane Imaris ImarisXT can load and execute ImageJ plugins bpImarisAdapter Windows only and requiring valid licenses for Imaris and ImarisXT allows the exchange of images between Imaris and ImageJ CellProfiler CellProfiler 17 features RunImageJ a module that allows ImageJ plugins to be run in a CellProfiler pipeline Icy Icy an open source community software for bio imaging executes ImageJ plugins with almost 100 plugin compatibility Knime Knime Konstanz Information Miner contains several image processing nodes KNIP that are capable of executing ImageJ plugins and macros Open Microscopy Environment All Open Microscopy Environment projects such as Bio Formats VisBio and OMERO integrate well with ImageJ RImageJ R bindings for ImageJ Bindings between ImageJ and R GNU S The free software environment for statistical computing and graphics The documentation for RImageJ is available at http cran r project org web packages RImageJ RImageJ pdf see also Bio7 in Software Packages Built on Top of ImageJ MIJ Matlab ImageJ bi directional communication A Java package for bi dir
215. is option is equivalent to using the Scale plugin from the TransformJ package to scale the stack in the z dimension by the slice spacing in pixels This checkbox is ignored if the slice spacing is less than or equal to 1 0 pixels 9 Last updated 2012 10 02 Image Stacks gt Help Opens http imagej nih gov ij docs menus image html project SEE ALSO Orthogonal Views H Z Project Grouped Z Project and 3D Viewer 101 Volume Viewer plugins 28 6 13 Plot Z axis Profile PET stack tif 63 57 Plots the ROI selection mean gray value versus slice number Requires a point or area selection Coordinates of the upper left corner of the selec tion or the bounding rectangle for non rectangular selections are displayed in the graph title SEE ALSO Profile Plot Options Plot Profile k h d ed E X 25 Y 27 0 Save Copy r 28 6 14 Label OO StackLabeler Adds a sequence of numbers e g timestamps and or a label IEA to a stack or hyperstack Numbers and label are drawn in Ba Mca the current foreground color cf Image Colors gt Color Picker Starting value D K Interval 00 00 00 The initial X Y location and Font size of the label are based X location pros on the existing rectangular selection if any Slices outside the eee Range are not affected Font size Range 1 36 Format Specifies the structure of the label 0 Un Text FLIM padded sequence 0000 Pads each
216. isplay options of ParticleAnalyzer Analyze gt Analyze Particles IJ 1 441 When displaying Overlays size color and background of text labels can be adjusted using Image Overlay gt Labels Display Results If checked the measurements for each particle will be displayed in the Results Table Clear Results If checked any previous measurements listed in the Results Table will be cleared Summarize If checked the particle count total particle area average particle size area fraction and the mean of all parameters listed in the Set Measurements dialog box will be displayed in a separate Summary table Note that while single images Summaries are output to the same Summary table stack Summaries are printed in dedicated tables named Summary of stack title Also note that descriptive statistics on Results measurements can be obtained at any time using the Summarize command Add to Manager If checked the measured particles will be added to the RO Manager Exclude on Edges If checked particles touching the edge of the image or selection will be ignored Include Holes If checked interior holes will be included Disable this option to exclude interior holes and to measure particles enclosed by other particles When this option is enabled ImageJ finds the extent of each particle by tracing the outer edge When it is disabled ImageJ finds the extent by flood filling 134 Last updated 2012 10 02 IMPROVED IN IJ
217. k as described for Image Adjust gt Brightness Contrast cat C Similarly to the Window Level tool if the B amp C UN Brightness UN window is opened it will be closed and the Color window Channel will be opened at the same location NB When switching from one color to another the changes made to one color will be lost unless Apply is Channel 3 ara 4 clicked before Also note that for 48 bit color images Channel 5 that load as a stack Brightness Contrast C works on Channel 6 single stack slices i e colors and the color settings of the Color panel are ignored SEE ALSO Brightness Contrast C Color gt submenu 28 2 4 Threshold T Use this tool to automatically or interactively set lower and upper threshold values segmenting grayscale images into features of interest and background Use Analyze gt Measure m with Limitto Threshold in Analyze gt Set Measurements checked to measure the aggregate of the 78 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Image gt Adjust gt XVI BRIGHTNESS CONTRAST OF HIGH BIT DEPTH IMAGES When displayed the intensity of each pixel that is written in the image file is converted into the grayness of that pixel on the screen How these intensities are interpreted is specified by the image type From the ImageJ website 16 bit and 32 bit grayscale images are not directly displayable on computer monitors which typically can show o
218. ker window XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts Temporary Activation of a Tool 36 Last updated 2012 10 02 IMPROVED IN IJ 1 46R CHANGED IN IJ 1 46R Tools More Tools Menu Y Startup Macros mo A menu e a or e ae Use this drop down menu to switch to alternative macro toolsets or to load additional plugin tools The toolsets listed in the menu are located in the Image macros toolsets folder and the plugin tools are the ones installed in the Plugins gt Tools submenu Lookup Tables Magic Montage ROI Manager Tools Stack Tools Toolset Creator Arrow Brush Developer Menu Flood Filler Toolsets LUT Menu Overlay Brush Pencil Pixel Inspector Spray Can Stacks Menu Hold the shift key down while selecting a Built in tools toolset to view its source code More macro toolsets are available at lt http imagej nih gov ij macros toolsets gt Plugin tools can be downloaded from the Tools section of the Plugins page at lt http imagej nih gov ij plugins gt More Tools Menu IJ 1 46n The menu lists tools from E StartupMacros txt in S ImageJ macros Toolsets installed in 5 ImageJ macros toolsets built in tools loaded from E ij jar Arrow Brush Developer Menu Flood Filler LUT Menu Overlay Brush Pencil Spray Can and Stacks Menu and Single Tools installed in GImageJ plugins Tools While toolsets replace all the eight slots in the toolbar single tools are inst
219. ks with a El tif extension open as virtual stacks when dragged and dropped on the toolbar icon A Image Baco Ja 1124 20 7 89 gt lt lt Open as Virtual Stack gt gt b Hyperstacks Hyperstacks are multidimensional images extending image stacks to four 4D or five 5D dimensions x width y height z slices c channels or wavelengths and t time frames Hyperstacks are displayed in a window with three labelled scrollbars see Stacks and Hyperstacks Similarly to the scrollbar in Stacks the frame slider t has a play pause icon SEE ALSO Image gt Hyperstacks gt submenu 13 Last updated 2012 10 02 Color Images 9 Color Images ImageJ deals with color mainly in three ways pseudocolor images RGB images RGB HSB stacks and composite images Pseudocolor Images A pseudocolor or indexed color image is a single channel gray image 8 16 or 32 bit that has color assigned to it via a lookup table or LUT A LUT is literally a predefined table of gray values with matching red green and blue values so that shadows of gray are displayed as colorized pixels Thus differences in color in the pseudo colored image reflect differences in intensity of the object rather than differences in color of the specimen that has been imaged 8 bit indexed color images such as GIFs are a special case of pseudocolor images as their lookup table is stored in the file with the image These images are limited to 256 colors
220. l selections it is also possible to specify the font size and text alignment Choose List coordinates to retrieve a dedicated table of XY coordinates from the active selection at evenly spaced one pixel intervals Note that while this command can only be applied to the active selection the ROI Manager s Properties command Analyze gt Tools ROI Manager can be applied to multiple ROIs SEE ALSO Selections Image gt Overlay gt Add Selection b File gt Import gt XY Coordinates File gt Save Asp XY Coordinates 27 12 13 Rotate IN IJ 1 46R MAO Rotate Selection Rotates the selection using floating point coordinates by the specified number of degrees A negative number indicates counter Angle 15 degrees E A clockwise rotation This command runs the RotateSelection macro Enter negative angle to ca Od rotate counter clockwise m E 1J Jar YET JA Cancel OK ee A SEE ALSO FlipSelection macro 27 12 14 Enlarge 0 Enlarge Selection Grows an area selection by a specified number of pixels Enter Enlarge by 15 pixels a negative value to shrink the selection This command runs the EnlargeSelection macro in Hlij jar ShrinkSelection is a variation of this macro that does not shrink polygonal selections from the edges of the image Enter negative number to shrink i HH A W Cancel OK y a e ee 65 Last updated 2012 10 02 Edit gt Selection gt XIII
221. le After Filtering If checked puts the lowest intensity to 0 and the highest intensity to 255 preserving all intensities Saturate Image when Autoscaling If checked allows some intensities to go into saturation and produces a better visual contrast Saturate Image when Autoscaling only has an effect when Autoscale After Filtering is enabled Display Filter If checked shows the filter generated Note that this disables Undo of the filter operation on the original image 29 10 6 Custom Filter PURO FFT Filter This command does Fourier space filtering of the active image using a user supplied spatial domain non FFT image as the filter Filter FFT Filter 05 06 tif a 3 AAA This image will be converted to 8 bit For pixels that have a value of 0 the corresponding spatial frequencies will be blocked Pixel with values of 255 should be used for passing the respective spatial frequencies without attenuation Note that the filter should be symmetric with respect to inversion of the center Points that are opposite of the center point defined as x width 2 y height 2 should have the same value Otherwise artifacts can occur For some examples see the FFT CustomPFilterDemo and FFTRemoveStreaks macros 29 10 7 FD Math This command correlates convolves or deconvolves two images Imagel FFT of Original SD 5 It does this by converting Image and Image2 to the fre Operation Deconvolve 4 quency d
222. lection in the active image 27 12 38 Restore Selection E Restores the previous selection to its original position A selection is saved when you Delete the selection by clicking outside of it Draw a new selection De activate the image containing the selection Close the image containing the selection Use a command that deletes or modifies the selection SEE ALSO Analyze Tools gt RO Manager 27 12 4 Fit Spline Fits a cubic spline curve to a polygon or polyline selection see ROI manipulations SEE ALSO Straighten Interpolate 63 Last updated 2012 10 02 Edit gt Selection gt XII TRANSFERRING SELECTIONS BETWEEN IMAGES You can transfer a selection from one image to another by activating the image with the selection activating the destination image then pressing the keyboard shortcut for Edit Selection gt Restore Selection E This shortcut can also be used to restore accidentally deleted ROIs Alternative ways to transfer ROIs across images involve the ROI Manager and the cursor synchronization features provided by Analyze Tools gt Synchronize Windows 27 12 5 Fit Circle Fits a circle to a multipoint with at least 3 points or area selection 14 see ROI manipulations Composite selections are not supported With open shapes lines and points the fitting algorithm Newton based Pratt fit described in Pratt V Direct least squares fitting of algebraic surfaces Co
223. les containing a configuration file Elplugins config specifying the location of the new commands implemented by the plugin You can rename reorganize or move commands implemented by external plugins by editing their Elplugins config file as described on the JAR demo documentation page If you don t know in which menu a plugin has been registered use Show full information in the command Finder Plugins gt Utilities gt Find Commands I to find out the location of the installed E jar files With Fiji Scripts and Macros can be registered in any menu by saving into SFiji app plugins Scripts menu name submenu name E g a certain macro El ijm file saved in SFiji app plugins Scripts File Import is registered in the Filetr gt Import sub menu SEE ALSO ImageJ s plugin architecture on the Fiji website 48 File gt 26 File 26 1 New Contains commands for creating new images stacks hyperstacks or text windows SEE ALSO Plugins gt New 26 1 1 Image n New Image Creates a new image window or stack A dialog box allows you to specify the image title type dimensions and initial content Name Text Image Type 8 bit Name is the title that will be used for the Window Type is the image type 8 bit grayscale 16 bit grayscale un signed 32 bit float grayscale or RGB color Fill With White Black or Ramp specifies how the image is initial ized Width and Height specify t
224. lor marks are only available with RGB images see XVII Embedding Color Annotations in Grayscale Images T Cancel OK A 00 e Auto Measure If checked clicking on the image records the pixel location and intensity Note that if Mark Width is not zero every time a point selection is measured a mark will be painted cf Measure m If unchecked Edit Draw d can be used to paint the mark Mark Width diameter at the location of each point Auto Nezxt Slice If checked ImageJ will automatically advance to the next stack slice Note that this feature will only allow one point per slice Add to ROI Manager If checked points will be automatically added to the RO Manager Label Points If checked each point selection will be displayed with an accompanying numeric label Selection Color Specifies Selections color chosen from one of the nine default colors red green blue magenta cyan yellow orange black and white The chosen color is highlighted in the center of the Point MultiPoint Tool It can also be specified using Edit gt Options gt Colors SEE ALSO Multi point Tool Using a Keyboard Shortcut to Change Selection Color Cell Counter plugin Toolbar Shortcuts 19 6 Multi point Tool The Multi point Tool selects multiple points behaving as the Point Tool when is pressed Label Points is checked and Auto Measure and Auto Next Slice are deselected As described for the Point Tool can also be used
225. ls If checked ROI names are used instead of the default numeric labels If unchecked the size selection count of the current overlay is used Selections can be renamed using the Edit gt Selection gt Properties y command or when using the ROI Manager either Rename or Properties Draw backgrounds If checked text will be displayed on complementary colored background This option produces similar labels to those produced by the ROI Manager Show All option when Labels is activated Bold If checked labels typeset in sans serif font are displayed in boldface SEE ALSO Overlay Options Add Selection b NEw IN 28 14 10 Overlay Options 7 7 Overlay Options Use this command to define the default overlay Stroke color Stroke width and Fill color As mentioned in Edit gt Selection gt Properties ly and Image Overlay gt Add Selection b Stroke contour color Width 1 and Width are ignored if a Full color is specified Set Stroke width to 0 to have selections drawn using a width of one pixel regardless of the image magnification see XVIII Working with Zoomed Canvases Stroke color yellow Fill color none Set stack positions As usual colors are specified using the name of one of the ImageJ UD default colors black blue cyan green magenta orange red white and yellow or using Hexadecimal Color Values With Stacks and Hyperstacks selecting the Set stack positi
226. lue Infinity o Divide by zero value Specifies the value used when LU Use pointer cursor O Hide Process Stack dialog Process gt Image Calculator e detects a divide by O Require command key for shortcuts zero while dividing one 32 bit real image by an Move isolated plugins to Misc menu other The default is infinity In addition to nu O Run single instance listener U Debug mode meric values infinity positive or negative infin o ity max largest positive value and NaN Not Cancel OK a Number can be entered as the Divide by zero value Use pointer cursor If checked ImageJ will use an arrow cursor instead of the default crosshair that is sometimes difficult to see on grayscale images in areas of medium brightness This option can also be used to work around a bug on Windows where the text cursor is sometimes used in place of the crosshair Note that the default crosshair can be customized as explained in Customizing the ImageJ Interface 13 Last updated 2012 10 02 Edit gt Options gt Hide Process Stack dialog If checked ImageJ will suppress the dialog that asks Process all xx slices only the current slice will be processed Require control command key for shortcuts If checked requires the Control key Com mand key on Macs to be pressed when using keyboard shortcuts for menu commands Move isolated plugins to Misc menu This option can reduce the size of the Plug
227. lues of the dividing lines between the cells are equal to the distance between the two nearest particles This is similar to a medial axis transform of the background but there are no lines in inner holes of particles SEE ALSO Find Maxima Segmented Particles output Delaunay Voronoi plugin 29 8 14 Options Specifies several settings used by Binary gt commands Iterations Specifies the number of times erosion dilation opening and closing are performed Iterations can be aborted by pressing Esc Count Specifies the number of adjacent background pixels necessary before a pixel is removed from the edge of an object during erosion and the number of adjacent foreground pixels necessary before a pixel is added to the edge of an object during dilation 116 Last updated 2012 10 02 Process gt Math gt Black background If checked binary images will be created without using an inverted LUT cf XX Creating Binary Masks and commands in the Process gt Binary gt submenu will assume that images contain white objects on a black background see XXII Interpreting Binary Im Iterations 1 100 1 Count 1 8 1 M Black background C Pad edges when eroding ages Macros can set this option using setOp tion BlackBackground true see XXII In terpreting Binary Images and Settings and Prefer ences Dilate Open esas Pad edges when eroding If checked Binary gt Erode Outline does not erode
228. ly The left half of the palette varies only in brightness while the right half varies only in saturation At the center of the color ramp are enlarged red green blue cyan magenta and yellow colors for quick selection To the left of the color palette is a grayscale ramp that goes from pure black to pure white 89 Last updated 2012 10 02 Image Stacks gt Double clicking on a color brings up the ColorChooser a widget with three sliders used to specify the RGB values of the foreground or background color The title of the ColorChooser widget Foreground Color or Background Color indicates the current selection mode To get precise colors manually change the values in the text boxes The hex value of the final color is also displayed offering a convenient way to retrieve custom colors to e g personalize Overlays see XIX Hexadecimal Color Values As mentioned earlier the Color Picker Tool tool can be used to pick up foreground background colors from an image canvas Foreground color can also be changed using the Color dropdown menu in the Options dialog of drawing tools such as Arrow Brush Overlay Brush and Pencil tools SEE ALSO XVII Embedding Color Annotations in Grayscale Images Draw d Fill f Clear Clear Outside Image Types and Formats Temporary Activation of a Tool XVII EMBEDDING COLOR ANNOTATIONS IN GRAYSCALE IMAGES Color marks are only available with color images or grayscale images that have been con
229. mage Types Lossy Compression and Metadata X Warning on JPEG Compression Acquisition plugins Input Output plugins 11 Last updated 2012 10 02 Stacks Virtual Stacks and Hyperstacks Il IMAGE TYPES LOSSY COMPRESSION AND METADATA Two critical aspects to keep in mind when converting images Lossy compression Transcoding an image into a format that uses lossy compression will alter the original data introducing artifacts see X Warning on JPEG Compression This is the case e g for JPEG formats with the exception of some JPEG2000 images that use lossless compression As such these types of data are intended for human interpretation only and are not suitable for quantitative analyses Metadata In ImageJ metadata associated with the image such as scale gray value calibration and user comments is only supported in tiff and zip compressed tiff images In addition selections and Overlays are also saved in the TIFF header cf File gt Save s None of the above is saved in other formats cf Native Formats 8 Stacks Virtual Stacks and Hyperstacks Stacks ImageJ can display multiple spatially or temporally related images in a single window These image sets are called stacks The images that make up a stack are called slices In stacks a pixel which represents 2D image data in a bitmap image becomes a voxel volumetric pixel i e an intensity value on a regular grid in a three dimensional space All the slices in a
230. mage Types and Formats 8 Stacks Virtual Stacks and Hyperstacks 9 Color Images 10 Selections 10 1 Manipulating ROIS 2 sew amp oe 10 2 Composite Selections 10 3 Selections With Sub pixel Coordinates 11 Overlays 12 3D Volumes 13 Settings and Preferences HI Extending ImageJ 14 Macros vil viii 1X 10 12 14 17 18 19 19 19 21 22 23 15 Scripts 24 16 Plugins 24 17 Scripting in Other Languages 26 18 Running ImageJ From the Command Line 26 IV ImageJ User Interface 19 Tools Tk Areas election Tools sorap aut oy A Wee SARS Ade Be wy Bubs Bh es 29 1972 Lime Selection Tools e es bu eee w ee e Dew RES SS ss 31 19 a Ole 2 2535 saties Sate Gs oe A A a ee ee 32 Io Manele TOO aaa ee ee Ss eae oe ce ae ee See aa 33 OOO Go Gan tech aes a A cae aes OEA A Gees Ge ee 33 O VU POM Tools deals a Ey Be he Ge ote aes Bk WG OS ee amp 34 A A AAA O et de Ge Bo a OB we o N E Y 34 19o Lex Tool orse Se ae o da II A ee Oo Ch ws 30 199 laca ASS y ed Gada ce A Ande a HO a St HP ees GAGE oes dea eds OY Ge 36 E crol NOM na oe oy ts e Aa Bk eB AA A 36 LIA Color Picker Tool ame E oe a ia a paca os o A 36 1912 More ROO LS UNC Z rda ects ve Be oe ci HOR ar Se RS Ge ee SSS ot LATA OW asa tia he OG A Ge ce BS PES ORS a Oe KS e 38 PERD a iee Mocs oe oc oe ee dd A a Be es a ees amp bee a 38 LO bor Developer Ment a detal amp Mee 4 6 bots a ee Sore tes Bas 38 POM IOOdE RMS s semea ri
231. mage or selection With 8 bit images results greater than 255 are set to 255 With 16 bit signed images results greater than 65 535 are set to 65 535 29 9 2 Subtract Subtracts a constant from the image or selection With 8 bit and 16 bit images results less than 0 are set to 0 Lg Last updated 2012 10 02 Process gt Math gt XXII INTERPRETING BINARY IMAGES Binary images are thresholded to only two values typically 0 and 1 but often as with ImageJ O and 255 that represent black and white on an 8 bit scale see Image Types and Formats The interpretation of binary images is not universal While some software packages will always perform binary operations on 255 values or 1 or any non zero value ImageJ takes into account the foreground and background colors of the binary image In ImageJ the Black background option in Process gt Binary gt 0 Black Background 255 White Options defines not only how new binary images will be created see XX Creating Binary Masks but also how previously created images are interpreted This means object s will be inferred on a image per image basis As such inverting the LUT see Invert LUT of a binary image without updating the Black background option may lead to unexpected results such as the aberrant thinning operation Process gt Binary gt Skeletonize depicted here This issue can be avoided by imposing adequate preferences at startup as described in Sett
232. med 0 allocate more than the default Note that specifying more than 75 of real RAM could result in virtual RAM being used which may cause ImageJ to become slow and unstable Also note that this dialog cannot be used to set the memory allocation if ImageJ is run from the command line or by double clicking on Elij jar Maximum memory 64 bit OS and a 64 bit version of Java are required to use more than 1700 MB of memory Windows users must be running a 64 bit version of Windows and must install a 64 bit version of Java Mac users must be running OS X 10 5 or later and may need to use the Java Preferences utility in Applications Utilities Java to select a 64 bit version of Java They may also need to switch to the ImageJ64 application Linux users need to be running 64 bit versions of Linux and Java The title of the Memory amp Threads dialog box changes to Memory 64 bit when ImageJ is running on a properly configured 64 bit system Parallel threads for stacks Determines the number of parallel threads used by commands in the Process Filters gt and the Process Math gt submenus when processing stacks The default value is the number of available processors Keep multiple undo buffers If checked the undo buffer will be preserved when switching images Edit Undo z remains restricted to the most recent operation but is available for each opened image as long as the buffer allows it If Keep multiple undo buffers is unchecke
233. mines the number of pixels in the image that are allowed to become saturated Increasing this value will increase contrast This value should be greater than zero to prevent a few outlying pixel from causing the histogram stretch to not work as intended Normalize If checked ImageJ will recalculate the pixel values of the image so the range is equal to the maximum range for the data type or 0 1 0 for float images The contrast stretch performed on the image is similar to the Auto option in the Brightness Contrast C window except that with stacks each slice in the stack is adjusted independently according to the optimal for that slice alone if Use Stack Histogram is unchecked The maximum range is 0 255 for 8 bit images and 0 65535 for 16 bit images With stacks another checkbox Normalize All n Slices is displayed If checked normaliza tion will be applied to all slices in the stack Note that normalization of RGB images is not supported and thus this option will not be available on RGB stacks Equalize Histogram If checked ImageJ will enhance the image using histogram equalization 30 Create a selection and the equalization will be based on the histogram of that selection 109 Last updated 2012 10 02 Process gt Noise gt Uses a modified algorithm that takes the square root of the histogram values Hold to use the standard histogram equalization algorithm The Saturated Pixels and Normalize parameters are ignored
234. mmand from the popup menu and enter the shortcut in the text field A shortcut can be a lower Shortcut or uppercase letter or F1 through F12 Use Pluginse f Yin 5 Cancel OK Utilities gt List Shortcuts to get a list of shortcuts that are already in use 157 Last updated 2012 10 02 Plugins gt Utilities gt 31 2 3 Install Plugin Installs a plugin in a user specified submenu Plug ins with a showAbout method are also automatically added to the Help About Plugins gt submenu Plugin Menu Use the first popup menu to select the plugin and the second to select the submenu it is to installed in The command must be different from any existing ImageJ Filters command Shortcut optional must be a single letter Kend or FI through F12 Argument optional is the string that will passed to the plugin s run method Command Shortcut Argument 31 2 4 Remove Removes commands added to the Shortcuts submenu by Create Shortcuts Also removes commands added by Install Plugin and removes plugins installed in the Plugins menu The menus are not updated until ImageJ is restarted 31 3 Utilities gt 31 3 1 Control Panel U 0 00 Tree amp Show Parent ES File 8 bit gt 3 Edit 16 bit Y Image 32 hi f Elve 8 hc Y Adjust es Brightness Contrast REUE Color This command 47 opens a window containing ImageJ
235. mputer Graphics Vol 21 pp 145 152 1987 is used With closed shapes the command creates a circle with the same area and centroid of the selection 27 12 6 Fit Ellipse Replaces an area selection with the best fit ellipse see ROI manipulations The ellipse will have the same area orientation and centroid as the original selection The same fitting algorithm is used to measure the major and minor axis lengths and angle when Fut Ellipse is selected in Analyze gt Set Measurements SEE ALSO DrawEllipse macro 27 12 7 Interpolate mM gt interpolate Converts the active selection into a sub pixel resolution ROI of floating Interval 0 05 pixel point coordinates spaced interval pixels apart If Smooth is checked traced and freehand selections see Area Selection Tools are first smoothed using a 3 point running average Refer to Selections With Cancel C k gt Sub pixel Coordinates for more details M Smooth SEE ALSO Floating point selections Fit Spline Edit Options gt Profile Plot Options 27 12 8 Convex Hull Replaces a polygon of freehand selection with its convex hull see ROI manipulations determined by the gift wrap algorithm The convex hull can be thought of as a rubber band wrapped tightly around the points that define the selection SEE ALSO Fit Ellipse E ConvexitySolidarity macro Convex Hull Plus plugin 27 12 9 Make Inverse Creates an inverse selection see ROI manipulations
236. n Per Line Smooth A z Cancel OK se Polygon Multiplier Adjusts the number of profiles used to generate the plot Draw Wireframe If checked the outline of each profile will be drawn in black Shade If checked a shaded plot will be generated using the LUT of source image Draw Axis If checked the three axis will be drawn and labelled Source Background is Lighter If checked lighter areas in the source image will represent lower elevations valleys while darker areas in the source image will represent higher elevations peaks Fill Plot Background with Black If checked the plot is drawn with a black background otherwise white will be used One Polygon Per Line If checked all polygons will be drawn Smooth If checked sharp fluctuations will be smoothed Note that some plots can be further improved by adjusting the contrast of the source image or smoothing it SEE ALSO Plot Profile k Interactive 3D Surface Plot plugin it works with all image types and viewing angle perspective scale lighting and smoothing can be interactively adjusted 3D Color Inspector Color Histogram 143 Last updated 2012 10 02 Analyze gt Gels gt XXIV USING SCANNERS IN DENSITOMETRY Electrophoretic gels such as Western blots need frequently to be quantified in order to translate biochemical results into statistical values see Gels gt Independently of the measurement method you should be familiar with the Beer L
237. n and Documentation Portal ImageJ wikipage http imagejdocu tudor lu doku php Video tutorials on the ImageJ Documentation Portal and the Fiji YouTube channel http imagejdocu tudor lu doku php id video start amp s video and http www youtube com user fijichannel New ImageJ users will probably profit from Christine Labno s video tutorial The ImageJ for Microscopy manual http www macbiophotonics ca imagej Several online documents most of them listed at http imagej nih gov ij links html and http imagej nih gov ij docs examples Mailing lists a ImageJ http imagej nih gov 1j list html General user and developer discussion about ImageJ Can be accessed via the Help gt Mailing List command This list is also mirrored at Nabble and Gmane You may find it easier to search and browse the list archives on these mirrors Specially useful are the RSS feeds and the frames and threads view provided by Gmane 6 Last updated 2012 10 02 Getting Help Help on ImageJ b Fiji users http groups google com group fiji users For user discussion specific to Fiji rather than core ImageJ c Fiji devel http groups google com group fiji devel For developer discussion specific to Fiji d ImageJ devel http imagejdev org mailman listinfo imagej devel For communication and coordination of the ImageJDev project e Dedicated mailing lists for ImageJ related project
238. n saving in JPEG or PNG format Name mri s i Format TIFF Start At 0 Digits 1 8 4 mitosis Name Ei Use slice labels AE ARA E hy Fa C Cancel _ Cancel O Name Specifies the leading string that will be common to all numeric filenames Start At Stacks only Specifies the starting number of the sequence Digits 1 8 The number digits of the incremental sequence Filenames are padded with leading zeroes Use slice labels as filenames Stacks only If checked each slice will be saved with its own label the image subtitle displayed above the image see Remove Slice Labels and no numeric sequence will be used With hyperstacks images are saved using Name td zd cd in which d is the incremental number of specified Digits t the frame z the slice and c the channel so e g for the depicted snapshot the first image would be saved as mitosis t001 z001 c001 tif 58 Last updated 2012 10 02 IMPROVED IN IJ 1 46R File gt Save Asp 26 10 8 AVI Exports a stack or hyperstack as an AVI file 12 Note that AVI files larger than 2 GB are not cor rectly written Compression Uncompressed Unco mpressed PNG JPEG Compression JPEG PNG or Uncompressed With IJ 1 44 and later Overlays are embed ded when saving in JPEG or PNG format The default compression is JPEG Frame Rate 5 fps Frame Specifies the frame frequency The proposed value is read from
239. n writing your own macros or to generate simple Session Logs In this case you would start the Recorder and let ImageJ keep track of the performed actions by generating macro code SEE ALSO Editor Extending ImageJ The ImageJ Macro Language Programmer s Reference Guide Fiji s Introduction into Macro Programming 31 2 Shortcuts This submenu contains commands for creating keyboard shortcuts and for installing and removing plugins SEE ALSO Toolbar Shortcuts 31 2 1 List Shortcuts This command generates a table with the ImageJ keyboard short Keyboard Shortcuts 1 h Hot Key command cuts in one column and the commands they call in another Com ASave As JPEG j 7 mands prefixed by refer to shortcuts created with Create Short refer to installed macros listed in Plugins gt Macros and override ImageJ default hot keys Plot Profile a cuts while commands prefixed by Find Commands Measure oa Note that unless Require control key for shortcuts in Edit gt Options gt en ei Misc 1s checked you do not have to hold down to use a Revert keyboard shortcut E g to open an image File Open o simply press O SEE ALSO Keyboard Shortcuts Create Shortcuts 31 2 2 Create Shortcuts Assigns a keyboard shortcut to an ImageJ menu com AON Create Shortcut mand and lists the shortcut in the Shortcuts submenu Command Add reference frame HA Select the co
240. nd is the Y coordinate of the selection and the third is the X coordinate Click on a label to restore the associated selection to the current image With stacks the selection is restored to the slice it came from Hold down while clicking Add to Add and Draw Hold down while clicking Add to Add and Rename Install the ROIManagerMacros macro set and you will be able to add a selection by pressing 1 add and rename by pressing E add and draw by pressing and add and advance to the next slice by pressing 4 J Update Replaces the selected ROI on the list with the current selection updating the z t position of the ROI in Stacks and Hyperstacks Delete Deletes the selected ROIs from the list Deletes all ROIs if none is selected Rename Renames the selected ROI The chosen string will be used as label Labels checkbox if Use ROI names as labels is checked in the More gt gt Options dialog The selected ROI can also be renamed using the Properties button Note that while it is not possible to rename multiple ROIs simultaneous you can use ROI Manager Tools to rename multiple ROIs Measure Measures the selected ROIs or if none is selected all ROIs on the list Use Analyze gt Set Measurements to specify the measuring parameters Deselect Deselects any selected ROIs on the list As mentioned in XXV Selecting ROIs in the ROI Manager when items are deselected subsequent ROI Manager comm
241. nds related to Stacks and Hyperstacks otherwise accessed through the hierarchy of Image gt Stacks gt Image Hyperstacks gt and File gt Open Samples gt submenus The list makes a particular emphasis on commands that have no keyboard shortcuts assigned SEE ALSO Plugins Shortcuts gt LUT Menu Stacks Menu Common Commands Menu Tool Developer Menu 20 Custom Tools Customized tools are add ons macros and plugins that allow custom interactions with the ImageJ toolbar and or the image canvas They are installed on the right side of the Toolbar between the Color Picker Tool and the More Tools Menu At startup the default set of tools is loaded from ElImageJ macros StartupMacros txt Later on tools can be appended or replaced using the More Tools Menu menu As mentioned custom tool configurations are saved in the preferences file and thus remembered across restarts see Settings and Preferences It is worth it to mention some differences between the installation of single tools and toolsets Single Tools Single tools are appended to the first available toolbar slot or installed in the last slot if no free slots are available Tools can be macros E txt and E ijm files or plugins E class and E jar files and are listed on the More Tools Menu menu if placed in the GImageJ plugins Tools directory In addition to the macro tools distributed with ImageJ and saved in GImageJ macros tools a vast repertoire of tools is available
242. nes Vertical Profile If checked row average plots of rectangular M Sub pixel resolution areas or line selections wider than 1 pixel will be gen erated instead of the default column average plots Note id encore that evoking Plot Profile k with will generate vertical profiles List values If checked the list of values will be automatically opened If Auto close is also checked the plot is closed and only the list of values remains open Interpolate line profiles If checked Analyzer Plot Profile k will use bilinear interpolation to retrieve intensity values along non straight line selections Draw grid lines If checked gray grid lines will be drawn in the plot Sub pizel resolution If checked line selections created on zoomed images will use floating point coordinates resulting in smoother curves see Selections With Sub pixel Coordinates and Edit Selection gt Interpolate Help Opens http imagej nih gov 1j docs menus edit html3 plot options 69 Last updated 2012 10 02 Edit gt Options gt 27 13 5 Rounded Rect Tool See Rounded Rectangular Selection Tool 27 13 6 Arrow Tool See Arrow Tool 27 13 7 Point Tool See Point Tool 27 13 8 Wand Tool See Wand Tool 27 13 9 Colors Displays a dialog box that allows you to set Foreground Back Foreground black e ground and Selection color As mentioned earlier the selection color is highlighted in the Point Tool and
243. ng the macro beware of any statements that may interfere with the normal operation of the batch processor such as Close or Open calls Test Tests the macro on the first image of the Input folder the processed image will be displayed Open Imports previously written macros Save Saves the assembled macro SEE ALSO Plugins Macros Record Batch Virtual Stack 29 12 4 Virtual Stack IN IJ 1 46R This command that shares the same interface of Batch gt Macro cf E BatchProcesser java allows virtual stack manipulations E g Cropping a virtual stack can be performed by executing the following steps 1 Open a virtual stack 2 Run Processp Batch gt Virtual Stack 3 Select an Output folder and Output format 4 Select Crop from the Add Macro Code drop down menu 5 Edit the macro code as needed and press the Test button to verify the macro 6 Click Process to create the cropped virtual stack Note that cropped images are not loaded into memory but are saved to disk as they are cropped see Virtual Stacks 29 13 Image Calculator Performs arithmetic and logical operations between two images selected from popup menus described in the Image operations table Image1 or both Imagel and Image2 can be stacks If both are stacks they must have the same number of slices Imagel and Image2 do not have to be the same data type or the same size With 32 bit float images pixels resul
244. nlarge to Fit Result and the image will be enlarged as needed to avoid clipping 28 12 7 Translate Translate X Offset pixels 15 0 Y Offset pixels 15 0 Interpolation None r M Preview a Cancel OK Translates moves the image in the x and y directions by a specified number of pixels With stacks you can translate either the current image or all the images in the stack Two resampling methods are possible Bilinear and Bicubic interpolation cf Imageb Size Check Preview to see how the translation will affect the image The background at the edges of the image will be set to 0 SEE ALSO Align Slice and Align RGB planes plugins NEw IN i 28 12 8 Bin O Image Shrink X shrink factor 2 Y shrink factor 2 Z shrink factor 4 Bin Method Average rH Cancel C ok Reduces the size of an image by binning groups of pixels of user specified size X Y Z shrink factor 27 The resulting pixel can be calculated as Average Median Maximum or Minimum Undo support is restricted to 2D images non stacks Z binning produces equivalent results to Image Stacks gt Tools gt Grouped Z Project However there are two main differences between the two commands While Bin replaces the original image Grouped Z Project creates a new substack and while Z shrink factor takes any value Group size must divide evenly into the stack size SEE ALSO Image Adjust gt Size Image
245. nly display 256 shades of gray Therefore the data are mapped to 8 bit by windowing The window defines the range of gray values that are displayed values below the window are made black while values above the window are white The window is defined by minimum and maximum values that can be modified using Imagep Adjust gt Brightness Contrast C It may happen that the initial windowing performed by ImageJ on these high bit depth or HDR images is suboptimal Please note that windowing does not affect image data cf the HDRexplorerTool Auto threshold methods GG Threshold un Highlighted range gt moves a fixed width Huang o O thresholding window 0 IsoData ate O i E 196 Min Max values Li o MaxEntropy Intermodes Red e Display mode Mean _ E Y Red MinError l _ Dark background y Stack histogram B amp W Minimum Auto Apply Reset Set Over Under Moments o ia Dinara Otsu Manual input Percentile gt Set Threshold Levels RenyiEntropy Are objects inthe Compute the whole Lower Threshold Level 0 Shanbhag image lighter than stack or treat single Upper Threshold Level 196 Triangle the background slices independently Yen Cancel Image gt Adjust gt Threshold T ImageJ 1 45m selected features Use Analyze gt Analyze Particles to measure features individually Use the Wand Tool to outline a single feature Upper slider Adjusts the minimum threshold val
246. ns ImageJ macros Macro functions and code snippets are typed in monospaced font e g resetMinAndMax Scripts and macros are numbered with arabic numerals included in parentheses e g 2 Replace Red with Magenta ijm Using Image Color gt Channels on page 17 and typeset with the same syntax markup provided by the Fiji Script Editor The full list of macro listings is available on page ix Selected highlights of version 1 46r are listed on page vii and flagged with colored marginal notes These should be interpreted as NEW IN IJ 146R A new feature implemented in ImageJ 1 46r A routine that has been improved since previous versions Typically a faster er Gal ae or more precise algorithm a command with better usability or a task that has been extended to more image types A pre existing command that has been renamed or moved to a different menu IN IJ 1 46R location in ImageJ 1 46r Part I Getting Started This part provides basic information on ImageJ installation troubleshooting and update strategies It discusses Fiji and ImageJ2 as well as third party software related to ImageJ Being impossible to document all the capabilities of ImageJ without exploring technical aspects of image processing external resources allowing willing readers to know more about digital signal processing are also provided 1 Introduction ImageJ is a public domain Java image processing and analysis program inspired by NIH Image for the Mac
247. nt e g 1 100 2 or a list e g 7 9 25 27 ranging 25 Slices 1 25 3 Extracted slices will be removed from the source M Delete slices from original stack stack if Deleted slices from original stack is checked Cancel CK Currently it does not work with hyperstacks and ps takes one of three types of input 1 A range of images E g 2 14 lextract slices 2 through 14 2 A range of images with increment which can be used to de interleave slices E g 2 14 2 extract slices 2 and 14 and every second slice im between 3 A list of images E g 2 4 7 9 14 SEE ALSO Imageb Duplicate D 28 6 15 8 Grouped Z Project IN IJ 1 46R Creates a substack of Stack size Group size slices with each slice being the result of a Z Projection performed over the range of Group size 94 Last updated 2012 10 02 Image gt Stacks gt Group size must divide evenly into the stack size This limitation can be overcome by the Image Projection method Average pet is Y Average Intensity ee Transftormb Bin that can also create grouped Group size 27 Max Intensity Min Intensity Z projections Valid factors 1 3 9 27 Sum Slices o Standard Deviation The first ten divisors of Stack size are suggested Median at the bottom of the dialog SEE ALSO Imager Stacks gt Z Project Image gt Adjust gt Size Image Adjust gt Scale E 28 6 15 9 Remove Slice Labels Removes slice labels from sta
248. nt computational gaint You can read more about the HSB color model here In ImageJ conversions between image types are performed using the Image Type gt submenu Segmentation on the HSB RGB CIE Lab and YUV color spaces can be performed by the Image gt Adjust gt Color Threshold command 20 Segregation of color components specially useful for quantification of histochemical staining is also possible using Gabriel Landini s Colour Deconvolution plugin In addition several other plugins related to color processing can be obtained from the ImageJ website Conveying Color Information People see color with significant variations Indeed the popular phrase One picture is worth ten thousand words may not apply to certain color images specially those that do not follow the basic principles of Color Universal Design Citing Masataka Okabe and Kei Ito Colorblind people can recognize a wide ranges of colors But certain ranges of colors are hard to distinguish The frequency of colorblindness is fairly high One in 12 Caucasian 8 one in 20 Asian 5 and one in 25 African 4 males are so called red green colorblind There are always colorblind people among the audience and readers There should be more than TEN colorblind in a room with 250 people assuming 50 male and 50 female There is a good chance that the paper you submit may go to colorblind reviewers Supposing that your paper will be reviewed
249. nvert to 8 bit Before converting disable Scale When Converting in Edit gt Options Conversions to prevent the image from being scaled to 0 255 SEE ALSO Text Images Pixel Inspector Image Transform gt Image to Results Results to Image Save Asp Text Image ElOpenTextImagesAsStack macro 26 6 5 Text File Opens a text file Note that text files can also be opened using File gt Open o or drag and drop 26 6 6 URL Downloads and displays known formats to Im ageJ specified by a URL Other URLs ending with or html are opened in the user s default browser The Input URL is saved in the ImageJ preferences file and retrieved across IJ restarts Enter a URL Enter URL of an image macro or web page URL http rsb info nih gov ij images clown gif It is also possible to open zip archives using a URL that contain multiple DICOM images Some example URLs are http imagej nih gov ij images ct dcm file Macintosh HD images Nanoprobes tif file D images neuron tif http imagej nih gov ij opens the ImageJ website 26 6 7 Results Opens an ImageJ table or any tab or comma delimited text file see Results Table Note that E csv and El x1s files can also be opened by drag and drop 54 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IJ 1 46R File gt Close w 26 6 8 Stack From List
250. nverted LUTs pixels with a value of zero are white and pixels with a value 255 are black Unlike the Edit Invert I command pixels values are not altered only the way the image is displayed on the screen SEE ALSO Image Color gt Show LUT Edit LUT 28 15 2 Apply LUT Applies the current lookup table function to each pixel in the image or selection and restores the default identity function This modifies the gray values so that when the image is viewed using the default grayscale lookup table it will look the same as it did before This command is equivalent to clicking on Apply in Image Adjust gt Brightness Contrast C For thresholded images it is equivalent to clicking on Apply in Image Adjust gt Threshold T SEE ALSO Image Coloro Show LUT Edit LUT 106 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Process gt 29 Process This menu lists all commands related to image processing including point operations filters and arithmetic operations between multiple images 11 The File gt Open Samples gt Blobs 25K B image will be used in most of the illustrations of this section LOOO blobsgif AO O A blobs gif Smooth 3x O O O blobs gif Sharpen 1x O O O blobs gif Find Edges 1x O O A blobs gif E Contrast 10 _ x pixels 6 bit inverting j 64 256x254 pixels 8 bit inverting LUT 64 x pixels bit inverting i x pixels 6 bit inverting i x pixels bit inverting 64 l
251. o Threshold false setOption BlackBackground true run Colors foreground white background black this line gt could be substituted by setBackgroundColor 0 0 0 setForegroundColor 255 255 255 run Profate Plot Options width 350 height 200 draw run Brightness7 Contrast Ji The setOption macro function can be used to set this and several other ImageJ options Calling this function from the AutoRun macro in the E StartupMacros txt file ensures preferences are set each time ImageJ starts The macro 3 Ensuring Specific Settings at Launch exemplifies this approach ensuring that the following settings are enforced at startup 1 TIFF tag values are displayed by ImageJ Debug Mode in Edit gt Options Misc 2 Bicubic interpolation is preferred over bilinear e g Edit gt Selection gt Straighten 3 The name of the measured image name is recorded in the first column of the Results Table Display Label in Analyze gt Set Measurements 4 Measurements are not restricted to thresholded pixels Limit to Threshold in Analyze gt Set Measurements 5 Binary images are processed assuming white objects on a black background Black back ground in Process Binary Options see XXII Interpreting Binary Images 6 Background color is black and foreground color is white Edit Options Colors 7 ImageJ plots contain grid lines and are always 350 x 200 pix
252. of complex ImageJ and Java APIs No support for batch mode Cannot create Custom Tools and toolbar menus Not compatible with Function Finder F and CodeBar No debugger CodeBar is a convenient ActionBar that retrieves snippets and common tasks frequently used in macro writing ActionBars provide one or many easy to use button bar s that extend ImageJ s graphical user interface You can read more about the ActionBar plugin at the ImageJ Documentation Portal Plugins are implemented as Java classes which means that you can use all features of the Java language access the full ImageJ API and use all standard and third party Java APIs in a plugin This opens a wide range of possibilities of what can be done in a plugin The most common uses of plugins are filters performing some analysis or processing on an image or image stack and I O plugins for reading writing not natively supported formats from to file or other devices But as you can see when looking at the plugins listed on the ImageJ plugins page there are many other things you can do with plugins such as rendering graphics or creating extensions of the ImageJ graphical user interface Plugins in the GImageJ plugins folder are listed at the bottom of the Plugins gt menu see VII Organizing Commands in the Menu Bar Only E class and El jar files in the plugins folder with at least one underscore in their name will be installed Note that with IJ 1
253. omain performing conjugate multiplication or Image2 FFT Filter 05 06 tif i division then converting the result back to the space do main These three operations in the frequency domain are Result Result equivalent to correlation convolution and deconvolution in M Do Inverse Transform the Space domain Cancel ox Refer to the DeconvolutionDemo and MotionBlurRemoval macros for examples 29 10 8 Swap Quadrants This command transforms between the user friendly display of Fourier transforms with the lowest frequencies at the center and the native form with the lowest frequencies at the four corners 123 Last updated 2012 10 02 Process gt Filters gt FFT Original ES Swap Quadrants swaps quadrants I with III and II with IV counter clockwise starting from Northeast so that points near the center are moved towards the edge and vice versa Another way to see this command is to imagine that the image is periodically repeated and the origin is shifted by width 2 in x and by height 2 in y Non FFT direction Original Swapped IV For Fourier transforms Swap Quadrants affects only the image o Pe Pe displayed not the actual FHT data Therefore editing an image m iv ro with swapped quadrants for filtering or masking may lead to undesired results 29 11 Filters This submenu contains miscellaneous filters 36 including those installed by the Plugins Utilities gt Install
254. ommand Note that the copied text will substitute any other information present in the file header and will only be available in images saved as TIFF see II Image Types Lossy Compression and Metadata Fonte This menu contains commands to adjust font size and type Macros This menu contains commands that allow you to run install or evaluate macro code Run Macro r Ctrl R Runs the macro or the selected line s of code Evaluate Line y Ctrl Y Runs the line of code that contains the insertion point Abort Macro Exits the macro 44 Last updated 2012 10 02 Editor Install Macros i Ctrl _ Adds the macro s contained in the editor to Plugins gt Macros gt submenu Plugins gt Help gt Macro Functions command Macro Functions M M Opens the Macro Functions reference page the indispensable guide to the built in functions that can be called from the ImageJ macro language alias for Help gt Macro Functions Function Finder F Ctr Shift F 3 Retrieves macro functions in the same way Find Commands I retrieves commands Functions are read from the E functions html file stored in the macros folder a local copy of http imagej nih gov ij developer macro functions html This file is deleted by Help Update ImageJ command every time ImageJ is updated to a release version i e not a daily build see Installing and Maintaining ImageJ forcing Function Finder to downlo
255. on Projection Type Maximum Intensity projection MIP creates an output image each of whose pixels con M All Time Frames tains the maximum value over all images in the stack at the particular pixel location Sum Slices projection creates a real image that is the sum of the slices in the stack Standard Deviation projection creates a real image containing the standard deviation of the slices Median projection outputs an image wherein each pixel stores median intensity over all images in stack at corresponding pixel location SEE ALSO Grouped Z Project 3D Project Plot Z axis Profile 28 6 12 3D Project 3D Projection This command creates a sequence of projections of a a rotating volume stack or hyperstack onto a plane Projection Method Brightest Point 14 using nearest point surface brightest point or mean Axis of Rotation Y Axis value projection or a weighted combination of nearest Slice Spacing pixels 1 00 point projection with either of the other two methods Spel cic Pence partial opacity The user may choose to rotate the Total Rotation 0 359 degrees 360 volume about any of the three orthogonal axes x y o or z make portions of the volume transparent using thresholding or add a greater degree of visual realism by employing depth cues Lower Transparency Bound 1 Upper Transparency Bound 255 A 0 Each frame in the animation
256. on History Date September October 2012 July 2012 June 2012 September 2011 January 2011 May 2010 April 2010 Acknowledgments We are extremely grateful to Alex Fernandez for his wonderful eLyXer Alex Gorbatchev for SyntaxHigh lighter Johannes Schindelin for assistance with the Git repository Norbert Vischer for critical corrections and Michael Schmid and Barry DeZonia for thorough revisions We are also thankful to all of those who submitted criticisms suggestions and encouragement to update this edition But above all our thanks Notes Reviewed by Michael Schmid Updated for v 1 46r Reviewed by Barry DeZonia Updated for v 1 46p Second major revision with new sections on overlays 3D volumes sub pixel selections curve fitting and interface customizations Improvements in layout and browsability Updated for v 1 45 Deeply revised edition with several new sections in Parts I IV Available as printable booklets Redesigned HTML version Updated for v 1 44 New sections on advanced ImageJ usage Fiji scripting command line usage and interoperability with other software packages First HTML version First edition v 1 43 go to the extraordinary ImageJ community for its dedication to the project
257. on Moffat Polina Golland and David M Sabatini Cellprofiler image analysis software for identifying and quantifying cell phenotypes Genome Biol 7 10 R100 Dec 2006 doi 10 1186 gb 2006 7 10 r100 Anne E Carpenter Lee Kamentsky and Kevin W Eliceiri A call for bioimaging software usability Nature Methods 9 7 666 70 Jun 2012 doi 10 1038 nmeth 2073 R Cathelin F Lopez and Ch Klopp AGScan a pluggable microarray image quantification software based on the ImageJ library Bioinformatics 23 2 247 248 Jan 2007 doi 10 1093 bioinformatics bt1564 Gary Chinga Per Olav Johnsen Robert Dougherty Elisabeth Lunden Berli and Joachim Walter Quan tification of the 3D microstructure of SC surfaces Journal of microscopy 227 Pt 3 254 65 Sep 2007 doi 10 1111 j 1365 2818 2007 01809 x B Choi C Nelson Y Tsunashima and P Balter Open source ImageJ based web accessible tool for treatment plan evaluation Med Phys 34 6 2477 2477 Jan 2007 doi 10 1118 1 2760991 Tony J Collins ImageJ for microscopy BioTechniques 43 1 Suppl 25 30 Jul 2007 URL www biotechniques com article 000112517 Clayton M Costa and Suann Yang Counting pollen grains using readily available free image processing and analysis software Ann Bot 104 5 1005 10 Oct 2009 doi 10 1093 aob mcp186 Evan C Crawford and James K Mortensen An ImageJ plugin for the rapid morphological characterization of separated particles and an initial application to placer
258. on level at which the images are copied and to select the layout of the resulting grid Label Slices If checked montage panels are labelled with slice labels Slice labels up to 60 characters correspond to the image subtitle the line of information above the image These labels are part of the stack metadata typically created by File gt Import gt Image Sequence or Stacks gt Images To Stack If no slice metadata exists the setMetadata Label string macro function can be used to customize slice labels images are labelled with slice numbers Note that the Stacksb Label command can be used to draw labels in stack slices Labels are typeset in sans serif typeface Use Foreground Color If checked borders and labels are drawn in the foreground color and blank areas of the panel are filled with the background color SEE ALSO Stacks gt Toolst gt Montage to Stack Stacks gt Tools gt Remove Slice Labels RC Montage plugin Magic Montage a macro toolset to reorder and manipulate images in the montage a video tutorial can be found here 88 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Image gt 28 6 9 Reslice IN IJ 1 46R Output spacing um 3 00 Stacks gt Reconstructs one or more orthogonal slices through the image volume represented by the current stack or hyper Start at Tp stack 21 O Flip vertically The estimated size of the output stack and the amount D Rotate 90 degrees of
259. ons checkbox will make Overlays visible only when browsing their respective slice or frame If unchecked overlays will be displayed throughout the stack see also ROI Manager More gt 0Options SEE ALSO Labels Remove Slice Info Analyze gt Tools gt ROI Manager Settings and Prefer ences 28 15 Lookup Tables This submenu contains a selection of color lookup tables that can be applied to grayscale images to produce Pseudocolor Images In addition it lists all the lookup tables installed in the GImageJ luts directory More than 100 additional lookup tables are available from the ImageJ website as individual files or in bulk as a ElZIP archive As explained earlier it is not possible to organize LUTs into subfolders However the most frequently used lookup tables can be renamed with a numeric prefix e g El01 glasbey lut 02 Termal lut etc so that they are listed earlier in the menu This submenu can also be accessed from the Toolbar by loading the LUT Menu When loading a lookup table is loaded and no image is open a 25632 ramp image is created to display the color table SEE ALSO Channels widget E Show_A11_LUTs a macro that creates a graphical palette of all the installed lookup tables 105 Last updated 2012 10 02 Image gt Lookup Tables gt 28 15 1 Invert LUT Inverts the current lookup table For 8 bit images the value v of each entry in the table is replaced by 255 v With i
260. ool allows to select the Display mode of composite images In addition several commands hosted in the Imageb Color gt submenu can easily be accessed through the Mores drop down menu The same drop down menu also provides a convenient list of primary colors additive red green and blue subtractive cyan magenta yellow that can be used to pseudocolor Color Composite Images A 7 Channels AAA flybrain tif AAA flybrain tif AB flybrain tif E Zz o pixels bit L 6x2 gt b pixels bit L FF b 6 pixels bit 11 Composite 2 AP Channels O OLO Channels A Channels Y Composite EE Piar a M Channel 1 O Channel 1 O Channel 1 Y M Channel 2 M Channel 2 a M Channel 2 Channel 4 A More More F More Convert to RGB y Split Channels gt W Merge Channels Edit LUT Red Green Blue C lt gt ge 4 lt gt E lt gt C lt gt g lt gt Cyan E z gt Q lt z4 gt O 4 z4 gt Mm 4 Channel manipulations in Color Composite Images using the Image gt Color gt Channels Tool Z tool 28 8 Crop X IN IJ 1 46R Crops the image or stack based on the current rectangular selection 28 9 Duplicate D IN IJ 1 46R Duplicate Creates a new window containing a copy of the active image or rectangular selection For stacks and hyperstacks it is possible to Duplicate hyperstack specify
261. ore selections are drawn in tones of gray While Analyze Plot Profile k requires a line or rectangular selection Multi Plot accepts all type of selections by first running Edito Selection gt Area to Line which converts area and freehand ROIs to line selections Sort Sorts the list in alphanumeric order Specify Alias for the Edit gt Selection gt Specify prompt that allows the creation of area ROIs at specific locations Remove Slice Info Removes the information in the ROI names that associates them with particular stack slices see ROI Manager illustration Help Opens http imagej nih gov ij docs menus analyze html manager Labels Alias for Image Overlay gt Labels which allow the customization of selection labels when Show All is active List Prints a table detailing the properties of the ROIs stored in the Manager including Index cf roiManager index macro function Name XY coordinates of ROI center pixels and stroke Color Options Displays a dialog box depicted in ROI Manager that allows you to set several ROI Manager settings Associate Show All ROIs with slices If checked Show All will only reveal ROIs when browsing their respective slice If unchecked ROIs are shown in all stack slices Restore ROIs centered If checked ROIs opened by More gt Open are centered on the image canvas This option avoids loaded ROIs to be displayed out of boundaries when the image has be
262. ound Sets non thresholded pixels in 32 bit float images to the NaN Not a Number value For float images the Apply option in Image Adjust gt Threshold T runs this command Pixels with a value of Float NaN 0f 0f Float POSITIVE INFINITY 1f 0f or Float NEGA TIVE_ INFINITY 1f 0f are ignored when making measurements on 32 bit float images 29 9 18 Abs Generates the absolute value of the active image or selection Works only with 32 bit float or signed 16 bit image images 29 9 19 Macro This command performs image arithmetic using an expression specified by the user 32 It can be used to create fully synthetic Code v v 50 sin d 10 v pixel value x y amp z pixel coordinates w image width h image height a angle d distance from center images or to perform precise pixel manip E Preview ulations on existing images The Math er TS mk MacroDemo macro demonstrates the usage Help Cancel OK e of this command SEE ALSO Expression plugin 29 10 FFT gt The commandes in this submenu support frequency domain display editing and processing They are based on an implementation of the 2D Fast Hartley Transform FHT contributed by Arlo Reeves the author of the ImageFFT spinoff of NIH Imaget 3D FHT can be performed using Bob Dougherty s 3D Fast Hartley Transform plugin The frequency domain image is stored as 32 bit float FHT attached to the 8 bit image that displays the power spectrum
263. over the histogram window FY Histogram of Cells Histogram Bins 256 With RGB images the default histogram is calculated by converting each pixel to grayscale using the formula gray red green blue 3 or gray 0 299 x red 0 587 x green 0 114 x blue if Weighted RGB Conversions is checked in Edit gt Options Conversions However single channel RGB histograms can be obtained by repeti tively clicking on the RGB button C Histogram Use the List or Copy buttons to save the histogram data Click on Log to display a log scaled version of the histogram overlaid in gray M Use min max or x Min 0 00 X Max 255 00 0 Count 64000 Min O Mean 76 448 Max 252 StdDev 61 524 Mode 5 282 Y Max Auto List Copy Log Live gt 4 Ctist Ccopy Ctoa Clive Comen Bins 256 Mi de With 16 bit images the range of gray values between the Min and X Min 0 00 Maz values is divided into 256 bins With 32 bit images the number X Max 255 00 of bins is specified in the depicted dialog box With any image type an options dialog can be called with or by holding while Y Max Auto clicking on Analyzep Histogram h Cancel ok Bins Specifies the number of bins 141 Last updated 2012 10 02 Analyze gt Plot Profile k Use min max If checked the X axis range is determined by the minimum and maximum values in the image or selection If unchecked X Min and
264. ow basic netiquette namely a Use descriptive subject lines Re Problem with Image gt Set Scale command is much more effective than a general Re Problem b Stay on topic Do not post off topic messages unrelated to the message thread 8 c Be careful when sending attachments Refrain from attaching large files Use e g a file hosting service instead d Edit replies You should include only the minimum content that is necessary to provide a logical flow from the question to the answer i e quote only as much as absolutely necessary and relevant 7 Last updated 2012 10 02 Part Il Working with ImageJ This part introduces some basic aspects of ImageJ so that you can use the software more efficiently It also introduces some important terms and concepts used throughout this guide You may skip it if you already use the program efficiently and are familiar with terms such as Virtual Stacks Hyperstacks Pseudocolor Images Color Composite Images or Composite Selections 4 Using Keyboard Shortcuts You ll learn more and more shortcut keys as you use ImageJ because almost all shortcuts are listed throughout ImageJ menus Similarly in this guide each command has its shortcut key listed on its name flanked by square brackets Please note that the notation for these key bindings is case sensitive i e Shift modifiers are not explicitly mentioned a capital A means Shift A and assumes that Require control ke
265. own images Scale Factor Specifies if images should be resized see Imager Scale E Runs a macro over a specified folder IMPROVED 29 12 3 Macro IN IJ 1 46R Batch Process The last used macro is stored in the Input Desktop Source ImageJ macros batchmacro ijm file en ee oem and remembered across restarts Output Desktop Destination a Output Format RIAN bd Input Selects the source folder contain ing the images to be processed Add Macro Code Scale Y Select from list eL Add Border Output Selects the destination folder w getWidth scale h getHeight scalg Convert to RGB where the processed images will be run Size width w height h inter Crop a A E a E stored Note that original files will Invert not be saved if this field is left Label empty Timestamp Max Dimension TEREE Output Format Specifies the output Resize format that can be set to TIFF 8 e cal ER bit TIFF JPEG GIF PNG PGM a e tte PO BMP FITS Text Image ZIP or Cancer 71 7 Raw cf Image Types and Formats and File gt Save As gt submenu 127 Last updated 2012 10 02 Process gt Image Calculator Add Macro Code This drop down menu contains macro snippets that can be combined to create the processing macro Other statements can be pasted from the macro recorder or ImageJ s editor while the dialog box is opened 38 Previously written macros can be imported using Open When editi
266. oxel depth temporal frame interval and luminance brightness information can be set globally i e can be applied to all images opened during the current session instead of just the active image The Global flag can be set in three dialog prompts 1 Image gt Properties P pixel width height voxel depth frame interval 2 Analyzeb Set Scale pixel width and height 3 Analyzep Calibrate pixel intensity Once Global calibration is set a G is displayed in all image titles until ImageJ is closed A warning message is displayed when a calibrated image with conflicting calibration is opened and the Global option is enabled mitosis tif The calibration of this image conflicts with the current global calibration M Disable Global Calibration O Disable these Messages Choose Disable Global Calibration to stop using global set Cancel OK tings or Disable these Messages to keep respecting global settings ignoring the calibration of the newly open image range of the image 41 The total pixel Count is also calculated and displayed as well as the Mean standard deviation StdDev minimum Min maximum Maz and modal Mode gray value Click on Live to monitor the histogram while browsing stacks or while moving a ROI Value Count pairs i e grayscale value cor responding to the X axis cursor position the number of pixels that have that intensity are displayed on the bottom right while mousing
267. p Adjust gt Window Level or B amp C Imager Adjust gt Brightness Contrast C window and the display range will be set to the minimum and maximum pixel values As an example the File Open Samples CT 420K 16 bit DICOM image has a Window Center of 50 and Window Width of 500 so the display range is set to 200 to 300 center width 2 to center width 2 Click Reset and the display range is set to 719 to 1402 Press Analyze gt Histogram h and you will see that the minimum pixel value in the image is 719 and the maximum is 1402 To display the DICOM tags press Imager Show Info i Press R File gt Revert r to revert to the initial display range SEE ALSO Enhance Contrast Color Balance XIV Applying Auto Brightness Contrast to Entire Stacks XVI Brightness Contrast of High Bit Depth Images XV Display Range of DICOM Images 28 2 3 Color Balance This panel makes adjustments to the brightness and contrast of a single color of a standard RGB image 8 bit per color channel For multi channels Stacks and Hyperstacks Color Com posite Images it adjusts each of the color channels in dependently Use the drop down menu to specify which color channel will be adjusted the histogram is drawn for the selected channel AA Color L 36 47 m E PEE Minimum Maximum and Minimum sliders Auto Set and Apply Y Red A Cree ELLE Channel 2 All 065 gt wor
268. pdated 2012 10 02 Process gt Binary gt 1984 in which a lookup table indexes all the 256 possible 3x3 neighborhood configurations for each foreground pixel The algorithm calculates the index number for each object pixel and uses the lookup table to decide if the pixel is eliminable This process is repeated until no pixel can be eliminated When debugging is enabled in Edit gt Options Misc Skeletonize creates an animation docu menting the iterations of the thinning algorithm SEE ALSO AnalyzeSkeleton plugin BinaryProcessor source code XXI Skeletonize vs Skeletonize 3D Skeletonize3D is a ImageJ plugin written by Ignacio Arganda Carreras 35 that offers several advantages over Process Binary gt Skeletonize the legacy skeletonization algorithm of ImageJ Skeletonize works only with binary 2D images Skeletonize3D works with 8 bit 2D images and stacks expecting the image to be binary If not Skeletonize3D considers all pixel values above O to be white 255 While Skeletonize relies on Black background value in Binary Options see XXII In terpreting Binary Images the output of Skeletonize3D always has a value of 255 at the skeleton and O at background pixels independently of the Black background option In Fiji Skeletonize 3D is already pre installed as Plugins gt Skeleton gt Skeletonize 2D 3D In ImageJ it can be downloaded and installed from the Skeletonize3D homepage Maximum projec
269. pixel wide straight lines 66 New command Edit gt Selection gt Image to Selection 2 a a 2 ee 67 New command Edit gt Optionsp gt Reset 2 2 2 a a 74 Image Color Merge Channels extended to 7 channels 83 ColorChoosers display hex color values a a 85 Improved Image Stacks gt Tools gt Concatenate o o o a a e 93 New command Imagep Transforme Bin aa a a a 99 New commands Image gt Transform gt Image to Results and Results to Image 100 New command Imagep Overlay gt Labels 2 n n aa aa e 104 New command Image Overlay gt Overlay Options 2 0 a a a 105 New command Edit gt Selection gt Gaussian Blur 3D eee 125 Live histograms s e s e e eas se ia dap Oe EEE AA a 140 Live profile plots lt lt ocios se sosa reas as os 142 Much improved Curve Fitter 22 i eit tet essere ee eee yee be ee od He 148 The ROI Manager now uses an overlay to display selections when in Show All mode 151 New command ROI Manager s Print List 1 1 o o 152 New command Analyze gt Tools gt Synchronize Windows a aoa a 154 New commands Plugins Newp gt Macro Plugin Tool oaa aaa 2 020000 161 Please note that this is not an extensive list Detailed release notes for version 1 46r are available on the ImageJ news web site http imagej nih gov ij notes html vii Notewor
270. pth WixHixD and We2xH2xD2 to create a new W W2xmax H H2 xmax D D2 stack E g a 256x256 40 and a 256x256x30 stack would be combined to create one 51225640 stack If Combine vertically is enabled creates a new max W1 W2 X H Hg xmax Dj D2 stack Unused areas in the combined stack are filled with background color cf Color Picker K SEE ALSO Concatenate 28 6 15 2 Concatenate Concatenator O All open windows Image 1 Stack tif ea Image4 Stack 4 tif b Title Concatenated Stacks wi Keep original images wi Open as 4D image Cancel ok 28 6 15 3 Reduce 055 Reduce Size Reduction Factor 2 M Reduce in Z Dimension Cancel Concatenates multiple images or stacks Images with mismatch ing type see Image Types and Formats and dimensions are omitted 24 Stacks with the same number of slices can be con catenated as a 4D Hyperstacks if Open as 4D image is checked In this case chosen stacks will be appended as time points SEE ALSO Combine Reduces the size of stacks and hyperstacks by the specified Reduction Factor E g For a 30 slices stack and a Reduction Factor of 2 the reduced stack will be be composed of 15 slices with every second slice being removed Virtual stacks hyperstacks are supported With Hyperstacks the default reduction is performed in the T Dimension but a choice is available to Reduce in Z Dimension instead SEE ALSO Hyperst
271. pts and plugins It also has a command that opens a text window of a specified size and a command that opens a table that macros can write to The editor windows opened by Plugin Plugin Filter and Plugin Frame contain prototype Java code for the three types of plugins supported by ImageJ SEE ALSO Editor Extending ImageJ 31 4 1 Macro Opens a blank editor window with the title Macro txt SEE ALSO Macros Editor Text Window File New Text Window N 31 4 2 Macro Tool Opens ElMacro_Tool txt an example macro tool that creates circular selections SEE ALSO Macro Tools Documentation 31 4 3 JavaScript Opens a blank editor window with the title Script js SEE ALSO Scripts Editor 31 4 4 Plugin Opens an editor window containing a prototype plugin that implements the PlugIn interface Plugins of this type open capture or generate images The prototype displays Hello world Press R File gt Run Macro to compile and run it Note that the name you choose for the plugin should include at least one underscore Another example is the Step Maker plugin 31 4 5 Plugin Filter Opens an editor window containing a prototype plugin that implements the PlugInFilter interface Plugins of this type process the active image The prototype inverts the active image twice Another example is the Image Inverter 161 Last updated 2012 10 02 Plugins gt Compile and Run 31 4 6 Plugin Frame Opens an editor windo
272. r to fill with background color see Color Picker K Modifier keys Alt Alt clicking makes the brush paint in background color SEE ALSO floodFill x y macro function Color Picker window XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 17 gt LUT Menu A drop down menu listing all the Imagep gt Lookup Tables gt commands It is a convenient way to deal with a large collection of lookup tables that otherwise would only be accessed through the menu bar Note that although it is not possible to organize LUTs into subfolders it is possible to rename the most frequently used lookup tables with a numeric prefix e g El01 glasbey lut 02 Termal lut etc so that they are listed earlier in the menu SEE ALSO Pseudocolor Images ElShow_A11_LUTs a macro that creates a graphical palette of all the installed lookup tables Stacks Menu Common Commands Menu Tool Developer Menu 19 18 gt 4 Overlay Brush A freehand paintbrush that draws OO Overlay Brush Options on a non destructive image overlay see Overlays as opposed to the Brush tool that draws invasively over the canvas Brush width pixels EJ Transparency 6 4 O gt co Color orange Double clicking on the tool icon Also set the color using Color Picker shift k opens its Options dialog box in which is possible to specify the Und cl e Brush width in pixels Trans parency and Color As previou
273. r in the polygon selection and the absence of point handlers in Composite Selections Overlays i e non active selections displayed in the non destructive image overlay are also displayed without handlers 10 1 Manipulating ROIs Most of commands that can be useful in defining or drawing selections are available in the Edit gt Selection gt submenu and summarized in ROI manipulations Listed below are the most frequent manipulations involving selections Adjusting Deleting Managing Moving Nudging Resizing Area selections can be adjusted with the Brush Selection Tool In addition vertexes of selections created with the Polygon Selection Tool and Segmented Line Selection Tool can be adjusted by Alt Shift clicking Choose any of the selection tools and click outside the selection or use Edit gt Selection gt Select None A Use Edit gt Selection gt Restore Selection E to restore a selection back after having deleted it With Overlays an activated ROI can be deleted by pressing the Delete on Mac key A selection can be transferred from one image window to another by activating the destination window and runnig Edit gt Selection gt Restore Selection E Alternatively Analyze gt Tools Synchronize Windows to create ROIs across multiple images Mul tiple selections can be stored as Overlays or in the ROI Manager list Analyze gt Toolst gt ROI Manager Selections can be moved by clicking and
274. ray value Most frequently occurring gray value within the selection Corresponds to the highest peak in the histogram Uses the heading Mode Min amp maz gray level Minimum and maximum gray values within the selection Centroid The center point of the selection This is the average of the x and y coordinates of all of the pixels in the image or selection Uses the X and Y headings Center of mass This is the brightness weighted average of the x and y coordinates all pixels in the image or selection Uses the XM and YM headings These coordinates are the first order spatial moments Perimeter The length of the outside boundary of the selection Uses the heading Perim With IJ 1 44f and later the perimeter of a composite selection is calculated by decomposing it into individual selections Note that the composite perimeter and the sum of the individual perimeters may be different due to use of different calculation methods Bounding rectangle The smallest rectangle enclosing the selection Uses the headings BX BY Width and Height where BX and BY are the coordinates of the upper left corner of the rectangle Fit ellipse Fits an ellipse to the selection Uses the headings Major Minor and Angle Major and Minor are the primary and secondary axis of the best fitting ellipse Angle is the angle between the primary axis and a line parallel to the X axis of the image The coordinates of the center of the ellipse are displayed as X and Y if Cent
275. rch source folder Extends the search scope to El ImageJ source if present Requires the ImageJ source code to be downloaded from http imagej nih gov ij download src and extracted into the ImageJ folder Note that you can perform searches in other directories by choosing none of the folders above mentioned In this case you will be asked to choose a target directory on a second dialog prompt SEE ALSO Search txt the macro in EJij jar implementing this command Find Commands I Finding Commands 31 3 4 Monitor Events By implementing the IJEventListener CommandListener and ImageLister interfaces this command is able to monitor foreground and background color changes tool switches Log window closings command executions and image window openings closings and updates SEE ALSO Debug mode Edit Options Misc 159 Last updated 2012 10 02 Plugins gt Utilities gt 31 3 5 Monitor Memory Displays a continuously updated graph of ImageJ s memory utilization which can be useful for detecting memory leaks Memory usage and running threads are displayed above the graph As for the IJ Status bar clicking on the window will reclaim unused memory by running the Java garbage collector Ideally you should be able to open several images process them close them and the amount of memory used will be the same as when you started SEE ALSO Edit Options gt Memory amp Threads 31 3 6 Capture Screen g
276. reas pixel value 255 cause the corresponding frequencies to be passed It is not however possible to both filter and pass during the same inverse transform Note that areas to be filtered in the frequency domain image must be zero filled and areas to be passed must be filled with 255 You can verify that this is the case by moving the cursor over a filled area and observing that the values displayed in the status bar are either 0 or 255 Thus you should always confirm that masked areas are not some other gray value by using the black amp white reset option in the Color Picker window widgets when defining foreground Edito Fill f and background Edit gt Clear colors Power spectrum with Power spectrum with a Result of inverse Result of inverse Original mask that filters low mask that passes low transform transform frequencies frequencies With off center selections the same spatial frequency appears twice in the power spectrum at points opposite from the center It is sufficient to fill clear only one of these o Edited power Power spectrum after Original Inverse transform spectrum filtering a ko NT FFT Example of low frequencies filtering 29 10 3 Redisplay Power Spectrum Recomputes the power spectrum from the frequency domain image 32 bit FHT This command allows you to start over after mis editing the 8 bit power spectrum image 121 Last updated 2012 10 02 Process gt FFT
277. regions of interest ROIs are typically created using the Toolbar Tools Although ImageJ can display simultaneously several ROIs see Overlays and ROI Manager only one selection can be active at a time Selections can be measured Analyze gt Measure m drawn Edit gt Draw d filled Edit Fill f or filtered Process Filters gt submenu in the case of area selections In addition it is also possible to hold multiple ROIs as non destructive Overlays Selections can be initially outlined in one of the nine ImageJ default colors Red Green Blue Magenta Cyan Yellow Orange Black and White Once created selections can be contoured or painted with any other color using Edit gt Selection gt Properties y Selection Color can be changed in Edit gt Options Colors by double clicking on the Point Tool or using hot keys see 7 Using a Keyboard Shortcut to Change Selection Color It is highlighted in the center of the Point Tool and Multi point Tool 17 Last updated 2012 10 02 Selections Rectangular Polygon Composite Manipulating ROIs Cursor outside selection 4 RK Selection can be moved h Selection can be resized iy Edge can be moved deleted or added Three types of area selections In ImageJ Notice the cursor changes to an arrow when it is within the selection to a cross hair when outside the selection to a hand when over a selection vertex or handler Notice also the filled handle
278. resholded images can be extracted by specifying suitable Size and Circularity ranges and or by choosing if particles should be traced by their outer edge or by flood filling Include Holes checkbox Circularity Particles with size circularity values outside the range specified in this field are also ignored Circularity 4r x Fees see Set Measurements ranges from 0 infinitely elongated polygon to 1 perfect circle Show This drop down menu specifies which image or overlay should ImageJ display after the analysis see Display options of ParticleAnalyzer Size color and background of text labels can be adjusted in the Imagep Overlay gt Labels prompt Non overlay outputs can be adjusted in a macro by using call ij plugin filter ParticleAnalyzer setFontSize size and call ij plugin filter ParticleAnalyzer setLineWidth width or Par ticleAnalyzer setFontSize size ParticleAnalyzer setLineWidth width in a script or plugin Nothing Neither images nor Overlays will be displayed Note that the particle analyzer will display a blank image when the count of detected particles is zero and Show is not Nothing Outlines 8 bit image containing numbered outlines of the measured particles gray levels Outlines 0 Labels 1 Background 255 If In situ Show is checked the original image will be replaced by this image Bare Outlines 8 bit image containing simple outlines of the measured particles without labels graylevels
279. roid is checked Note that ImageJ cannot calculate the major and minor axis lengths if Pixel Aspect Ratio in the Analyzep Set Scale dialog is not 1 0 There are several ways to view the fitted ellipse 1 The Edito Selection gt Fit Ellipse command replaces an area selection with the best fit ellipse 2 The DrawEllipse macro draws destructively the best fit ellipse and the major and minor axis 3 Select Ellipses from the Show drop down menu in the particle analyzer Analyze gt Analyze Particles and it will draw the ellipse for each particle in a separate window Shape descriptors Calculates and displays the following shape descriptors Circularity 47 x pee with a value of 1 0 indicating a perfect circle As the value approaches 0 0 it indicates an increasingly elongated shape Values may not be valid for very small particles Uses the heading Circ Aspect ratio The aspect ratio of the particle s fitted ellipse i e A If Fit Ellipse is selected the Major and Minor axis are displayed Uses the heading AR Esa jz Or the inverse of Aspect Ratio Uses the heading Round Tx Major axis Solidity o selection convex Roundness 4 x Note that the Edit gt Selection gt Convex Hull command makes an area Feret s diameter The longest distance between any two points along the selection boundary also known as maximum caliper Uses the heading Feret The angle 0 180 degrees of 137 Last updated 2012 10
280. rom ImageJ tables including the Summarize table cf Analyzep Analyze Particles In addition it allows to specify the file extension to be used when saving data Custom extensions e g El csv E x1ls or E ods allow ImageJ tables to be imported seamlessly by spreadsheet applications ImageJ tables are saved in CSV format if File extension for tables is El csv SEE ALSO Plugins gt New Table 43 Last updated 2012 10 02 Editor Menu Commands Debug Window Debug AAA Debug Debug Macro D Name Step HE Trace T nimages 1 Fast Trace TET getTitle blobs gif Run variable 1 Ij Run to Insertion Point Main Window variable2 1 43p Macros Abort OOO Untitled txt Run Macro SER variablel 1J 3 Evaluate Line gy variable2 getVersion Abort Macro showMessage I am running variablel variable2 Install Macros l Function Finder EN Evaluate JavaScript 38 Macros gt Function Finder ADA Built in Functions message Edit gt Go to Line Goto Line Edit gt Find Go to line number Cut xX aA Dialog addMessage string Copy C Find exit or exit error message Paste gV getBoolean message Find fC l redirectErrorMessages Find a6 F showMessage message Find Next G O Case Sensitive L Whole Words showMessage title message Go to Line L showMessageWithCancel title message Cancel C OK gt showStatus message
281. rong scientific background Frequently the most demanding tasks in scientific image processing relate to experimental design Even if you do not have special expertise in image processing by participating on the mailing list discussions your experience will be valuable to others Do you want ImageJ to improve You can report bugs or request new features using the mailing list Do you have experience in graphic web design If you are able to to improve the look and feel of the guide we welcome your skills The ImageJ Icon The Hartnack microscope ca 1870 s depicted on the front page inspired the ImageJ icon for Mac OS X It is based on a photograph by Tom Grill at arsmachina com Edmund Hartnack 1826 1891 was a renown microscope manufacturer that pioneered the use of correction collars in water immersion lenses and the adoption of the substage condenser The precision and robustness of Hartnack optics played a pivotal role in the groundbreaking research by the Nobel laureates Robert Koch Camillo Golgi and Santiago Ram n y Cajal Merico G Microscopy in Camillo Golgi s times J Hist Neurosci 2003 8 2 113 20 Brock TD Robert Koch A life in medicine and bacteriology ASM 1999 Brenni P Gli strumenti di fisica dell Istituto Tecnico Toscano Ottica IMSS 1995 DeFelipe amp Jones Santiago Ram n y Cajal and methods in neurohistology Trends Neurosci 1992 15 7 237 46 Document Revisi
282. s 8 bit inverting LUT 64 pixels 8 bit inverting y gt Adie gt A EZ on de 6 A hi y Cd Shadows North South East and West kernels 29 8 Binary This submenu contains commands that create or process binary black and white images They assume that objects are black and background is white unless Black Background is checked in the Process Binary Options dialog box see XXII Interpreting Binary Images Summary of morphological operators Process gt Binary gt submenu Original Make Binary Erode Dilate Open Close Outline Skeletonize nageJ ImageJ ImagcJ ImageJ ImageJ ImageJ ImageJ ImageJ Adjust gt Threshold T Minimum Maximum Erode then Dilate then 1pixel wide 1 pixel wide grayscale grayscale Dilate Erode outline skeleton 29 8 1 Make Binary Make Zinan Converts an image to black and white If a threshold has A leek nes ees reed eal been set using the Image Adjust gt Threshold T tool M Remaining pixels to background color the depicted dialog is displayed The value of the Black foreground white background checkbox reflects and sets the global Black Background value of Process Binary gt Cancel oK gt Options M Black foreground white background If a threshold has not been set Make Binary will analyze the histogram of the current selection or of the entire image if no selection is present and set an automatic thres
283. s Described at http imagejdev org mailing lists Using Mailing lists If you are having problems with ImageJ you should inquiry about them in the appropriated list The ImageJ mailing list is an unmoderated forum subscribed by a knowledgeable worldwide user community with 2000 advanced users and developers To have your questions promptly answered you should consider the following 1 Read the documentation files described earlier in this section before posting Because there will always be a natural lag between the implementation of key features and their documentation it may be wise to check briefly the ImageJ news website Help ImageJ News 2 Look up the mailing list archives Help Mailing List Most of your questions may have already been answered 3 If you think you are facing a bug try to upgrade to the latest version of ImageJ Help gt Update ImageJ You should also check if you are running the latest version of the Java Virtual Machine for your operating system Detailed instructions on how to submit a bug report are found at http imagej nih gov ij docs faqs html bug 4 Remember that in most cases you can find answers within your own ImageJ installation without even connecting to the internet since the heuristics for finding commands or writing macros have been significantly improved in later versions see Finding Commands and Extending ImageJ 5 As with any other mailing list you should always foll
284. s Ne dor oaks a de Se a a we de OO ee e E 118 AAMT Glopa albratons eee e e See e hoe eee oA OM SS 141 XXIV Using Scanners in Densitometry e ace acae 41 as ea Rede A 144 XXV Selecting ROIs in the ROI Manager e 153 XXVI Focus on Bioimage Informatics e re a ds Sg ie OS we A 173 vill Macro Listings COO N O 10 11 Replace Red with Magenta ijm Using Process gt Image Calculator 17 Replace Red with Magenta ijm Using Image Color gt Channels 17 Ensuring Specie Settings at LANCI an ae ee be AS we ee ER we eee OES 22 Customizing the Image Popup Mentes dem s he 8 3 2 42 Customizing the Float Behavior of IJ s Main Window 47 Setting File Open Samples gt for Offline Usage oa aaa 51 Using a Keyboard Shortcut to Change Selection Color 70 Obtains istopram last e ra a ewe Ge A ia 142 Assigning Keyboard Shortcuts to ImageJ Tools 169 Cycling Through the Toolbar Using Keyboard Shortcuts 170 Temporary Activation ofa Tool ce brit ran Hind ew ee Swe oe BS eR 170 1X Guide Formats This guide is available in the following formats Enhanced PDF Optimized for electronic viewing and highly enriched in hypertext links see Conventions Used in this Guide Available at http imagej nih gov ij docs guide user guide pdf HTML document available online at http imagej nih gov ij docs guide For offline us age a
285. s Reference Guide by J r me Mutterer and Wayne Rasband This booklet compiles most of the documentation dispersed throughout the web related to ImageJ s macro programming It provides an up to date printable manual for the ImageJ macro language http imagej nih gov ij docs macro reference guide pdf 23 Plugins 2 The Built in Macro Functions webpage Help gt Macro Functions and Macros Function Finder F in the Editor is the indispensable guide to the built in functions that can be called from the ImageJ macro language It is thoroughly documented and constantly updated http imagej nih gov 1j developer macro functions html 3 Tutorials on the Fiji webpage http fiji sc wiki index php Introduction_ into Macro Programming 4 How tos and tutorials on the ImageJ Documentation Portal http imagejdocu tudor lu SEE ALSO Scripts Plugins Editor Fiji Script Editor 15 Scripts JavaScript scripting was introduced in ImageJ 1 41 in order to bring full access to ImageJ and Java APIs see Advantages and disadvantages of JavaScript ImageJ uses the Mozilla Rhino interpreter built into Java 1 6 for Linux and Windows to run JavaScript Mac users and users of earlier versions of Java must download E JavaScript jar into the plugins folder This JAR file is available on the ImageJ website and is included with the Mac version of ImageJ in GiImageJ plugins jars Example JavaScript programs are available at imagej
286. s required It proceeds by 1 Making first guesses of all the non linear parameters 2 Computing the model comparing it to the data set and calculating a fitting error 3 If the fitting error is large the CurveFitter will systematically change parameters and return to step 2 The loop stops when the fitting accuracy is met which in difficult cases may never happen In the latter case the procedure terminates after an imposed number of iterations or restarts The typical usage of this command is listed below 1 Tabular data is entered or copied in the input window or alternatively a two column text file is opened by clicking on the Open button Values may be separated by spaces tabs commas or semicolons 2 The function to be fit is selected from the drop down menu Several built in functions are available see CurveFitter s built in functions User defined functions with up to six parameters are also possible by choosing User defined Note that elimination of parameters by linear regression does not take place for user defined functions As a consequence the performance of custom functions does not fully match that of built in functions 3 Once the Fit button is pressed ImageJ displays a graph of the data with the fitted curve as depicted in CurveFitter If Show Settings is checked detailed information about the fit including measures of goodness of fit is printed to the Log Window and the user is prompted to re adjust th
287. s this issue Sort Names Numerically When checked the stack will be opened in NUMERIC file name order e g namel tif name2 tif namel0 tif instead of alphanumeric order e g namel tif namel0 tif name2 tif DICOM files in the same series tag 0020 0011 are always sorted by the image number tag 0020 0013 The List Stack Tags macro part of the ListDicomTags macro set lists the values of the image number and image series tags Use Virtual Stack When checked images are opened as a read only virtual disk resident stack using a version of the Virtual Stack Opener plugin This allows image sequences too big to fit in RAM to be opened but access time is slower and changes are lost when switching to a different image in the stack see Virtual Stacks Note the following consequences of enabling this option Image Overlays are not loaded If the folder contains tiff stacks only the first slice of those stacks will be imported with RAM resident stacks all slices are imported and concatenated into the sequence see Image gt Stacks gt Tools Concatenate Help Opens http imagej nih gov 1j docs menus file htmlseq1 SEE ALSO File Save Asp Image Sequence OpenSeriesUsingFilter macro Image Overlay gt Regular expressions basic syntax summary For more information on regex filtering see http download oracle com javase tutorial essential regex Regex Syntax
288. s txt The same file can be opened by holding while selecting Startup Macros from the gt drop down menu see More Tools Menu 31 1 4 Record Opens the ImageJ command recorder To create a macro open the recorder use one or more ImageJ commands then click Create When the recorder is open each menu command you use generates a macro run function call The run function has one or two string arguments The first is the command name The optional second argument contains dialog box parameters Examples Create a rectangular oval or line selection and the recorder will generate a makeRectan gle makeOval or makeLine function call Click on Auto or Set in the Image Adjust gt Threshold T window to generate a set Thresold call and on Reset to generate a resetThresold call Select an image from the Window menu to generate a selectWindow call Click in the Image Color Color Picker K window to generate setForegroundColor and setBackgroundColor calls 156 Last updated 2012 10 02 Plugins gt Shortcuts gt j E il j 5 El 5 Record Macro Macro ijm Create setp l make border k run Select All JavaScript run Line Width run Draw setp 2 save saveAs Tiff Desktop blobs tif Plugin Note that you can interact with the recorder window by deleting or commenting lines of code or pasting text from Editor This may be specially useful whe
289. selection is moved or resized To obtain profiles of several selections in a single plot use the ROI Manager s Multi Plot command Analyze gt Toolst gt RO Manager Other types of area selections such as oval or freehand ROIs can be profiled by first running Edit gt Selection Area to Line which will convert these ROIs to line selections Use the List Save or Copy buttons to view and save the profile data Use Edit gt Options gt Profile Plot Options to adjust how plots are generated SEE ALSO Rectangular Selection Tool Line Selection Tools Plot Z axis Profile Surface Plot Dynamic Profiler Oval Profile Plot Radial Profile Plot Radial Profile Extended plugins StackProfilePlot macro 142 Last updated 2012 10 02 IMPROVED IN IJ 1 46R Analyze gt Surface Plot 30 12 Surface Plot Displays a three dimensional graph of the intensities of pixels in a grayscale or pseudo color image non RGB images The plot is based on the existing rectangular selection or on the entire image if no selection is present A stack of plots can be produced when the source image is a stack or hyperstack In this case closing the plot stack window will abort the plotting process AAA Surface Plot Sx SoU KGE A A hi E 2738 0 Surface Plotter Polygon Multiplier 10 200 100 M Draw Wireframe M Shade M Draw Axis O Source Background is Lighter O Fill Plot Background with Black M One Polygo
290. selections containing one or more lines of text Note the following when using the Text Tool Use the keyboard to add characters to the text and the backspace key to delete characters Use to type special unit symbols such as y Alt M or A Alt Shift A Note that menu shortcuts require holding down while using the Text Tool see Using Keyboard Shortcuts 30 Last updated 2012 10 02 Tools Magnifying Glass Use Edit gt Selection Properties y to re adjust font color and size text justification and to specify a background color for the text selection XIX Hexadecimal Color Values provides instructions on how to define semi transparent colored backgrounds see also E DrawlextWithBackground macro Use Image Overlay gt Add Selection b to create non destructive text anno tations see Overlays EJOverlayDrawStringDemo E TextOverlay macros Alternatively use Ctrl D Edit gt Draw d to permanently draw the text on the image In the latter case the background of the text selection is not drawn see also XVII Embedding Color Annotations in Grayscale Images SEE ALSO Arrow Tool Brush Overlay Brush Pencil E TextDemo macro Toolbar Shortcuts 19 9 Magnifying Glass Magnifies and reduces the view of the active image Activate the tool and click on the image to zoom in Right click or Alt click to zoom out The current magnification is shown in the image s title bar Double click on the magn
291. sequence is the result of Surface Depth Cueing 0 100 1 projecting from a different viewing angle To visualize Interior Depth Cueing 0 100 50 this imagine a field of parallel rays passing through O Interpolate a volume containing one or more solid objects and Cana COR striking a screen oriented normal to the directions of the rays Each ray projects a value onto the screen or projection plane based on the values of points along its path Three methods are available for calculating the projections onto this plane nearest point brightest point and mean value The choice of projection method and the settings 90 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Image Stacks gt of various visualization parameters determine how both surface and interior structures will appear Projection Method Select Nearest Point projection to produce an image of the surfaces visible from the current viewing angle At each point in the projection plane a ray passes normal to the plane through the volume The value of the nearest non transparent point which the ray encounters is stored in the projection image Brightest Point projection examines points along the rays projecting the brightest point encountered along each ray This will display the brightest objects such as bone in a CT computed tomographic study Mean Value projection a modification of brightest point projection sums the values of
292. sing commands like Image Crop X because any stack generated from commands that do not generate virtual stacks will be RAM resident 12 Last updated 2012 10 02 Stacks Virtual Stacks and Hyperstacks B A mitosis HEN AAR MAX_ mitosis tif s MAX mitosis Stacks and Hyperstacks in ImageJ File Open Samples Mitosis 26MB 5D stack Hyperstacks dimensionality can be reduced using Image Hyperstacks gt Reduce Dimensionality Image Stacks gt Z Project or Image Hyperstacks gt Channels Tool Z The V on the window title denotes a virtual image see Virtual Stacks TIFF virtual stacks can usually be accessed faster than JPEG virtual stacks A JPEG sequence can be converted to TIFF by opening the JPEG images as a virtual stack and using File gt Save Asp Image Sequence to save in TIFF format ImageJ appends a V to the window title of virtual stacks and hyperstacks see Hyperstacks Several built in ImageJ commands in the File gt mport gt submenu have the ability to open virtual stacks namely TIFF Virtual Stack Image Sequence Raw Stack From List AVI cf Virtual Stack Opener In addition TIFF stacks can be open as virtual stacks by drag and drop cf III Opening Virtual Stacks by Drag amp Drop SEE ALSO LOCI Bio Formats and RegisterVirtualStackSlices plugins Process Virtual Stack and VirtualStackFromList macros III OPENING VIRTUAL STACKS BY DRAG amp DROP TIFF stac
293. site Display Mode 28 5 6 Show LUT Displays a plot of the active image s lookup table LUT The lookup table or color table describes the color that is displayed for each of the 256 possible pixel values For 16 and 32 bit images the range of displayed pixel values is mapped to 0 255 A bar under the plot displays the color representation of the pixel values Note that RGB color images do not use a lookup table Use the List radio button to export the LUT as a CSV file Look Up Table SEE ALSO Edit LUT 84 Last updated 2012 10 02 Image Color gt 28 5 7 Edit LUT LUT Editor Opens the ImageJ LUT Lookup Table Editor A Co 2AaE lookup table in ImageJ has up to 256 entries The entry index and the three values red green and blue associated with it are displayed in the ImageJ status bar as you move the cursor over the LUT Editor window Click on an entry to edit the red green and blue values for that entry using a Color Selector window cf Color Picker K _ _ _ Ep E BREE Ll BeBe A O a EE ld BeBe lL BeBe E O a a A O E PEE 5 50 REAST BeBe REAST SEE ALSO Show LUT 28 5 8 Color Picker K The Color Picker 17 enables the user to select foreground and background colors which affects Edit gt Fill f Draw d Clear and other drawing commands It displays current foreground and background colors in the selection boxes at th
294. sly described see Brush and Pencil tools the Color dropdown menu changes the foreground color bypassing the Color Picker window activated by K Press Undo to remove the last painted stroke from the overlay Overlay manipulations are described in Image Overlay gt SEE ALSO Freehand Line Selection Tool XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 39 Last updated 2012 10 02 Tools Pencil 19 19 gt Pencil A freehand painting tool that draws invasively in foreground color It is in all similar to the Brush tool but it is typically used with thinner strokes Double clicking on the tool icon opens its Options dialog box in which is possible to specify the Pencil width in pixels and Color Refer to the Brush tool tools for details Modifier keys Shift dragging on the canvas will adjust the brush size Alt Holding Alt makes the brush paint in background color see Color Picker KD SEE ALSO Freehand Line Selection Tool Overlay Brush Color Picker window XVII Embedding Color Annotations in Grayscale Images Toolbar Shortcuts 19 20 M Pixel Inspector Pixel Inspector Prefs Radius 2 1 10 Grayscale readout Raw H RGB readout R G B rs Copy to clipboard Data Only H o move red outline data to clipboard 17 The Pixel inspector displays the values of a square neighborhood around the current cursor position as a table 9 Values are update
295. software J Anat 215 6 698 704 Dec 2009 doi 10 1111 j 1469 7580 2009 01150 x Gene Myers Why bioimage informatics matters Nat Meth 9 7 659 660 Jun 2012 URL http www nature com nmeth journal v9 n7 full nmeth 2024 html doi 10 1038 nmeth 2024 Christopher A Myrick A low cost system for capturing and analyzing the motion of aquatic organisms J N Am Benthol Soc 28 1 101 109 Jan 2009 doi 10 1899 08 067 1 Martha L Narro Fan Yang Robert Kraft Carola Wenk Alon Efrat and Linda L Restifo NeuronMetrics software for semi automated processing of cultured neuron images Brain Res 1138 57 75 Mar 2007 doi 10 1016 j brainres 2006 10 094 Mahdad Noursadeghi Jhen Tsang Thomas Haustein Robert F Miller Benjamin M Chain and David R Katz Quantitative imaging assay for NF KB nuclear translocation in primary human macrophages J Immunol Methods 329 1 2 194 200 Jan 2008 doi 10 1016 j jim 2007 10 015 Francesca Papadopulos Matthew Spinelli Sabrina Valente Laura Foroni Catia Orrico Francesco Alviano and Gianandrea Pasquinelli Common tasks in microscopic and ultrastructural image analysis using ImageJ Ultrastruct Pathol 31 6 401 7 Jan 2007 doi 10 1080 01913120701719189 Eckard Picht Aleksey V Zima Lothar A Blatter and Donald M Bers SparkMaster automated calcium spark analysis with ImageJ Am J Physiol Cell Physiol 293 3 C1073 81 Sep 2007 doi 10 1152 ajpcell 00586 2006 Madeline Pool Joachim Thiem
296. stack must be the same size and bit depth A scrollbar provides the ability to move through the slices and the slider is preceded by a play pause icon that can be used to start stop stack animation Right clicking on this icon runs the Animation Options Alt dialog box Most ImageJ filters will as an option process all the slices in a stack ImageJ opens multi image TIFF files as a stack and saves stacks as multi image TIFFs The File gt Import gt Raw command opens other multi image uncompressed files A folder of images can be opened as a stack either by dragging and dropping the folder onto the ImageJ window or or by choosing File gt Import gt Image Sequence To create a new stack simply choose File gt Newp Image n and set the Slices field to a value greater than one The Imagep Stacks gt submenu contains commands for common stack operations SEE ALSO Stacks Menu Stack Manipulations on Fiji website Image5D Virtual Stacks Virtual stacks are disk resident as opposed to RAM resident and are the only way to load image sequences that do not fit in RAM There are several things to keep in mind when working with virtual stacks Virtual stacks are read only so changes made to the pixel data are not saved when you switch to a different slice You can work around this by using macros e g Process Virtual Stack or the Process Batch gt Virtual Stack command You can easily run out of memory u
297. stant are replaced by the constant 29 9 10 Gamma Applies the function f p 255 Y x 255 to each pixel p in OQO Gamma the image or selection where 0 1 lt y lt 5 0 For RGB images Value loas this function is applied to all three color channels For 16 bit EI Preview images the image min and max are used for scaling instead of 209 KA 3 Cancel OK Mee ia a SEE ALSO GammaCorrection Tool macro 29 9 11 Set Fills the image or selection with the specified value 29 9 12 Log For 8 bit images applies the function f p In p x 255 1n 255 to each pixel p in the image or selection For RGB images this function is applied to all three color channels For 16 bit images the image min and max are used for scaling instead of 255 For float images no scaling is done To calculate log of the image multiply the result of this operation by 0 4343 1 In 10 29 9 13 Exp Performs an exponential transform on the active image or selection 29 9 14 Square Performs a square transform on the active image or selection 29 9 15 Square Root Performs a square root transform on the active image or selection 29 9 16 Reciprocal Generates the reciprocal multiplicative inverse of the active image or selection transforming each pixel p into 1 p Requires 32 bit float images see Image Types and Formats 119 Last updated 2012 10 02 Process gt FFT gt 29 9 17 NaN Backgr
298. stration at different zoom levels SEE ALSO XII Transferring Selections Between Images ROI Manager 155 Last updated 2012 10 02 Plugins gt 31 Pluginsp 31 1 Macros This submenu contains commands for installing running and recording macros as well as any macro commands added by Plugins Macros Install Macros contained in a file named El StartupMacros txt in the macros folder are automatically added to this submenu when ImageJ starts up By design only one set of macros can be installed at a time As such the last set of macros installed by Plugins Macros Install or by the More Tools Menu will always replace previously installed macros 31 1 1 Install Adds one or more macros contained in a file to the bottom of this submenu To install a set of macros and at the same time view their source code open the macro file with File Open and use Editor s Macros Install Macros command Macros in the file E ImageJ macros StartupMacros txt are automatically installed when ImageJ starts up Similarly with ImageJ 1 44f and later newly opened macro sets with two or more macros are also automatically installed in this menu SEE ALSO More Tools Menu 31 1 2 Run Loads and runs a macro without opening it in Editor To run a macro and at the same time view its source code open it with File Open and use the editor s Macros gt Run Macro command 31 1 3 Startup Macros Opens E ImageJ macros StartupMacro
299. stribution 30 4 Distribution Produces a relative frequency histogram from the data of a chosen column of the Results table 40 Use the List or Copy buttons to save the histogram data Mouse over the histogram bars to read the counts for each bin on the window s lower right corner Analyzeb Histogram h describes in more detail ImageJ s histogram window This command is also available by right clicking on the Results Table Parameter Specifies the parameter in the Results Table to be analyzed Data points The number of rows that will be analyzed informative Automatic binning If checked ImageJ will use the method described by David Scott to assess the optimal histogram bin width see Scott DW Optimal and data based histograms Biometrika 66 3 605 610 Jan 1979 If unchecked the number of bins can be set with Specify bins and the start ing and ending limits of the histogram with range Distribution j Parameter Area Data points 223 O Count 223 Min 5 Mean 73 946 Max 1 StdDev 31 866 Mode Bins 8 O Automatic binning or specify bins 8 and range 0 200 o 08 y A s r C L L List Copy Log 4 F he at a e 135 Last updated 2012 10 02 Analyze gt Label SEE ALSO Distribution Plotter a macro that plots relative and cumulative frequencies on a double Y axis graph 30 5 Label This command labels the active selection
300. t for statistical calculations done by Analyze gt Measure m and Analyze gt Analyze Particles commands This feature allows you to outline a structure on one image and measure the intensity of the corresponding region in another image Decimal places This is the number of digits to the right of the decimal point in real numbers displayed in the Results Table and in Histogram windows Analyzeb Histogram h 138 Last updated 2012 10 02 IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Analyze gt Set Scale 30 8 Set Scale Use this dialog to define the spatial scale of the active image so measurement results can be presented in calibrated units such Pa as mm or pm Distance in Pixels 1 Known Distance 0 33348 Before using this command use the straight line selection Pixel Aspect Ratio 1 0 tool to make a line selection that corresponds to a known Unit of Length ym distance Then bring up the Set Scale dialog enter the M Global Known Distance and unit of measurement then click OK The Distance im Pixels field will be automatically filled in based on the length of the line selection Click to Remove Scale y Scale 2 999 pixels um T As described in Image Properties P p and A symbols can be typed using and Alt Shift A respectively ym can also be defined as um or micron Setting Pixel Aspect R
301. tacks gt Tools gt Start Animation Hyperstacks gt Channels Tool Z V ARGU gt O In 00 or gt LK WN 3 gt Hyperstacks or Hyperstacks or Hyperstacks Hyperstacks Hyperstacks Hyperstacks Crop X Shift Duplicate D D Scale E Zoom ln O O A e Zoom Out Zoom Original Scale 4 Zoom gt View 100 5 Overlay gt Add Selection b tele Process gt Smooth S Repeat Command R UN gt BG Analyze gt Measure m Histogram h Plot Profile k Gels gt Select First Lane Gels gt Select Next Lane Plugins gt Utilities gt Control Panel U Utilities gt Capture Screen g Utilities gt Find Commands I Window gt Show All Put Behind tab Main ImageJ window Enter BE Mm AIA N SE p p FP 34 Key Modifiers 34 1 Alt Key Modifications File gt Open Next O Open previous File gt Revert r Skip dialog prompt 167 Description Open Color Picker Go to next stack slice Go to previous stack slice Reslice stack Toggle orthogonal view display Start stop stack animation Open the Channels tool Next hyperstack channel Previous hyperstack channel Next hyperstack slice Previous hyperstack slice Next hyperstack frame Previous hyperstack frame Crop active image or selection Duplicate active image or selection Scale image or selection Make image larger Make image smaller Revert
302. tart Animation Note that setting the Frame Interval in Imagep Properties P sets the animation speed as long as the unit used is sec Cancel OK This dialog can also be accessed by right clicking on the play pause icon that precedes the slice slider in Stacks and frame slider in Hyperstacks see Stacks and Hyperstacks SEE ALSO File gt Save Asp Gif AVI 95 Last updated 2012 10 02 Image gt 28 7 Hyperstacks gt Hyperstacks gt This submenu hosts commands specifically related Hyperstacks images that have four 4D or five 5D dimensions General operations related to Stacks are listed in the Image Stacks gt submenu 28 7 1 New Hyperstack Display Mode Width Height Channels c Slices z Frames t M Label Images 28 7 2 Stack to Hyperstack gt Convert to HyperStack Order Channels c Slices z Frames t Display Mode 28 7 3 Hyperstack to Stack Creates a new hyperstack Hyperstacks have Width Height Channels c dimension Slices z dimension and time Frames t dimension Image Type see Image Type and Image Types and For mats and Display Mode see Channels Tool Z can be specified Checking Label Images will draw the channel number slice number and frame number on each image in the hyperstack SEE ALSO File gt New Hyperstack an alias of this com mand and File gt Newp Image n
303. the difference between the two images using Process gt Image Calculator By adjusting the Brightness Contrast C you will notice that the imperceptible JPEG artifacts are most pronounced along regions of higher contrast changes In addition to this edge artifact the JPEG algorithm may shift colors to improve compression which may lead to artificial colocalization These artifacts are intrinsic to the format and may persist even if JPEG Quality has been increased to 100 in Edit gt Options gt Input Output Once an image has been lossy compressed there is no way of reverting it to the original Given all this and since Metadata is poorly supported in lossy formats it is unreasonable to use JPEG in image processing Original JPEG copy 75 quality Difference 26 10 4 Text Image Saves the active image as a spreadsheet compatible tab delimited text file Calibrated and floating point images are listed with the precision specified by Decimal places in Analyze gt Set Measurements For RGB images each pixel is converted to grayscale using the formula gray red green blue 3 or the formula gray 0 299 x red 0 587 x green 0 114 x blue if Weighted RGB to Grayscale Conversion is checked in Edit gt Options gt Conversions SEE ALSO Text Images Pixel Inspector Image Transform gt Image to Results Results to Image Import gt Text Image 26 10 5 Zip Saves the active image or stack as a TIFF file inside
304. then selects a contiguous area under the condition that all pixel values in that area must be in the range initial value tolerance to initial value tolerance 4 connected Only the four neighbors of a pixel are considered neighbors E g the wand does not follow a one pixel wide diagonal line because the pixels of that line are not four connected 8 connected Each pixel is considered to have eight neighbors So the wand follows a diagonal line if you click onto it On the other hand if you have an area of constant value dissected by a one pixel wide diagonal line the 8 connected wand will jump over the line and include the other part of that area Legacy In this mode no neighbor is checked and no tolerance is used This is the default mode of the Wand Tool in ImageJ 1 42 and earlier Modifier keys Shift clicking appends the traced area to previously traced selections Alt Alt clicking removes the traced area from previously traced selections SEE ALSO Analyzep Analyze Particles Flood Filler Versatile Wand plugin Composite Selec tions Toolbar Shortcuts 19 8 Text Tool Enter text then press rctri b to add to overlays or ctri d to draw Font style and text alignment is specified in the Fonts widget activated by double clicking on Alor by running Edit gt Options Fonts Text is drawn in foreground color see Color Picker K Use this tool to add text to images It creates text ROIs rectangular
305. thy I Frontmost Window and Window Activation 00 9 II Image Types Lossy Compression and Metadata 12 HI Opening Virtual Stacks by Draw amp Drop x 4 004 we ae Aw eo SS 13 IV Replacing Red with Magenta in RGB Images 16 V Toce ine Calibrated UMIS s 20D ek oles ESO 29 VI Opening File Paths in the Log Window 4 444 4 46 e Rb dw A 46 VIL Organizing Commands in the Menu Bar sa woema lg ox a e ae te 48 VIII Opening Files File gt Open File gt Import gt and Drag amp Drop 50 IX Reducing Memory Requirements When Importing Images 93 X Warne on JPEG Compressi li 2 4 aoe ari a a Seek ea a eed e ae N 57 XI Drawing Lines Wider Than One Pixel a 62 XII Transferring Selections Between Images ooa a a 64 XIII Converting Composite Selections a 66 XIV Applying Auto Brightness Contrast to Entire Stacks 76 XV Display Range of DICOM Images vasca ee hoe Eh e ro di 18 XVI Brightness Contrast of High Bit Depth Images 79 XVII Embedding Color Annotations in Grayscale Images 00 86 XVIII Working with Zoomed Canvases 2 2 222 2 eS HS kee eR eAS ERE OE 101 XALA Hexadecimal Color Vall s s asc Soe s dd ea ae dl e ee Ee es 103 Xe Oreste Binary Masky s 2 2 3 64 a tie hw Ree es wR a 8 HB 113 XXI Skeletonize vs Skeletonize 3D e 115 AAI Interprete Dinary Image es
306. ting from division by zero are set to Infinity or to NaN Not a Number if a zero pixel is divided by zero The divide by zero value can be redefined in Edit gt Options Misc 128 Last updated 2012 10 02 Process gt Image Calculator Subtract Multiply Divide Image2 mri stack 2 M Create New Window C 32 bit float Result ee Y Help Cancel Average Difference Copy Transparent zero Image Calculator Subtract Background Operation Selects one of the thirteen available operators see Image operations Create New Window If checked a new image is created to hold the result If unchecked the result of the operation is applied directly to Imagel 32 bit float Result If checked source images will be converted to 32 bit floating point be fore performing the operation Help Opens http imagej nih gov ij docs menus process html calculator operations On these examples source and destination images 8 bit grayscale are displayed with inverted LUTs White 0 Black 255 cf Lookup Tables gt submenu Note that calculations between images can also be performed using copy and paste and the Edit Paste Control command Source image imgl Operator Add imgl imgl img2 Subtract imgl imgl img Multiply imgl imgl x img Divide imgl imgl img AND imgl imgl A img2 OR imgl img1 V img2 XOR imgl img1 img2 SEE ALSO Calc
307. tion Tool Elliptical Selection Tool 10 Magnifying Glass and Brush Selection Tool 11 Scrolling Tool Polygon Selection Tool 12 Color Picker Tool Freehand Selection Tool 13 More Tools Menu Straight Line Selection Tool Segmented Line A H Customized tools installed Selection Tool Freehand Line Selection Tool from ElStartupMacros txt and Arrow Tool Gmacros toolsets Angle Tool Gimacros tools or Point Tool and Multi point Tool Splugins Tools Toolbar The ImageJ toolbar contains tools for making selections drawings zooming and scrolling etc In addition the right side of the toolbar contains seven slots that can host any of the 60 tools and 15 toolsets available on the ImageJ website see Custom Tools All ImageJ tools share common features The on the bottom right corner of some icons in the toolbar depicts a contextual menu that can be accessed by right clicking on the tool icon e g Stacks Menu If an Options dialog is available for a particular tool it can be accessed by double clicking on the tool icon e g Wand Tool Status bar When the cursor is over an image pixel intensities and coordinates are displayed in the status bar After running a filter elapsed time and processing rate in pixels second are also displayed When clicking on the status bar the ImageJ version the Java version memory in use memory 28 Tools available and percent memory used will be displayed As Selections
308. tion in Edit gt Selection gt Properties y that tabulates ROI coordinates at evenly spaced one pixel intervals SEE ALSO Selections With Sub pixel Coordinates File gt Importo XY Coordinates 99 Last updated 2012 10 02 Filep Revert r 26 11 Revert r Reloads the active image stack or hyperstack from disk reverting it to its last saved state It is actually a shortcut for closing the window without saving and then reopening it Note that it may not work with Non native Formats opened through external plugins such as the OME Bio Formats library SEE ALSO Undo and Redo 26 12 Page Setup Page Setup The Page Setup dialog allows you to control the size of printed output plus other printing options Scale 100 Draw border Scale Values less than 100 reduce the size of printed images and M Center on page E Print title values greater than 100 increase the size 100 corresponds O Selection only to 72 pixels per inch ppi about the unzoomed screen size tad wo of the image The size of the printed image is determined Print actual size f a A A by the Scale value and the width and height of the image in a cence pixels Spatial calibration is ignored Draw border If checked ImageJ will print a one pixel wide black border around the image Center on page If checked the image will be printed in the center of the page instead of in the upper left corner Print title If checked
309. tions Image gt Stacks gt Z Project of skeletons produced by Skeletonize middle and Skele tonize3D right The left image is the maximum projec tion of the original stack File gt Open Samples gt Bat Cochlea Volume 19K Topographic skeletons can be analyzed us ing the AnalyzeSkeleton plugin 29 8 10 Distance Map Generates a Euclidian distance map EDM from a binary image 69 Each foreground pixel in the binary image is replaced with a gray value equal to that pixel s distance from the nearest background pixel for background pixels the EDM is 0 The Ultimate Points Watershed and Voronoi operations are based on the EDM algorithm The output type Overwrite 8 bit 16 bit or 32 bit of this command can be set in the Binary gt Options dialog box Note that when selecting Overwrite or 8 bit output distances larger than 255 are labelled as 255 29 8 11 Ultimate Points Generates the ultimate eroded points UEPs of the Euclidian distance map EDM see Distance Map from a binary image Ultimate Eroded Points are maxima of the EDM In the output the points are assigned the EDM value which is equal to the radius of the largest circle that fits into the binary particle with the UEP as the center The output type Overwrite 8 bit 16 bit or 32 bit of this command can be set in the Binary gt Options dialog box 115 Last updated 2012 10 02 Process gt Binary gt Original Segmentation Movie W
310. to original zoom level Zoom to 1 1 Adds active selection to image overlay 3x3 unweighted smoothing Repeat previous command Display statistics of active image selection Display histogram of active image selection Display density profile plot of active selection Select first gel lane Select next gel lane Open Control Panel Grab screenshot with if a dialog box is active List find and launch commands Make all windows visible Switch to next image window Bring ImageJ window to front Last updated 2012 10 02 Key Modifiers Shift Key Modifications Edit gt Copy c Copy to system clipboard Image gt Color gt Split Channels Keep original image Image gt Stacks gt Add Slice Insert before current slice Image gt Stacks gt Next Slice gt Skip ten slices Image gt Stacks gt Previous Slice lt Skip ten slices Image gt Stacksp Start Animation 1 Show options dialog Image gt Duplicate D Skip dialog prompt Image gt Overlay gt Add Selection b Show options dialog Process gt Enhance Contrast Do classic histogram equalization Analyze Histogram h Show dialog prompt Analyze gt Plot Profile k For rectangular selections generate row average plot For wide straight lines display rotated contents Analyze gt Gels gt Select First Lane Assumes lanes are horizontal Analyze gt Tools gt Analyze Line Graph Show intermediate image Analyze gt Tools gt ROI Manager Add Name and add sel
311. tputs Single Points Results in one single point per maximum Maxima Within Tolerance All points within the Noise Tolerance for each maximum Segmented Particles Assumes that each maximum belongs to a particle and segments the image by a watershed algorithm applied to the values of the image in contrast to Process Binary Watershed which uses the Euclidian distance map EDM See Process gt Binary gt Voronoi for EDM based segmentation of binary images Point Selection Displays a multi point selection with a point at each maximum List Displays the XY coordinates of each maximum in the Results window Count Displays the number of maxima in the Results window Exclude Edge Mazima Excludes maxima if the area within the noise tolerance surrounding a maximum touches the edge of the image edge of the selection does not matter Above Lower Threshold Thresholded images only Finds maxima above the lower threshold only The upper threshold of the image is ignored If Segmented Particles is selected as Output Type the area below the lower threshold is considered a background This option cannot be used when finding minima image with light background and inverted LUT Light Background To be checked if the image background is brighter than the objects to be found as it is in the Cell Colony image in the illustration below Help Opens http imagej nih gov ij docs menus process html find maxima 108 Last updated 2012 10 02
312. trol as well as mouse wheel control The Imager Stacks gt 3D Project was written by Michael Castle and Janice Keller of the University of Michigan Mental Health Research Institute MHRI Bill Mohler added suport for hyperstacks and 16 32 bit images in IJ 1 44m The Imager Stacks gt Tools gt Concatenate command implemented in IJ 1 46e is based on the Concatenate plugin by Jonathan Jackson The Imager Stacks Tools gt Make Substack command is based on the Substack Maker plugin by Anthony Padua Daniel Barboriak and Ved Sharma 171 C26 C27 C28 C29 C30 C31 C32 C33 034 C35 C36 C37 C38 C39 C40 C41 C42 C43 C44 C45 C46 C47 C48 C49 C50 The Image Hyperstacks gt Reduce Dimensionality command is based J r me Mutterer s Reduce HyperStack macro The Imagep Transformb Bin command is based on Nico Stuurman s Binner plugin The Image Zoome Set command is based on Albert Cardona s Zoom Exact plugin The Processp Find Maxima command is based on a plugin contributed by Michael Schmid The equalization code implemented in Process gt Enhance Contrast was contributed by Richard Kirk The Process gt Noise gt Remove NaNs was contributed by Michael Schmid The Process gt Math gt Macro command is modeled after Ulf Dittmer s Expression plugin The Process gt FFT gt Bandpass Filter is a built in version of Joachim Walter
313. ty 136 Spatial scale 138 Spray Can 40 Square root transformation 118 Square transformation 118 Stacks 11 Brightness Contrast 75 Export 57 58 From List 54 Hyperstacks 13 95 Labeling 91 93 Montage 87 93 Profile 91 Projection see Projection 89 93 Reduce 92 93 95 Substacks 93 Tools 92 Virtual 12 54 127 Stacks Menu 40 StartupMacros 21 36 40 41 49 155 Statistics 133 134 Status bar 28 Straight line selection 31 Straighten 65 Sub pixel accuracy 68 Sub pixel selections 19 63 Subtract see Math 117 Subtract Background 128 Subtract images see Image calculator 127 Surface 142 Swing 67 Syntax highlighting vi 26 Synthetic images 119 Tessellation see Voronoi 115 Text 35 Thinning 113 Threads 159 3D Projection 89 3D Filters 20 124 3D Gaussian blur 124 3D Object Counter 20 3D ROIs 20 3D Viewer 20 Threshold 77 119 137 Binary images 111 Color 79 TIFF 10 55 Time stamper 91 Tolerance Wand Tool 34 Toolbar 28 Tools Angle 33 Area Selection Brush 30 Ellipse 30 Freehand 31 Oval 30 Polygon 31 Rectangle 29 Rounded Rectangle 30 Wand 34 Arrow 32 36 Brush 37 Color Picker 36 Custom tools 40 Developer Menu 37 Flood Filler 38 Line Selection Arrow 32 36 Freehand Line 32 Segmented Line 32 Straight Line 31 LUT Menu 38 Magnifying Glass 35 More Tools Menu 36 Multi point 34 Overlay Brush 38 Pencil 39 Pixel Inspector 3
314. ual Machine that includes the javac compiler which is contained in the E Tools jar archive E Tools jar is included with the Windows and Linux versions of ImageJ bundled with Java and it is pre installed on Mac OS X Troubleshooting information can be found under Compiling Plugins in the Linux and Windows release notes The Edit gt Options gt Compiler command can be used to configure the javac compiler Since ImageJ 1 44c and later Compile and Run adds the Bio Formats plugin E loci_tools jar to the Java compiler s classpath 162 Last updated 2012 10 02 IJ 1 46R Window gt 32 Window This menu contains four commands plus a list of all open windows The currently active image will have a checkmark next to its name To activate a window pull down this menu and select the window by name 32 1 Show All Makes all the windows associated with ImageJ visible 32 2 Put Behind tab Displays the next open image Repeatedly press the tab key to cycle through all open images Note that pressing on any image will bring the Main ImageJ window to the foreground SEE ALSO I Frontmost Window and Window Activation 32 3 Cascade Moves all open images to the left side of the screen slightly offset from each other and displayed in the order they are listed at the bottom of this menu 32 4 Tile Shrinks all open image windows and repositions them to fit on the screen without overlapping 163 Last updated
315. ue Hold while adjusting the minimum to move a fixed width thresholding window across the range of gray values Lower slider Adjusts the maximum threshold value Method Allows any of the 16 different automatic thresholding methods to be selected 18 These methods are described on Fiji s Auto Threshold website The Default method is the modified IsoData algorithm used by ImageJ 1 41 and earlier Note that these are global thresholding methods that typically cannot deal with unevenly illuminated images such as in brightfield microscopy In these cases local algorithms are more appropriated by allowing the threshold to smoothly vary across the image These are implemented by the Auto Local Threshold plugin pre installed in Fiji Display Selects one of three display modes 19 Last updated 2012 10 02 Image Adjust gt Red Displays the thresholded values in red BE W Features are displayed in black and background in white This mode respects the Black background flag set in Process Binary gt Options Over Under Displays pixels below the lower threshold value in blue thresholded pix els in grayscale and pixels above the upper threshold value in green These colors can be changed from a macro by calling the ImageProces sor setOverColor and setUnderColor methods example Dark background To be checked when features are lighter than the background The state of the checkbox is remembered across restarts
316. ulator Plus plugin 29 14 Subtract Background 1 Result wale akl i GAS a Destination image img2 Operator Result Min imgl min imgl img2 Max imgl max imgl1 img2 Average imgl imgl img2 2 Difference imgl limgl img2 Copy mgl img Transparent zero mujm gt lo oles ol Removes smooth continuous backgrounds from gels and other images 39 Based on the concept of the rolling ball algorithm described in Sternberg Stanley Biomedical image processing EEE 129 Last updated 2012 10 02 Process gt Subtract Background Computer Jan 1983 Imagine that the 2D grayscale image has a third dimension height by the image value at every point in the image creating a surface A ball of given radius is rolled over the bottom side of this surface the hull of the volume reachable by the ball is the background to be subtracted Subtract Background Rolling ball radius 50 0 pixels v Light background O Create background don t subtract v Sliding paraboloid O Disable smoothing M Preview Process gt Subtract Background This command uses a sliding paraboloid or a legacy rolling ball algorithm that can be used to correct uneven illuminated background as shown in the profiles Analyze gt Plot Profile k below each image Rolling ball radius should be set to at least the size of the largest obj
317. upMacros trt else run cmd So e g to add the ability to run the Process gt Subtract Background command from the contextual menu one can simply add that command to the list of items defining the PopUp Menu Note that defines menu separators var pmCmds newMenu Popup Menu newArray Help Rename Duplicate Original scale Baste Control eS Record Capture Dcroen Monitor Memory Startup Macros Search u Find Maxima m Subtract lt Background 00 42 Last updated 2012 10 02 Results Table Results Save As 38S Min Rename 2 320E2 1 280E2 2 480E2 1 805E2 2 302E2 1 137E2 cold Cut 2 160E2 1 280E2 2 240E2 4 654E1 2 410E2 5 453E1 Copy Sec 59 blobs Cut 4 619E0 1 280E2 1 280E2 1 360E2 7 683E1 2 428E2 5 657E0 Clear 60 blobs Copy D 000E0 1 280E2 1 280E2 1 280E2 1 105E2 2 465E2 2 828E0 Select All l 61 blobs Clear 3 468E1 1 280E2 1 280E2 2 480E2 1 790E2 2 518E2 4 238E1 Results 62 blobs Select All 560E1 1 840E2 1 280E2 2 160E2 1 280E2 2 519E2 4 956E1 Clear Results 63 blobs 3 880E1 1 360E2 1 280E2 2 480E2 2 348E2 2 522E2 3 556E1 Summarize 64 blobs Clear Results 874E1 1 760E2 1 280E2 2 240E2 7 417E1 2 525E2 3 673E1 Distribution Summarize l lt gt Set Measurements Distribution Options a Duplicate ImageJ Results table version 1 44k Columns width can be adjusted
318. urity or vibrancy characterizes the shade of color i e how much white is added to the pure color Brightness also know as Value HSV system describes the overall brightness of the color see e g the color palette of Color Picker window In terms of digital imaging processing using the HSB system over the traditional RGB is often advantageous e g since the Brightness component of an HSB image corresponds to the grayscale version of that image processing only the brightness channel in This section is partially extracted from the MBF ImageJ online manual at http www macbiophotonics ca imagej colour_image_processi htm 14 Last updated 2012 10 02 Color Images Representation of an eight pixel color image in the RGB and HSB color spaces The RGB color space maps the RGB color model to a cube with Red R values increasing along the x axis Green G along the y axis and Blue B along the z axis In the HSB cylindrical coordinate system the angle around the central vertical axis corresponds to Hue H the distance from the axis corresponds to Saturation S and the distance along the axis corresponds to Brightness B In both cases the origin holds the black color The right panel shows the same image after brightness reduction easily noted by the vertical displacement along the HSB cylinder Images produced using Kai Uwe Barthel s 3D Color Inspector plugin routines that require grayscale images is a significa
319. us This option allows any value of the radius gt 0 0001 the rolling ball algorithm requires a radius of at least 1 The sliding paraboloid typically produces more reliable corrections since the rolling ball a legacy algorithm only kept for backward compatibility is prone to edge artifacts To reduce the computing time the rolling ball algorithm downscales the image in a incon sistent way The sliding paraboloid algorithm does not use downscaling and thus produces no downscaling artifacts Nevertheless the sliding paraboloid is also an approximation since it does not use a de facto paraboloid an exact implementation would require a great computing effort but it rather slides parabolae in different directions over the image Disable Smoothing For calculating the background rolling the ball images are maximum filtered 3x3 pixels to remove outliers such as dust and then smoothed to reduce noise average over 3x3 pixels With Disable Smoothing checked the unmodified image data 130 Last updated 2012 10 02 Process gt Repeat Command R are used for creating the background Check this option to make sure that the image data after subtraction will never be below the background Help Opens http imagej nih gov ij docs menus process html background SEE ALSO How to correct background illumination in brightfield microscopy by G Landini Auto Local Threshold command s source co
320. useful are the platform specific Troubleshooting and Known Problems sections Fiji installation is described at http fiji sc wiki index php Downloads The downloaded package may not contain the latest bug fixes so 1t is recommended to upgrade ImageJ right after a first installation Updating IJ consists only of running Help Update ImageJ which will install the latest Elij jar in the ImageJ folder on Linux and Windows or inside the ImageJ app on Mac OSX Help Update ImageJ can be used to upgrade or downgrade the E ij jar file to release updates or daily builds Release updates are announced frequently on the IJ news website and are labelled alphabetically e g v 1 43m Typically these releases contain several new features and bug fixes described in detail on the ImageJ News page Daily builds on the other hand are labelled with numeric sub indexes e g v 1 43n4 and are often released without documentation Nevertheless if available release notes for daily builds can be found at http imagej nih gov ij source release notes html When a release cycle ends v 1 42 ended with 1 42q v 1 43 with 1 43u etc an installation package is created downloadable from http imagej nih gov ij download html Typically this package is bundled with a small list of add ons Macros Scripts and Plugins SEE ALSO Luts Macros and Tools Updater a macro toolset that performs live updating of macros listed on the ImageJ
321. uted radiography Radiol Med 111 8 1156 67 Dec 2006 doi 10 1007 s11547 006 0113 5 Janos Roszik Duarte Lisboa Janos Szollosi and Gyorgy Vereb Evaluation of intensity based ratiometric FRET in image cytometry approaches and a software solution Cytometry A 75 9 761 7 Sep 2009 doi 10 1002 cyto a 20747 Janos Roszik Janos Sz llosi and Gy rgy Vereb AccPbFRET an ImageJ plugin for semi automatic fully corrected analysis of acceptor photobleaching FRET images BMC Bioinformatics 9 346 Jan 2008 doi 10 1186 1471 2105 9 346 Stephan Saalfeld Albert Cardona Volker Hartenstein and Pavel Tomanc k CATMAID collaborative annotation toolkit for massive amounts of image data Bioinformatics 25 15 1984 6 Aug 2009 doi 10 1093 bioinformatics btp266 Stephan Saalfeld Albert Cardona Volker Hartenstein and Pavel Tomancak As rigid as possible mosaicking and serial section registration of large sstem datasets Bioinformatics 26 12 i57 63 Jun 2010 doi 10 1093 bioinformatics btq219 Daniel Sage Franck R Neumann Florence Hediger Susan M Gasser and Michael Unser Automatic tracking of individual fluorescence particles application to the study of chromosome dynamics IEEE Trans Image Process 14 9 1372 83 Sep 2005 Sascha Meyer Dos Santos Ute Klinkhardt Reinhard Schneppenheim and Sebastian Harder Using ImageJ for the quantitative analysis of flow based adhesion assays in real time under physiologic flow conditions
322. verted to RGB Image gt Type gt submenu For non RGB images background foreground color will be drawn in equivalent gray levels e g For a 8 bit image if the foreground color is red RGB 255 0 0 intensity of drawn selections will be 255 0 0 3 85 Colored Overlays see Image Overlayt gt Overlay Brush tool on the other hand can be created on all image types becoming the easiest way to annotate grayscale images 28 6 Stacks gt This submenu contains commands related to Stacks Operations specifically related to Hyper stacks are listed in the Image Hyperstacks gt submenu 28 6 1 Add Slice Inserts a blank slice after the currently displayed slice Holding m inserts a blank slice before the current slice With Hyperstacks a E dialog prompt allows to insert either a channel z slice or t frame SEE ALSO Delete Slice 28 6 2 Delete Slice gt i Delete i Deletes the currently displayed slice With Hyperstacks it can Delete current chassa Al delete the current channel z slice or t frame E SEE ALSO Add Slice 86 Last updated 2012 10 02 IMPROVED IN IJ 1 40R IMPROVED IN IJ 1 46R IMPROVED IN IJ 1 46R Image gt 28 6 3 Next Slice gt Stacks gt Displays the slice that follows the currently displayed slice Holding will skip ten slices forward SEE ALSO Arrow Keys 28 6 4 Previous Slice lt Displays the slice that precedes
323. w containing a prototype plugin that extends the PlugInFrame class Plugins of this type displays a window containing controls such as buttons and sliders The prototype opens a window containing a text area Another example is the IP Demo plugin 31 4 7 Plugin Tool Opens a prototype plugin tool demonstrating EJij plugin tool PlugInTool 49 A plugin tool is a Java plugin that installs in the ImageJ toolbar to interact with the image canvas see Custom Tools Plugin tools with names ending in E Tool are listed on the More Tools Menu if placed in the GImageJ plugins Tools directory SEE ALSO Plugin Tools 31 4 8 Text Window New Text Window ae Opens a text window of a specified size that macros can write to Width 60 characters PrintTo Text Window Clock and ProgressBar are examples of macros Height 16 lines that write to a text window M Menu Bar f E l T eE Eon SEE ALSO Plugins gt New Macro File gt Newb Text Window N Te Cancel OK M A 31 4 9 Table New Table Name Opens a blank table similar to the Results table that macros can write to SineCosineTable2 is an example of such a macro Width 350 pixels Height 250 pixels SEE ALSO Plugins gt New Text Window SS RATA Cancel OK Me AAA 31 5 Compile and Run Compiles and runs a plugin Runs a plugin if the name of the selected file ends in class Requires that ImageJ be running on a Java Virt
324. web site 2 1 ImageJDistributions ImageJ alone is not that powerful it s real strength is the vast repertoire of Plugins that extend ImageJ s functionality beyond its basic core The many hundreds probably thousands freely available plugins from contributors around the world play a pivotal role in ImageJ s success 22 Running Help Update ImageJ however will not update any of the plugins you may have installed ImageJ add ons Plugins Scripts and Macros are available from several sources ImageJ s plugins page Helpo Plugins ImageJ Information and Documentation Portal and Fiji s webpage among others making manual updates of a daunting task This reason alone makes it extremely convenient the use of ImageJDistributions bundled with a pre organized collection of add ons Below is a list of the most relevant projects that address the seeming difficult task of organizing and maintaining ImageJ beyond its basics If you are a life scientist and have doubts about which distribution to choose you should opt for Fiji It is heavily maintained offers an automatic updater improved scripting capabilities and ships with powerful plugins More specialized adaptations of ImageJ are discussed in Software Packages Built on Top of ImageJ Fiji Fiji Fiji Is Just Image J Batteries included is a distribution of ImageJ together with Java Java 3D and several plugins organized into a coherent menu structure Citing its developers
325. when Equalize Histogram is checked Use Stack Histogram If checked ImageJ will use the overall stack histogram instead of individual slice histograms that allow optimal adjustments for each slice alone This option may be specially relevant when performing enhancements based on a ROI Help Opens http imagej nih gov ij docs menus process html enhance SEE ALSO Brightness Contrast C XIV Applying Auto Brightness Contrast to Entire Stacks 29 6 Noise Use the commands in this submenu to add noise to images or remove it AAA Original tif OOO Salt Pepper tif Salt Pepper gt Despecle tif OOO Add Noise tif 256x254 pixels 8 bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 x254 pixels 8 bit inverting LUT Process gt Noise Salt and Pepper Despeckle Add Noise and Remove Outliers SEE ALSO RandomJ Binomial Exponential Gamma Gaussian Poisson and Uniform a Java package for image randomization by Erik Meijering 29 6 1 Add Noise Adds random noise to the image or selection The noise is Gaussian normally distributed with a mean of zero and standard deviation of 25 SEE ALSO Filters Gaussian Blur 29 6 2 Add Specified Noise M 5 Gaussian Noise Adds Gaussian noise with a mean of zero and a chosen standard Standard Deviation 30 deviation Cancel SEE ALSO Filterst Gaussian
326. when saving Results Tables Files with txt and El x1s extensions are saved in tab delimited format and files with E csv extensions are saved in comma delimited format Use JFileChooser to open save Enables versions of File Open and File gt Save As that use the Java Swing s JFileChooser instead of the native OS dialogs The main advantage of JFileChooser is the ability to open multiple files by Shift clicking to select multiple contiguous files and control clicking to select more than one individual file On the other hand it is slower uses more memory and does not behave like the file open and save dialogs used in other applications It requires Java 2 which is included with the Linux and Windows distributions of ImageJ and is built into Mac OSX Save TIFF and raw in intel byte order Specifies the byte order used when saving 16 bit and 32 bit images using File gt Save Asp Raw Data or File Save Asp Image Sequence when Raw is chosen as the format Check this option to export images using the order used by Intel x86 based processors little endian This Wikipedia article has more information Results Table Options Specifies if column headers and row numbers should be saved or copied from ImageJ tables such as the Results and Summarize windows see Results Table 68 Last updated 2012 10 02 Edit gt Options gt 27 13 3 Fonts Opens a small widget with three pop up menus for specifying the typeface size styl
327. wikipage Luke Miller s tutorial Dot Blot Analysis Dot Blot Analyzer toolset 30 14 Tools This submenu provides access to various image analysis plugins 30 14 1 Save XY Coordinates eT ae Writes to a text file the XY coordinates and pixel value of all non background pixels in the active image Background is assumed to be the This plugin writes to a text file the XY coordinates and pixel value of all non background pixels Backround defaults to be the value of the pixel in the upper value of the pixel at the upper left corner of left corner of the image f the image For grayscale images writes three Background value 40 values per line x y and value separated by A a o eine spaces For RGB images writes five values per C Suppress Log output line x y red green and blue The origin of A h rdin m i he lower left corner beane the coordinate system is at the lower left corne of the image The number and percentage of non background pixels is printed to the Log Window if Suppress Log output is not checked SEE ALSO Edite Selection gt Properties y 146 Last updated 2012 10 02 Analyze gt Tools gt 30 14 2 Fractal Box Count Estimates the fractal dimension D of a binary image D can be used as a measure of pattern complexity D 1 8792 cell shape vascularization textures etc and is specially relevant in cases in which Euclidean measures such as diameter or length are not good des
328. with the current measurement counter value i e the number of rows present in the Results Table Selection outline and label at the selection centroid are drawn invasively using current foreground background colors As for Edit gt Draw d use the Edit Options Line Width command or double click on the line tool to change the width of selection s outline Selections can be labelled if they were previously analyzed Analyze Particles or Measure m commands and the parameter Centroid cf Set Measurements extracted in the Results table SEE ALSO Color Picker Tool XVII Embedding Color Annotations in Grayscale Images 30 6 Clear Results Erases the results table and resets the measurement counter This command is also available by right clicking on the Results Table 30 7 Set Measurements Use this dialog box to specify which measurements are recorded by Analyze gt Measure m ROI Manager s Measure command and Analyze gt Analyze Particles Measurements are performed on the current selection the entire active image if no selection is present For thresholded images Image gt Adjust gt Threshold T measurements can be restricted to highlighted pixels if Limit to Threshold is checked This command is also available by right clicking on the Results Table The dialog contains two groups of checkboxes The first group controls the type of measurements that are printed to the Results table
329. x bytes per pixel xn to skip n images for each image read White is Zero Should be checked if black pixels are represented using numbers that are less than the numbers used for white pixels If your images look like photographic negatives changing this field should fix the problem Little Endian Byte Order Probably needs to be checked when importing 16 bit or 32 bit grayscale images from little endian machines such as Intel based PCs Open All Files in Folder If checked ImageJ will import all the images in the folder as a stack The images must all be the same size and type Use Virtual Stack Images are imported as virtual stacks 53 Last updated 2012 10 02 File gt Import gt Help Opens http imagej nih gov 1j docs menus file html raw SEE ALSO Image Types and Formats 26 6 3 LUT Opens an ImageJ or NIH Image lookup table or a raw lookup table The raw LUT file must be 768 bytes long and contain 256 reds 256 blues and 256 greens If no image is open a 256x32 ramp image is created to display the LUT Note that lookup tables with file names ending in lut can also be opened using File gt Open o or drag and drop 26 6 4 Text Image Opens a tab delimited text file as a 32 bit real image see Text Images The image s width and height are determined by scanning the file and counting the number of words and lines For text files with integer values no larger than 255 use Image Typeb 8 bit to co
330. y 132 136 Clipboard 48 60 Clojure 26 CodeBar 25 Color Background 36 84 Balance 77 Blindness 15 Composites 15 Deconvolution 15 Foreground 36 84 Models 14 Quantization 14 74 Quantization see Heckbert quantization 74 Separation see Color Deconvolution 15 Settings 69 Threshold 79 Color filter array 14 182 Color palette see LUT 14 Color Picker 36 Combine 92 Command key 8 Command launcher 157 Compile 71 161 Concatenate 92 Contextual Menu 41 Control Panel 157 Conversions 70 126 Convex hull 63 Convolution 106 110 122 123 Coordinates 28 33 58 107 ido Coronal see Orthogonal views 88 Counting objects 33 34 106 Crop 96 127 CSV 42 53 58 CT 77 90 Cursorsee Pointer 45 72 Curve fitting 147 Built in functions 148 Data mining see TrakEM2 4 De interleave 93 Dead pixels 110 Debug 44 71 72 114 115 158 Deconvolution see Convolution 106 Deflate see Zip compression 10 Delaunay see Voronoi 115 Despeckle 110 Developer Menu 37 Developer resources 163 DICOM 10 72 77 Digitized graphs 146 Dilation 113 Dirichlet see Voronoi 115 Display range 74 Distribution 134 Divide see Math 117 Divide images see Image calculator 127 Dot blot analysis 137 Downsizing 80 97 DPI 55 59 Drag amp Drop 49 Duplicate 93 96 Eclipse 25 Editor 43 EDM see Euclidian distance map 114 Elliptical selection 30
331. y See eo a eS oh ee ea a 165 9111 Refresh Menisi 5 4 42 3 db Seek oA oS Sm eK amp dS eS 165 Sool Abou PICAS seka ran A Brae oe eB oa eee da sr E E 165 Sol ABOUT IMAGEN eso a a y Sd amp A oS BS Ge in wen 165 VI Keyboard Shortcuts 34 Key Modifiers AT ale Key Modilicatiols msc a a ee ts Boe Ee a EA 167 DAS on er Modica ons arde ib a ee a e we ek Sd hae 168 Aa Culore Key Nodiiemtions gt nero AE 169 del wie bal e asa ee ee Ae eee A a SS 169 Bao AON ISVs o oats a He Se he oes We eS ee Oe ds Se ee N 169 35 Toolbar Shortcuts Credits 171 ImageJ Related Publications 174 List of Abbreviations and Acronyms 181 Index 182 Colophon 186 vi Summarized Release Notes for ImageJ 1 46r Selections with sub pixel resolution a e a e a a 19 Improved handling of Overlays ooo a a a 19 Fourtneen new macro functions ooo e e Bo we ESS era 24 New tools Arrow Brush Developer Menu Flood Filler LUT Menu Overlay Brush Pencil Pixel Inspector Spray Can and Stacks Menu 38 PIUCUEOO S amp eae E todas rara daa 41 Customizable crosshair cursor 43 oe e 47 Improved AVI SUPPO stenske 8e 64484 SESEE S henee hannt 599 New command File gt Import gt XY Coordinates a a 55 Undo support extended to Edit gt Selection gt commands a a a a 63 New command Edito Selection gt Interpolate ooo aa a o 64 Edit gt Selection gt Line to Area now works with one
332. y for shortcuts in Edit Options Misc is unchecked i e except when using the IJ Editor or the Text Tool you won t have to hold down the Control key to use menu shortcuts For example the command Edit Invert I can be evoked by 1 or 1 if Require control key for shortcuts is checked The full list of ImageJ shortcuts see Keyboard Shortcuts can be retrieved at any time using the Plugins gt Utilitiest gt List Shortcuts command There are three modifier keys in ImageJ Control Command Key on Apple keyboards Denoted by Ctrl or in this document Although a control key is typically present on Apple keyboards on a Macintosh computer running ImageJ the Command key replaces the functionality of the Control key of other operating systems For sake of simplification Ctrl will always refer to both throughout this guide Shift Denoted by Shift or in this document Alt Denoted by Alt or in this document This is also the Option or Meta key on many keyboards In ImageJ it is also used to type special unit symbols such as p AM or A Ale Shite A SEE ALSO Keyboard Shortcuts Plugins gt Shortcuts gt 5 Finding Commands Navigating through the extensive list of ImageJ commands macros and plugins may be quite cumbersome Through its built in Command Finder Launcher 48 ImageJ offers an expe dite alternative that allows you to retrieve commands extremely fast Plugins Utilities
333. y tion to a text file or copy it to the system clipboard Height 384 00 mm 256 Clear Depth 193 50 mm 129 seat AI SEE ALSO Image Properties P 82 Last updated 2012 10 02 Image gt 28 4 Properties P organ of corti tif Channels c Slices z Frames t Unit of Length Pixel Width Pixel Height Voxel Depth Frame Interval Origin pixels O Global 15 1 Note c z t must equal 60 um 0 3150001 0 3150001 0 3150001 O sec 0 0 A APA Cancel OK T E Properties P Use this command to display and set various properties of the current image or stack The number of Channels c Slices z and Frames t in the image can be changed as long as the product of c z and t is equal to the number of images in the stack Unit of Length is a string describing the measuring unit of Pixel Width Pixel Height and Voxel Depth These three dimensions are automatically converted if Unit of Length is changed from one of ImageJ s known unit nm um or um or micron mm cm meter km or inch to another y and A symbols can be typed using and Alt Shift A respectively With t series stacks the Frame Interval in seconds reciprocal of the frame rate can be viewed and set If the unit is sec setting the Frame Interval will also set the frame rate used by Animation Options Alt Origin is
334. your own ImageJ bundle by gathering the plugins that best serve your needs probably someone else at your institution already started one or create symbolic links to share plugins between different installations SEE ALSO Description of all ImageJ related projects at ImageDev 2 2 Related Software 2 2 1 Software Packages Built on Top of ImageJ Bio7 Bio7 is an integrated development environment for ecological modeling with a main focus on individual based modeling and spatially explicit models Bio7 features Statistical analysis using R Spatial statistics Fast communication between R and Java BeanShell and Groovy support Sensitivity analysis with an embedded flowchart editor and creation of 3D OpenGL Jogl models see also RImageJ in ImageJ Interoperability BoneJ BoneJ is a collection of tools for trabecular geometry and whole bone shape analysis uManager Micro Manager is a software package for control of automated microscopes It lets you execute common microscope image acquisition strategies such as time lapses multi channel imaging z stacks and combinations thereof Manager works with microscopes from all four major manufacturers most scientific grade cameras and many peripherals used in microscope imaging MRI CIA MRI Cell Image Analyzer developed by the Montpellier RIO Imaging facility CNRS is a rapid image analysis application development framework adding visual scripting interface to ImageJ s capabilities
335. ys these two values as Display range Note that this scaling is not done if Scale When Converting is not checked in Edit Options Conversions RGB images are converted to grayscale using the formula gray red green blue 3 or gray 0 299 x red 0 587 x green 0 114 x blue if Weighted RGB Conversions is checked in Edit Options gt Conversions 16 bit Converts to unsigned 16 bit grayscale 82 bit Converts to signed 32 bit floating point grayscale 8 bit Color Converts to 8 bit indexed color using Heckbert s median cut color quantization algorithm A dialog box allows the number of colors 2 256 to be specified The active image must be RGB color RGB Color Converts to 32 bit RGB color RGB Stack Converts to a 3 slice red green blue stack The active image must be RGB color HSB Stack Converts to a 3 slice hue saturation and brightness stack The active image must be RGB color 28 2 Adjust gt This submenu contains commands that adjust brightness contrast threshold levels and image size 15 Last updated 2012 10 02 Image gt Adjust gt XIV APPLYING AUTO BRIGHTNESS CONTRAST TO ENTIRE STACKS The Process Enhance Contrast command can be used to adjust the brightness and contrast of each slice in a stack according to either the optimal for each individual slice if Use Stack Histogram is unchecked or the overall stack by ticking Use Stack Histogram The default behavior of the B amp

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