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The ImageJ User Guide

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1. Display options of Particle Analyzer Analyze gt Analyze Particles IJ 1 441 27 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 _ Jlabel_ Area Mean Mode Min Max Perim 63 blobs gif 5 3001 1 884E2 1 360E2 1 280E2 2 480E2 3 556E1 5 268E 1 1 603E1 9 984E3 1 840E2 1 000E2 Z 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 347 547 187 727 203 375 128 230 375 67 902 0 634 24 875 67786 250193 500 100 217 766 19 042 32 933 0 26 096 26 562 0 144 9 516 44012 25 3 111 0 1 128 128 128 128 2 828 0 406 1 414 128 128 100 902 219 915 248 128 248 132 225 1 52 202 178720 240 100 SEE ALSO Analyze Particles Distribution 27 4 Distribution Produces a relative frequency histogram from the data of a chosen column of the Results table 33 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 120 Latest revision 2011 08 06 Analyze gt Label Parameter Specifies the parameter in p i uN ES E O pixels 8 bit 70 the Result
2. Width pixels 186 e Height pixels 226 M Constrain aspect ratio M Ave rage when downsizing Bilinear Check the Average when downsizing checkbox for better results when scaling down images 16 Two resampling methods are possible Bilinear and Y Bilinear be i Cancel OK Bicubic Bicubic interpolation The implementation of the bicubic method Catmull Rom interpolation is derived from Burger and Burge 2008 51 Interpolation SEE ALSO Imager Scale E 70 Latest revision 2011 08 06 Image gt 25 2 7 Canvas Size 5 Resize Image Canvas Width pixels Height 172 pixels Position Center Top Center Top Right Center Left Y Center Center Right Bottom Left U Zero Fill 1 Cancel Bottom Center Bottom Right 25 2 8 Line Width Line Width O Spline Fit Show Info i Changes the canvas size of an image or stack without scaling the actual image Width and Height may be either expanded or contracted If the canvas size is in creased the border is filled with the current background color or if Zero Full is checked the border is filled with pixels that have a value of zero The position of the old image within the new canvas may also be specified SEE ALSO Size Color Picker K XVII Embedding Color Annotations in Grayscale Images This widget is used to adjust the width of line selec tions in pixel
3. 2 Open the Threshold T tool Shift T Adjust the threshold so the graph is highlighted in red 3 Click on Apply make sure foreground is black and background is white Y pixels 4 Use the Elliptical Selection Tool as an eraser press Backspace to erase to isolate a single curve NB background color must be white cf Color Picker 0 K 0 X pixels 132 y e 9 Select the curve by clicking to the left of it with the List Save Copy Wand Tool 6 Use Edito Clear Outside to erase everything but the curve 7 Use Analyze gt Tools Analyze Line Graph to get the XY coordinates AB Curve Fitter ABEL y a b x c x x User defined 34 Fit Open Apply M Show settings Straight Line p 2nd Degree Polynomial 20 3rd Degree Polynomial 4th Degree Polynomial Ar User defined y a b x c x x a 0 2107 b 0 4318 0 00 0 1 00 0 2 00 4 8 1 2 ou wo 3 00 4 00 15 AN 5 00 e Exponential Formula c 0 9125 Power gt R 2 0 987 A e d Formula y a b x c x x 7 Simplex Fitting Options 10 Gamma Variate Function name User defined y a b In x c Formula y a b x c x x 5 Formula y a b x c x x Rodbard NIH Image Initial a 1 00 Time 31ms Exponential with Offset Number of iterations 179 4500 Gaussian Initi
4. Grayscale C Shift JC to adjust the brightness and contrast of the current channel 25 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 0 the LUT as a CSV file Look Up Table SEE ALSO Edit LUT 13 Latest revision 2011 08 06 Merges 1 4 grayscale images or stacks into an RGB image or stack creating composite images i e when Create Composite is chosen IMPROVED IN IJ 1 45M Image Color gt 25 5 7 Edit LUT LUZ Edito Opens the ImageJ LUT Lookup Table Editor A TOS lookup table in ImageJ has up to 256 entries The a entry index and the three values red green and blue SEE pp EEE joa associated with it are displayed in the ImageJ status 90989898998 bar as you move the cursor over the LUT Editor win AHAHHH Set dow Click on an entry to edit the red green and blue EE invert values for that entry using a Color Selector window cf mammaa Color Picker K SEE ALSO Show LUT 25 5 8 Color Picker K The Color Picker tool 14 enables the user to select foreground and back
5. Rolling Ball Radius 50 0 Pixels M Light Background O Separate Colors M Create Background Don t Subtract O Sliding Paraboloid M Disable Smoothing M Preview re Help Cancel OK Removes smooth continuous backgrounds from gels and other images Based on the rolling ball algorithm by Stanley Sternberg see Sternberg S Biomedical image processing IEEE Com puter Jan 1983 Imagine a 3D surface with the pixel values of the image being the height then a ball rolling over the back side of the surface creates the background If Sliding Paraboloid is checked the command will use a more robust implementation of the algorithm that uses an approximation of a paraboloid of rotation instead of a ball 114 Latest revision 2011 08 06 Process gt Subtract Background Original Image Corrected Image Process gt Subtract Background command This command uses a sliding paraboloid or a legacy rolling ball algorithm that can be used to correct for uneven illuminated background as shown in the profiles Analyzer Plot Profile k below each image The radius should be set to at least the size of the largest object 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 u
6. SEE ALSO Freehand Selection Tool V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 3 Arrow Tool OO ___ Arrow Tool This tool shares the same toolbar slot with the Line Selection Width lt las Tools Double clicking on the tool icon opens the Options Size RP E prompt depicted on the left 6 Because it is an annotation tool changing Color will change foreground color and not Corot selection color see Point Tool Color Picker K Style Notched B O Outline Double head Width and Size in pixels can be adjusted by dragging the respective sliders or by direct input Apart from the arrow Cancel ok styles listed here a Headless option is also available As with other tools add arrows to a non destructive overlay using Image Overlay gt Add Selection b or Editt Draw d to permanently draw an arrow see XVII Embedding Color Annotations in Grayscale Images Straight Line Selection Tool key modifiers can be used when creating an arrow e g holding down will force the arrow to be horizontal or vertical 29 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45 Tools Angle Tool 18 4 4 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
7. 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 Stack setActiveChannels t1000 Jf Select first channel i e Red run Magenta Run Image gt Lookup Tables gt Magenta Stack setAcotiveChannels O I Select all the three channels run RCB Color Run Image gt Type gt RGB Color rename title MGB Rename the image setBatchMode false Restore GUI mode 16 Latest revision 2011 08 06 O oan aa fF WwW WD Rh Rp O Selections 2 REPLACE RED WITH MAGENTA IJM USING PROCESS gt IMAGE CALCULATOR 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 green b title blue run Split Channels imageCalculator Add b r run Merge Channels red amp r green amp g blue amp b rename title MGB setBatchMode false Color Composites In a composite image colors are handled through channels The advantages with this type of image over plain RGB images are 1 Each channel is kept separate from the others a
8. Copies the contents of the current image selection to the internal clipboard and fills it with the current background color SEE ALSO Edit gt Copy to System Paste Control 24 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 amount of image data copied is shown in the status bar SEE ALSO Filet gt Internal Clipboard Paste Control 24 4 Copy to System Copies the contents of the current image selection to the system clipboard SEE ALSO File New System Clipboard V Copy c Paste Control 24 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 Undo z to abort the paste operation SEE ALSO Paste Control 24 6 Paste Control pa pan Paste Control Y Copy Blend Difference Transparent white Transparent zero AND OR After pasting use the Paste Control pop up menu to con trol 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 descrip tion of Process gt Image Calculator The Blend mode is th
9. Saves the selected ROI as a roi file If no ROIs are selected saves all the ROI Manager selections in a ZIP archive Fill Alias for Edit gt Fill f Draw Alias for Edit gt Draw d Multi Plot Sort Specify Remove Slice Info Help Options AND Uses the conjunction operator on the selected ROIs to create a composite selection All ROIs are considered if none is selected OR Combine Uses the union operator on the selected 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 36 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 Analyzep 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 oo i m Multi Measure Multi Measure Measures all the ROIs on all slices in the stack creating a Results table with either one row per slice if One Row Per Slice is checked in the dialog or one row per measurement 37 M Measure All 27 Slices M One Row Per Slice Enabling both options will result in a table with 90 columns Cancel
10. Search plugins folder Extends the search scope to ImageJ plugins Search scripts folder Extends the search scope to ImageJ scripts if present Search source folder Extends the search scope to 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 2j jar implementing this command Find Commands lI Finding Commands 28 3 4 Monitor Events IJ 1 45M 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 gt Options Misc 141 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Plugins gt Utilities gt 28 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 Clicking on the window will reclaim unused memory by running the Java garbage collector cf Status bar Ideally you should be able to open sev
11. 2 Latest revision 2011 08 06 Image gt Color gt 25 5 2 Merge Channels Arr Color Merge Select None to keep a channel empty filled with 0 Red C 1 tif e as Ss Check Keep Source Images if you wish to keep the originals Check Green C 2 tif 4 Create Composite to convert 2 4 grayscale images or stacks into a Blue C 3 tif F composite image or hyperstack see Color Composites Gray None F Merge Channels will preserve color LUTs and display ranges when W Create Composite O Keep Source Images You can avoid this behavior either by not checking Create composite Ignore source LUTS in the dialog box or by checking Ignore source LUTs checkbox es 1 i Cancel SEE ALSO Channels Tool Z 25 5 3 Channels Tool Z Alias for Image gt Hyperstacks gt Channels Tool Z 25 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 gt HeLa Cells 1 3M 48 bit RGB into RGB 25 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 Make Composite Display Mode ds Y Composite Color the channels of a composite image Use Brightness Contrast
12. 3 Fill Color none SA Cancel O A E E With text selections see Text Tool it is also possible to specify the font size and text alignment Opens a dialog box that allows the user to assign a contour color Stroke Color and a contour width Width to the active selection or a filling color Note that selections can be either filled or contoured but not both The nine default selection colors black blue cyan green Any other color must be typed using hex notation see XIX Hexadecimal Color Values Y Note that while this command can only be applied to the active selection the ROI Manager s Properties command Analyzeb Tools RO Manager can be applied to multiple ROIs SEE ALSO Selections Image Overlay gt Add Selection b 24 12 12 Rotate MP Rotate Selection Angle degrees Enter negative angle to rotate counter clockwise F E k Cancel OK ee 0 E 24 12 13 Enlarge Enlarge Selection Enlarge by as pixels Enter negative number to shrink SS Cancel OK 4 A P Rotates the selection by the specified number of degrees negative number indicate counter clockwise rotation This command runs the RotateSelection macro in 27 7ar SEE ALSO FlipSelection macro Grows an area selection by a specified number of pixels Enter a negative value to shrink the selection This command runs the EnlargeSelection macro in 27 jar ShrinkSelecti
13. 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 Shift D d only the key and assumes that Require control key for shortcuts in Edit gt Optionse Misc is unchecked Useful tips and reminders are placed in Noteworthy notes numbered with upper case roman numerals e g I Frontmost Window and Windows Activation The full list of these notes is available on page viii Filenames directory names and file extensions are indicated in italic e g the Application s ImageJ macros folder 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 1 Replace Red with Magenta ijm Using Image Color gt Channels on page 16 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 45m are listed on page vii and flagged with colored marginal notes These should be interpreted as a ay A new feature implemented in ImageJ 1 45m A routine that has been improved since previous versions Typically a faster a Tal den or more precise algorithm a command with better usability or a task that has been extended to mo
14. 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 This 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 SEE ALSO Orthogonal Views H Z Project Grouped Z Project and 3D Viewer 13 Volume Viewer plugins 80 Latest revision 2011 08 06 Image gt Stacks gt 25 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 selection or the bounding rectangle for non rectangular selections are displayed in the graph title SEE ALSO Profile Plot Options Plot Profile fk z h o NA NN j X 25 Ye27 0 Save Copy 25 6 14 Label OO 0 StackLabeler Adds a sequence of numbers e g timestamps and or a label Format birmi to a stack or hyperstack Numbers and label are drawn in the current foreground color cf Image gt Colors gt Color Picker Starting value Interval 90 00 00 K l A REENE Text The initial X Y location and Font size of the label are based y location on the existing rectangular selection if any
15. Michael Schmid contributed improvements to the AVI reader and AVI writer plugins Karen Collins contributed improvements to the FITS Writer File gt Save Asp FITS command The Edit gt 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 The Color Picker Image Color gt 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 gt 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 control as well as mouse wheel control The Imager Stacks gt 3D Project w
16. 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 in the plugins folder are listed at the bottom of the Plugins gt menu Only class and jar files in the plugins folder with at least one underscore in their name will be installed Note that with IJ 1 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 ij 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 15 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 Alber
17. 82 Contextual Menu 34 Control Panel 140 Conversions 45 47 60 64 93 112 117 122 127 Convex hull 54 Convolution 93 97 108 109 Coordinates 25 30 49 94 124 131 Coronal see Orthogonal views 78 Counting objects 30 93 Crop 85 113 CSV 35 45 49 CT 67 79 Curve fitting 132 163 D Data mining see TrakEM2 3 Dead pixels 97 Debug 37 62 63 100 101 141 Deconvolution see Convolution 93 Deflate see Zip compression 10 Delaunay see Voronoi hyperpage 101 Despeckle 96 Developer resources 146 DICOM 11 62 67 Dilation 99 Dirichlet see Voronoi 101 Display range 64 Distribution 121 Divide see Math 103 Divide images see Image calculator 113 Dot blot analysis 123 Downsizing 70 86 DPI 47 50 Drag amp Drop 41 Duplicate 83 86 E Editor 36 EDM see Euclidian distance map 100 Elliptical selection 27 Eroded points 100 Erosion 99 Ethics see Acceptable manipulation hyperpage 5 Euclidian distance map 94 100 101 Exponential transformation 104 Eye dropper 32 F Fast Fourier transform 105 Fast Hartley Transform 105 Feret 123 Pili Devons los Dl 2202 Filters 107 109 111 Find see Search 140 Fit Circle 54 Cubic spline 54 56 Curve see Curve fitting 132 Ellipse 54 122 FITS 11 49 Flatten 90 Fractal box count 131 Frame interval 72 Freehand area selection 28 Freehand line selection 29 Frequency domain 105 G Gamm
18. Bicubic interpolation is preferred over bilinear e g Edit 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 background in Process Binary Options see XXI Interpreting Binary Images 6 Background color is black and foreground color is white Edit Options Colors are set properly at startup the following AutoRun macro could be appended to the Startup Macros txt file 3 ENSURING SPECIFIC SETTINGS AT LAUNCH macro AutoRun setOption DebugMode true setOption Bicubic true setOption Display Label true setOption Limit to Threshold false setOption BlackBackground true setBackgroundColor 0 0 0 setForegroundColor 255 255 255 run Colors foreground white background black SEE ALSO FAQ s on ImageJ Documentation Wiki 19 Latest revision 2011 08 06 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 T
19. Dawson A Simple Quantitative Bedside Test to Determine Methemoglobin Ann Emerg Med Oct 2009 J nos Roszik Duarte Lisboa J nos Sz llosi and Gy rgy Vereb Evaluation of intensity based ratiometric FRET in image cytometry approaches and a software solution Cytometry A 75 9 761 7 Sep 2009 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 Stephan Saalfeld Albert Cardona Volker Hartenstein and Pavel Tomanc k CATMAID collabo rative annotation toolkit for massive amounts of image data Bioinformatics Oxford England 25 15 1984 6 Aug 2009 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 156 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 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 G Land
20. Multi Plot Runs Analyzep Plot Profile k on the selected ROIs on a single graph 38 All selections are plotted if none is selected When plotting less than seven selections colored lines are drawn blue ROI1 green ROI2 magenta ROI3 red ROI4 cyan ROI5 and yellow ROI6 Profiles with more selections are drawn in gray tones While Analyze Plot Profile k requires a line or rectangular selection Multi Plot accepts all type of selections by first running Edit gt Selection gt Area to Line which converts oval and freehand ROIs to line selections Sort Sorts the list in alphanumeric order Specify Alias for Edito Selection gt Specify Remove Slice Info Removes the information in the ROI names that associates them with particular stack slices see ROI Manager Help Opens http imagej nih gov ij docs menus analyze html manager Options Displays a dialog box depicted in ROI Manager that allows you to set several ROI Manager settings 135 Latest revision 2011 08 06 IJ 1 45M N IJ 1 45M Analyze gt Tools gt Logical operations using the ROI Manager Analyze gt Tools ROI Manager Show All Color Defines the color in which the ROIs are highlighted when Show All is pressed in ROI Manager panel 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 Resto
21. Slices outside the ECE ETA kange are not affected oo 1 36 Format Specifies the structure of the label 0 Un DO LM padded sequence 0000 Pads each number with lead Use overlay ing zero s 00 00 Converts the label into a min a taoi font utes seconds timestamp 00 00 00 Converts the label into a hours minutes seconds timestamp Text Stamps Help _ Cancel only the contents of the Text field 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 Use text tool font If checked labels will be created using the type face and style specified in the Fonts widget Help Opens http imagej nih gov ij docs menus image html label SEE ALSO Make Montage Stacks Tools gt Insert 81 Latest revision 2011 08 06 Image gt 25 6 15 Tools Stacks gt 25 6 15 1 Combine Pl Combiner Combines two stacks Width x Height x Depth W xH xD and W 2xH xD2 to create a new Stackl mri stack 1 tif 14 W W gt xmax H Ho xmax Dy Do stack E g a 256x256 40 Stack2 mri stack 2 tif F and a
22. This command does not alter pixel values as long as Normalize 7 Equalize Histogram or Normalize All n Slices in the case of stacks Cancel ok are not checked Saturated Pixels Determines 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 images Equalize Histogram If checked ImageJ will enhance the image using histogram equalization 25 Create a selection and the equalization will be based on the histogram of the selection Uses a modified algorithm that takes the square root of the
23. angle point multipoing text macro Cycle Tools Fwd Fi setTool tools tIndx GCindxs e if tIndx tools length tIndx 0 macro Cycle Tools Rwd F2 tIndx if tIndx lt 0 tIndx tools length 1 setTool tools tIndx 152 Latest revision 2011 08 06 Credits C1 C2 C3 C4 C5 C6 C7 Cs C9 C10 011 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 ij 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 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 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 gt Importe Stack From List command in IJ 1 45f
24. detects a divide by Hide Process Stack dialog zero while dividing one 32 bit real image by an A epee other The default is infinity In addition to nu _ Move isolated plugins to Misc menu o O Run single instance listener meric values infinity positive or negative in J Debug mode finity max largest positive value and NaN A AAA Not a Number can be entered as the Divide by Cancel OK zero value Use pointer cursor If checked ImageJ will use an arrow cursor instead of the default cross hair 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 cross hair 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 Plugins menu preventing it from running off the bottom of the screen When this option is enabled plugins that attempt install themselves in a submenu with only one command are instead installed in the Plugins gt Miscellaneous submenu An example of such a plugin is TurboReg which normally creates a Pl
25. jfly iam u tokyo ac jp color accessed 2009 01 15 15 Latest revision 2011 08 06 O ana a FP W WD Rh Rp 12 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 IV REPLACING RED WITH MAGENTA IN RGB IMAGES For two channel images magenta can be obtained by selecting the same source for both the red and the blue channels in the Image Color gt Merge Channels dialog RGB images can be converted to MGB using Image Colort 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 1 and 2 Once saved in the ImageJ plugins folder these macros are treated as regular ImageJ commands see Macros In Fiji as expected the procedure is even more simple one just needs to run Image 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 1 REPLACE RED WITH MAGENTA IJM USING IMAGED COLOR gt CHANNELS
26. l ImageJ User Guide ImageJ User Guide IJ 1 45m Tiago Ferreira Wayne Rasband August 6 2011 FOREWORD The ImageJ User Guide provides a detailed overview of ImageJ and inherently Fiji the standard in scientific image analysis 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 45m Noteworthy Macro Listings Guide Conventions I Getting Started 1 What is ImageJ 2 Installing and Maintaining ImageJ 2 1 ImageJ Distributions 2 2 Software Packages Built on Top of ImageJ Zio AMAT rd a 3 Getting Help 3 1 Help on Image Analysis owe Help onlmaded oee e ads s II Working with ImageJ 4 Using Keyboard Shortcuts 5 Finding Commands 6 Undo and Redo 7 Image Types and Formats 8 Stacks Virtual Stacks and Hyperstacks 9 Color Images 10 Selections 11 Settings and Preferences HI Exten
27. 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 Options gt 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 numbers 10 Latest revision 2011 08 06 Stacks Virtual Stacks and Hyperstacks 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 Filer gt Import 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 DI
28. 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 Shift F Flatten F to create an RGB image with the overlay embedded in it The overlay is preserved when an image is saved in TIFF format cf II Image Types Lossy Compression and Metadata Non destructive image overlays Outputs from Grid Overlay MakeOverlay and ROI Color Coder macros that exemplify the usage of most Image Overlay gt submenu commands 25 14 1 Add Selection b Adds the current selection to the overlay displaying the dialog box O Add to Overlay depicted on the left Stroke Color Stroke Width and Fill Color gt gt to be set Except for text selections and as explained in Edit Selection gt Properties y the stroke line color and width are a ignored if a fill color is specified Fill Color none Colors are specified using the name of one of the eight default colors black blue cyan green magenta red white and yellow or using hex notation see XIX Hexadecimal Color Values O New Overlay Use the alt b shortcut POSER Pa en Holding will immediately add the selection to the overlay Cancel C o gt skipping this dialog Previously added overlays are removed if New Overlay is checked SEE ALSO Selectio
29. white yellow can be typed textually Any other color must be specified using hex notation see XIX Hexadecimal Color Values If multiple ROIs have been selected from the Manager s list see XXIII Selecting Multiple 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 a non destructive overlay that outlines all ROI Manager selections If Edit Mode is active selections will also be labelled In this case click on a label selection number in the overlay to activate the corresponding selection That selection can then be moved or edited and the overlay will be dynamically updated The color and behavior of the Show All overlay can be changed in the Mores Options dialog box Edit Mode Toggles selection labels displayed by Show All As mentioned clicking on a label activates its selection allowing edit operations Mores Displays a drop down menu with several additional commands this menu is also available when right clicking on the ROI Manager s list area 134 Latest revision 2011 08 06 Analyze gt Save Fill Draw AND OR Combine XOR Split Add Particles Multi Measure Tools gt Open Opens a roi file and adds it to the list or opens a ZIP archive zip file and adds all the selections contained in it to the list Use the Open All macro to add all the roz files in a folder Save
30. 1 280E2 2 480E2 1 805E2 2 302E2 1 137E2 ee Cut 2 160E2 1 280E2 2 240E2 4 654E1 2 410E2 5 453E1 Copy C 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 61 blobs Clear 3 468E1 1 280E2 1 280E2 2 480E2 1 790E2 2 518E2 4 238E1 Results 62 blobs Select All 2 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 ui 874E1 1 760E2 1 280E2 2 240E2 7 417E1 2 525E2 3 673E1 Distribution Ja ummarize 7 TEE Set Measurements Distribution Options me Duplicate ImageJ Results table version 1 44k Columns width can be adjusted by clicking on and dragging the vertical lines that separate the column headings Selected lines can be deleted by pressing the backspace key The arrow keys can be used to vertically scroll the window 20 Results Table Most of ImageJ analyses are printed to the Results table Table commands are organized in four menus File gt Edit Font 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 exclusi
31. 1j developer api ij measure CurveFitter html Function Straight Line 2 Degree Polynomial 3 Degree Polynomial 4 h Degree Polynomial 5 Degree Polynomial Exponential Power Log Gamma Variate Rodboard Exponential with Offset Gaussian Exponential Recovery Formula y aw bzx y a br cr y a bz cz dr y a bz cx dr ext y a bz cz dr ex fa y ax e y ax y a x ln be y a x x b x eT _ d a d y a H y axe 4 x c y a b a xe 2a y ax 1 e Dd e Comments cf Pixel Inverter Calibrate Via the User defined option 35 35 cf FRAP Profiler plugin 4 Click Apply to create a 32 bit copy of the current image transformed with the chosen function SEE ALSO Profile Plot Options CurveFittingDemo RodbardSigmoidFit and PlotSigmoid Derivatives macros 27 14 5 ROI Manager IMPROVED IN IJ 1 45M The ROI Region of Interest Manager is a tool for working with multiple selections The 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 T 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 second is the
32. 29 Straight Line 28 Macro Tools 33 Macro Toolset Switcher 33 Magnifying Glass 32 Multi point 30 Point 30 Scrolling 32 Text 31 Toolset 33 Topological Skeleton see Skeleton 100 Tracing see Wand tool 31 TrakEM2 3 Transform 55 87 Translate 87 Transparency see Blend 92 Tutorials 6 U Undo 9 50 61 Unsharp mask 111 Updates 2 147 y Variance 111 Virtual stacks see Stacks Virtual 13 Voronoi 101 Voxel depth 72 W Wand tool 31 Watershed 94 101 Web browser 146 Window management 145 Windows OS 2 X XOR see Logical operations 114 135 166 Y Yen see Threshold 68 YUV 11 14 69 70 Z Z Profile 58 79 81 128 Z Projection see Projection 18 19 80 83 Z1P 41 47 135 Archived TIFF files 10 Compressed TIFF 47 Lossless compression 10 Zoom 32 88 Fit to Screen 88 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 website ImageJ Documentation Portal Fiji webpage or Tony Collins ImageJ for Microscopy manual Nevertheless because there has never been accompanying documentation for some of the 300 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
33. 45m has been intentionally omitted The guide was typeset with T XLive 2011 on Mac OS 10 6 8 All illustrations were created with Grab 1 5 and Fiji ImageJ 1 43 1 45m loaded with G Landini s IJ Robot and J Schindelin s Tutorial Maker plugins The HTML version was produced with eLyXer 1 2 and formatted using CSS code from Alex Fern ndez and Michael Hiineburg JavaScript code from Stuart Langridge and Ciar n O Kelly 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 strong 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 your experience may be valuable to others Do you want ImageJ to improve You can report
34. 68 Linear regression see Curve fitting 132 Linux 2 LOCI Bio Formats 2 11 Log 104 Logical operations Images 114 ROIs 135 Lossless compression 10 Lossy compression 12 47 LUT 14 44 49 91 LZW compression 10 M Mac OS X 2 Macro functions 37 146 Macro recorder 138 Macro Tools 33 Macro Toolset Switcher 33 Macros 20 105 112 138 Install 138 Magenta Green Blue MGB 16 Magnifying Glass Tool 32 164 Mailing lists see Help resources 6 Mask 54 98 99 111 Math 102 MATLAB see Interoperability 24 Max 104 MaxEntropy see Threshold 68 Maxima 93 MBF ImageJ 3 6 13 Measure 112 117 Measurements 122 Median cut algorithm see Color quantization 64 Memory 25 60 142 Metadata 12 71 pManager 3 MIJ see Interoperability 24 Min 104 MinError see T hreshold 68 Minima 94 MIP see Projection 79 MJPG 11 Modeling see TrakEM2 and Bio 3 Modifier keys 8 148 150 Moments see Threshold 68 Montage see Stacks Montage 76 Morphological operators 98 Multi Plot 135 Multiplicative inverse see Reciprocal 105 Multiply see Math 103 Multiply images see Image calculator 113 Multi point tool 30 N NaN 97 105 Noise 93 94 96 110 114 Non background pixels 131 O Opacity see Blend 92 Optical density see Calibration Optical density 125 Options see Settings 19 57 OR see Logical operations 114 135 Orthogon
35. 8 bit images to have inverted pixel values where white O and black 255 This is done by both inverting the pixel values and inverting the LUT Use the Image Lookup Tablest 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 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 OS X Help Opens http imagej nih gov ij docs menus edit html fappearance 24 13 11 Conversions Conversion Options V Scale When Converting Use this dialog to set options that control how wi Weighted RGB Conversions 0 30 0 59 0 11 images are converted from one type to another EE TA Cancel j OK AAA 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
36. 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 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 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 Jun Xu and Dong Chen Desing and Implementation of the Image Format Batch Conversion Software Based on ImageJ Astronomical Research and Technology Publications of National Astronomical Observatories of China ISSN 1672 7673 5 270 Sep 2008 157 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 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 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 Madeline Pool Joachim Thiemann Amit Bar Or and Alyson E Fournier NeuriteTracer a
37. Drag amp Drop sd da ls ae A 13 IV Replacing Red with Magenta in RGB Images 16 V Loeeling Calibraved UMSS AR ee 26 VI Opening File Paths in the Log Window e 38 VI Organizme Commands in the Menu Bar 0 os eo ea a RR AA 39 VIII Opening Files File Open File Import and Drag amp Drop 41 IX Reducing Memory Requirements When Importing Images 43 X Warming on JPEG compressi se ites one oe Teie E E 47 XI Drawing Lines Wider Than One Pixel 0002 wees 52 XII Transferring Selections Between Images 2 0000000 53 XIII Converting Composite Selections o sooo ee 55 XIV Applying Auto Brightness Contrast to Entire Stacks 65 XV Display Range of DICOM Images aaa a a id a 67 XVI Brightness Contrast of High Bit Depth Images 67 XVII Embedding Color Annotations in Grayscale Images 15 XVII Serollime Zoomed Mares st aea aaa a E e be BSE RS Ge BE 8 eR 88 ALX Hexadecimal Color Vall s o oo o a a 92 XM Creatine Binary Masks coa e da Rk a a a ds S 99 LAS Amterpretine Binary Maltes esta cd a A Ba e AA 103 XAM Glopa Calibrations ta sd a ob eee A e ee G 126 XXIII Selecting Multiple ROIs in the ROI Manager 136 vili Macro Listings WwW CO N O OH Replace Red with Magenta ijm Using Image Color gt Channels 16 Replace Red with Magenta ijm Using
38. 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 6 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 7 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 8 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 Platelets 21 1 60 6 Feb 2010 9 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 10 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 Physi
39. Green Blue Cc lt gt am lt gt E lt gt amm lt gt C lt gt E lt gt Cyan z 4 gt Q lt ze Q lt z gt 3 4 Channel manipulations in composite images using the Image Color gt Channels Tool Z tool 25 9 Duplicate D ANO Duplicate Title mitosis 1 tif 7 o aa Creates a new window containing a copy of the active image or rectangular selection For stacks and hyperstacks it is possible to specify the range of Channels c Slices z and Frames t to be Y Duplicate hyperstack Channels c 1 2 Slices z 1 5 duplicated Frames t 1 51 With single images hold to skip the dialog box E Cancel OK 25 10 Rename Rename Title mri stack tif a A Cancel OK 25 11 Scale E IN IJ 1 45M X Scale 1 0 Renames the active image 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 Y Scale 1 0 Z Scale 1 0 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 16 Also when downsizing smoothing the source image prior to scaling Width pixels 186 Height pixels 226 Depth images Interpolation Bicubic O Fill with background color wi Average when downsizing M Process
40. Mort Quantitative analysis of patch patterns in mosaic tissues with ClonalTools software J Anat 215 6 698 704 Dec 2009 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 Emel Bulut and B nyamin Sahin A new method of assessing the size of mandibular cysts on orthopantomograms projection area fraction J Craniofac Surg 20 6 2020 3 Nov 2009 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 Jason R Swedlow and Kevin W Eliceiri Open source bioimage informatics for cell biology Trends in Cell Biology 19 11 656 60 Nov 2009 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 Nicola Goodall Lilian Kisiswa Ankush Prashar Stuart Faulkner Pawe 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 Fathima Shihana Dhammika Menike Dissanayake Nicholas Allan Buckley and Andrew Hamilton
41. Note that when selecting Overwrite or 8 bit output distances larger than 255 are labelled as 255 26 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 100 Latest revision 2011 08 06 Process gt Binary gt Original Segmentation Movie Watershed result og y Process gt Binary gt Watershed running in Debug mode 26 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 of the region of another growing UEP Watershed segmentation works best for smooth convex objects that don t overlap too much Enable debugging in Edit Options Misc and the Watershed command will create an anima tion that shows how the watershed algorithm works cf online exam
42. O_o Gamma the image or selection where 0 1 lt y gt 5 0 For RGB images Value 1 loz this function is applied to all three color channels For 16 bit e images the image min and max are used for scaling instead of M Preview 255 a e Cancel OK Me sy a SEE ALSO GammaCorrection Tool macro 26 9 11 Set Fills the image or selection with the specified value 26 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 26 9 13 Exp Performs an exponential transform on the active image or selection 26 9 14 Square Performs a square transform on the active image or selection 26 9 15 Square Root Performs a square root transform on the active image or selection 104 Latest revision 2011 08 06 Process gt FFT gt 26 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 26 9 17 NaN Background Sets non thresholded pixels in 32 bit float images to the NaN Not a Number value For float images the Apply opti
43. 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 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 qual ity 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 in creased 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
44. Popup Menu macro From this point you can decide what to do according to what item was chosen 4 CUSTOMIZING THE IMAGE POPUP MENU 1 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 macros StartupMacros txt and several other macro toolsets 2 3 4 var pmCmds newMenu Popup Menu newArray Help Rename Duplicate gt Original Scale Paste Control T n Record Capture Screen Monitor Memory Startup Macros Search Rind Maxima 5 6 macro Popup Menu 7 cmd getArgument s if cmd Help 9 showMessage About Popup Menu 10 To customize this menu edit the line that starts with n t 11 we Yar pmemds in Image macros startupMacros trt 12 else 13 run cmd 14 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 3 var pmCmds newMenu Popup Menu newArray Help Rename Duplicate A 0riginal scale Paste Control A Record C Capture Screen Monitor Menor sotarcup Macros Wy search 3 hind Maxima 2 Subtract Background 34 Latest revision 2011 08 06 Results Table Results Save As 38S Min Rename 2 320E2
45. R Learte and Alicia Hidalgo DeadEasy Caspase Automatic Counting of Apoptotic Cells in Drosophila Plos One 4 5 e5441 Jan 2009 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 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 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 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 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 Ricardo 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
46. Save As 50838 MetaDataByteCounts va Cut 50839 MetaData value 22022 Copy Most of the general shortcuts described in ImageJ Metadata Clear l 0 6c61626c 336 slice labels Select All Editor apply to the Log window import files are printed to the log window when ImageJ runs in Debug Mode In the Log Window double click on a file path 1 ImageJ samples mitosis test data zip to have it open by ImageJ 38 Latest revision 2011 08 06 Part V Menu Commands As described in the 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 images including geometric transformations Process 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 ImageJ plugins directory Window 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 cl
47. Saves the active image in GIF format RGB images must first be converted to 8 bit color using using Image Type 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 23 10 3 Jpeg Saves the active image in JPEG format Edit JPEG Quality Edit gt Optionsp 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 files 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 in Pixels to 300 Known Distance to 1 and Unit of Length to inch will set the DPI to 300 Since IJ 1 43 and later overlays are embedded when saving in Jpeg format cf Flatten F SEE ALSO II Image Types Lossy Compression and Metadata X Warning on JPEG compression X WARNING ON JPEG COMPRESSION The JPEG format uses lossy compression that can result in artifacts You should never used this format on images to be
48. Specific documentation on advanced uses of ImageJ macro programming plugin development etc is discussed in Extending ImageJ 1 The ImageJ online documentation pages Can be accessed via the Help gt Documentation command 2 The Fiji webpage http fiji sc 5 Latest revision 2011 08 06 Getting Help Help on ImageJ 3 The ImageJ Information and Documentation Portal ImageJ wiki http imagejdocu tudor lu doku php 4 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 5 The ImageJ for Microscopy manual http www macbiophotonics ca imagej 6 Several online documents most of them listed at http imagej nih gov ij links html and http imagej nih gov ij docs examples 7 Mailing lists a ImageJ http imagej nih gov ij 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 b Fiji users http groups google com group fiji users For user discussion specific to Fiji rather than core ImageJ
49. 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 stacks with a tif extension open as virtual stacks when dragged and dropped on the tool bar icon AAO Image Aa os A4 007141412 10 7 8 a gt gt lt lt Open as Virtual Stack gt gt E 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 Hyperstacks gt submenu 9 Color Images ImageJ deals with color mainly in three ways pseudocolor images RGB images RGB HSB stacks and composite images This section is partially extracted from the MBF ImageJ online manual at http www macbiophotonics ca imagej colour_image_processi htm 13 Latest revision 2011 08 06 Color 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 tabl
50. This menu contains commands related to statistical measurements on image data profile and histogram plotting and plugins related to image analysis 27 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 first four tools in the toolbar cf Area Selection Tools For linear selections Straight Segmented and Freehand lines cf Line Selection Tools length and angle straight lines only are also calculated For Point selections cf 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 cf Point Tool 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 green 0 114 x blue if Weighted RGB Conversions is checked in Edit gt Options gt Conversions Intensity statistics Mean Modal Median Min amp Max Gray Value Standard Deviation and Integrated Density can be performed on area line and mu
51. 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 2 add and draw by pressing and add and advance to the next slice by pressing 4 Update Replaces the selected ROI on the list with the current selection With IJ 1 45 it also updates the z t position of the ROI in stacks hyperstacks 2 i 133 Latest revision 2011 08 06 Analyze gt Tools gt ROI Manager Rename As Triangle Comas ROI Manager 0013 0057 0051 addi gt 0026 0175 0067 e Show All in Edit Mode 0026 0112 0054 Update 0026 0186 0141 0001 0092 0095 Delete 0024 0098 0156 Rename 0026 0018 0141 fe Measure X coo rdinate Da Deselect Y coordinate s Slice number Show All mri stack 1 tif AOA Annotatian X OO O mri stack 1 tif Properties Name Some Annotation Stroke Color white Font Size 16 More Fill Color 477000088 Options LO Options Flatten F iv Show All M Edit Mode Show All Col
52. 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 handler Notice also the filled handler in the polygon selection and the absence of point handlers in Composite Selections 17 Latest revision 2011 08 06 Selections Most of commands that can be useful in defining or drawing selections are available in the Edit gt Selection submenu 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 polygon selections can be adjusted by Alt Shift clicking cf Polygon Selection Tool 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 A selection can be transferred from one image window to another by activating the destination window and using Edit gt Selection gt Restore Selection E Selections can be saved to disk using File gt Save Asb Selection and restored using File gt Open Use the ROI Manager Analyze gt Tools gt ROI Manager to work with multiple selections Selections can be moved by clicking and dragging as long as the cursor is
53. also saved in the TIFF header cf File gt Save s None of the above is saved in other formats cf Native Formats ABE FAA MAX mitosis tif MAX mitosis E l a ME idas x17 35 um Stacks and Hyperstacks in ImageJ File Open Samples Mitosis 26MB 5D stack Hyperstacks dimensionality can be reduced using the Image gt Hyperstacks gt Reduce Dimensionality Image Stacks gt Z Project or Image Hyperstacks gt Channels Tool Z commands The V on the window titles denotes a virtual image see Virtual Stacks pixel which represents 2D image data in a bitmap image becomes a voxel volumetric pixel 1 e an intensity value on a regular grid in a three dimensional space All the slices in a stack must be the same size and bit depth A scrollbar provides the ability to move through the slices and in ImageJ 1 43 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 c
54. 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 24 13 12 Memory amp Threads AOLA Memory 64 bit Use this dialog to specify the maximum amount of IS de memory available to ImageJ and the number of threads Parallel threads for stacks 2 used by filters when processing stacks Java applications vi Keep multiple undo buffers such as ImageJ will only use the memory allocated to Run garbage collector on status bar click them typically 640 MB but this dialog allows the user Help Cancel OK gt to 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 60 Latest revision 2011 08 06 Edit gt Options gt set the memory allocation if ImageJ is run from the command line or by double clicking on 2 JQr Maximum memory 64 bit OS and a 64 bit version of Java are requir
55. being created or adjusted Press Analyze gt Measure m to record the angle in the Results Table 18 5 Point Tool Use this tool to create a point selection to count objects or to record pixel coordinates The following modifier keys apply to the Point Tool Shift Shift clicking will add more points Point count is displayed on the Status bar Alt clicking on a point will delete it Alt clicking and dragging with the rectangular or oval selection tool will delete multiple points Double clicking on the point tool icon or running Edit E la Options Point Tool displays its configuration dialog box e ae ee Mark Width If greater than zero a mark of the specified O Auto Next Slice diameter will be permanently drawn in the current fore L Add to RO Manager ground color cf Color Picker K Note that marks E Label la modify the image it may be wise to work with a copy Selection Color magenta WN and color marks are only available with RGB images see XVII Embedding Color Annotations in Grayscale Images A FS Cancel OK A a 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 gt 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 auto
56. biased towards the life sciences To reference ImageJ one of the following citations is possible 1 Rasband W S ImageJ U S National Institutes of Health Bethesda Maryland USA imagej nih gov ij 1997 2011 2 Abramoff M D Magalh es P J and Ram S J Image Processing with ImageJ Biophotonics International 11 7 36 42 2004 PDF 102 To reference this document Ferreira T and Rasband W 2010 2011 The mageJ User Guide IJ 1 45 imagej nih gov ij docs guide 1 Janos Kriston Vizi Ng Wee Thong Cheok Leong Poh Kwo Chia Yee Joan Sim Poh Ling Rachel Kraut and Martin Wasser Gebiss an imagej plugin for the specification of ground truth and the performance evaluation of 3d segmentation algorithms BMC Bioinformatics 12 232 2011 2 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 3 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 4 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 5 Michael J M
57. checkbox SEE ALSO Settings and Preferences XX Creating Binary Masks 26 9 2 Subtract Subtracts a constant from the image or selection With 8 bit and 16 bit images results less than O are set to 0 26 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 26 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 Options Misc 26 9 5 AND Does a bitwise AND of the image and the specified binary constant 103 Latest revision 2011 08 06 Process gt Math gt 26 9 6 OR Does a bitwise OR of the image and the specified binary constant 26 9 7 XOR Does a bitwise XOR of the image and the specified binary constant 26 9 8 Min Pixels in the image with a value less than the specified constant are replaced by the constant 26 9 9 Max Pixels in the image with a value greater than the specified constant are replaced by the constant 26 9 10 Gamma Applies the function f p 255 Y x 255 to each pixel p in
58. clipboard Clear Backspace Erase selection to background color Fill f IF Fill selection in foreground color Draw d D Draw selection Invert I Shift T Invert image or selection Selection gt Select All a A Select entire image Selection gt Select None A Shift A Remove selection Selection gt Restore Selection E Shift E Restore previous selection Selection gt Properties y Y Defines selection properties Selection gt Add to Manager t IT Add selection to ROI Manager Image Adjust gt Brightness Contrast C Adjust gt Threshold T Shift C Shi T Adjust brightness and contrast Adjust threshold levels List of ImageJ 1 45m built in shortcuts This table can be obtained within ImageJ using the Plugins Shortcuts gt List Shortcuts command Command Operation Shortcut Description Show Info i T Display information about active image Properties P Shift P Display image properties Color gt Color Picker K Shift K Open Color Picker Stacks Next Slice gt gt or 2 Go to next stack slice Stacks gt Previous Slice lt I lt or Ie Go to previous stack slice Stacks Reslice TA Reslice stack Stacks gt Orthogonal Views H Shift H Toggle or
59. composite ROI into a polygon using the Edit gt Selection gt Enlarge command see XIII Converting Composite Selections SEE ALSO ROI2PolylineROI macro 18 Latest revision 2011 08 06 Settings and Preferences 11 Settings and Preferences ImageJ preferences are automatically saved in a preferences file the IJ prefs txt text file This file is stored in the Library Preferences folder on Mac OS X in the imagej folder on Linux and in the ImageJ folder on Windows Several macros and plugins also write parameters to this file If the IJ prefs txt is erased ImageJ creates 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 The setOption macro function can be used to set this and several other ImageJ options cf Built in Macro Functions setOption Calling this function from the AutoRun macro in the StartupMacros txt file ensures preferences are set each time ImageJ starts So e g to make sure that 1 TIFF tag values are displayed by ImageJ Debug Mode in Edit gt Options Misc 2
60. 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 dimensions 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 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 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 25 6 7 Stack To Images Converts the slices in the current stack to separate image windows SEE ALSO Stacksp gt Images To Stack 25 6 8 Make Montage Produces a single image which contains the images from a stack displayed in a grid format This can be useful for visual comparisons of a series of images stored in a stack and to create panel figures With hyperstacks that are not directly supported instructions are displayed that ex
61. current E Box Ub pixels 16 b 206 pixels stack or hyperstack 19 E g if a stack displays sagittal sections 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 Shift 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 Imagep Properties P SEE ALSO 3D Project and 3D Viewer 13 Volume Viewer Stack Slicer Display3_ TP plugins Edit gt Options gt DICOM 25 6 11 Z Project Projects an image stack along the axis perpendicular to image plane the so called z axis 18 With hyperstacks the projection s 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 Average Intensity projection outputs an image wherein each pixel stores average intensity over all images in stack at corresponding pixel location 78 Latest revision 2011 08 06 Image Stacks gt ZProjection Averag
62. eG ee ey ee oe SG He a es 151 O10 JATTOW Keys oa amp bw ene Dee oh Seo ed OSS Ree ae eR RSS 151 32 Tools Shortcuts Credits 153 ImageJ Related Publications 155 List of Abbreviations and Acronyms 162 Index 163 Colophon 167 vi Summarized Release Notes for ImageJ 1 45m Undoable Edite Undo J2 gt lt lt o e c cooonsae rs rro rada 9 ImageJ can now open ZIP compressed TIFFs o ee eae 10 New macro functions drawString text x y background IJ pad Overlay set_ Position roiManager select indexes Stack getFrameInterval Stack _ getUnits and Stack setFrameInterval 1 2 eee eee ee ee 2l Improved Rounded Rectangular Selection Tool a 27 Improved Arrow Tool oaoa a 29 File gt Import gt Stack From List accepts URLs o ooo a 45 Improved Edit gt Selection gt Make Band e 55 New command Editi gt Selection gt Area to Line 57 Multiple undo buflers s s s sa sensorial 61 Improved handling of DICOM images 0 0 00 eee ee ee eee 62 Auto threshold based on stack histograms o 69 Improved Imager Color gt Merge Channels 1 73 Improved Imager Stacks Tools gt Make Substack 2 aaa 1 83 New command Imager Stacks Toolst gt Remove Slice Labels 83 Improved zooming commands ss s sesi y rra 88 Improved handling of image selec
63. eval return getVersion run command Runs an ImageJ menu command Example run About ImageJ debug Runs ImageJ in debug mode SEE ALSO ImageJ Documentation Portal Command line Running ImageJ in headless mode Linux installation 17 ImageJ Interoperability Several packages exist that allow ImageJ to interact with other applications environments CellProfiler CellProfiler 78 features RunImageJ a module that allows ImageJ plugins to be run in a CellProfiler pipeline 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 Bio in Software Packages Built on Top of ImageJ Bitplane Imaris ImarisXT can load and execute ImageJ plugins from within the Imaris application In addition the ImageJ plugin bpImarisAdapter Windows only and requiring valid licenses for Imaris and ImarisXT allows the exchange of images between Imaris and ImageJ MIJ Matlab ImageJ bi directional communication A Java package for bi directional 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
64. features Miji m which makes even more convenient to use the libraries and functions provided by Fiji s components from within Matlab 24 Latest revision 2011 08 06 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 A window version 1 441 10 11 12 A B C D E F G H 13 Bolalol denia els lal a gt Status bar bar 1 Rectangular Selection Tool and Rounded is Point Tool and Multi point Tool Rectangular Selection Tool 8 Wand Tool 2 Oval Selection Tool Elliptical Selection Tool 9 Text Tool and Brush Selection Tool 10 Magnifying Glass Polygon Selection Tool 11 Scrolling Tool Freehand Selection Tool 12 Color Picker Straight Line Selection Tool Segmented Line A H Macro Tools loaded from Selection Tool Freehand Selection Tool and Startup Macros txt macros Arrow Tool tools or macros toolsets 6 Angle Tool 13 Toolset Switcher Toolbar The ImageJ toolbar contains tools for making selections drawings zooming and scrolling etc In addition the right side of the
65. histogram values Hold to use the standard histogram equalization algorithm The Saturated Pixels and Normalize parameters are ignored when Equalize Histogram is checked 95 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Process gt Noise gt 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 SEE ALSO Brightness Contrast C XIV Applying Auto Brightness Contrast to Entire Stacks 26 6 Noise Use the commands in this submenu to add noise to images or remove it AOO Original tif OOGO Salt amp Pepper tif Salt Pepper gt Despecle tif 256x254 pixels 8 bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 AOO Add Noise tif OA pixels E bit inverting LE 04 Process gt Noise gt 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 26 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 26 6 2 Add Specified Noise 53 Gaussian Noise Standard Devia
66. in the selection divided _ Skewness J Kurtosis ge ere O Stack position by the number of pixels Reported in cali brated units e g optical density if Analyze gt O Limit to threshold M Display label Sa 9p y y O Invert Y coordinates J Scientific notation Calibrate was used to calibrate the im Y Add to overlay age For RGB images the mean is calculated Redirect to None 4 by converting each pixel to grayscale using Decimal places 0 9 3 the formula gray red green blue 3 Or gray 0 299 x red 0 587 x green 0 114 x blue i E Help_ Cancel if Weighted RGB Conversions is checked in Edit gt Options Conversions Standard deviation Standard deviation of the gray values used to generate the mean gray value Uses the Results Table heading StdDev Modal gray value Most frequently occurring gray value within the selection Corresponds to the highest peak in the histogram Uses the heading Mode Min 3 max 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 bounda
67. 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 ImageJ Window or copy it to the clipboard Ctrl Al Ctrl Cc switch to ImageJ and run File Newb System Clipboard V Ctrl Shift IV pasting the macro into a new editor window cf ImageJ Editor Run it using the editor s Macros Run Macro command Ctrl 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 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 20 Plugins 2 The Built in Macro Functions webpage Help Macro Functions the indispensable guide to the built in functions that can be called from the ImageJ macro language It is T thoroughly documented and constantly updated http imagej nih gov ij developer macro functions html
68. novel ImageJ plugin for automated quantification of neurite outgrowth J Neurosci Methods 168 1 134 9 Feb 2008 Wilhelm Burger and Mark James Burge Digital image processing An algorithmic introduction using Java ISBN 978 1 84628 379 6 Springer Jan 2008 C Igathinathane LO Pordesimo EP Columbus WD Batchelor and SR Methuku Shape identifi cation and particles size distribution from basic shape parameters using ImageJ Comput Electron Agr 63 2 168 182 Jan 2008 Anna Kirilova Gina Lockwood M Math Perry Choi Neelufer Bana Masoom A Haider Kristy K Brock Cynthia Eccles and Laura A Dawson Three dimensional motion of liver tumors using cine magnetic resonance imaging Int J Radiat Oncol 71 4 1189 1195 Jan 2008 Mahdad Noursadeghi Jhen Tsang Thomas Haustein Robert F Miller Benjamin M Chain and David R Katz Quantitative imaging assay for NF B nuclear translocation in primary human macrophages J Immunol Methods 329 1 2 194 200 Jan 2008 Janos Roszik Janos Sz llosi and Gyorgy Vereb AccPbF RET an ImageJ plugin for semi automatic fully corrected analysis of acceptor photobleaching FRET images BMC Bioinformatics 9 346 Jan 2008 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 S Sieuwerts FAM de Bok E Mols WM de Vos and JETvan Hylckama Vlieg A simpl
69. of interest and background Use Analyze gt Measure m with Limit to Threshold in Analyze gt Set Measurements checked to measure the aggregate of the selected features Use Analyzer Analyze Particles to measure features individually Use the Wand Tool to outline a single feature Auto threshold methods OO Threshold _ Highlighted range i p moves a fixed width F gt d A thresholding window ntermodes 0 IsoData E l O gt q 196 Min Max values Li MaxEntropy Intermodes Si Red Display mode Mean _ Y Red MinError I _ Dark background Stack histogram B amp W Minimum Auto Apply Reset Set Over Under Moments Otsu Manual input Percentile gt Set Threshold Levels RenyiEntropy Are objects in the Compute the whole Lower Threshold Level 0 Shanbhag Ha stad a bala Paa e a T Trianale the background slices independently Yen 3 Cancel _ok_ Image gt Adjust gt Threshold T ImageJ 1 45m Upper slider Adjusts the minimum threshold value Hold Shift 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 15 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
70. or comma delimited csv text y file Prior to ImageJ 1 44b this command used to be named Measurements 23 10 13 Selection Saves the current area selection boundary to a file Use File Open to restore the selection Since IJ 1 43 active selections are saved in the TIFF header by default cf File Save Asp Tiff 23 10 14 XY Coordinates Exports the XY coordinates of the line selection or area selection boundary as a two column tab delimited text file Coordinates of rectangular oval and straight line selections cannot be exported Use the Path Writer plugin to save coordinates at evenly spaced one pixel intervals 49 Latest revision 2011 08 06 File gt Revert r 23 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 SEE ALSO Undo and Redo 23 12 Page Setup The Page Setup dialog allows you to control the size of printed output plus other printing options Scale 100 ae border Scale Values less than 100 reduce the size of printed images and A eee as values greater than 100 increase the size 100 corresponds O Selection only to 72 pixels per inch ppi about the unzoomed screen size Pl 90 l of the image The size of the printed image is determined Print actual size by the Scale value and the width and height of the imag
71. 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 web site 2 1 ImageJ Distributions 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 64 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 Helpp 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 ImageJ Distributions bundled with a pre organized collection of a
72. summary For more information on regex filtering see http download oracle com javase tutorial essential regex Regex Syntax 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 l Zero or more of the preceding item T 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 j Negation 0 9 Any character that is not a digit amp amp AND Intersection 0 9 amp amp 3 A digit that is not 3 OR Alternation 0 9 a zA Z A digit or lower or upper case letter IX REDUCING MEMORY REQUIREMENTS WHEN IMPORTING IMAGES Since ImageJ 1 44d the File gt 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 gt Type gt 8 bit Memory requirements can also be reduced by using the Scale Images option SEE ALSO Edit Options Memory Threads 43 Latest revision 2011 08 06 File gt Import gt Use this command to import images that are not in a file format directly supported by ImageJ You will need to know certain informati
73. switches focus back to the prompt Double clicking on a command will run that command Pressing closes the window 140 Latest revision 2011 08 06 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 Enter Export Prints the filtered list of commands to an ImageJ table SEE ALSO Control Panel U Search Finding Commands 28 3 3 Search IN IJ 1 45M Searches for macros tzt ijm scripts js py rb clj bsh plugins source java and html files containing a particular Threshold string ada contents Search is performed recursively subdirectories are included Ignore case Ei cearch macros Folder 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 O Search source folder Search contents Specifies if the search should be restricted to Coancel A COK A 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 ImageJ macros
74. the filter operation on the original image 26 10 6 Custom Filter ANO FFT Filter Filter FFT Filter 05 06 tif This command does Fourier space filtering of the active image using a user supplied spatial domain non FFT image as the filter a 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 FFTCustomFilterDemo and FFTRemoveStreaks macros 26 10 7 FD Math This command correlates convolves or deconvolves two images Imagel FFT of Original SD It does this by converting Image1 and Image2 to the fre Operation Deconvolve 1 quency domain performing conjugate multiplication or Image2 FFT Filter 05 06 tif 4 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 M Do Inverse Transform in the Space domain Cancel Refer to the DeconvolutionDemo and MotionBlurRemoval macros for examples 26 10 8 Swap Quadrants This comman
75. the brightest objects such as bone in a CT computed tomographic study Mean Value projection a modification of brightest point projection sums the 19 Latest revision 2011 08 06 Image Stacks gt values of 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 specially on overlapping objects of different colors and dimensions The surface
76. to the hand tool 31 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 Alt 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 Shift 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 Shift or 32 Tools 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 setTool1 function Once copied to the to the ImageJ macros Startup Macros txt file ImageJ will automatically install them at start up The set of macros listed below taken from the Rename and Save ROI Sets toolset exemplify the use of the setTool function by assigning the numeric keys 1 4 to the first four area selection Area Selection Tools The macro Refine selection activates temporarily the brush selection tool when is pressed Once the mouse is released the previously selected tool is restored 7 ASSIGNING KEYBOARD SHORTCUTS TO IMAGEJ TOOLS This set of macros exemplifies how to use the setTool functi
77. unit can be set Pixel Width 0 3150001 Pixel Height 0 3150001 Voxel Depth 0 3150001 Frame Interval 0 sec Width Height and Depth are automatically converted if Unit of sees 0 0 Length is changed from one known unit nm um ym micron O Global mm cm meter km or inch to another With t series stacks y the Frame Interval in seconds reciprocal of the frame rate can A E Cancel OK M e be viewed and set Since IJ 1 43 setting the Frame Interval sets the frame rate used by Animation Options Alt as long as the unit used is sec Origin always in pixels is the reference point 0 0 of the image coordinate system see also Analyzeb gt Set Measurements Check Global to make the current settings global i e apply to all images opened during the current session cf XXII Global Calibrations SEE ALSO Imager Show Info i Imager Scale E Analyze gt Set Scale 25 5 Colore This submenu contains commands that deal with color images 25 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
78. 011 08 06 IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M Process gt Math gt Black background If checked binary images will be created without using an inverted LUT cf XX Creating Binary Masks and com mands in the Process gt Binary submenu will assume that images contain white objects on a black background see XXI Interpreting Bi nary Images Macros can set this option using setOption BlackBackground true see XXI Interpreting Binary Images and Settings and Preferences Iterations 1 100 1 Count 1 8 1 M Black background C Pad edges when eroding Dilate Open esas Pad edges when eroding If checked Binary gt Erode Outline does not erode from the edges of the image This Fill Holes setting also affects Binary gt Close which erodes from the edges unless this checkbox is selected Skeletonize Black background If checked binary images will be created without using an inverted LUT cf XX Creating Binary Masks and commands in the Processp Binary gt submenu will assume that images contain white objects on a black background see XXI Interpreting Binary Images Macros can set this option using setOption BlackBackground true see XXI Interpreting Binary Images and Settings and Preferences Pad edges when eroding If checked Binary gt Erode does not erode from the edges of the image This setting also affects Binary Close which erodes from the edges unless this check
79. 256x256x30 stack would be combined to create one O Combine vertically 512x 256x40 stack SOUTO If Combine verticall is enabled creates a new Help Cancel ok y gt max W W2 X Hy H2 X max D D2 stack Unused areas in the combined stack are filled with background color cf Color Picker K SEE ALSO Concatenate IMPROVED 25 6 15 2 Concatenate IN IJ 1 45M Concatenator Stackl mri stack 1 tif Stack2 mri stack 2 tif T Concatenates two images or stacks that have the same width height and data type Title Concatenated Stacks C Keep Source Stacks SEE ALSO Combine Caral A Cancel ok 25 6 15 3 Reduce Reduce Size Reduction Factor 2 M Reduce in Z Dimension e Ko Cancel Reduces the size of stacks and hyperstacks by the specified Reduc tion 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 Hyperstacks gt Reduce Dimensionality 25 6 15 4 Reverse Alias for the Image Transforme Flip Z command 25 6 15 5 Insert Stack Inserter Source Image or Stack Destination Stack X Location Y Location microscope icon p
80. 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 and Plugins 13 Scripts ImageJ 1 41 added support for JavaScript scripting 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 JavaScript jar into the plugins folder This JAR file is available at rsb info nih gov ij download tools JavaScript jar It is also included with the Mac version of ImageJ 1 41 and later in the ImageJ plugins tools folder Example JavaScript programs are available at rsb info nih gov ij macros js Since ImageJ 1 43 thread safe JavaScript code can be generated using the Recorder Plugins Macros Record Scripts can be opened in the editor as any other macro cf Macros Scripts with the extension js can be run using the Macros Run Macro command otherwise Macros Evaluate JavaScript J must be used JavaScript Programming Resources on ImageJ JavaScript scripting include 1 The ImageJ web site with growing documentation http imagej nih gov 1j developer javascript html 2 Tutorials on the Fiji webpage http fiji sc wiki index php Javascript Scripting 14 Plugins Plugins are a much more powerful concept than macros and scripts and most of ImageJ s built in
81. 79 81 Aug 2004 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 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 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 160 102 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 103 F Leymarie and M D Levine Fast raster scan distance propagation on the discrete rectangular lattice CVGIP Image Understanding 55 1 84 94 1992 161 List of Abbreviations and Acronyms Alt Option or Meta key DIC ias Application Programming PBM rca Icentage PGM a n American Standard Code for a Information Interchange pace eee pieediek PNG cava Bitmap Image File Device Pee ear Independent Bitmap DIB PPM sens Bits per pixel RAM Charge Coupled Device regex Color Picker RGB Comma S
82. B 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 Adjust gt Brightness Contrast C Image Show Info i displays 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 gt Options Conversions 16 bit Converts to unsigned 16 bit grayscale 32 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 25 2 Adjust gt This submenu contains commands that adjust brightness contrast threshold levels and image size 64 Latest revision 2011 08 06 Image Adjust gt XIV APPLYING AUTO BRIGHTNESS CONTRAST
83. 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 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 it is recommended to upgrade ImageJ right after a first installation Updating IJ consists only of running Help Update ImageJ which will install the latest ij jar in the ImageJ folder on Linux and Windows or inside the ImageJ app on Mac OS X Help gt Update ImageJ can be used to upgrade or downgrade the 3j jar file to release updates or daily builds Release updates are announced frequently 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
84. COM Dictionary txt More information can be found here FITS Flexible Image Transport System image is the format adopted by the astronomical community for data interchange and archival storage Use Image Show Info il 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 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 LOCI Bio Formats library if present a remarkable plugin that supports around eighty 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 y
85. Convert to Mask XX Creating Binary Masks 98 Latest revision 2011 08 06 Process gt Binary gt 26 8 3 Erode Removes pixels from the edges of objects in a binary image Use Filters Minimum to perform grayscale erosion on non thresholded images SEE ALSO Binary Options XXI Interpreting Binary Images 26 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 Binaryb Options XXI Interpreting Binary Images 26 8 5 Open Performs an erosion operation followed by dilation This smoothes objects and removes isolated pixels SEE ALSO Binary Options XXI Interpreting Binary Images 26 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 gt Close w SEE ALSO Binaryb Options XXI Interpreting Binary Images 99 Latest revision 2011 08 06 IMPROVED IN IJ 1 45 Process gt Binary gt Original Voronoi Original Distance Ma Ultimate Points Watershed Voronoi 8 p Ultimate Points AOA AAA OOO Original EDM UEP Watershed OOO Voronoi APP Orig torong JEPS 200x180 pixels 8 bit 35K 200x180 pixels 8 bit inverting 200x181 pixels 8 bit inverting 200x180 pixels 8 bit 35K x180 pixels 8 bit inverting v
86. Documentation Wiki 24 13 13 Proxy Settings Proxy Setting ae oy ens Use this dialog to modify the proxy settings of the Java Proxy server Oe 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 Port 8080 M Or use system proxy settings Help Cancel OK or to open the images in the File gt Open Samples gt submenu 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 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 JJ_ Prefs tat 24 13 14 Compiler Displays a dialog box with options for the Plugins Compile and Run command Target Specifies the Java version of the class files Plugins compiled with a Target of 1 6 will not run on earlier versions of Java A Target of 1 4 should be used to de Target 11 5 L Generate debugging info javac g create plugins capable of running on all ImageJ versions Target cannot be set higher than the version of Java ImageJ is currently running on a RETA Help Cancel OK y O A a A 61 Latest revision 2011 08 06 I
87. Height X Coordinate Y Coordinate O Oval C Centered an This command is also available through the ROI Manager More gt Cancel _ok_ drop down menu see Analyze gt Tools gt ROI Manager 24 12 16 Straighten This command straightens a curved object in an image The curved object must first be outlined using the segmented line tool Use the Image gt Adjust gt Line Width widget opened by double clicking on the line tool icon to adjust the width of the line selection Check Spline Fit in the Line Width widget to fit a cubic spline curve to the points that define the line With IJ 1 44k and later it is not necessary to check the status of the Spline Fit checkbox since Edit gt Selection gt Straighten will assess if the line has already been prepared for straightening by a run Fit Spline straighten call Drag the points along the line selection to reposition them Alt click on a point to delete it Shift click on a point to duplicate it Press Shift E Edit gt Selection gt Restore Selection E to restore accidentally deleted lines Straighten also works with straight line selections In this case the object defined by the line selection is rotated to be horizontal Edit gt Selection gt Straighten 24 12 17 To Bounding Box Converts a non rectangular selection to the smallest rectangle that completely contains it 24 12 18 Line to Area Converts a line selec
88. INTA image is displayed with logarithmic scaling enhancing ans 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 26 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 press horizontal or vertical stripes that were suppress Stripes q created by scanning an image line by line 28 Filter Small Structures Up to 3 pixels Tolerance of Direction 5 Horizontal E Vertical Y 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 a E Cancel OK inal image thus no jumps occur at the edges Filter Large Structures D
89. 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 wiki Luke Miller s tutorial Dot Blot Analysis Dot Blot Analyzer toolset 27 14 Tools gt This submenu provides access to various image analysis plugins 130 Latest revision 2011 08 06 Analyze gt Tools gt 27 14 1 Save XY Coordinates a a EE 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 the image For grayscale images writes three Background value 40 values per line x y and value separated by EE E E A TR spaces For RGB images writes five values per _ Suppress Log output line x y red green and blue The origin of _ Y the coordinate system is at the lower left corner Cancel y of the image The number and percentage of non background pixels is printed to the Log Window if Suppress Log output is not checked 27 14 2 Fractal Box Count This command is used to estimat
90. ImageJ News Opens the News section of the ImageJ website 30 3 Documentation Opens the Documentation section of the ImageJ website 30 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 30 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 30 6 Dev Resources Opens the Developer Resources section of the ImageJ website 30 7 Plugins Opens the Plugins page on the ImageJ website which lists more than 500 ImageJ plugins 30 8 Macros Opens the macros directory on the ImageJ website which contains more than 400 ImageJ macros 30 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 language SEE ALSO Macros 146 Latest revision 2011 08 06 Help gt Update ImageJ 30 10 Update ImageJ Image Updater Upgrades ImageJ to the latest ij jar at http imagej nih gov ij upgrade or downgrades to one of the earlier versions You are currently running v1 430 at http imagej nih gov ij download jars Select daily build from the drop down menu and ImageJ
91. ImageJ website FFT Measurements and FFT Filtering 26 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 Bob Dougherty s 3D Fast Hartley Transform plugin performs 3D FHTs 105 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Process gt FFT gt 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 Analyzeb Set Scale 26 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 areas 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 domai
92. J 1 45M IMPROVED IN IJ 1 45M Edit gt Options gt Target Specifies the Java version of the class files created by Plugins Compile and Run Plugins compiled with a Target of 1 6 will not run on earlier version 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 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 24 13 15 DICOM IN IJ 1 45M Mo DICOM Options This dialog sets options related to the handling of DICOM images Namely if ImageJ should open DICOM images as 32 bit float if voxel as 32 bit float depth should be calculated based on the distance between the first at aes EER 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 Orthogonal Views _ Rotate YZ O Flip XZ Qa y A SEE ALSO Image Types and Formats 24 13 16 Misc Miscellaneous Options Displays a dialog box containing miscellaneous options Divide by zero value Infinity Divide by zero value Specifies the value used when E E Process gt Image Calculator
93. O Global Calibrations Image Types and Formats Curve Fitting XXII GLOBAL CALIBRATIONS Calibration settings related to spatial pixel width height and voxel 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 Analyze gt Set Scale pixel width and height 3 Analyzep Calibrate pixel intensity Once Global calibration is set a GY 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 a Choose Disable Global Calibration to stop using global set tings or Disable these Messages to keep respecting global settings ignoring the calibration of the newly open image canral Cancel OK 27 10 Histogram h ONM Histogram of Cells Calculates and displays a histogram of the distribution of gray values in the active image or selection The X axis represents the possible gray val ues and the Y axis shows the number of pix els found for each gray value The horizontal LUT bar be
94. 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 143 Latest revision 2011 08 06 Plugins gt Compile and Run 28 4 6 Text Window 055 New Text Window ae Opens a text window of a specified size that macros can write miso es to PrintToTextWindow Clock and ProgressBar are examples of Height 16 lines macros that write to a text window M Menu Bar eae ee SEE ALSO Plugins gt New Macro File gt New Text Window N o si Cancel OK Me e A 28 4 7 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 1350 pixels Height 250 pixels SEE ALSO Plugins gt Newe Text Window AT TIT Cancel OK a 28 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 Virtual Machine that includes the javac compiler which is contained in the Tools jar archive 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 Editt Options Compiler command can be used to configure the javac com
95. Process gt Binary commands 26 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 26 8 8 Fill Holes This command fills holes 4 connected background elements in objects by filling the background 29 26 8 9 Skeletonize Repeatably removes pixels from the edges of objects in a binary image until they are reduced to single pixel wide shapes topological skeletons There are several skeletonization algorithms The one implemented by ImageJ is based on a thinning algorithm by T Y Zhang and C Y Suen 1984 A fast parallel algorithm for thinning digital patterns Commun ACM 27 3 March 1984 236 239 DOI 10 1145 357994 358023 Enable debugging in Edit gt Options Misc and the Skeletonize command will create an animation that shows how the thinning algorithm works SEE ALSO Skeletonize3D and AnalyzeSkeleton plugins BinaryProcessor source code 26 8 10 Distance Map Generates a Euclidian distance map EDM from a binary image 103 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
96. Process gt Image Calculator 16 Ensuring Specific settings at Launch scsi dais a a a ees 19 Customizing the lmase Popup Ment ec ac ra 6244 62945 8 9 iae 62 44 34 Setting File Open Samples for Offline Usage oaoa a a 42 Obtaining Histogram Lists o es s oo nde ee oe bm Oe a he ee eS 127 Assigning Keyboard Shortcuts to ImageJ Tools e nas rr we RR ee 151 Cycling Through ImageJ Tools Using Keyboard Shortcuts 152 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 user guide pdf HTML document available online at http imagej nih gov ij docs guide For offline us age a 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 user guide A4booklet pdf and letter size paper http imagej nih gov ij docs 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
97. Rotation Angle Increment 10 x y or z make portions of the volume transparent A PE using thresholding or add a greater degree of visual A 255 realism by employing depth cues Axis of Rotation Y Axis 5 Opacity 0 100 Surface Depth Cueing 0 100 Interior Depth Cueing 0 100 Each frame in the animation sequence is the result of projecting from a different viewing angle To visualize this imagine a field of parallel rays passing through O Interpolate a volume containing one or more solid objects and A strikine a screen oriented normal to the directions of Cancel 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 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
98. TO ENTIRE STACKS The Process gt 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 C tool Image gt Adjust gt Brightness Contrast C is to use the overall stack histogram 25 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 E values define the display range or window ImageJ displays images Minima by linearly mapping pixel values in the display range to display ae E values in the range 0 255 Pixels with a value less than the min cal imum are displayed as black and those with a value greater th
99. a correction 104 Gaussian Blur 110 1 D Gel analysis 129 GIF 47 Transparency 57 Gift wrap algorithm see Convex hull 54 Global calibration see Calibration 126 Graph 58 128 132 Grayscale morphology 99 Grouped Z Projection 79 80 83 H HandleExtraFile Types 42 Hartnack see IJ icon 167 Heat maps 89 Heckbert s median cut algorithm see Color quantization 64 Help resources 4 6 39 146 Hexadecimal Hex colors 92 Histogram 126 127 Histogram measurements T21 Holes 100 119 Hot pixels 97 HSB 14 64 69 70 Huang see Threshold 68 Hyperstacks 84 I IJ icon 167 Image calculator 113 Image formats Native 10 Non native 11 Image processing help 5 Image Selection see ImageROI 89 Image sequence 42 48 Image types 10 13 64 ImageDev see ImageJ2 4 ImageJ for Microscopy see MBF ImageJ 3 ImageJ2 4 ImageROI 89 90 Imaris 24 Immunohistochemistry see Histochemical staining 15 Import 42 Installation 147 Integrated density 123 Interoperability 24 Interpolation 70 IsoData see Threshold 68 J Java 20 21 58 60 61 142 JavaScript 20 21 JFileChooser 58 JPEG 11 13 47 Quality 57 JPEG2000 12 Jython see Python 22 K Keyboard see Shortcuts and Modifier keys 8 Kurtosis 123 L Label ROIs 134 136 Lanes see 1 D Gel analysis 129 Layers see ImageROI Overlays 89 Levels see Brightness amp Contrast 65 66 Li see Threshold
100. ack or hyperstack In this case closing the plot stack window will abort the plotting process PEEN GE e E JE 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 Polygon Per Line M Smooth A Cancel OK T 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 128 Latest revision 2011 08 06 Analyze gt Gels gt 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 ad
101. al b 1 00 ol Number of restarts 2 2 Exponential Recovery Initial c 1 00 0 2 i Sum of residuals squared 5 9469 Maximum iterations 4500 OO Standard deviation 1 0906 Number of restarts 2 SE a edad ll Error tolerance 1 10A x 10 a 0 2107 b 0 4318 Cancel c 0 9125 ImageJ s curve fitter Analyze gt Tools gt Curve Fitting ImageJ 1 441 27 14 4 Curve Fitting ImageJ s curve fitter provides a very simple tool for fitting various functions to X and Y data 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 Fourteen built in functions are available User defined functions with up to six parameters are also possible by choosing User defined In this case Formula and Fitting Options must be specified by the user 3 Once the Fit button is pressed ImageJ displays a graph of the data with the fitted curve as well as a log window containing information about the fit and the values of the fitted parameters if Show Settings is checked 132 Latest revision 2011 08 06 Analyze gt Tools gt Summary of ImageJ s CurveFitter functions The complete documentation for the CurveFitter class is available at http imagej nih gov
102. al views 78 Otsu see Threshold 68 Outliers 97 Outline 100 Oval selection 27 Overlay 89 134 136 P PackBits compression 10 Panel figures see Stacks Montage 76 Paste 51 Performance see Memory hyperpage 9 25 26 43 60 102 109 142 Perimeter 122 PGM 11 Pixel size 72 Planar views see Orthogonal views 78 Plot profile 58 79 81 127 128 133 139 Plugins 2 20 21 146 plugins config 39 PNG 11 49 Transparency 57 Point tool 30 Polar coordinates 105 Polygon selection 28 Polynomial regression see Curve fitting 132 Pop up menu see Contextual menu 34 Power spectrum 106 PPI 50 Preferences see Settings 19 57 Print 50 Processing 93 Progress bar 26 Projection 78 80 83 Proxy server 61 Python 22 Q Quit 50 R R GNU 5 see Interoperability 4 24 RAM see Memory 25 60 Raw 48 58 Raw density 123 Reciprocal 105 Record see Macro recorder 138 Rectangular selection 26 Redo 10 Reduce 82 85 Reflex angles 30 Regex 42 43 Renyi see Threshold 68 Resampling see Interpolation 70 Reset 142 Results table 35 Revert 50 RGB 14 64 69 70 ROI see Selection 17 18 ROI Manager 90 Rolling ball 115 Rotate 87 Rounded rectangle 27 Roundness 123 Ruby 22 S Sagittal see Orthogonal views 78 SalsaJ 4 Sample Images 41 Scale 86 Scale bar 136 Scientific notation 124 Screenshot 142 Scrolling 32 Searc
103. alibrations 124 Latest revision 2011 08 06 Analyze gt Calibrate Calibrate Function Rodbard NIH Straight Line 2nd Degree Polynomial 10 02 0 3rd Degree Polynomial 21 194 12218576 69 52 4th Degree Polynomial ee 2 Exponential d 365 424344 118 72 Power 130 63 R 2 0 981385 179 95 mer 1886 Rodbard 245 93 Gamma Variate 252 35 y a b In x c Rodbard NIH Image Exponential with Offset i EEE Gaussian i 1 a i A AA AAA Open Save Exponential Recovery List a on Y Copy y X 187 6 Y 0 41 sl te M Global Calibration Unit pmol mg protein Calibration Function Log Gray Value a Pixel Inverter Uncalibrated OD EAS Click to Remove Scale resets Distance in Pixels field and Know Distance to zero and the Unit of Length to pixel SEE ALSO Three 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 27 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 to create a calibration curve the left column must contain a list of measured mean pixel values and the right column must contain the same number of calibration standard values The ImageJ web site has a tutorial on Optical Density Calibration exp
104. an sl Es i the maximum are displayed as white cy Contrast Minimum and Maximum sliders Control the lower and upper limits of the display range Holding down Shift will simultaneously adjust all channels of a composite image e g File Open Samples HeLa Cells 1 3M 48 bit RGB Brightness slider Increases or decreases image brightness by moving the display range Hold ing down Shift 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 Shift 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 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 do
105. and lines For text files with integer values no larger than 255 use Image Type 8 bit to convert to 8 bit Before converting disable Scale When Converting in Edito Options Conversions to prevent the image from being scaled to 0 255 SEE ALSO OpenTextImagesAsStack macro Save As Text Image 23 6 5 Text File Opens a text file Note that text files can also be opened using File Open or drag and drop 23 6 6 URL Downloads and displays known formats to ImageJ specified by a URL Other URLs end ing 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 With IJ 1 44g and later it is 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 23 6 7 Results Opens an ImageJ table or any tab or comma delimited text file see Results Table Note that csv and zls files can also be opened by drag and drop 23 6 8 Stack From List Opens a stack or virtual stack from a text file or URL containing a list of image file paths 9 The images can be in different
106. as 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 Tools Make Substack command is based on the Substack Maker plugin by Anthony Padua Daniel Barboriak and Ved Sharma The Image Hyperstacks gt Reduce Dimensionality command is based on J r me Mutterer s Reduce HyperStack macro 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 153 C26 C27 C28 C29 C30 031 C32 C33 034 C35 C36 037 C38 C39 C40 C41 C42 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 s FFT Filter plugin The Process Binary gt Fill Holes algorithm was contributed by Gabriel Landini 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 c
107. ay image quantification software based on the ImageJ library Bioinformatics 23 2 247 248 Jan 2007 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 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 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 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 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 Joel B Sheffield ImageJ a useful tool for biological image processing and analysis Microsc Microanal 13 200 201 Jan 2007 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 Jason Moffat Polina Golland and David M Sabat
108. ayed in the Results Table and in Histogram windows Analyzeb Histogram h 27 8 Set Scale Use this dialog to define the spatial scale of the active image Distance in Pixels 1 so measurement results can be presented in calibrated units Known Distance 0 33348 such as mm or pm Pixel Aspect Ratio 1 0 i r Before using this command use the straight line selection tool to make a line selection that corresponds to a known distance Then bring up the Set Scale dialog enter the Known Click to Remove Scale Distance and unit of measurement then click OK Scale 2 999 pixels um Unit of Length pm M Global ImageJ will have automatically filled in the Distance in Pixels Cancel field based on the length of the line selection Setting Pixel Aspect Ratio 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 XXII Global C
109. blel 1J Evaluate Line gy variable2 getVersion Abort Macro showMessage I am running variablel variable2 Install Macros 36l Function Finder EN Evaluate JavaScript 3 Macros gt Function Finder ALDEA Built in Functions message Edit gt Go to Line Goto Line Cut 32X ad a Go to line number Dialog addMessage string Copy C Find exit or exit error message Paste eV 4 getBoolean message Find fC redirectErrorMessages Find HF showMessage message Find Next WG O Case Sensitive L Whole Words showMessage title message Go to Line L showMessageWithCancel title message Cancel OK showStatus message Select All A SS a waitForUser title message Zap Gremlins Copy to Image Info The ImageJ editor version 1 43n 21 ImageJ Editor Macros gt Scripts and Plugins can be opened and executed in the ImageJ editor The editor commands are organized in five menus File gt Edit gt Fontt gt Macros gt and Debug File gt Basic file operations Open Save Print etc are listed in this menu The last saving directory is kept in J prefs tat the IJ preferences file 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 This dialog box enables you to quickly go to a specified line of code Zap Gr
110. box is selected EDM output Determines the output type for the Binary gt 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 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 options 26 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 P Er E ene Roces 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 E i the active slice The dialog is not displayed if Hide Process a A e ra E Pam 2 i 5 a i 1 1 No Cancel Yes Stack dialog is checked in Edit gt Options Misc SEE ALSO Memory amp Threads 26 9 1 Add Adds a constant to the image or selection With 8 bit images results greater than 255 are set to 255 With 16 bit signed images results greater tha
111. 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 Revision History Date Notes 01 September 2011 Updated for v1 45 Deeply revised edition with several new sections in Parts I IV Available as printable booklets HTML version completely redesigned 31 January 2011 Updated for v 1 44 New sections on advanced ImageJ usage Fiji Scripting command line usage and interoperability with other softwar
112. 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 As for hex triplets alpha values range from 00 solid color to FF fully transparent Alpha values can be omitted for fully opaque colors The table below provides some hexadecimal color values in 10 transparency increments Transparency value optional Col RGB triplet oe CPOE O 10 20 30 40 50 60 70 80 90 100 Hex triplet Blue 0 0 255 Cyan 255 200 Green 0 255 0 OC HE5 FF 4B2 CC E5 FF PCO E5 FF Magenta 255 0 255 Bo Co E5 4FF Red 255 0 0 99 B2 CC E5 FF Orange 255 150 0 on HB HOC E5 FF Yellow 255 255 0 TF 499 B2 HOC E5 FF White 255 255 255 00 19 33 4C 66 7F 99 B2 CC E5 FF FFFFFF Gray 127 127 127 POs AS 66 47F 499 B2 ACC E5 FF B2 HE5 FF Bren Black 0 0 0 00 19 33 440 Wes Alpha blending values can be added to the beginning of hex triplets to modulate the color transparency e g 7F FF 0000 defines red at 50 transparency In ImageJ the hash prefix is optional when specifying overlay colors see Image Overlay gt The built in macro function toHex returns hexadecimal representations of decimal numbers and can be used to convert RGB color values into hex color notation see RGBtoHEX macro Several other macros e g MakeOverlay exemplify how to annotate images using hex colors 92 Latest
113. c IJ Macro Support Group http listes inra fr wws info imagejmacro The ImageJ macro support group connects a network of ImageJ users who are specifically interested in improving their skills in writing macros and plugins for ImageJ The membership base includes experienced programmers and new users who are interested in learning to write their very first macros d Fiji developers http groups google com group fiji devel For developer discussion specific to Fiji e ImageJX http groups google com group imagejx Highly technical developer discussion about ImageJ future directions f ImageJDev http imagejdev org mailman listinfo imagej devel For communication and coordination of the ImageJDev project 8 Dedicated mailing lists for ImageJ related projects 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 Helpt gt ImageJ N
114. 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 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 will be removed Overlay Masks Displays filled outlines of the measured particles in the image overlay removing previously added overlays will be removed 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 d
115. cs 11 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 Oxford England Jan 2010 155 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 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 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 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 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 Richard L
116. ctangular selections or line selections wider than one pixel displays a column average plot where the X axis represents the horizontal distance through the selection and the Y axis the vertically averaged pixel intensity 50 100 Distance pixels 127 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Analyze gt Surface Plot To average horizontally hold or check Vertical Profile in Edit gt Options Profile Plot Options To obtain profiles of several selections in a single plot use the ROI Manager s Multi Plot command Analyze gt Tools ROI Manager Other types of area selections such as oval or freehand ROIs can be profiled by first running Edit gt Selection gt 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 27 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 st
117. ctors shown on the right in the illustration Use the sliders to specify the RGB values of the foreground or background color To get precise colors manually change the values in the text boxes The title of the Color Selector window Foreground Color or Background Color indicates the current selection mode 14 Latest revision 2011 08 06 Image Stacks gt SEE ALSO XVII Embedding Color Annotations in Grayscale Images Draw d Fill f Clear Clear Outside Image Types and Formats XVII EMBEDDING COLOR ANNOTATIONS IN GRAYSCALE IMAGES Color marks are only available with color images or grayscale images that have been converted 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 Overlay gt on the other hand can be created on all image types becoming the easiest way to annotate grayscale images 25 6 Stackse This submenu contains commands that work with stacks 25 6 1 Add Slice IN IJ 1 45M Inserts a blank slice after the currently displayed slice stacks or channel composite images Holding inserts a blank slice before the current slice channel 25 6 2 Delete Slice IMPROVED IN IJ 1 45M Deletes the currently displayed slice channel 25 6 3 Next Slice gt Displays the s
118. cuts 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 cf Edit gt Options Misc SEE ALSO Using Keyboard Shortcuts Finding Commands Tools Shortcuts KeyboardShort cuts txt macro demonstrating how assign shortcuts to custom macros List of ImageJ 1 45m built in shortcuts This table can be obtained within ImageJ using the Plugins Shortcuts gt List Shortcuts command Command Operation Shortcut Description File gt Newb Image n Create new image or stack Newb Text Window N New gt System Clipboard V Open Open Next O Open Samples Blobs 25K Create new text window IN Shift IN Shift V Create image from system clipboard Open file any format recognized by ImageJ S il Shift O Shift B Open next image in folder Opens the Blobs gif example image Close w wW Close the active window Save s iS Save active image in Tiff format Revert r R Revert to saved version of image Print p P Print active image Edit gt Undo z Z Undo last operation Cut x x Copy selection to internal clipboard and clear Copy c c Copy selection to internal clipboard Paste v Vv Paste contents of internal
119. d amp ada e da eee we Bees 144 29 Window gt 145 DOM sO AE tr da ecg eth a Shes ead eet aes 145 292 Rue Behind tab 2 sra Pek Boe Se ee Se OD ae Se es 145 DOs Cascades 4 6 ooh 64 ees aoe ald a A SRSA ES A ee 145 DAs WNC ote She sa Saas ts ee ee et em Seca ee ee eS ee ee 145 30 Help gt 146 aL mages Webs tue hg ecards He GO oe Sr ete ae eae See Ae a eas 146 02 imace NeWSc o ce nba Bat BBA ee EE ee eS A See 146 Si Doc mentat Ole ma rap are vce e Ged cg ea dee ee Be ee Ts 146 BO etalase a 0 amp a 3 c0 amp amp 4 2 amp amp AAA 146 SU Wahine List 4 mee bw bee Be ee ee ERA 146 30 6 Dev RESOULCCS crc aone AP me ae tte et Bs dr de oe a He nen Pe ee Le a 146 IN ce kuti hse ceca ee see Ge Se es ES GN Be ie Bs e 146 SS COS das E e a Ge a ein a Gg ns Ge th aa 146 SOKO Macro FUNCHONS ue a de hee cae we Gece Se BR ea Bere we ae ees Ge es a de a 146 SOS a o ia ah dh be St Kk eke Avo oat Ge ne Re ea ws Ba eS 147 SUL Re rrestulVIEnUS a i o t a ho gy Sn Me ts Bees oe Boe ck Wh HR a 147 SOA Aboul MOMS IS s m a ae ste ede Sok Bt ey O A Be a a ea E i 147 A DOUG Mae deca et cn Gate egy ce ener Gee bea alate eg ae ee ee ce eat ea et et a 147 VI Keyboard Shortcuts 31 Key Modifiers Sl AE key MO di AOS seue ek A amp argh eB ok ee a de OL a BE 150 old DME Rey NIOCINCALIONG a e OR SE ee ee ges 150 Slo trl Key or Cid Modihcations as 4 25 aa Bae goo BOER He Poa Hg Oe Ge A 151 ley Cee a cat ae Se ae oo et Ae
120. d lists the shortcut in the Shortcuts submenu Command Add reference frame Select the command from the popup menu and enter the shortcut in the text field A shortcut can be a lower Shortcut T1 c gt l or uppercase letter or F1 through F12 Use Plugins gt a fC rancealA C Cancel OK Utilities gt List Shortcuts to get a list of shortcuts that are already being used 139 Latest revision 2011 08 06 Plugins gt Utilities gt 28 2 3 Install Plugin Install Plugin Installs a plugin in a user specified submenu Plug ins with a showAbout method are also automatically Plugin Bleach Correction 4 added to the Help About Plugins submenu Menu Shortcuts Use the first popup menu to select the plugin and the Command AntiBleach in second to select the submenu it is to installed in The Shortcut CN command must be different from any existing ImageJ Argument command Shortcut optional must be a single letter ias or Fl through F12 Argument optional is the string that will passed to the plugin s run method 28 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 28 3 Utilities 28 3 1 Control Panel U SA OY Control Panel 4 8 bit 16 bit This comma
121. d 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 108 Latest revision 2011 08 06 Process gt Filters gt FFT Swapped Original 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 periodi cally repeated and the origin is shifted by width 2 in x and by height 2 in y direction Non FFT Original Swapped ra lil a Filters gt For Fourier transforms Swap Quadrants affects only the image displayed not the actual FHT data Therefore editing an image with swapped quadrants for filtering or masking may lead to undesired results 26 11 This submenu contains miscellaneous filters 30 including those installed by the Plugins Utilities gt Install 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 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 ot
122. dd 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 ImageJ Batteries included is a distribution of ImageJ together with Java Java 3D and several plugins organized into a coherent menu structure Citing its developers Certain plugins however provide self updating mechanisms e g ObjectJ and the LOCI Bio Formats library 2 Latest revision 2011 08 06 Installing and Maintaining ImageJ Software Packages Built on Top of ImageJ 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 publicl
123. de Overlay 25 14 5 From ROI Manager Creates an overlay from the selections on the ROI Manager list see RO Manager Note that previously added overlays will be removed 25 14 6 To ROI Manager Copies the selections and images in the current overlay to the ROI Manager where they can be edited moved resized or re colored see ROI Manager Note that previous items in the ROI Manager list will be deleted 25 14 7 Remove Overlay Permanently clears the overlay so that it cannot be restored using Show Overlay 25 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 90 Latest revision 2011 08 06 Image Lookup Tables gt 25 15 Lookup Tables gt This submenu contains a selection of color lookup tables that can be applied to grayscale images to produce false color images If no image is open a 256 x32 ramp image is created to display the color table More than 100 additional lookup tables are available at imagej nih gov ij download luts or as a ZIP archive at http imagej nih gov ij download luts luts zip SEE ALSO Toolset Switcher 25 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 Wit
124. ding ImageJ 12 Macros 13 Scripts 14 Plugins 15 Scripting in Other Languages 16 Running ImageJ From the Command Line Vil vill mewn Nd A Ae 10 11 13 17 19 20 21 21 22 23 17 ImageJ Interoperability IV ImageJ User Interface 18 Tools 18 1 Area Selection Tools 18 2 Line Selection Tools 18 3 Arrow Tool sites 18 4 18 5 18 6 18 7 18 8 18 9 18 10 Angle Tool ROME LOOM gt ae See a Multi point Tool Wand Tol speti s aad Text Tool usa 4 see x Magnifying Glass SCTOUIMe Tool a cn 15 11 Color Picker 4 sn ses 18 12 Toolset Switcher im 18 13 Macro Tools ee a 19 Contextual Menu 20 Results Table 21 ImageJ Editor 22 Log Window V Menu Commands 23 Filer 23 4 LOL 23 3 23 4 235 23 6 23 4 23 8 23 9 23 10 29 11 2 L2 2313 Zo LA 24 Edit gt 24 1 24 2 24 3 24 4 OAS Pe fussed giao da Open Next O Open Samples gt Open Recentb IDOL dh Shanes hg oct Clos W as hy cain hte ia Close All oy oe we sd DAVE S sa de SAVES cae ew eee Reverte rre Page Setup Copy to System PASE pda ek BO 24 34 35 36 38 111 24 6 Paste Control DAs Clea soaa oh oy eS dee a 24 8 Clear Outside DAD Fille corridos DANO Draw ld sir 2411 Invert I esee ers 24 12 Selection o e s so smaa JAAS ODUONS sa e e y e a 25 Image gt Zou WEE s
125. dows visible Put Behind tab Tab Switch to next image window ImageJ window Enter Bring ImageJ window to front 149 Latest revision 2011 08 06 Key Modifiers 31 Key Modifiers 31 1 Alt Key Modifications File gt Open Next Open previous File gt Revert Don t show dialog Edit gt Copy 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 Skip nine slices Image gt Stacks gt Previous Slice Skip nine slices Image gt Stacks gt Start Animation Show options dialog Image gt Duplicate Don t show dialog Image gt Overlay gt Add Selection Don t show dialog Process gt Equalize Do classic histogram equalization Analyze gt Histogram Show dialog Analyzer Plot Profile For rectangular selections generate row average plot Analyzer Plot Profile For wide straight lines display rotated contents Analyze gt Gels gt Select First Lane Assume lanes are horizontal Analyze gt Tools gt Analyze Line Graph Show intermediate image Analyze gt Tools gt Roi Manager Add Name and add selection Plugins gt Utilities gt ImageJ Properties List all Java properties Any user plugin Load using new class loader Area selection tools Subtract current selection from the previous one Rectangle and Oval selection tools Current aspect ratio is maintained while resizing Polygon selection tool Alt click on a nod
126. e Scale taining the images to be processed Select from list Add Border Convert to RGB Crop Gaussian Blur Invert Label Timestamp h Max Dimension Measure Resize Scale Show File Info Unsharp Mask scale 1 5 w getWidth scale h getHeight scale runCsize width w height h inter Output Selects the destination folder where the processed images will be stored 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 Save As gt submenu E EEE o li i ij j T gto Test Open Save 112 Latest revision 2011 08 06 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 32 Previously written macros can be imported using Open When editing 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 Help Opens http imagej nih gov 1j docs menus file html fHraw SEE ALSO Pluginst Macros Record Batch gt Virtual Stac
127. e Intensity projection outputs an im age wherein each pixel stores average inten sity over all images in stack at corresponding pixel location Start slice 1 Stop slice 5 Average v Average Intensity Max Intensity Min Intensity Sum Slices Standard Deviation Median Projection Type Maximum Intensity projection MIP creates an output image each of whose pixels con tains the maximum value over all images in the stack at the particular pixel location M All Time Frames 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 25 6 12 3D Project 3D Projection This command creates a sequence of projections of a projection Method Brightest Point rotating volume stack or hyperstack onto a plane using nearest point surface brightest point or mean value projection or a weighted combination of nearest Slice Spacing pixels 1 00 point projection with either of the other two methods Initial Angle 0 359 degrees 0 partial opacity 20 The user may choose to rotate Total Rotation 0 359 degrees 360 the volume about any of the three orthogonal axes
128. e and fast method for determining colony forming units Lett Appl Microbiol 47 4 275 278 Jan 2008 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 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 Gary Chinga Per Olav Johnsen Robert Dougherty Elisabeth Lunden Berli and Joachim Walter Quantification of the 3D microstructure of SC surfaces Journal of microscopy 227 Pt 3 254 65 Sep 2007 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 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 David Stepensky FRETcalc plugin for calculation of FRET in non continuous intracellular compartments Biochem Biophys Res Commun 359 3 752 8 Aug 2007 Tony J Collins ImageJ for microscopy Bio Techniques 43 1 Suppl 25 30 Jul 2007 Martha L Narro Fan Yan
129. e g for the depicted snapshot the first image would be saved as mitosis t001 z001 c001 tif 48 Latest revision 2011 08 06 File gt Save Asp 23 10 8 AVI Save as AVI Exports a stack or hyperstack as an AVI file 10 Compression Uncompressed Ye Uncompressed Compression JPEG PNG or Uncompressed Frame Rate 5 fps With IJ 1 44 and later overlays are embedded when saving in JPEG or PNG format The default compression is JPEG Frame Specifies the frame frequency The proposed value is read from Image Stacks Tools gt Animation Options Alt and Imagep Properties P as long as the unit of Frame Interval is sec SEE ALSO Filet gt Import AVI 23 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 Edit gt Options Input Output dialog box With IJ 1 44 and later overlays are embedded when saving in PNG format 23 10 10 FITS Saves the active image in FITS Flexible Image Transport System format 11 23 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 23 10 12 Results Exports the contents of the Results window as a tab delimited
130. e 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 Each measured selection will be displayed with an accompanying numeric label matching its row in the Results Table Selections color can be changed in Edit gt Options Colors or by double clicking on the Point Tool The numeric label is defined by the Show All color in ROI Manager s Mores Options widget Redirect to The image selected from this popup menu will be used as the target 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 displ
131. e in Print Cancel OK l ee 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 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 23 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 With 1J 1 44 and later overlays are embedded when printing 23 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 50 Latest revision 2011 08 06 Edit gt 24 Edit 24 1 Undo z Described in Undo and Redo 24 2 Cut x
132. e in Preview mode Two resam wi Enlarge Image to Fit Result El Preview A pling methods are possible Bilinear aid gt aa 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 Enlarge to Fit Result and the image will be enlarged as needed to avoid clipping 25 12 7 Translate Banite Translates moves the image in the x and y directions by a X Offset pixels 150 specified number of pixels With stacks you can translate either ea 1 the current image or all the images in the stack Two resampling methods are possible Bilinear and Bicubic interpolation cf Imagep Size Y Offset pixels 15 0 Interpolation None Ke M Preview a Check Preview to see how the translation will affect the image Cancel OK _ The background at the edges of the image will be set to 0 87 Latest revision 2011 08 06 IMPROVED IN IJ 1 45 Image gt Zoom gt SEE ALSO Align Slice and Align RGB planes plugins 25 13 Zoome 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 When a selection exists zooming with the arrow keys requires holding down either Shift or Ctrl 25 13 1 In Zooms in to next higher magnification level and if possible enlarges the wind
133. e 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 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 gt Type gt menu which in most cases allows indexed color images to look nearly identical to their 24 bit originals SEE ALSO Image gt Lookup Tables gt submenu 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 mos
134. e packages 13 May 2010 First HTML version 17 April 2010 First edition covering v 1 43 Acknowledgments We are extremely grateful to Alex Fern ndez for his wonderful eLyXer and Johannes Schindelin for fruitful discussions and assistance with the Git repository We are also thankful to all of those who submitted material criticisms suggestions and encouragement to update this edition But above all our thanks go to the extraordinary ImageJ community for its dedication to the project
135. e 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 Overlayb Add Image 51 Latest revision 2011 08 06 Edit gt Clear 24 7 Clear Erases the contents of the selection to the current background color Backspace and Del 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 Backspace to avoid this dialog SEE ALSO Clear Outside Fill f Color Picker 24 8 Clear Outside Erases the area outside the current image selection to the background color SEE ALSO Clear Fill f Color Picker 24 9 Fill A 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 24 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 Options 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 ima
136. e the fractal dimen sion D of a binary image D can be used as a measure of pattern complexity cell shape vascular ization textures etc and is specially relevant in cases in which Euclidean measures such as diameter or length are not good descriptors of complexity log count 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 tal Methods and Results in Cellular Morphology J Neurosci Methods 69 1123 126 1996 log box size List 4 Save y Copy y A S AA 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 27 14 3 Analyze Line Graph ImageJ can be used to recover numeric coordinate data from scanned line graphs using the following procedure Steps 2 and 3 are not necessary for binary black and white graphs For practice use the File gt Open Samples Line Graph sample image 131 Latest revision 2011 08 06 Analyze gt Tools gt OOO bine Graph 1 Open the image containing the graph 161
137. e to be horizontal or vertical 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 28 Latest revision 2011 08 06 Tools Arrow Tool Ctrl While moving either end of the line rotates resizes it about its center SEE ALSO Calibration Bar Set Scale V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 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 The following modifier keys apply to the Segmented Line Tool Shift Clicking on an existing point with the Shift key down adds a point Alt Clicking on an existing point with the Alt key down deletes it SEE ALSO Polygon Selection Tool Freehand Selection Tool V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 2 3 77 Freehand Line Selection Tool Select this tool and drag with the mouse to create a freehand line selection
138. e to delete it Straight line 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 tool Alt click on a node to delete it Point tool Alt click on a point to delete it Color picker tool Alt click to pick up background color All tools Show location and size in pixels 31 2 Shift Key Modifications Image gt Adjust gt Threshold Adjusting Min also adjusts Maz Image gt Adjust gt Brightness Contrast Apply adjustments to all channels of a composite image Installed Macros and Scripts Open instead of run Rectangle and Oval selection tools Forces 1 1 aspect ratio Area selection tool Add this selection to previous one Polygon selection tool Shift click on a node to duplicate it Straight line tool Forces line to be horizontal or vertical Segmented line selection tool Shift click on a node to duplicate it Point selection tool Shift click to add a point 150 Latest revision 2011 08 06 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Tools Shortcuts Ctrl Key or Cmd Modifications 31 3 Ctrl Key or Cmd Modifications Rectangle and Oval selection tools With Alt Current aspect ratio is maintained while resizing Rectangle and Oval selection tools Selection is resized around the center Straight line tool Rotate resize the line about its center 31 4 Space Bar Any Tool Switch
139. e to open it 23 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 Open File gt Import and Drag amp Drop IMPROVED 23 6 1 Image Sequence IN IJ 1 45M Sequence Options Opens a series of images in a chosen folder as a stack Images may have different dimensions and can be of any format supported by ImageJ see Image Types and Formats and HandleExtraFileTypes plugin Invisible files Thumbs db or ie O RoiSet zip files created by the ROI Manager are ignored Scale Images Number of Images 28 Starting Image 1 Information width x height x depth 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 Convert to RGB M Sort Names Numerically O Use Virtual Stack 198 x 255 x 28 5 4MB Increment If set to 2 every other image will be opened SS if set to 3 to every third image will be opened etc Starting Image If set to n import will start with the nt image in the folder File Name Contains Enter a string into this field and ImageJ will only open files whose name contains that string Enter Pattern Regular expressions regex can be typed here for advanced filtering cf Basic syntax of regular ex
140. ection 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 18 Tools 18 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 18 1 1 Rectangular Selection Tool Location width height and aspect ratio are displayed in the status bar during drawing see V Toggling Calibrated Units The following modifier keys apply to the Rectangular Tool Shift Selection is constrained to a square Alt Width height are changed with arrow keys one pixel at a time Ctrl Selection is resized around the center Ctrl Alt Current aspect ratio is maintained while resizing SEE ALSO Rounded Rectangular Selection Tool Specify V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 26 Latest revision 2011 08 06 Tools Area Selection Tools 18 1 2 AJRounded Rectangular Selection Tool Rounded Rectangle Tool This tool creates rectangular shapes with rounded corners It shares the same toolbar slot a
141. ed 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 Defaults to the number of available processors determines the number of parallel threads used by commands in the Process Filters gt and the Process gt Matht submenus when processing stacks 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 unchecked 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 ImageJ status bar which may help to reclaim unused memory cf Status bar Help Opens http imagej nih gov ij docs menus edit html memory SEE ALSO FAQ s on the ImageJ
142. ed y axis Scale If checked the Y axis range is fixed and the specified Minimum Y and Maximum Y values are used otherwise plots are scaled based on the minimum Minimum Y 0 00 Maximum Y 0 00 Fixed y axis scale and maximum gray values _ Do not save x values 7 Auin cione Do not Save x values If checked List Save and _ Vertical profile i Seen Copy buttons will appear in profile plot windows wi interpolate line profiles M Draw grid lines Auto close If checked profile plot windows will be automat _ _ ically closed when List Save and Copy are clicked Help Cancel oK on Vertical Profile If checked row average plots of rectangular areas or line selections wider than 1 pixel will be generated instead of the default column average plots Note that evoking Plot Profile k with will generate vertical profiles 58 Latest revision 2011 08 06 Edit gt Options gt 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 profile values will be interpolated along the line selection Draw grid lines If checked gray grid lines will be drawn in the plot Help Opens http imagej nih gov ij docs menus edit html plot options 24 13 5 Rounded Rect Tool IJ 1 45M See Rounded Rectangular Selectio
143. edicated 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 ROI 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 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 ij docs menus analyze htmlFap SEE ALSO Particle Remover plugin 4 8 Connected Particle Analysis 119 Latest revision 2011 08 06 Analyze gt Summarize Original thresholded Outlines Bare Outlines Masks Count Masks Count Masks of blobs gif 4
144. eiret ove wo 25 2 Adjust keto eka eas 25 3 Show Infor i ses 25 4 Properties P Zas MEDIO 4 Sie yn he Zo Sides heh E 25 7 Hyperstacks gt 200 OPA aa oH 25 9 Duplicate D 25 10 Rename e 2 4 why ae acd 20 LU Scale Ele ce ole ath eA 25 12 Tr nsiorMbr ais amp waka 2k OOM oh oe oe a a re OS Os ZONA Overlay dos adds 25 15 Lookup Tables gt 26 Process gt ZO Ll SHOES sag eat 2 2x 20 2 MADERA dde 20 3 Find Edges p e la 26 4 Find Maxima Leds 26 5 Enhance Contrast 200 NOED 04 0 a A 20 SWAGOWSE A ie aar at a ZOO BINA edi 200 WIKRE cor a a eed 20 L0 A E 201 Filters a in aia 20 12 Baten c a e De Se en a 26 13 Image Calculator 26 14 Subtract Background 26 15 Repeat Command R 27 Analyze gt 27 1 Measure m 27 2 Analyze Particles 27 3 Summarize a aeaa 27 4 Distribution 4 3 5 4 2o NESDEl ica e ele ay Y 27 6 Clear Results 27 7 Set Measurements 279 Set SCl seal ek ee 1V A AI O A a a a a a e ds 125 210 istoer anmni ilies 300 Au bo ara eo 6 n oe hoe a 126 EL Plot Promelk et db o EE a RA ORN E R 127 ARS AA eas Se eo Se ee S 128 E bs nas ey Bs A A dk 129 Peels MOISES 3 us sas gee oh A a A his ee ERE E LA eee eS 130 28 Plugins gt 138 Deel AI 138 De MORGUE ae a a a e a as Be Ae a e o a a 139 De WEG bx das Be ao a dr 140 2 E INS do E a a A O SU AA a e 143 28 0 Compie and RUN A er a
145. emlins 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 command 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 Font gt 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 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 Install Macros i Ctrl 1 Adds the macro s contained in the editor to Plugins gt Macros gt submenu Plugins gt Macros Install command 36 Latest revision 2011 08 06 ImageJ Editor Function Finder F Ctrl Shift F 3 Retrieves macro functions in the same way Find Commands I retrieves commands Functions are read from the functions html file stored in the macros folder a local copy of http imagej nih g
146. enerates a stack containing examples of the circular masks used by the Median Mean Minimum Maximum and Variance filters for various neighborhood sizes 26 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 images 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 111 Latest revision 2011 08 06 version of the image the unsharp mask from the original IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M IMPROVED 26 12 3 Macro IN IJ 1 45M Process gt Batch gt 26 12 1 Measure This command measures all the images in a user specified folder by running the Analyze gt Measure m command in all images of the chosen directory Note that measurements are performed o
147. ent 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 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 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 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 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 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 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 8
148. ent J Cheng and Gregory W Schmidt Analysis and documentation of progression of Fuchs corneal dystrophy with retroillumination photography Cornea 25 4 485 9 May 2006 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 XMa and MSHutson Quantifying the intercellular forces during drosophila morphogenesis Amer can Physical Society page 29003 Mar 2006 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 M Doube ImageJ and analysis of correlated confocal and BSE SEM imaging Scanning 28 2 93 94 Jan 2006 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 Jennifer L West and Ian D Cameron Using the medical image processing package ImageJ for astronomy arXiv astro ph Jan 2006 A M Bell K Parton and E Smith EarthTutor An Interactive Intelligent Tutoring System for Remote Sensing American Geophysical Union 52 08 Dec 2005 c 2005 American Geophysical Union J r me N Feige Daniel Sage Walter Wahli B atrice Desvergne and Laur
149. entire stack M Create new window Title mri stack 1 tif Cancel ok may produce better looking results Scaled image selection are copied to a new image named Title if Create new window is checked If scaling a selection that will not be copied to a new image check Fill with Background 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 86 Latest revision 2011 08 06 Image gt Transform gt 25 12 Transform gt This submenu contains commands that perform geometrical image transformation on the active image or stack 25 12 1 Flip Horizontally Replaces the image or selection with a x mirror image of the original 25 12 2 Flip Vertically Turns the image or selection upside down y mirror 25 12 3 Flip Z Reverses the order of the slices in a stack z mirror 25 12 4 Rotate 90 Degrees Right Rotates the entire image or stack clockwise 90 25 12 5 Rotate 90 Degrees Left Rotates the entire image or stack counter clockwise 90 25 12 6 Rotate Use this dialog to rotate the active im RGB of Color48 E 1206 age or selection clockwise the specified xX162 pixels E Angle degrees 8 0 E pume B A co oiece Grid Lines 3 di Interpolation Bicubic Set Grid Lines to a value greater than zero to superimpose a grid on the im 4 Fill with Background Color ag
150. eparated Values ROI Computed Tomography Shift Control key In this guide also the TIFF Command key in Apple keyboards MES uote Digital Imaging and URL o Communications in Medicine voxel Dots Per Inch WYSIWYG Euclidian Distance Map Frequently Asked Questions Fast Fourier Transform Fast Hartley Transform Fiji Is Just ImageJ Flexible Image Transport System Frames Per Second Graphics Interchange Format Graphical User Interface High Dynamic Range Hexadecimal Hypermedia Image Processing Reference Hue Saturation Brightness HyperText Markup Language ImageJ Java ARchive Joint Photographic Experts Group Lookup table Lempel Ziv Welch Maximum Intensity Projection Motion JPEG Not a Number Operating System 162 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 A Acceptable manipulation 5 ActionBar 22 Add see Math 102 Add images see Image calculator 113 Alpha blending value see Blend 92 Analyze 117 AND see Logical operations 114 135 Angle tool 30 Animation 47 84 Annotations 29 31 74 Gray
151. eral images process them close them and the amount of memory used will be the same as when you started 28 3 6 Capture Screen g Copies the screen to an RGB image and displays that image a new window Holding Ctrl Shift G will capture the screen while a modal dialog box is active if the dialog is based on ImageJ s GenericDialog class 28 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 28 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 Alt lists all Java properties SEE ALSO Status bar Help About ImageJ 28 3 9 Threads This command lists in a text window the currently running threads and their priorities 28 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 FAQ s on the ImageJ Documentation Wiki 28 3 11 Reset Use this command to unlock a locked Image or to reclaim x Locked Image memory used by the clipboard and undo buffers cf Undo R Clipboard and Redo Undo Buffer 142 Latest revision 2011 08 06 Plugins gt New gt 28 4 New This submenu conta
152. esets the measurement counter This command is also available by right clicking on the Results Table 27 7 Set Measurements Use this dialog box to specify which measurements are recorded by Analyze gt Measure m ROI Managers Measure command cf ROI Manager and Analyze gt Analyze Particles Measurements are performed on the current selection the entire active image if no selection is present For thresholded images 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 The second group controls measurement settings 121 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Analyze gt Set Measurements The eighteen checkboxes of the first group are Area Area of selection in square pixels or in cali Set Measurements brated square units e g mm yum 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 M Center of mass M Perimeter M l A a th J Bounding rectangle Fit ellipse el gray Gante eam eo Vane wa ee M Shape descriptors O Feret s diameter selection This is the sum of the gray val U Integrated density Median ues of all the pixels
153. ews 2 Look up the mailing list archives Help gt Mailing List Most of your questions may already been answered 6 Latest revision 2011 08 06 Getting Help Help on ImageJ 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 follow 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 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 Latest revi
154. examples AutoThresholdingDemo txt Cancel OK Image macros examples SetOverUnderThresholdColors txt Plugins gt Utilities gt Search 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 40 ImageJ offers an expe dite alternative that allows you to retrieve commands extremely fast Plugins Utilities gt Find Commands I In addition ImageJ features a find function that locates macros scripts and plugins source java files on your computer the Plugins Utilities Search command Because most of IJ source files contain circumstanced comments you can use this utility to retrieve files 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 SourceCodeRetriever a macro that searches for a menu entry and tries to retrieve the java source file of the respective plugin 6 Undo and Redo Probably the first thing you will notice is that ImageJ does not have a large undo redo buffer Undo Edi
155. expected dimensions and size of the reduced stack are displayed in the dialog SEE ALSO Hyperstack to Stack 25 7 5 Channels Tool Z Opens the Channels widget or brings it to the front if it is already open Shift Z is the keyboard shortcut for this command This tool allows to select the Display mode of composite images In addition several commands hosted in the Image gt 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 composite channels Composite images are discussed with more detail in Color Composites 25 8 Crop X Crops the image or stack based on the current rectangular selection 85 Latest revision 2011 08 06 Image Duplicate D Channels flybrain tif flybrain tif AAA flybrain tif 296x296 pixels bit dll losis 2 32 51 230x230 pixels bit LI cicis z 32 gt 250x220 pixels 6 bit LI Composite 2 Channels A Channels A Channels Y Composite e cone ES M Channel 1 O Channel 1 O Channel 1 M Channel 4 M Channel 2 M Channel 2 M Channel 2 More More 3 More Convert to RGB Split Channels gt W Merge Channels Edit LUT Red
156. f bins is specified in the depicted dialog box With any image type an options dialog can be called with or by holding while clicking on Analyze gt Histogram fh Bins 256 Bins Specifies the number of bins M Use min max or X Min 0 00 Use min maa If checked the X axis range is determined by the X Max 255 00 minimum and maximum values in the image or selection If unchecked X Min and X Maz values can be or specified to fix the X axis range Y Max Auto Cancel ok 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 6 Obtaining Histogram Lists 6 OBTAINING HISTOGRAM LISTS 1 Single images run Blobs 25K getHistogram values counts 256 for i 0 i lt values length i print values i countsli l Vf 2 Entire stacks run UT Head 2 4M 16 bite r n Histogram stack Plot getValues values counts for i 0 i lt values length i print values i counts il 27 11 Plot Profile k Displays a two dimensional graph of the in tensities of pixels along a line or rectangular selection The X axis represents distance along the line and the Y axis is the pixel intensity NaNs values in 32 bit images see Image Types and Formats are ignored For re
157. 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 23 6 9 TIFF Virtual Stack Opens a TIFF file as virtual stack see Virtual Stacks and III Opening Virtual Stacks by Drag amp Drop 45 Latest revision 2011 08 06 IMPROVED IN IJ 1 45Mm File gt Close w 23 6 10 AVI Uses a built in version of the AVI reader plugin to open an AVI file Fist Frame 1 JPEG or PNG compressed or uncompressed as a stack or virtual Last Frames 27 stack one slice per video frame AVI files can also be opened using l File Open or drag and drop but macros must use this command _ Use Virtual Stack i i Zon lo to gain access to the dialog box options C Flip Vertical Please note that ImageJ only supports a restricted number of AVI Cancel ok ec formats cf plugin source code Since version 1 41b an improved version of the AVI reader 10 retrieves animation speed from image frame rate adds support for YUV formats the ability to specify the starting and ending frame an option to convert to 8 bit grayscale an option to open the movie as a virtual stack along with many bug fixes 23 7 Close w Closes the active image 23 8 Close All ANO Close All There are 2 images w
158. from being enlarged Alt Will zoom out right click behavior SEE ALSO Zoomb XVIII Scrolling Zoomed Images 18 10 Scrolling Tool Allows you to scroll through an image that is larger than its window When using other tools except the text tool you can temporarily switch to this tool by holding down the space bar see XVIII Scrolling Zoomed Images 18 11 4Color 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 gt Color Picker K window 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 Clear Clear Outside and Cut x use the background color Double clicking on the tool icon will display the Color Picker window The following modifier key applies to the Color Picker Tool Alt clicking with the Color Picker Tool on an image will change the background color SEE ALSO Image Types and Formats XVII Embedding Color Annotations in Grayscale Images 32 Latest revision 2011 08 06 Tools Toolset Switcher Image Baco esa am 4 Switch to alternate macro tool sets Dev Stk tent my y Startup Macros S MAA rrow Labelling Tools ale O 7 2 Drawing Tools Maa EM Example Icons
159. g 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 158 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 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 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 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 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 R Carmona D Mac as J A Guadix V Portillo J M P rez Pomares and R Munoz Chapuli A simple technique of image analysis for specific nuclear immunolocalization of proteins Journal of microscopy 225 Pt 1 96 9 Jan 2007 R Cathelin F Lopez and Ch Klopp AGScan a pluggable microarr
160. g three dimensional modeling and image stitching registration editing and annotation 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 Relational 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 ObjectJ ObjectJ the successor object image of supports graphical vector objects that non destructively mark images on a transparent layer Vector objects can be placed manually or by macro commands and composite objects can encapsulate different color coded marker structures in order to bundle features that belong togetherObjectJ provides back and forth navigation between results and images The results table supports statistics sorting color coding qualifying and macro access MRI CIA MRI Cell Image Analyzer developed by the Montpellier RIO Imaging facility CNRS is a rapid image analysis application development framework adding visual 3 Latest revision 2011 08 06 Getting Help ImageJ2 scripting interface to ImageJ s capabilities It can create batch applications as well as in 2 E teractive applications The applications include the topics DNA combing quantification of stained proteins in cells comparison of intensity ratios between nuclei and cytoplas
161. g does not affect image data cf the HDRexplorerTool 25 2 3 Color Balance This panel makes adjustments to the brightness and con trast of a single color of a standard RGB image 8 bit per color channel For multi channels stacks and hyper stacks it adjusts each of the color channels independently Use the drop down menu to specify which color channel will be adjusted the histogram is drawn for the selected Minimum E h E Maximum color EE Ed gt l ed Brightness Maximum and Minimum sliders Auto Set and Apply work as described for Image gt Adjust gt Brightness Contrast A o C Similarly to the Window Level tool if the B amp C C Y Channel 1 A F window is opened it will be closed and the Color window Channel 1 Channel 3 will be opened at the same location Channel 4 Channel 5 NB When switching from one color to another the Channel 6 Al changes made to one color will be lost unless Apply 67 Latest revision 2011 08 06 Image Adjust gt is clicked before Also note that for 48 bit color images that load as a stack Brightness Con trast C works on single stack slices i e colors and the color settings of the Color panel are ignored SEE ALSO Brightness Contrast C Color gt submenu 25 2 4 Threshold T Use this tool to automatically or interactively set lower and upper threshold values segmenting grayscale images into features
162. ges Draw the selection by pressing to avoid this dialog SEE ALSO Analyze gt Label Color Picker 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 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 92 Latest revision 2011 08 06 Edit gt Invert I 24 11 Invert I Creates a reversed image similar to a photographic negative of the entire image or selection SEE ALSO Image Lookup Tables Invert LUT 24 12 Selection gt Original Fit Fit Fit Convex Make Create Area to Make cont S Spline Circle Ellipse Hull Inverse Mask Line Band Box 8 5 SOO Oh 50 ROI manipulations using the Edit gt Selection gt submenu commands 24 12 1 Select All a Creates a rectangular selection that is the same size as the image 24 12 2 Select No
163. 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 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 30 Magenta is the equal mixture of red and blue Colorblind people that 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 ImageJ macro language see IV Replacing Red with Magenta in RGB Images It is also possible to simulate color blindness using the Vischeck plugin or in Fiji using the Image Color gt Simulate Color Blindness command 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
164. ground colors which affect Edito Fill f Edito Draw d and other color drawing commands It displays current foreground and background colors in the selection boxes at the 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 Ramp Algorithm Red H Color Selectors ALOE d Col y Yellow oreground Color Pr Background Color Green Red lt gt rire Green lt gt Dal 255 i Cyan Blue lt gt E nre m Black White 0 Ramp Blue Cancel OK Magenta a Foreground Color EL Foreground Background Switcher Background Color TL Black White Reset The Color Picker K tool The color palette is based on HSB Hue Saturation and Brightness color model Hue increases as you go down the palette while saturation and brightness values are split horizontally 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 Double clicking on a color brings up one of the Color Sele
165. h 141 Segmented Line selection 29 Selection 17 Composite 18 55 Properties 55 Refine 27 Restore 53 ROI Manager 133 Settings 19 57 59 62 88 101 107 Shadows 97 Shanbhag see Threshold 68 Shape descriptors 123 Sharpen 93 Shortcuts 8 139 148 64 bit 61 Skeleton 100 Skewness 123 Sliding paraboloid see Rolling ball 115 Smooth 93 Sobel edge 93 Solidity 123 Spatial scale 124 Square root transformation 104 Square transformation 104 Stacks 11 Brightness Contrast 65 Export 48 49 From List 45 Hyperstacks 13 84 Labeling 81 82 165 Montage 76 77 83 Profile 58 79 81 128 Projection see Projection 78 79 80 83 Reduce 79 80 82 83 85 Substacks 83 Tools 82 Virtual 13 45 113 StartupMacros 33 34 41 138 Statistics 119 121 Status bar 25 Straight line selection 28 Straighten 56 Subtract see Math 103 Subtract Background 114 Subtract images see Image calculator 113 Surface 128 Syntax highlighting x 23 Synthetic images 105 T Tessellation see Voronoi 101 Text 31 Thinning 100 Threads 142 3D Projection 79 3D viewer 78 Threshold 68 105 123 Binary images 98 Color 69 TIFF 10 46 Time stamper 81 Tolerance Wand Tool 31 Toolbar 25 Tools Angle 30 Area Selection Brush 27 Ellipse 27 Freehand 28 Oval 27 Polygon 28 Rectangle 26 Rounded Rectangle 27 Wand 31 Color Picker 32 Line Selection Arrow 29 Freehand Line 29 Segmented Line
166. h 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 Shift in this document Alt Denoted by Alt or in this document This is also the Option or Meta key on many keyboards SEE ALSO KeyboardShortcuts txt macro demonstrating how assign shortcuts to custom macros I FRONTMOST WINDOW AND WINDOWS 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 pressing its keyboard shortcut Shift C will activate it In addition pressing on any image will bring the ImageJ window to the foreground Undo and Redo Plugins Utilities gt Find Commands I Command Finder Adaptive 3DThreshold Colocalisation Threshold Color Threshold DynamicThreshold 1d Entropy Threshold Show full information EJ Close when running Threshold _ i a pe Run Export J Close M Search contents wi lgnore case M Search macros folder O Search plugins folder U Search scripts folder O Search source folder ImageJ macros AutoThresholdAndSegment txt Image macros
167. h inverted LUTs pixels with a value of zero are white and pixels with a value 255 are black Unlike the Edito Invert I command pixels values are not altered only the way the image is displayed on the screen 25 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 91 Latest revision 2011 08 06 Image Lookup Tables gt 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 0123456780910 11 12 13 14 15 16 17 181920 30 40 50 60 70 80 90 100 110 255 Hex 012345678924 BC D FE F 10111213141 IE 28 323046 50 5A 64 0E 4 2 FEF Decimal RGB color values that typically range from 0 to 255 are succinctly represented by two hexadecimal digits ranging from 00 through FF Hex color values are 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
168. h the current ImageJ 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 duplication of efforts To provide a central online resource for ImageJ program downloads a plugin repository developer resources and more Right now ImageJ remains a highly experimental application but a finalized released is expected by the end of 2011 Be sure to follow the project news and the ImageDev blog for further developments 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 4 Latest revision 2011 08 06 Getting Help Help on ImageJ 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 manipulatio
169. 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 Wand Tool Mode Legacy Tolerance 0 0 pt tt tt 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 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
170. he File Import submenu one would save it in Fiji app plugins Scripts File Import SEE ALSO ImageJ s plugin architecture on the Fiji website 39 File gt 23 Filer 23 1 New Contains commands for creating new images stacks hyperstacks or text windows SEE ALSO Plugins New 23 1 1 Image n gt New Image Creates a new image window or stack A dialog box allows you to specify the image title type dimensions and initial Name Text Image content 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 the image dimensions in pixels Set Slices to a value greater than one to create a stack Fill With Width Height 40 pixels 1 Slices SEE ALSO Image Hyperstacks gt New Hyperstack Image Types and Formats 23 1 2 Hyperstack Alias for the Image Hyperstacks gt New Hyperstack command 23 1 3 Text Window N Creates a new text window with the title Untitled txt SEE ALSO Plugins Newb Text Window Macro Table 23 1 4 Internal Clipboard Opens the contents of the internal ImageJ clipboard SEE ALSO Edit gt Copy c Cut x Paste Control 23 1 5 System Clipboard V Opens the contents of the operating system cli
171. he 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 txt and 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 java source files and compiled to 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 the simplicity of macro language and feel less integrated in ImageJ 12 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 txt or ijm extension and once installed executed by selecting the macro name in the Plugins Macros gt 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 the ImageJ plugins folder with an ijm extension will be installed in the Plugins gt menu
172. he squares 1 2 1 1 0 l 0 0 0 2 06 2 2 l 1 0 l 26 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 24 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 Conversions 93 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Process gt Find Maxima 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 Noise Tolerance 50 Single Point l Single Points A exclude Edee Mama Maxima Within Tolerance Above Lower Threshold M Light Background Point Selection the threshold is analyzed For List i ae accepting a maximum this area Output type tolerance and the contiguous M Preview Point Selection must not contain any point with a value higher at than the maximum Only one maximum within this area is accepted 65 Maxima S a Cancel OK 3 ee e Output Type Can be see Process Find Max
173. he surrounding the median should be replaced SEE ALSO Despeckle 26 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 26 It does not remove patches of NaNs Help Cancel larger than the kernel size however Radius 2 0 i M Preview Note that some ImageJ filters such as Process Filters gt Gaussian Blur Mean and Vari ance 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 26 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 1 2 1 1 2 1 1 0 1 1 0 1 Original 0 1 0 0 1 0 2 1 2 2 1 2 1 2 1 1 2 1 1 0 1 1 0 1 AOO Original tif OOO North tif AB South tif AB East tif x 256x254 pixels 8 bit inverting LU y 64 pixels bit inverting y o gt X pixels bit
174. hecked Zoom Factor Magnifies the entire calibration bar canvas 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 SEE ALSO Calibrate XXII Global Calibrations 137 Latest revision 2011 08 06 Plugins gt 28 Plugins 28 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 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 gt Macros Install or by the Toolset Switcher will always replace previously installed macros 28 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 gt Open and use ImageJ Editor s Macros Install Macros command Macros in the file ImageJ macros StartupMacros tat 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 Toolset Switcher 28 1 2 Run Loads and runs a macro without opening it in I
175. her Red Black White or Black amp White cf Threshold T Color space Selects the color space HSB RGB CIE Lab or YUV Dark background To be checked when features are lighter than the background With IJ 1 441 and later 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 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 rest of the slices in a stack if any using the 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 Color Threshold s built in help SEE ALSO 3D Color Inspector Color Histogram IMPROVED 25 2 6 Size IN IJ 1 45M Scales the active image or selection to a specified Width and Height in pixels Width pixels 186 Height pixels 226 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 Resize
176. her elements correspond to neighboring pixels The destination pixel is calculated by multiplying each source pixel by its corresponding kernel 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 of the kernel but it must be square and have an odd width de de de de Cu oh k Co Ch 6 6 3 3 3 3 olvido i i Ll Dto A Open Save wi Normalize Kernel Preview 8 5 i Cancel 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 Ctrl V 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 be saved as a text file by clicking on the Save button displayed as an image using File gt Import gt Text Image scaled to a reasonable size using Image gt Adjust Size and plotted using Analyze gt Surface Plot SEE ALSO ConvolutionDemo macro 109 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M Process gt Filters gt IMPROVED 26 11 2 Gaus
177. htness of the color 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 routines that require grayscale images is a significant computational gaint You can read more about the HSB color model here In ImageJ conversions between image types are performed using the Image lype gt submenu Segmentation on the HSB RGB CIE Lab and YUV color spaces can be performed by the Image Adjust gt Color Threshold command 17 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
178. ima outputs 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 gt 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 Maxima 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 Output is a b
179. 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 23 10 7 Image Sequence Arr Save Image Sequence Format TIFF Saves a Stack or a hyperstack as an image se quence 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 when saving in JPEG or PNG format Name mri s Format TIFF Start At 0 Digits 1 8 4 mitosis Name Muse slice labels SE o Can Cancel _ Cancel 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
180. in the hyperstack Frames t M Label Images File gt New Hyperstack is an alias of this command 84 Latest revision 2011 08 06 Image gt Crop X 25 7 2 Stack to Hyperstack Converts a stack into a hyperstack RGB stacks are converted into 3 channel hyperstacks Arr Convert to HyperStack Order 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 Channels c Slices z Frames t Display Mode SEE ALSO Hyperstack to Stack 25 7 3 Hyperstack to Stack Converts a hyperstack into a stack in czt order SEE ALSO Stack to Hyperstack 25 7 4 Reduce Dimensionality This command 22 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 Create Image With and time point O Channels 2 M Slices 5 M Frames 51 Uncheck Channels n to delete all but the current channel Slices n 1x5x51 L6MB to delete all but the current z slice and Frames n to delete all but the current time point Check Keep Source and the original stack will not v Keep Source he deleted Cancel The
181. inary image with foreground 255 and background 0 using an inverted or normal LUT depending on the Black Background option in Process Binary gt Options see XXI Interpreting Binary Images The number of particles as obtained by Analyze gt 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 94 Latest revision 2011 08 06 Process gt Enhance Contrast Ss Renae S e n J TO e e Process Find Maxima ImageJ 1 43s Six outputs are possible Single Points Maxima Within Tolerance Segmented Particles Point Selection List and Count Analyze Particles 26 5 Enhance Contrast Enhances image contrast by using either histogram stretching or Saturated Pixels o4 histogram equalization Both methods are described in detail in the Ss Hypermedia Image Processing Reference Contrast Stretching and O Equalize Histogram Histogram Equalization Normalize All 27 Slices O Use Stack Histogram
182. ini Cellprofiler image analysis software for identifying and quantifying cell phenotypes Genome Biol 7 10 R100 Dec 2006 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 FRE T acquired by confocal microscopy and available as an ImageJ Plug in Microsc Res Tech 69 12 941 56 Dec 2006 R Ropolo O Rampado P Isoardi A Izzo L Savio T Cammarota O Davini R De Lucchi and G Gandini Automated quality control in computed radiography Radiol Med 111 8 1156 67 Dec 2006 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 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 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 159 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 1101 John D Gottsch Olof H Sundin Erik V Rencs David G Emmert Walter J Stark Clem
183. ini 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 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 Stephan Preibisch Stephan Saalfeld and Pavel Tomancak Globally optimal stitching of tiled 3D microscopic image acquisitions Bioinformatics Oxford England 25 11 1463 5 Jun 2009 Delphine 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 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 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 Evan C Crawford and James K Mortensen An ImageJ plugin for the rapid morphological characterization of separated particles and an initial application to placer gold analysis Computers ES Geosciences 35 347 Feb 2009 Manuel G Forero Jenny A Pennack Anabel
184. ins commands opening editor windows that can be used to edit and run macros scripts 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 ImageJ Editor Extending ImageJ 28 4 1 Macro Opens a blank editor window with the title Macro txt SEE ALSO Macros ImageJ Editor Text Window File Newp Text Window N 28 4 2 JavaScript Opens a blank editor window with the title Script js SEE ALSO Scripts ImageJ Editor 28 4 3 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 in the ImageJ window Press Ctrl 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 28 4 4 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 28 4 5 Plugin Frame Opens an editor window containing a prototype plugin that extends the PlugInFrame class
185. ith 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 23 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 Imagep Duplicate D command Note that Save s and File gt Save Asp Tiff are redundant commands 23 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 results lookup tables selections and selection XY coordinates 23 10 1 Tiff Saves the active image or stack in TIFF format in redundancy with File gt 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 with IJ 1 43 and later 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 Optionse Input Output dialog box to save using little endian byte order SEE ALSO Native Formats II Image Types Lossy Compression and Metadata X Warning on JPEG compression 46 Latest revision 2011 08 06 File gt Save Asp 23 10 2 Gif
186. justed 3D Color Inspector Color Histogram 27 13 Gels gt Use the commands in this submenu to analyze one dimensional electrophoretic gels 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 AA Lane 1 of gel gif E a b OE AP Plot of Lane 1 of gel gif gel gif Uncalibrated OD 1 0 1 0 Vertical scale factor Horizontal scale factor M Uncalibrated OD M Label with percentages C Invert peaks A Results of Lane 1 4820 175 36 631 8338 731 63 369 Cancel X OK The commands listed in the this submenu are Select First Lane Requires a rectangular selection Note that lanes are assumed to be vertical unless the width of the initial selection is at least twice its height 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 co
187. k 26 12 4 Virtual Stack This command that shares the same interface of Batch Macro cf 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 Run Process Batch gt Virtual Stack Select an Output folder and Output format Select Crop from the Add Macro Code drop down menu Edit the macro code as needed and press the Test button to verify the macro E A E p 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 26 13 Image Calculator Performs arithmetic and logical operations between image Eiculcios two images selected from popup menus Imagel Image1 or both Image1 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 Operation Subtract Image2 mri stack 2 Multiply the same data type or the same size M Create New Window Divide With 32 bit float lti f O 32 bit float Result AND i it float images pixels resulting from es division by zero are set to Infinity or to NaN Help Cancel Min Not a Number if a zero pixel is divided by zero Max The divide by zero value can be redefined in Edit gt peerage Options Misc Difference Cop
188. kground Adjusts the filling color of Cancel the label text box 136 Latest revision 2011 08 06 Analyze gt Tools gt 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 Image Overlay gt submenu If unchecked the scale bar is drawn invasively Note that previously added overlays are removed when using this option SEE ALSO Analyzep Set Scale Image gt Properties P XXII Global Calibrations 27 14 7 Calibration Bar Creates an RGB copy of the current image and displays a la belled calibration bar on it gt yf Calibration Bar Location At Selection a a cia Location Defines the position Fill Color None of the bar If an area se Label Color Black lection is active the bar is initially drawn at that Number of Labels 5 selection Decimal Places 2 Font Size 14 Fill Color Defines the bar s Zoom Factor 1 0 background color U Bold Text Label Color Adjusts the text Cancel color Number of Labels Adjusts the total number of values displayed Decimal Places Adjust the number of decimal places present in the labels Font Size Adjusts labels font size labels are drawn bold if Bold Text is c
189. laining how to calibrate an image to an optical density step tablet 1 Before using this command use Analyzep Clear Results to reset the measurement counter and use one of the selection tools and Analyze gt Measure m to record the mean gray value of each of the standards 2 When finished making the measurements select Analyze gt Curve Fitting to display the Calibrate dialog box To calibrate the image enter the known standard values in the 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 ImageJ will then display the calibration function on a separate window 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 fourteen functions that can be chosen from the drop down menu described in Curve Fitting two other functions are available that do not require any measurement of OD standards 125 Latest revision 2011 08 06 Analyze gt Histogram h Uncalibrated OD Causes ImageJ to convert gray values from 8 bit images to uncalibrated optical density values using the function Unc OD log 255 Pixel value 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 16 bit images SEE ALS
190. le Save Asp Jpeg Requesting a GIF and PNG transparent index 1 higher degree of compression a lower value will File asa r pe 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 M Copy row numbers transparent color used for images saved in GIF M Save column headers M Save row numbers and PNG formats Use 1 for none Note that PNG and GIF transparency only works with 8 bit Cancel images File extension for tables Sets the default extension to be used when saving tables see Results Table Files with txt and xls extensions are saved in tab delimited format and files with csv extensions are saved in comma delimited format 57 Latest revision 2011 08 06 Edit gt Options gt Use JFileChooser to open save Enables versions of File Open and File gt Save As that use the Java JFileChooser instead of the native OS file open and save 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
191. les smaller than that value will be ignored Circularity Particles with size circularity values outside the range specified in this field are also ignored Circularity 4r x pe cf 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 Particle Analyzer Note that with IJ 1 44e and later the particle analyzer will display a blank image when the count of detected particles is zero and Show is not Nothing The font size and line width used can be change in a macro by using call ij plugin filter ParticleAnalyzer setFontSize size and call ij plugin filter ParticleAnalyzer setLineWidth width ina macro or ParticleAnalyzer setFontSize size ParticleAnalyzer setLineWidth width in a script or plugin Nothing Neither images nor overlays will be displayed 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 Outlines 0 Background 255 If In situ Show is checked the original image will be replaced by this image 118 Latest revision 2011 08 06 Analyze gt Analyze Particles Masks 8 bit binary image
192. 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 that ImageJ deals with ImageJ2 will support many more types of image data are 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 2 gray levels integers and fractional values 32 bit images pixels can have ANY intensity value i e any real number including NaN Not a Number In computing these are called floating point images RGB Color Images that can display 256 values in the Red Green and Blue channel These are 24 bit 2x8 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 SEE ALSO Color Images 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 SEES Images can be 1 bit 8 bit 16 bit unsigned 32 bit real
193. lice that follows the currently displayed slice Holding will skip nine slices forward SEE ALSO Arrow Keys 25 6 4 Previous Slice lt Displays the slice that precedes the currently displayed slice Holding will skip nine slices backward SEE ALSO Arrow Keys 25 6 5 Set Slice Slice Number 1 27 1 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 Cancel OK of slices in the stack 15 Latest revision 2011 08 06 IMPROVED IN IJ 145M Image gt 25 6 6 Images To Stack Stacks gt 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 i Y Copy center Copy top left Scale smallest Scale largest Images to Stack Name Stack Title Contains C Bicubic Interpc M Use Titles as L Name Stack U Keep Source Ir Title Contains M Use Titles as Labels Keep Source Images a A 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
194. lick 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 The following modifier keys apply to the Polygon Tool Shift Clicking on a point with with the Shift key pressed adds a new point Alt Clicking on a point with with the Alt key pressed removes it SEE ALSO Segmented Line Selection Tool Enlarge V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 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 drawing SEE ALSO Freehand Line Selection Tool Polygon Selection Tool Enlarge V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 2 Line Selection Tools Use these tools to create line selections Double click on any line tool to specify the line width by opening the Image Adjust gt Line Width widget 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 18 2 1 Straight Line Selection Tool Length and line angle are displayed in the status bar during drawing see V Toggling Calibrated Units The following modifier keys apply to the Straight Line Tool Forces the lin
195. llipse 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 Fit Ellipse is selected in Analyze gt Set Measurements SEE ALSO DrawEllipse macro 24 12 7 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 ConvexitySolidarity macro Convex Hull Plus plugin 24 12 8 Make Inverse Creates an inverse selection see ROI manipulations What is inside the selection will be outside and vice versa 24 12 9 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 Options is checked SEE ALSO Process gt Binary Convert to Mask XX Creating Binary Masks XXI Interpreting Binary Images 24 12 10 Create Selection Creates a selection from a thresholded image or a binary mask 13 94 Latest revision 2011 08 06 Edit gt Selection gt 24 12 11 Properties y Properties Name MySelection Stroke Color magenta Width
196. low the X axis is scaled to reflect Histogram Bins 256 M Use min max or X Min 0 00 0 Count 64000 Min O Mean 76 448 StdDev 61 524 X Max 255 00 Y Max Auto the display range of the image 34 The total pixel Count is also calculated and displayed as well as the Mean standard deviation Std Dev minimum Min maximum Maz and modal Mode gray value 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 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 OSX 126 Latest revision 2011 08 06 o an aa RA Ww Ne Rh a pa N e O Analyze gt Plot Profile k Value Count pairs i e grayscale value corresponding to the X axis cursor position the number of pixels that have that intensity are displayed on the bottom right while mousing over the histogram window With RGB images the 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 Options gt Conversions With 16 bit images the range of gray values between the Min and Maz values is divided into 256 bins With 32 bit images the number o
197. lt opens the previous image if any in its directory 23 4 Open Samples gt 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 http imagej nih gov ij download sample images zip The AutoRun macro in the Startup Macros tat file can be used to change the default path of sample images allowing a complete off line usage of the File Open Samples gt submenu 41 Latest revision 2011 08 06 File gt Open Recent gt 5 SETTING FILE gt OPEN SAMPLESD gt FOR OFFLINE USAGE 1 This macro calls the Prefs setImageURL method to change the default path of Sample Images http imagej nih gov ij images to a local subfolder of ImageJ s directory named samples Note that Fiji provides this feature by default 2 3 4 macro AutoRun 5 dir getDirectory imagej samples 6 if File exists dir 7 dir replace dir 720 8 if startsWith getInfo os name Windows 9 dir replace dir File separator 10 call ij Prefs setImagesURL file dir 11 23 5 Open Recent The submenu shows a list of the 15 recently opened files Click on a filenam
198. lti point selections With area selections the following parameters can be recorded Area Center of Mass Centroid Perimeter Bounding Rectangle Shape Descriptors Fitted Ellipse Feret s Diameter Skewness Kurtosis and Area Fraction With line selections the following parameters can be recorded Length Angle straight lines only and Bounding Rectangle The mean standard deviation etc are calculated from the values of the pixels along the line SEE ALSO Results Table Analyze Particles Summarize Distribution Set Measurements Batch gt Measure 27 2 Analyze Particles This command counts and measures objects in binary or thresholded images Image gt Adjust gt Threshold T or Color Threshold Anal ysis is performed on the existing area selection or on the entire image if no selection is present MO Analyze Particles Size umaA2 0 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 Y Clear results A cars Outines lines the object using the Wand Tool measures i Ellipses M Summarize O Record start Masks it using the Measure m command fills it to M Add to Manager C In situ Sho Help Cancel Count Masks Overlay Outlines Overlay Masks make it invi
199. m and counting nuclei stained in different channels 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 Bio7 Bio 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 SEE ALSO BiolmageXD Endrov Image SXM and VisBio 2 3 ImageJ2 ImageJ Dev is a federally funded multi institution project dedicated to the development of the next generation version of ImageJ ImageJ2 ImageJ2 will be a complete rewrite of ImageJ that will include 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 wit
200. mageJ Editor To run a macro and at the same time view its source code open it with File gt Open and use the editor s Macros gt Run Macro command 28 1 3 Startup Macros Opens ImageJ macros StartupMacros tat The same file can be opened by holding Shift while selecting Startup Macros from the drop down menu see Toolset Switcher 28 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 Adjustp gt Threshold T window to generate a setThresold call and on Reset to generate a resetThresold call Select an image from the Window menu to generate a selectWindow call 138 Latest revision 2011 08 06 Plugins gt Shortcuts gt Record Macro setp l make border k run Select All JavaScript run Line Width run Draw setp 2 save saveAs Tiff Desktop blobs tif Plugin Click in the Image Color gt Color Picker K window to generate setForegroundColor a
201. matically 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 the selection color chosen from one of the nine ImageJ default colors Red Green Blue Magenta Cyan Yellow Orange Black and White Note that the selection color is highlighted in the center of the Point MultiPoint Tool cf Selections SEE ALSO Multi point Tool Cell Counter plugin 18 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 to remove points Similarly when using Edit Draw d marks are painted with the diameter of Mark Width Double clicking on the tool icon runs the Image Overlay gt Add Selection b command 30 Latest revision 2011 08 06 Tools Wand Tool SEE ALSO Point Tool Cell Counter plugin 18 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
202. mber of bytes from the end of one image to the beginning of the next Set this value to width x height x bytes per pixelxn to skip n images for each image read White 1s 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 Help Opens http imagej nih gov ij docs menus file html fraw SEE ALSO Image Types and Formats 23 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 or drag and drop 44 Latest revision 2011 08 06 File gt Import gt 23 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
203. me 25 6 15 11 Stop Animation 1 Terminates animation of the active stack see Start Animation 1 25 6 15 12 Animation Options Alt Animation Options A Use this dialog to set the animation speed in frames per second Pore a pi j 5 the starting and ending frame or to enable oscillating animations E Selecting Start Animation animates the stack as soon as the dialog Last Frame 27 is dismissed C Loop Back and Forth M Start Animation Note that setting the Frame Interval in Imageb Properties P sets the animation speed as long as the unit used is sec E y Cancel A 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 25 7 Hyperstacks gt This submenu contains commands that work with Hyperstacks images that have four 4D or five 5D dimensions 25 7 1 New Hyperstack Title Creates a new hyperstack Hyperstacks have Width Height Channels c dimension Slices z dimension and time Frames t dimension Type Display Mode Width Height Channels c Image Type see Image Type and Display Mode see Channels Tool Z can be specified Checking Label Images will draw the channel number slice Slices 2 number and frame number on each image
204. menu commands are in fact implemented as plugins Quoting Werner Bailer 87 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 pro cessing 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 21 Latest revision 2011 08 06 Scripting in Other Languages Advantages and disadvantages of JavaScript in ImageJ A thorough comparison between different scripting languages is available on the Fiji webpage JavaScript Advantages JavaScript Disadvantages Full access to ImageJ and Java APIs Slower especially starting up Standardized No equivalent of macro sets Richer language objects operator break Cannot use most of ImageJ s 360 built in macro continue etc functions Extensive documentation Requires knowledge of complex ImageJ and Java APIs No support for batch mode Cannot create tools and toolbar menus Not compatible with Function Finder and CodeBar
205. mmand 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 is not available Reset Resets the analysis Label Peaks Uses the area measurements obtained with the Wand Tool to label lane peaks see Gel Analyzer Options 129 Latest revision 2011 08 06 Analyze gt Tools gt Gel Analyzer Options Use this dialog to control the behavior of the Gel Analyzer Vertical Horizontal scale factor Specifies the scale factor at which the lane profile plots are displayed Uncalibrated OD If checked ImageJ will convert gray values to uncalibrated optical density values using the function Unc OD logjo 255 pizel value Note that images can be calibrated to e g a calibrated optical density step tablet using the Analyze gt Calibrate command Label With Percentages If checked the Label Peaks command will print to the Results 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 gt Invert LUT For practice refer t
206. n 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 imagingbook com Web site of Digital Image Processing An Algorithmic Introduction using Java by Wilhelm Burger and Mark Burge 51 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 wiki The Imaging Facility Network IFN in Biopolis Dresden provides access to advanced microscopy systems and image processing Its wiki 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 155 3 2 Help on ImageJ Below is a list of the ImageJ help resources that complement this guide see Guide Formats
207. n sc lt gt gt Lookup Tables BIC SEs Magic Montage Plugins 3D A E F M_ JS T_ Scale Bar Tools for Microscopes AO EM Ol gt Stack Tools Toolset Creator Help Macro Toolsets distributed with ImageJ 1 43 42 While the StartupMacros tat file is located in the ImageJ macros directory all other toolsets must be located in the ImageJ macros toolsets subfolder Note that you can use Toolset Creator to create Menu Tools for running plugins macros and scripts see Extending ImageJ 18 12 Toolset Switcher Use this drop down menu to switch to alternative macro tool sets located in the ImageJ macros toolsets folder or to revert to the default ImageJ macros StartupMacros tat toolset 42 Macro toolsets can be obtained at http imagej nih gov ij macros toolsets or created e g using Toolset Creator The following modifier key applies to the Macro Toolset Switcher S 2 Shift Shift clicking on the Toolset Switcher will open the selected toolset file on the drop down menu 18 13 Macro Tools The tools on the right side of the toolbar all slots between Color Picker and Toolset Switcher are defined by macros in the StartupMacros txt file ImageJ macros folder which can be opened using the Plugins Macros Startup Macros command These tools can be customized by editing the StartupMacros txt file or replaced entirely using the Toolset Switcher menu The macr
208. n 65 535 are set to 65 535 102 Latest revision 2011 08 06 Process gt Math gt XXI INTERPRETING BINARY IMAGES Binary images are thresholded to only two values typically 0 and 1 but often as with ImageJ 0 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 Binary gt 0 Bleck Backeround 2 gt While 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 Settings and Preferences You can use the Process gt 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
209. n Properties y ROI Manager XIX Hexadecimal Color Values ROI Color Coder and RGBtoHEX macros 25 14 2 Add Image Adds an image to the overlay Overlays two open images 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 89 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Image Overlay gt Select Create image selection to create an overlay image that can be moved around the canvas Opacity of image selec tions ImageROIs can be adjusted at any time using Edit gt Image to add blobs gif 1 Selection gt Properties y Use Shift E Edit gt Selection gt Restore Selection E to recover the blending image after click ing outside its limits Use Shift F Flatten F to finally embed the imageROI Add Image X location Y location Opacity 0 100 5 Note that image selections behave only partially as regular selections e g can be added to the ROI Manager list can Cancel be moved beyond image boundaries but cannot be resized or rotated In addition image selections are not saved in the TIFF header M Create image selection SEE ALSO Paste Control Blend transfer mode ROI Manager 25 14 3 Hide Overlay Causes ImageJ to stop displaying the overlay displayed by Show Overlay 25 14 4 Show Overlay Displays an overlay that was hidden by Hi
210. n Tool 24 13 6 Arrow Tool See Arrow Tool 24 13 7 Point Tool See Point Tool 24 13 8 Wand Tool See Wand Tool 24 13 9 Colors EET Displays a dialog box that allows you to set Foreground Color Background white KF Background Color and Selection Color Selection magenta SEE ALSO Color Picker Image Color Color Picker K Point Tool ATTE TIT Cancel OK a 0 A 24 13 10 Appearance Appearance This dialog contains options that control how images are dis O Interpolate zoomed images played an option to display better looking toolbar icons and Open images at 100 an option to set the menu font size Black canvas Seals E Interpolate zoomed images Uses interpolation instead of O Use inverting lookup table E Antialiazed tool icons pixel replication when displaying zoomed images Menu font size points Open Images at 100 Newly open images are displayed e SEAE using 100 magnification 1 image pixel 1 screen pixel Help Cancel OK 8 8 ge p pP Black Canvas Causes the 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 59 Latest revision 2011 08 06 Edit gt Options gt No image border Displays images without the default one pixel wide black border Use inverting lookup table Causes newly opened
211. n 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 Power spectrum with mask that passes low frequencies Power spectrum with Original mask that filters low frequencies Result of inverse transform Result of inverse transform FFT Example of low frequencies filtering 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 Edited power Power spectrum after Inverse transform Original spectrum filtering FFT Example of low frequencies filtering 26 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 106 Latest revision 2011 08 06 Process gt FFT gt 26 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 com Display mand Mi FFT Window ae cat podras Display FFT Window The standard output It consists of la Fast Hartey Fansrorm alas a a an 8 bit image of the power spectrum and the actual data Hine Foard Ta ana which remain invisible for the user The power spectrum p
212. n 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 Analyzep gt Set Measurements Batch gt Macro 26 12 2 Convert Batch Convert Batch converts and or resizes multiple images from a specified folder A input Desktop Raw Output X f Desktop Converted Output Format TIFF me Input Selects the source folder containing the images to be processed Interpolation Bilinear TN Output Selects the destination folder where the processed images will be stored Scale Factor EE Cancel Convert y A 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 Save Asp submenu Interpolation The resampling method to be used in case Scale Factor is not 1 00 see Im agebSize and Imagep Scale E For better results Average when downsizing is automatically selected when scaling down images Scale Factor Specifies if images should be resized see Imager Scale E Batch Process Runs a macro over all the images in a specified folder T he macro is stored in the ImageJ macros batchmacro ijm file E A l Input Desktop Source n Output Desktop Destination Output Format TIFF Input Selects the source folder con Add Macro Cod
213. nalysis 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 created 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 123 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Analyze gt Set Scale Display label If checked th
214. nd 39 opens a window containing ImageJ commands in a hierarchical tree structure Click on a gt 3 File gt D Edit leaf node to launch the corresponding ImageJ command aa ER or plugin Double click on a tree branch node folder E re 8 bit Color to expand or collapse it Click and drag on a tree branch RGB Color RGB Stack HSB Stack node folder to display its descendants in a separate child window In a child window click on Show Parent to re open the parent window Brightness Contrast Window Level Color Balance Threshold SEE ALSO Find Commands I 28 3 2 Find Commands I Command Finder The quickest way to find a command without Type part of a command having to navigate through all the menus 40 Adaptive3 DThreshold Evoke the prompt by pressing L as in command Colocalisation Threshold c ali Launcher or Locator If you type part of a DynamicThreshold 1d command name the list will only show commands Entropy Thresholo that match that substring If only a single com C Show full information M Close when running mand matches then that command can be run by Run Export Close 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 Backspace
215. nd can be turned on and off using the Channels tool Image 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 10 Selections Selections are typically created using the Toolbar Tools Although ImageJ can display simul taneously several selections or regions of interest ROIs only one selection can be active at a time Selections can be measured Analyze gt Measure m drawn Edit gt Draw d filled Edito Fill f or filtered Process gt Filters submenu in the case of area selections In addition since ImageJ 1 43 it is also possible to create non destructive image overlays Image Overlay gt submenu 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 cf Edit Selection gt Properties y Selection Color can be changed in Edit gt Optionsb Colors or by double clicking on the Point Tool It is highlighted in the center of the Point Multi point Tool cf The ImageJ window Rectangular Polygon Composite Cursor outside selection Sin Rh Selection can be moved h Selection can be resized tH Edge can be moved deleted or
216. nd set an automatic threshold 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 Edit Selection gt Convert to Mask XX Creating Binary Masks XXI Interpreting Binary Images 26 8 2 Convert to Mask Converts an image to black and white Convert to Mask The mask will have an inverting LUT white is 0 and black is 255 unless Black Background is checked in the Process gt Y Calculate Threshold for 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 TP gt Cancel OK A 0 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 gt Options SEE ALSO Make Binary Edit Selection gt
217. nd setBackgroundColor calls Note that you can interact with the recorder window by deleting or commenting lines of code or pasting text from ImageJ Editor This may be specially useful when 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 ImageJ Editor Extending ImageJ The ImageJ Macro Language Programmer s Reference Guide Fiji s Introduction into Macro Programming 28 2 Shortcuts gt This submenu contains commands for creating keyboard shortcuts and for installing and removing plugins 28 2 1 List Shortcuts Keyboard Shortcuts This command generates a table with the ImageJ keyboard short not Key CENDANA B cuts in one column and the commands they call in another Com diee EG 111 mands prefixed by refer to shortcuts created with Create Short Find Commands L cuts Commands prefixed by refer to installed macros listed Measure in Plugins gt Macros gt submenu and override default ImageJ hot keys Image Open Note that as mentioned previously you do not have to hold down Print Ctrl t keyboard shortcut E g t i 10 de cnt ody E eyboard shortcu g to open an image press SEE ALSO Keyboard Shortcuts Create Shortcuts 28 2 2 Create Shortcuts Assigns a keyboard shortcut to an ImageJ menu com Create Shortcut mand an
218. nd 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 Corner diameter Fill color Stroke width The width of the contour Cancel 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 gt 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 Embedding Color Annotations in Grayscale Images 18 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 The following modifier keys apply to the Oval Tool Shift Selection becomes circular Alt Width height are changed with arrow keys one pixel at a time Ctrl Selection is resized around the center Ctrl Ale Current aspect ratio is maintained while resizing SEE ALSO Elliptical Selection Tool Specify V Toggling Calibrated Units XVII Embedding Color Annotations in Grayscale Images 18 1 4 O Elliptical Selection Tool Ellipse proper
219. ne A Deactivates the selection in the active image 24 12 3 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 Analyzep gt Tools gt ROI Manager 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 Shift E the keyboard shortcut for Edit gt Selection gt Restore Selection E 53 Latest revision 2011 08 06 Edit gt Selection gt 24 12 4 Fit Spline Fits a cubic spline curve to a polygon or polyline selection see ROI manipulations 24 12 5 Fit Circle Fits a circle to a multipoint with at leas 3 points or area selection 12 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 Computer 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 24 12 6 Fit Ellipse Replaces an area selection with the best fit e
220. ng Inserts an image or stack into a stack at the specified X and Y Location The Source Im age or Stack must be of the same type as the Destination Stack SEE ALSO Image Stacks Label 82 Latest revision 2011 08 06 Image gt Stacks gt 25 6 15 6 Montage to Stack OO Stack Maker 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 SEE ALSO Demontager plugin 25 6 15 7 Make Substack Extracts selected images from the active stack copy Substack Maker ing them to a new stack in the order of listing or Enter a range e g 2 14 a range with increment ranging 21 e g 1 100 2 or a list e g 7 9 25 27 Ese It takes one of three types of input 1 a range of images e g 2 14 extract slices 2 through 14 2 a range of images with increment e g 2 14 2 extract Cancel OK slices 2 and 14 and every second slice in between or 3 a list of images e g 2 4 7 9 14 M Delete slices from original stack Extracted slices will be removed from the source stack if Deleted slices from original stack is checked Currently it does not work with hyperstacks SEE ALSO Image Duplicate D 25 6 15 8 Grouped Z Project Creates a substack of Stack size Group
221. nge of 0 65535 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 and stacks and with 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 XIV Applying Auto Brightness Contrast to Entire Stacks XV Display Range of DICOM Images 25 2 2 Window Level Window Width 188 im na Level 94 a Pp a y Window 188 Cancel HL 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 zL window will be opened at the same location 66 Latest revision 2011 08 06 Image 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 Imager Adjust gt Window Level or B amp C Image gt Adjust gt Brightness Contrast C window and the dis
222. nless 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 radius 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 is prone to edge artifacts E g to reduce the comp
223. nt 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 and 25 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 experimental This command implemented in version 1 431 is a built in version of the Threshold Colour plugin 17 Note that the final thresholded image type is RGB not 8 bit gray a Thresholding method Default 34 Threshold color Black Color space RGB 4 M Dark background Original Filtered Select Sample Segmentation of DAPI stained nuclei using Image gt Adjust gt Color Threshold 69 Latest revision 2011 08 06 IJ 1 45M 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 cf Threshold T Threshold Color Selects the threshold color eit
224. nyverting l X pixels bit inverting i Shadows gt North South East and West kernels 97 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M IJ 1 45M Process gt Binary gt 26 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 XXI Interpreting Binary Images Summary of morphological operators Process gt Binary gt submenu Original Make Binary Erode Dilate Open Close Outline Skeletonize ageJ ImageJ ImageJ ImageJ ImageJ ImageJ ImageJ ImageJ Adjust gt Threshold T Minimum Maximum Erode then Dilate then 1pixel wide 1pixel wide grayscale grayscale Dilate Erode outline skeleton 26 8 1 Make Binary Make Binary Converts an image to black and white If a threshold has A E E color 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 Cook 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 there is no selection is present a
225. o language documentation at http imagej nih gov ij developer macro macros html tools has more information about macro tools SEE ALSO Toolset Switcher Luts Macros and Tools Updater macro 33 Latest revision 2011 08 06 Contextual Menu 19 Contextual Menu IN IJ 1 45M Rename Duplicate Original Scale Paste Control Record Capture Screen Monitor Memory Startup Macros Search Find Maxima Subtract Background As mentioned earlier macros and macro tools in the StartupMacros txt are automatically installed in the Plu gins gt Macros gt submenu and in the toolbar when ImageJ starts up In addition the StartupMacros 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 special ized ones In this case re installing the StartupMacros using the Toolset Switcher will revert the contextual menu to its default The ImageJ Macro Language Programmer s Reference Guide explains how this menu can be customized 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
226. o the video tutorial on the ImageJ wiki page and use the File gt Open Samples gt 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 the left most lane if the lanes are vertical or the top lane if the lanes are horizontal 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 gt 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
227. on about the layout of the image 16 bit Signed Width een 16 bit Unsigned i Height 32 bit Signed file including the size of the image and the Offset to First Image 0 32 bit Unsigned offset to the beginning of the image data Number of Images 32 bit Real 00 64 bit Real Interleaved RGB images have pixels stored Cap Between Images _hit r PARE RAE contiguously rgbrgbrgb in a single im ETR 24 bit RGB Planar White is Zero 24 bit BCR age plane Planar RGB images have the red U Little Endian Byte Order dE p ore 24 bit Integer green and blue image data stored in sepa _ Open All Files in Folder 32 bit ARCB O Use Virtual Stack NE ABC rate 8 bit sample planes ImageJ saves RGB images both TIFF and raw in interleaved A A 1 bit Bitma 3 format 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 nu
228. on 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 NEG ATIVE INFINITY 1f 0f are ignored when making measurements on 32 bit float images 26 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 26 9 19 Macro This command performs image arithmetic using an expression specified by the user 27 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 El Preview ulations on existing images The Math Help Cancel OK gt MacroDemo macro demonstrates the usage of this command SEE ALSO Expression plugin 26 10 FFT gt The commands 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 Image The frequency domain image is stored as 32 bit float FHT attached to the 8 bit image that displays the power spectrum 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
229. on is a variation of this macro that does not shrink from the image edges XIII CONVERTING COMPOSITE SELECTIONS Enter zero in the Editt Selection gt Enlarge dialog box to convert a composite selection into a polygon selection Note however that the conversion may fail if the composite ROI is composed of more than one piece and or contains internal holes 24 12 14 Make Band Make Band Band Size pixels i E h Cancel OK M E Takes an area selection and creates a band with a thickness of the specified number of pixels see ROI manipulations If you imagine the band as a doughnut shape then the original selection corresponds to the hole i e the band is made by growing out the original selection SEE ALSO MakeSelectionBand the macro that implemented this command in previous IJ versions 55 Latest revision 2011 08 06 magenta orange red white yellow can be typed textually IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M Edit gt Selection gt 24 12 15 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
230. on to assign hotkeys to ImageJ tools macro Rectangle 1 setTool rectangle macro Ellipse 2 setTool elliptical macro Polygon 3 setTool polygon macro Freehand 4 setTool freehand macro Refine Area Selection 5 4 tooli TI eetTool Nane setTIool brush while true 4 getCursorLoc x y 2 flags if flags amp 16 0 setTool tool exit 151 Latest revision 2011 08 06 1 oO AN Q A 17 Tools Shortcuts This approach however requires the user to memorize as many shortcuts as the number of tools in the toolbar In addition it may be difficult to assign so many hot keys without conflicting with previously defined ones see List Shortcuts Probably a more efficient way to control the toolbar using the keyboard is to create macros that can cycle through all the tools in the ImageJ toolbar The next example demonstrates such an approach It is composed of two macros activated by and that iterate through the toolbar items from left to right forward cycle and right to left reverse cycle 8 CYCLING THROUGH IMAGEJ TOOLS USING KEYBOARD SHORTCUTS These two macros loop through the tools listed in an array using Fi and F2 as keyboard shortcuts forward and reverse cycling var tiIndx 0 var tools newArray rectangle roundrect oval ellipse brush polygon freehand line freeline polyline arrow wand dropper
231. ontributed by Michael Schmid The NonBlockingGenericDialog class used by the Process Batch gt Macro command was added by Johannes Schindelin The Analyzeb Distribution command was written by Gabriel Landini The scaled color bar implemented in Analyzep Histogram h was contributed by Bob Dougherty The Rodboard and Gaussian functions implemented in Analyze gt Tools gt Curve Fitting were contributed by David Rodbard NIH and Stefan W rz DKFZ respectively The ROI Manager XOR command Analyze Tools gt ROI Manager was added by 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 ROI Manager was contributed by Philippe Gendre 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 Other additions improvements and bug fixes have been contributed by Adrian Daerr Albert Cardona Alberto Duina Alden Dima Andreas Maier Andrew French Andrii Savchenko Arttu Miettinen Barry DeZonia Bill Mohler Bruno Vellutini Carne Draug Christian Moll Daniel Kalthoff Daniel Senff David McDonald Daniel Kalthoff Dimiter Pro danov Divakar Ramachandran Duncan Mak Erik Meijering Francis Burton Frank Sprenger Frederic He
232. or Deselect Select All Y Associate Show All ROIs with slices O Restore ROIs centered O Use ROI names as labels Misc operations via drop down Cancel OK menu also available when right clicking on list area Show All with without labels The ROI Manager Analyze Tools gt ROI Manager ImageJ 1 45i Delete Deletes the selected ROIs from the list Deletes all the ROIs if none is selected Rename Renames the selected ROI The chosen string will be used as Edit Mode label 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 Measure Measures the selected ROIs or if none is selected all ROIs on the list Use Analyze gt Set Measurements to specify the parameters to measure Deselect Deselects any selected ROIs on the list Delete Measure More gt Save and Mores Combine work with all ROIs on the list when none are selected Properties Opens a dialog box depicted in ROI Manager in which is possible to assign a contour color Stroke color of a certain Width or a Filling color With text selections see Text Tool it is also possible to specify the Font size and Justification As explained for Edit gt Selection Properties ly selections can be either filled or con toured but not both The nine default selection colors black blue cyan green magenta orange red
233. other applications 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 gt Save As 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 24 13 3 Fonts Opens a small widget with three pop up menus for specifying the type face size style and antialiasing Smooth checkbox of the font used by the Text Tool and Image Stacks 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 he 118 MH M Smooth 24 13 4 Profile Plot Options Use this dialog to control how plots generated by ImageJ are displayed Imagep gt Stacks p gt Plot Z axis Profile Analyze Plot Profile k Analyze gt Calibrate Analyze gt Tools Curve Fitting Multi Plot Analyze gt Tools gt ROI Manager etc Plot Width and Plot Height Specify the length in pixels Width pixels 350 of the X axis Plot Width and Y axis Plot Height Height pixels 350 Fix
234. 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 18 8 Text Tool Use this tool to add text to images It creates a rectangular selection containing one or more lines of text Use the keyboard to add characters to the text and the backspace key to delete 31 Latest revision 2011 08 06 Tools Magnifying Glass characters Use Image gt Overlay gt Add Selection b to add the text non destructively to the overlay Use Edit Draw d to permanently draw the text on the image Use Edit gt Options gt Fonts or double click on the text tool to specify the typeface size alignment and style Alternatively use Edit gt Selection gt Properties y to adjust font size and text alignment SEE ALSO TextDemo DrawTextWithBackground OverlayDrawStringDemo and TextOverlay macros 18 9 Magnifying Glass Click on the image with this tool 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 magnifying glass icon to revert to the image s original magnification 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 percent The following modifier keys apply to the Magnifying Glass Tool Shift When zooming in will prevent the window
235. ou keep them updated In addition the ImageJ web site lists more than fifty plugins that recognize more exotic file formats The ImageJ Documentation Portal maintains a list of file formats that are supported by ImageJ SEE ALSO Native Formats Filet gt Import gt II Image Types Lossy Compression and Metadata X Warning on JPEG compression Acquisition plugins Input Output plugins 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 11 Latest revision 2011 08 06 Stacks Virtual Stacks and Hyperstacks II 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 with IJ 1 43 and later selections and overlays are
236. ov 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 forcing Function Finder to download 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 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 Executes the highlighted statement and advances to the next The variable names and values in the Debug window are updated Trace t Ctrl T J 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 gt Run Macro Run to Inser
237. ow 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 XVIII SCROLLING ZOOMED IMAGES Hold down the space bar and drag to scroll a zoomed image 25 13 2 Out Zooms out to next lower magnification level and if needed shrinks the window 25 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 25 13 4 View 100 5 Displays the image using 100 magnification 1 image pixel I screen pixel Enable Open Images at 100 in the Edit Options Appearance dialog to have images automatically opened at 100 magnification 25 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 25 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 gt Zoom 25 13 1 The zoomed Y center 127 canvas will be centered at X Y center pixel coordinates 23 Cancel 88 Latest revision 2011 08 06 Image Overlay gt 25 14 Overlay gt This submenu contains commands for creating and working with non destructive image overlays An overlay consists of one or more selections arrows lines points
238. own 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 107 Latest revision 2011 08 06 Process gt FFT p gt Autoscale 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
239. particle in a separate window Shape descriptors Calculates and displays the following shape descriptors Circularity 4r x Py 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 SS If Fit Ellipse is selected the Major and Minor axis are displayed Uses the heading AR Area Major axis Roundness 4 x FI z Or the inverse of Aspect Ratio Uses the heading Round Solidity eE Note that the Edit gt Selection gt Convex Hull command makes an area selection convex 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 the Feret s diameter is displayed as FeretAngle as well as the minimum caliper diameter MinFeret The length of the objects projection in the X FeretX and Y Feret Y direction is 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 A
240. pboard SEE ALSO Editt gt Copy to System Cut x Paste Control 40 Latest revision 2011 08 06 File gt Open 23 2 Open 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 lut extension Tables in tab delimited text format als or csv extension see Results Table Selections roi or zip extension Text files txt ijm js and java extensions SEE ALSO File gt Importe Image Types and Formats Virtual Stacks VIII Opening Files File gt Open File gt Import and Drag amp Drop VIII OPENING FILES FILED OPEN FILED IMPORT AND DRAG amp DROP While the File gt Open command opens formats natively supported by ImageJ images and non images files the Filet gt Import 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 ImageJ Window E g in the illustration below a remote macro file is opened by dragging its URL directly from a Web browser 23 3 Open Next O Closes the current image and opens the next image if any in its directory Holding A
241. piler Since ImageJ 1 44c and later Compile and Run adds the Bio Formats plugin loci_ tools jar to the Java compiler s classpath 144 Latest revision 2011 08 06 Window gt 29 Window gt This menu contains four commands plus a list of open ImageJ 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 29 1 Show All Makes all the windows associated with ImageJ visible 29 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 ImageJ window to the foreground SEE ALSO I Frontmost Window and Windows Activation 29 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 29 4 Tile Shrinks all open image windows and repositions them to fit on the screen without overlapping 145 Latest revision 2011 08 06 CHANGED IN IJ 1 45M Help gt 30 Help gt 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 30 1 ImageJ Website Opens the ImageJ home page 30 2
242. plain how to create c z or t montages A dialog box allows you to specify the magnification level at which the images are copied and to select the layout of the resulting grid 76 Latest revision 2011 08 06 Image Stacks gt Montage Columns Rows Scale Factor First Slice Last Slice Increment Border Width Font Size M Label Slices O Use Foreground Color Te Cancel OK 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 Image Sequence or Stacksp 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 Stacksp Label command can be used to draw labels in stack slices 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 Tools 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 25 6 9 Reslice Reconstructs one or more orthogonal slices through the image volume represented by the current
243. play 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 File Revert r to revert to the initial display range 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 only 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 Image 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 windowin
244. ple SEE ALSO Find Maxima Segmented Particles output for watershed segmentation of grayscale images 26 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 82 bit of this command can be set in the Process gt Binary Options dialog box In the output the value inside the Voronoi cells is zero the pixel values of the dividing lines between the cells are equal to the distance to 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 26 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 101 Latest revision 2
245. ports 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 via 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
246. pressions 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 75 42 Latest revision 2011 08 06 File gt Import gt 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 circumvents 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 tagf0020 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 cf Virtual Stacks Help Opens http imagej nih gov 1j docs menus file htmlFseql SEE ALSO OpenSeriesUsingFilter macro Regular expressions basic syntax
247. problems all the time if startswWith getInfo os name Windows W 15 path z BN 16 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 SEE ALSO IJ ED a plugin by J r me Mutterer that binds jEdit to ImageJ 16 Running ImageJ from the Command Line ImageJ recognizes the following command line options file name Opens a file Examples DLODS tai Users wayne images blobs tif es81lx t1f ijpath path Specifies the path to the directory containing the plugins directory Example ijpath Applications ImageJ port Specifies the port ImageJ uses to determine if another instance is running Examples port1 use default port address 1 port2 use default port address 2 port0 do not check for another instance 23 Latest revision 2011 08 06 ImageJ Interoperability macro path arg Runs a macro or script passing it an optional argument which can be retrieved using getArgument Examples macro analyze i3jm macro analyze Users wayne images stackl batch path arg Runs a macro or script in batch mode no GUI passing it an optional argument ImageJ exits when the macro finishes eval macro code Evaluates macro code Examples eval print Hello world
248. re 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 been resized Use ROI names as labels If checked ROI names are used as selection labels when in Edit Mode If unchecked the ROI position in the Manager s list is used Selections can be renamed using either kename or Properties SEE ALSO Editp gt Selection gt submenu Image Overlay gt submenu XIX Hexadecimal Color Val ues ROI Color Coder macro XXIII SELECTING MULTIPLE 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 with a single Shift click Non contiguous ROIs can be selected by Control click Command click on Mac OS X IMPROVED 27 14 6 Scale Bar IN IJ 1 45 1 MAX_confocal series tif MCB Draws a labelled spatial calibration bar Width Length of the bar in calibrated ScaleBar Plus units Width in ym 5 Height Height of the bar in pixels Height in pixels 4 Font Size 16 Font Size Adjusts the font size of the Color White TA scale bar label Background None Color Adjusts the text color see XVII Location Upper Left F Embedding Color Annotations in Grayscale Images M Bold Text O Hide Text O Serif Font O Overlay Bac
249. re image types A pre existing command that has been renamed or moved to a different menu IN IJ 1 45M location in ImageJ 1 45m 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 What is ImageJ ImageJ is a public domain Java image processing and analysis program inspired by NIH Image for the Macintosh 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 sup
250. reate a new stack simply choose File Newe Image n and set the Slices field to a value greater than one The ImagepStacks gt submenu contains commands for common stack operations SEE ALSO Stack Manipulations on Fiji website Image5D 12 Latest revision 2011 08 06 Color Images 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 implemented in ImageJ 1 43 You can easily run out of memory using commands like Image Crop X because any stack generated from commands that do not generate virtual stacks will be RAM resident 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 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
251. revision 2011 08 06 Process gt 26 Process This menu lists all commands related to image processing including point operations filters and arithmetic operations between multiple images 51 The File Open Samples gt Blobs 25K B image will be used in most of the illustrations of this section LOOO blobs gif LO O blobs gif Smooth 3x O blobs gif Sharpen 1x LO O O blobs gif Find Edges 1x LO blobs gif E Contrast 10 256x254 pixels amp bit inverting LUT 64 256x254 pixels 8 bit inverting LUT 64 x pixels 8 bit inverting R x pixels 8 bit inverting i 256x254 pixels 8 bit inverting LUT 64 Process gt submenu Smooth S Sharpen Find Edges and Enhance Contrast 26 1 Smooth S Blurs the active image or selection This filter replaces each pixel with the average of its 3x3 neighborhood 26 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 1 l l 12 1 1 26 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 t
252. ry 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 Fut 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 Centroid is checked Note that ImageJ cannot calculate the major and minor axis lengths if Pixel Aspect Ratio in the AnalyzebSet Scale dialog is not 1 0 There are several ways to view the fitted ellipse 1 The Edit gt Selection gt Fit Ellipse command replaces an area selection with the best fit ellipse 122 Latest revision 2011 08 06 Analyze gt Set Measurements 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
253. s 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 SEE ALSO Edit Options Line Width Edit Selection gt Fit Spline 25 3 Show Info i ALALA Info for t1 head tif These comments were added to the TIFF file Z using this macro info File openAsString Title tl head tif Cut Width 384 00 mm 256 Copy Height 384 00 mm 256 Clear Depth 193 50 mm 129 Select All Opens a text window containing information about the active image including the pixel or voxel size since IJ 1 44k For DICOM and FITS images also displays file header information Use the popup menu right click in the Info window to save the informa tion to a text file or copy it to the system clipboard SEE ALSO Image Properties P 71 Latest revision 2011 08 06 Image Properties P 25 4 Properties P organ of corti tif Use this command to display and set various properties of the Channels c current image or stack ecos EE The number of Channels c Slices 2 and Frames t in the image Frames t 1 can be changed as long as the product of c z and t is equal to the spark ust Note c z t must equal 60 number of images in the stack Unit of Length pm The Unit of Length a string describing the measuring unit Pixel Width Pixel Height and Voxel Depth all in the Unit of Length
254. s Table to be analyzed Data points The number of rows that will be analyzed informative Distribution Automatic binning If checked ImageJ will use the method described by E a David Scott to assess the opti Count 223 Data points 223 mal histogram bin width see Scott ae ace O Automatic binning DW Optimal and data based his Bins 8 or specify bins 8 tograms Biometrika 66 3 605 610 es 0 200 Jan 1979 If unchecked the number of bins can be set with Specify bins and the starting and ending limits of the histogram with range List Copy Log Cancel gt OK SEE ALSO Distribution Plotter a macro that plots relative and cumulative frequencies on a double Y axis graph 27 5 Label This command labels the active selection 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 Draw d Use the Edit gt 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 XVII Embedding Color Annotations in Grayscale Images 27 6 Clear Results Erases the results table and r
255. scale images 75 Appearance 59 88 Area 122 Area under the curve see Definite integral 129 Arrows 29 Aspect ratio 26 27 70 123 AutoRun 19 41 AVI 11 46 49 B Background see Subtract Background 114 Background pixels 131 Batch processing 111 138 BeanShell 22 Benchmark 142 Bicubic interpolation see Interpolation 70 Bilinear interpolation see Interpolation 70 Binary 98 99 Bio7 4 Bio formats see LOCI 11 Bitwise operations 103 104 Black background 68 99 102 Blend 89 92 Bounding box 53 56 Brightness Contrast 65 67 95 Brush selection tool 27 Bug reporting see also Debug 7 C Calibration 126 Optical density 125 137 Spatial 72 124 136 Calibration bar 137 Caliper length see Feret 123 Canvas 71 Capture image 142 Catmull Rom see Interpolation 70 CCD 14 97 Cell Counter plugin 30 31 CellProfiler see Interoperability 24 Center of mass 122 Centroid 122 Channels 72 85 CIE 70 CIE Lab 14 69 Circularity 118 123 Clipboard 40 51 Clojure 22 CodeBar 22 Color Background 32 74 Balance 67 Blindness 15 Composites 17 Deconvolution 15 Foreground 32 74 Models 14 Quantization 64 Separation see Color Deconvolution 15 Settings 17 19 52 59 124 Threshold 69 Color filter array 14 Color palette see LUT 14 Color Picker 32 Combine 82 Command key 8 Command launcher 9 37 39 140 141 149 154 Compile 61 144 Concatenate
256. segmented or measured 23 10 4 Text Image Saves the active image as a spreadsheet compatible tab delimited text file For calibrated images and floating point images the Decimal Places field in Analyze gt Set Measurements determines the number of digits to the right of the decimal point 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 Import Text Image 23 10 5 Zip Saves the active image or stack as a TIFF file inside a compressed ZIP archive AT Latest revision 2011 08 06 IMPROVED IN IJ 1 45M File gt Save Asp AA Text Image txt Jer ez es ea es c Jer cs co cio Jer erz cis cia cis cae box It UR o oS oS oS oS oS Qo o o OOOO ODO ooo OOOO ODOoO ooo Coo oor ee eo ee eo amp OOOO ODO OOOO 0 O 0 O 0 O 0 O 0 O 0 O O OOO ODO O O ao ao amp Coo eo A A eo DO OQO O OOOO ODO Oo Text Images File gt Import Text Image and File gt Save Asp Text Image 23 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 Open Samples CT 420K 16 bit DICOM are saved as signed shorts 32 bit
257. sian Blur IN IJ 1 45M IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M IMPROVED IN IJ 1 45M Gaussian Blur This filter uses convolution with a Gaussian function for smoothing 31 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 7 of the Gaussian this is the same as in Adobe Photoshop AS 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 107 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 SEE ALSO Accurate Gaussian Blur plugin AnimatedGaussianBlur macro 26 11 3 Median Reduces noise in the active image by replacing each pixel with the median of the neighboring pixel values 26 11 4 Mean Smooths the c
258. sible then resumes scanning until it reaches the end of the image or selection Press to abort this process 117 Latest revision 2011 08 06 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 thresholded 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 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 partic
259. sion 2011 08 06 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 Composites 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 key for shortcuts in Edit gt Options gt Misc is unchecked i e except when using ImageJ Editor 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 or Ctrl Shift 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 Utilities gt List Shortcuts command There are three modifier keys in ImageJ Control Command Key on Apple keyboards Denoted by Ctrl or in this document Althoug
260. size slices with each slice being the result of a Z Pro Ere pr EEEE Intensity jection performed over the range of Group size Group size 27 Max Intensity Group size must divide evenly into the stack sli Min Intensity Sum Slices Standard Deviation Median size SEE ALSO ImagebStacks gt Z Project 25 6 15 9 Remove Slice Labels Removes slice labels from stacks 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 Stacksp Label 25 6 15 10 Start Animation 1 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 83 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M IJ 1 45M Image gt Hyperstacks gt 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 ti
261. ssman Gilles Carpentier Gabriel Landini Ingo Bartholomaeus Jan Eglinger Jan Funke Jarek Sacha Jay Unruh Jerome Mutterer Joachim Wesner Johannes Hermen Johannes Schindelin Johannes Weissmann John Oreopoulos John Pearl Jonathan Silver Juan Grande Julian Cooper Kai Uwe Barthel Karen Collins Kevin Moll Kris Sheets Mark Longair Mat Al Tamimi Matthew Smith Michael Cammer Michael Doube Michael Ellis Michael Schmid Nico Stuurman Norbert Vischer Olaf Freyer Peter Haub Reinhard Mayr Richard Cole Robert Dougherty Shannon Stewman Simon Roussel Stefan Starke Stephan Saalfeld Tiago Ferreira Tomas Karlsson Tseng Qingzong Ulf Dittmer Uwe Walschus Valerio Mussi Ved Sharma Vytas Bindokas Wilhelm Burger Macro Toolsets distributed with ImageJ have been contributed by Gilles Carpentier Arrow Labelling Tools txt Scale Bar Tools for Microscopes txt J r me Mutterer Example Icons txt Magic Montage txt Tiago Ferreira Toolset Creator txt 154 ImageJ Related Publications The following references are a small sample 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 This list appears in chronological order rather than alphabetical and is particularly
262. stack or hyper stack 18 O Flip vertically The estimated size of the output stack and the amount S iba of available memory are displayed at the bottom of the CAYO IMterpolacion es aa dialog Increase Output spacing to reduce the size of the output stack Voxel size 3x3x2 5 um A E eee A dialog allows you to specify the spacing of the recon e i Ganceind structed slices 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 T Latest revision 2011 08 06 Image gt Stacks gt 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 25 6 10 Orthogonal Views H PPP ti head ti i ed mand einen Ut 102 l Provides an orthogonal view display of the
263. t Cardona http www ini uzh ch acardona fiji tutorial 2 Dedicated tutorials on the Fiji webpage http fiji sc wiki index php Scripting comparisons 22 Latest revision 2011 08 06 Running ImageJ From the Command Line Fiji Script Editor Fiji features a more powerful script editor than ImageJ s built in one see ImageJ Editor The Fiji editor is an invaluable help when writing 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 see the Fiji website at http fiji sc wiki index php Script Editor Edit Language Templates Run Startup 1 StartupMacros txt 2 See file About Startup Macros i 3 4 5 _ eteeeteteetetetetetetetetetatetetatetetatatefatatoter AutoRun macro ajajajaja jajajajaja a a a a a a a a a a a a a a a 7 macro AutoRun fldr getDirectory imagej samples 9 if File exists fldr 10 fldr getDirectory imagej Sample Images 1 if File exists fldr 12 path replace fldr 20 4 o o File Open Help on Macro Functions Extract source jar Create new plugin Focus on the main Fiji window Find Next Calibration0 C Match Case Regex M Search forward M Mark All M Whole Word in windows backslashes give
264. t 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 Memory amp Threads If you cannot recover from a mistake you can always use File 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 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Image Types and Formats In ImageJ the equivalent to Redo is the Process Repeat Command R that re runs the previous used command skipping Edit Undo z and File Open commands SEE ALSO Plugins Utilities 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 i 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
265. t 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 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 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 14 Latest revision 2011 08 06 Color Images distinguishes between colors Saturation sometimes called purity 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 brig
266. thogonal view display Stacks Tools gt Start Animation AJ Start stop stack animation Hyperstacks gt Channels Tool Z Shift Z Open the Channels tool Hyperstacks gt or Next hyperstack channel Hyperstacks I lt or Previous hyperstack channel Hyperstacks Ctr Next hyperstack slice Hyperstacks Ctrl Previous hyperstack slice Hyperstacks Alt gt Next hyperstack frame Hyperstacks Alt I lt Previous hyperstack frame Crop X Shift X Crop active image or selection Duplicate D Shift D Duplicate active image or selection Scale E El Scale image or selection Zoomb In or Make image larger Zoom Out or Y Make image smaller Zoom Original Scale 4 4 Revert to original zoom level Zoom gt View 100 5 B Zoom to 1 1 Process gt Smooth S Shift S 3x3 unweighted smoothing Repeat Command R Shift Repeat previous command Analyze gt Measure m M Display statistics of active image selection Histogram hl H Display histogram of active image selection Plot Profile k K Display density profile plot of active selection Gels gt Select First Lane Ti Select first gel lane Gels gt Select Next Lane 2 Select next gel lane Plugins gt Utilitiest gt Control Panel U Shift U Open Control Panel Utilities gt Capture Screen g Shift G Grab screenshot Utilities gt Find Commands I E List find and launch commands Window gt Show All 1 Make all win
267. 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 Red Displays the thresholded values in red BEW Features are displayed in black and background in white This mode respects the Black background flag set in Process gt Binary Options 68 Latest revision 2011 08 06 Image Adjust gt Over Under Displays pixels below the lower threshold value in blue thresholded pixels in grayscale and pixels above the upper threshold value in green These colors can be changed from a macro by calling the ImageProcessor setOverColor and setUnderColor methods ex ample Dark background To be checked when features are lighter than the background The state of the checkbox is remembered across restarts 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 curre
268. ties 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 18 1 5 O Brush Selection Tool Adjusts the shape of any area selection 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 Brush diameter can be adjusted by double clicking on the tool icon Location width height and aspect ratio are displayed in the status bar during drawing The following modifier keys apply to the Brush Tool 27 Latest revision 2011 08 06 IMPROVED IN IJ 1 45M Tools Line Selection Tools Shift Brush is always added to the selection Brush is always subtracted from selection SEE ALSO Composite Selections V Toggling Calibrated Units 18 1 6 E 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 c
269. tion 30 Adds Gaussian noise with a mean of zero and a chosen standard deviation Cancel ok 26 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 26 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 96 Latest revision 2011 08 06 Process gt Shadows gt 26 6 5 Remove Outliers A g O Replaces a pixel by the median of the pixels in the surrounding acini 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 4 Radius Determines the area uncalibrated i e in pixels used Y Preview for calculating the median Run Process Filters gt Show oe A i e Cancel Circular Masks to see how radius translates into an 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 t
270. tion Point Ctrl Shift 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 The ImageJ Macro Language Program mer s Reference Guide Fiji s Introduction into Macro Programming Plugins gt Macros Record Fiji Script Editor IJ ED 37 Latest revision 2011 08 06 Log Window 22 Log Window The Log window is used to display useful infor openimage zip samples mitosis test data zip mation about ongoing operations It is frequent mitosis testi oyenitiy for plugins and macros to send messages to the 254 NewSubfileType value 0 Log window reporting progress errors or trou 256 ImageWidth value 256 bleshooting information 257 ImageLength value 256 258 BitsPerSample value 8 If you are troubleshooting a problem you can 262 Photolnterp value 1 heck D i in Edit gt ions gt Misc 270 ImageDescription value 194 count 10 naa ebug mode dit gt Options AS ED 273 StripOffsets value 768 have ImageJ outputting messages to the Log 277 SamplesPerPixel value 1 window ImageJ will exit debug mode as soon 278 RowsPerStrip value 256 as the Log window is closed 279 StripByteCount value 65536 282 XResolution value 297 In addition Tiff tags and information needed to 283 YResolution value 305 296 ResolutionUnit value 1
271. tion to an area traced ROI Straight lines must be wider than one pixel 96 Latest revision 2011 08 06 Edit gt Options gt 24 12 19 Area to Line IJ 1 45M Converts a non composite area selection to its enclosing outline see ROI manipulations The obtained line will have the width specified in the Image gt 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 24 12 20 Add to Manager t Adds the current selection to the ROI Manager Analyze gt Tools gt ROI Manager If there is no selection the ROI Manager is open 24 13 Options Use commands in this submenu to change various ImageJ user preference settings 24 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 Line Width lines generated by the Edit gt Draw d command This legacy command has been superseded by the Image Adjust Line Width widget e sa but required since the later is not recordable see Plugins gt Macros gt Record 24 13 2 Input Output 1 0 Options JPEG quality 0 100 Specifies the compression JPEG quality 0 100 75 level used by Fi
272. tions ImageROIs o o 89 New command Process gt Noise gt Remove NaNs 2 2 ee ee 97 Multithreaded Process gt Math Macro 105 Multithreaded ProcesspFilters gt a aoaaa ee 109 Reduced memory fooprint when running Process Filters gt on non float images 110 Added more macro snippets to Process gt Batch gt Macro 0 2000 112 More accurate calculation of Feret s diameter 0 0 eee ee ee ee 123 Measured ROIs can be automatically added to the image overlay 124 Improveded handling of ROIs in stacks and hyperstacks by the ROI Manager 133 New command ROI Manager s Multi Plot 2 0000 o 135 ROI Manager can now use ROI names as labels 0 136 Scale bars can now be added as overlays 136 Improved Plugins Utilities Search e 141 New command Plugins Utilities gt Monitor Events 0 141 Redesigned Plugins Utilities gt Monitor Memory o 142 This list is not extensive Detailed release notes for version 1 45m are available on the ImageJ News web site http imagej nih gov ij notes html vil Noteworthy I Frontmost Window and Windows Activation 00082 eee 8 JI Image Types Lossy Compression and Metadata 12 MI Opening Virtual Stacks by
273. toolbar contains seven slots that can host any of the 60 tools and 15 tool sets available on the ImageJ website cf Macro 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 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 is also displayed When clicking on the status bar the ImageJ version the Java version memory in use memory 20 Tools available and percent memory used will be displayed As selections 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 cf Edit gt Options Memory amp Threads Aaa 4 07141413 10 7 8 gt Image 1 4305 Java 1 6 0_17 64 bit 3344K of 1166MB lt 1 hk SEE ALSO Plugins Utilities gt ImageJ Properties Helpt About ImageJ V TOGGLING CALIBRATED UNITS If a spatial scale has been defined cf Image Properties P or Analyze gt Set Scale sel
274. ugins 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 On Windows this avoids the problem where another copy 62 Latest revision 2011 08 06 Edit gt Options gt 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 Close the Log window to disable display of debugging messages Help Opens http imagej nih gov 1j docs menus edit html misc 63 Latest revision 2011 08 06 Image gt 25 Imager 25 1 Typecr 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 bi B HSB a me 2a S color color stack stack 8 bit T I S I S I S 16 bit I S ee LS I S 32 bit I S I S LS 8 bit color I S eae I RGB color I S I S e LS LS RG
275. urrent image by replacing each pixel with the neighborhood mean 26 11 5 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 gt Erode 26 11 6 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 Binaryb Dilate 110 Latest revision 2011 08 06 Process gt Batch gt 26 11 7 Unsharp Mask ii Sharpens and enhances edges by subtracting a blurred 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 review low frequency structures as in the input image This is equivalent to adding a high pass filtered image and thus sharpens the image a 3 Cancel OK e 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 26 11 8 Variance Highlights edges in the image by replacing each pixel with the neighborhood variance 26 11 9 Show Circular Masks G
276. uting time the rolling ball algorithm downscales the image in a way that is not really consistent 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 really use a 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 are used for creating the background Check this option to make sure that the image data after subtraction will never be below the background 115 Latest revision 2011 08 06 Process gt Repeat Command R 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 Background subtractor Rolling Ball Background Subtraction the plugin that implemented this command in versions up to 1 39e 26 15 Repeat Command R Reruns the previous command The Edit gt Undo and File gt Open commands are skipped SEE ALSO Undo and Redo 116 Latest revision 2011 08 06 Analyze gt 27 Analyze gt
277. uttered after the installation of several plugins Since Plugins reflects the hierarchy of directories in ImageJ plugins up to two subfolders submenus 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 EPS_ Writer class into ImageJ 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 files containing a configuration file plugins 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 plugins 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 jar files With Fiji Scripts and Macros can be registered in any menu by saving into Fijt app plugins Scripts menu name submenu name E g to have a certain macro installed in t
278. vely 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 Analyzeb Distribution command Results gt Set Measurements Alias for the Analyze gt Set Measurements command Results gt Options Opens the Edit gt Options Input Output dialog in which is possible to specify if column headers and row numbers should be saved or copied from ImageJ tables including the Summarize table cf Analyze gt Analyze Particles In addition it allows to specify the file extension to be used when saving data Custom extensions e g csu tls or ods allow ImageJ tables to be imported seamlessly by spreadsheet applications ImageJ tables are saved in CSV format if File extension for tables is csv SEE ALSO Plugins gt New Table 30 Latest revision 2011 08 06 ImageJ Editor Menu Commands Debug Window Debug Macro D Step aE Trace eT nimages 1 Fast Trace CHET getTitle blobs gif Run variable 1 Ij Run to Insertion Point Main Window variable2 1 43p Macros Abort OOO Untitled txt Run Macro SER varia
279. will be upgraded Upgrade To daily build 5 If you click OK Image will quit i ie and you will be running the upgraded to the latest daily build at http imagej nih gov ij ij jar version after you restart Image A m SEE ALSO Installing and Maintaining ImageJ Cancel OK 30 11 Refresh Menus 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 30 12 About Plugins gt 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 30 13 About ImageJ About Image Image 1 4 o E 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 gt ImageJ Prop erties Status bar 147 Latest revision 2011 08 06 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 StartupMacros txt You can also assign a function key to a menu command using Plugins gt Shortcuts gt Create Short
280. 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 Copy c Edit gt 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 Rectangular and oval selections see Area Area Selection Tools can be resized by holding Alt while using the arrow keys Composite Selections Composite selections are non contiguous ROIs containing more than ROIs are typically originated with the Brush Selection Tool but they O C2 one cluster of pixels and or ROIs containing internal holes Composite can be defined with any other selection tool using key modifiers The following modifier keys can be use to create composite selections Shift 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 Drawing inside current selection while pressing Alt creates a hole removing content from ROL Note that some operations may not be performed properly on complex ROIs In these cases it may be useful to convert a
281. wn 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 Check 65 Latest revision 2011 08 06 Image gt Adjust gt Propagate to all open images to apply these values to the rest of the images currently open 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 ADD Set Display Range Maximum displayed value 255 Unsigned 16 bit range _ Propagate to all open images Minimum displayed value X 8 bit 0 255 10 bit 0 1023 12 bit 0 4095 15 bit 0 32767 16 bit 0 65535 Cancel 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 ra
282. y Operation Selects one of the thirteen available op erators see Image Calculator operations Tran sparent zero 113 Latest revision 2011 08 06 Process gt Subtract Background Create New Window If checked a new image or stack will be created to hold the result Otherwise the result of the operation replaces some or all of Imagel 32 bit float Result If checked the source images will be converted to 32 bit floating point before the specified operation is performed Help Opens http imagej nih gov ij docs menus process html calculator Image Calculator operations On these examples source and destination images 8 bit grayscale are displayed with inverted LUTs White 0 Black 255 cf Lookup Tables 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 amgl mgl img Subtract amgl mgl img Multiply imgl imgl x img Divide imgl imgl img AND imgl imgl A img2 OR imgl mgl V img2 XOR imgl imgl 6 img2 I Destination image img2 Result Operator Result Min imgl min mgl img2 Max imgl max imgl img2 Average imgl imgl img2 2 Difference imgl img1 img2 Copy imgl img Transparent zero solo ole Es a mujm gt jo nla alal 26 14 Subtract Background Ari Subtract Background
283. y 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 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 Software Packages Built on Top of ImageJ 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 TrakEM2 TrakEM2 is a program for morphological data minin

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