Look@NanoSIMS - Max-Planck-Institut für Marine Mikrobiologie
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1. 13 b otherwise f Classify ROls 14 D Figure 19 GUI for manual A and automated B ROI classification C Examples of ROI classification file cells dat When the number of ROIs is large and the classification conditions can be formu lated by a logical expression ROI classification can be done automatically First display and export ROI ratios in a scatter plot to identify the classification conditions Fig 18 Subsequently select Classify ROIs Automatically based on conditions from the ROIs menu In a dedicated GUI Fig 19B associate data exported as de scribed in Sec 5 7 with one of the classification variables x y or z This is done by 24 selecting the filename containing the data e g a 19F 12C 13C dac file containing the ME C 18C ratios and specifying which data column should be considered as the classification variable usually mean or size This can be done for up to 3 independent variables Subsequently specify the classification conditions in the Condition fields using Matlab like syntax characters amp and represent logical AND and OR respec tively and the single character names of the classes During classification invoked by clicking Classify ROIs the assignment to classes is done sequentially all ROIs for which condition 1 is satisfied are assigned to the first class all ROIs for which condition 2 is satisfied are assigned to the second class irrespective whether some of the2. 13 4 099e 001 68 349e1000 4 162e 001 1 06 16 3 66 28 116 53 6 433e 001 1 734e 001 6 852e 001 2 60 g7 4 68 73 105 83 5 439e 001 1 375e 001 5 392e 001 2 23 71 C home lpolerec nanosims E3T6lightCF319a 8 13C 12C i Xi Yi MEANi Poiss Ei Foiss Ei SIZE i PIXELS1 LWratio 1 18 48 195 02 1 522e 02 1 335e 03 8 770e 00 2 01 ag 1 25 2 39 00 112 12 2 608e 02 2 412e 03 5 24 7e 00 1 06 le 1 31 3 66 28 116 53 1 168e 02 7 216e 04 6 179e 00 2 60 of 2 34 68 73 105 83 2 214e 02 8 903e 04 4 022e 00 2225 Fi 1 95 Figure 14 Examples of data files exported by Look NanoSIMS A Depth profiles of 3C 12C ratios B Accumulated 12G14N ion counts C 2C 12C ratios calculated from accumulated 13C7 and 12C7 ion counts When exporting values derived from total ion counts accumulated over all selected planes the ion counts in ROI k Fig 14B are calculated as m my l 1 is calculated by Eq 2 and L is the number of selected planes Furthermore the mean ratio of masses M and N in ROI k Fig 14C is calculated as 6 where m r m fn 7 whereas the Poisson percentage and standard error are calculated as 6 r 1 m 1 nk x 100 PE r r 6 r 100 8 9 Additionally to these values the exported dac file contains information about the ROI position size and shape Fig 14C The size is given both as the amount of pixels in the ROI and as the diameter of a circle in um with the equivalent amount of pixel
3. 6 we ow we we amp aM OS 5 6 ROI definition 4 6 3 6 ew ee he eee DO HE ee ee ws 5 6 1 ROI definition based on an external image Oot be eke ee ee ee eRe ee ea Rew ee DS SataprieslOutpue Il ce ce et eH setaa Ewe EDS Sw Ook RCI i ce A SERED EERE EK BE 5 8 2 Depth profiles ok i ew EP ER RRP ERR ROR DEY HA 5 8 3 Lateral profiles 2 266 i daw nde BEERS RHE De RHEE EM 58a OCA plots 2a ons Beate Rae EE eH EDD we OOS Poe POF OUI nc eee eee eee rora eei 29 ROI CSCO se A eK RRO SRO 5 10 Statistical comparison of ROIs and ROI classes 022 5 11 Metafile processing o oo ooa a a a a o a A A A WwW W 1 Installation instructions Look NanoSIMS can be run on a variety of operating systems including Linux Microsoft Windows and Mac OS X It requires core Matlab installation and the ac companying Image Processing and Statistical Toolboxes minimum version 7 11 R2010b Furthermore to allow export of images and data in PDF format it requires installation of BT X a freely available text processing program To display EPS and PDF output files generated by the program it is recommended to use freeware programs Ghostview and Acrobat Reader 1 1 Installation under Microsoft Windows Download the MikTeX distribution of TeX HTRX from miktex org and install it on your computer Download Ghostscript and GSview from pages cs wisc edu ghost and Adobe Acrobat Reader from www adobe com and ins
4. Furthermore this will also export the graphical representation of the result as an EPS 26 eraphics in the eps directory ROI classes are usually compared to test for differences in substrate uptake between cell populations To do this select Classes in the Compare pull down menu of the GUI dedicated to statistical analysis check multiple classes that should be compared check one treatment select the type of test that should be performed and the ratio based on which classes should be compared and click on Run test The graphical representation of the results of the test is displayed in the graph Fig 20B with a more detailed description listed in the Matlab s command window To export the test results click on Export results This will generate a text file in the tests directory containing various types of statistics calculated for the compared classes including the mean 95 confidence interval of the mean standard error of the mean and class ranking Rmin Rmax Fig 20D Furthermore this will also export the graphical representation of the result as an EPS graphics in the eps directory Comparison of treatments is usually done to test for differences between the incu bated and control samples or between incubation time points To do this select Treatments in the Compare pull down menu of the GUI dedicated to statistical analysis check multiple treatments and one class that should be compared select the type of test t
5. detected planes Fig 6 Check the Deselect checkbox to mark the plane that will be excluded from drift corrected accumulation At the end select the alignment region based on which the alignment of each plane relative to the base plane will be calculated It is recommended to define it as a minimum rectangular region in the image that contains pronounced spatial heterogeneities such as a cell or a group of cells Close the Alignment mass GUI and select the identification number of the base 11 on ea ese 7 35 _ Deselect lt sl all 100 150 200 eal Define alignment region Close Figure 6 GUI for the selection of accumulated planes and alignment region plane b Check Align images when accumulating and select Accumulate plane images to start drift corrected plane accumulation Fig 1A B For a given plane l the optimum alignment relative to the base plane b is defined as such a translation T a by a whole number of pixels in the x and y directions that minimizes the sum Apo LAN ye Taye 1 i l where N is the number of pixels in the alignment region and su is the base mass image in plane normalized such that its mean and variance over that region is 0 and 1 respectively The accumulation progress is displayed in the Matlab s command window After suc cessful completion of this step the alignment information is automatically stored in the xyalign mat file Fig 2B and will be used for future a
6. expdata birgit E3T6lightCF319a_6 0 05 alias cells a ae Ga eer cells b Re selst ee zt cells c O cells d eae D a 2 cells d 0 03 cellse EOE i cellse 0 04 cells f PERS es 2 pe cells f cells g 3 ae 8e cells g 0 02 m G cells k 12C15N 12C14N 0005 0 01 0 015 0 02 0025 0 03 0 035 13C 12C 43C 12C 12C15N 12C14N Figure 18 Examples of 2D and 3D scatter plots 5 9 ROI classification ROI classification is done after ROI definition by selecting Classify ROIs in the ROIs menu ROI classes are defined by names that are one character long e g a b c etc and stored in a text file with the recommended name cells dat Fig 19C When the number of ROIs is low classification is best done manually First display the outlines of the currently defined ROIs by selecting Export ROIs image from the ROIs menu Fig 12 Subsequently select Manually ROI by ROI from the ROIs menu and assign a class name to each ROI identification number in the provided GUI Fig 19A Pay attention that the number of classified ROIs is the same as the number of defined ROIs Save the classification file to disk when done Fig 19C 2 Variable Dag POISS_SE STD com ob Ww bh ee Oo SF af a amp T iy lt 0 015 amp x lt 0 03 POISS_PE MED Ziy lt 0 015 amp x gt 0 03 9 a 5 10 b i hi E Wrst Load conditions d conditions 12 k Soll fe Saye conditions
7. in a directory with read write access rights e g home user programs LANS matlab 5 Read the notes in the lookatnanosims m file to modify system specific settings e g paths to pdflatex and ghostscript 1 4 Updating Look NanoSIMS When updating the Look NanosSIMS program all you need to do is to download the updated distribution file see Installation instructions above and extract its content in the same directory where you have your older version overwriting the older files Naturally it is wise to keep a back up of the older version just in case This is especially true for the lookatnanosims m file under Mac OS X which contains settings adjusted for your system Alternatively you can join the lookatnanosims folder at www dropbox com and have the files automatically updated by the Dropbox program Please send an e mail to LP to be invited to join the folder 1 5 Adjusting program appearance obsolete from 24 04 2012 The graphical user interface GUI does not appear the same on all platforms when defined on one platform For example when defining a GUI the file with extension fig under Linux it will look quite different and often rather distorted when using it under Windows Thus all GUI files are defined separately for each platform and are stored in separate sub directories figs_win figs_lnx figs_lng sgd figs_mac If you wish to change anything in the layout of the GUI you need to modify the fig fi
8. in a log10 transformed scale in this case the minimum scale should not be 0 By checking the Include ROI outlines the outlines of defined regions of interest see Sec 5 6 will be included in the image The color of the ROI outline is specified in the corresponding field w for white r for red b for blue g for green k for black Additionally the width of the ROI outline can be specified by adding the line width in pixels after the line color e g w1 5 for a white outline of thickness 1 5 pt By checking Zero values outside ROIs all pixels that do not belong to defined ROIs will be displayed in black Use this last option wisely and not to hide unwanted information By checking the Graph image title checkbox the title containing the file name and the ratio string will be automatically added to the displayed image If you wish to exclude the file name from the title type space in the title field 14 Figure 11 13C 12C Figure 13 log 12C15N 12C14N MBR Figure 12 19F 12c ata birgit E3T6lightCF319a_6 13C 12C E3T6lightCF319a_6 log 12C15N 12C14N ct Tum by 0 02 0 03 i i 24 22 2 18 16 1 4 13C 12C log 12C15N 12C14N 19F 12C wo Figure 9 Display of ratios in publishing quality 5 5 3 Composite RGB images Select Combine images as RGB to combine specific mass or ratio images in a com posite RGB image Fig 10 The images that should be combined are specified by entering their i
9. masses of the detected ions raster size stage position etc Fig 3 This information is essential for the Look NanoSIMS program to correctly load the binary file Make sure with the operator of the Nanosims instrument that this file is generated for each measurement Nanosims MPI IMAGE Sample 15 08 11 17 10 Stage Position gt x 8096 um y 16463 um z 1300 um Raw data file export home Ecoli Azo Gam42a Ecoli Azo Gam42a_5 im Frame 1 1 256 256 Raster 20 0um Tuning Mode Multicollection Regulation Mode NMR Check every fr 0 Secondary Ion Beam Centering OFF Primary Current before acq 25260 pA after acq 25220 pA Mass Species Mass Det Tc s BField Radius Peak RefPeak Method i 126 12 019 Tr2 131 072 1054 802 423 231 2 13 13 023 Tr3 131 072 1054 802 440 556 3 19F 19 026 Tr4 131 072 1054 802 532 499 4 12C 14N 26 077 Tr5 131 072 1054 802 623 409 5 0 000 SE 131 072 1054 802 0 000 6 32S 32 094 Det7 131 072 1054 802 691 607 Figure 3 Format of the chk_im file containing essential information about the structure of the binary ion counts file A A typical data processing session This section describes steps of typical data processing sessions The description is very brief to give you an idea about the data processing sequence Refer to Sec 5 for a more detailed description of each implemented data processing function A 1 Analysis of a single dataset from scratch Define dead time and
10. stored preferences ficin 2 4 5 o2ess1 AE cinici 1010 Me Bees een Aa 00 1 gaa C E oor Post processing About aut Display template for ROI definition 1245 i 001 E my 32S Bil 2 4 5 0223 E Aa eana an E go o Eo ol E i A INTERACTIVE ROIs definition tool Load ROIs from disk Translate ROIs by x and y Export ROIs image Manually ROI by ROI Clear ROIs image Automatically based on conditions D Output Post processing About Display masses Display ratios Generate LaTeX PDF output Store Preferences Close all figures Generate processing metafile Process metafile Combine processed files into a single Cameca like file Figure 1 Main graphical user interface of the Look NanoSIMS program 3 Organization of the input and output data To facilitate efficient data processing especially through the metafile processing function datasets belonging to a given experiment including the raw image stack files files with extension im and the information files files with extension chk_im need to be copied to a single directory Fig 2A The names of the directory and dataset files must not contain spaces The processed information for a given dataset is stored in a directory of the same name as the input file name This directory is further structured into sub directories that contain exported information in the Matlab mat vector graphics eps and pdf bitmap tif and text dat tests and tex
11. 0 01885 0 01783 0 01987 0 00050 2 2 TRS e 0 01109 0 00829 0 03048 0 00450 3 4 Mitog t0 transform E z z ay g 0 00844 0 00721 0 00967 0 90059 3 3 _ Show multiple comparison graph 3 Eua f 0 00782 0 00598 0 00966 0 00078 34 P dz 12C15N 12014N so k 0 00406 0 00384 0 00428 0 00011 4 4 7 x10 a 0 00494 0 00388 0 00420 0 00008 4 4 Export results b 0 00389 0 00380 0 00399 0 00005 4 4 Figure 20 GUI for statistical analysis of individual ROIs A ROI classes B and treatments C D Example of the text based output generated by the statistical analysis Individual ROIs are usually compared to identify heterogeneity of substrate uptake by cells within a specific population To do this select ROIs in the Compare pull down menu of the GUI dedicated to statistical analysis check the class for which ROIs should be compared check one treatment select the type of test that should be performed and the ratio based on which ROIs should be compared and click on Run test The graphical representation of the results of the test is displayed in the graph Fig 20A with a more detailed description listed in the Matlab s command window To export the test results click on Export results This will generate a text file in the tests directory containing various types of statistics calculated for each ROI including the mean 95 confidence interval of the mean standard error of the mean and ROI ranking Rmin Rmax
12. 03 4 07e 003 pixel count k 5 54e 003 1 66e 003 gt all cells 0 02 0 03 0 04 0 05 13C0 12C accumul of co 19F mean Poisson error per pixel 45 20 plane number plane number Figure 16 GUI for ploting depth profiles of masses A and ratios B in selected ROls 5 8 3 Lateral profiles To display ion counts Eq 2 and ratios Eq 3 along a lateral profile check the Display lateral profiles option and select Display masses and Display ratios from the Output menu In the provided GUI Fig 17 define the profile by clicking Define lateral profile and selecting the starting and ending points and specify the width of the profile in pixels Click on Display lateral profiles to display the profile for the selected mass or ratio in a separate window or for all masses or ratios in a single window Fig 17 If 22 Display lateral profile Define lateral profile Profile thickness pixels Ei 13C 12C 13C 12C 13C 12C 12C15N 12C14N 13C 12C 100 150 200 normalized values min max gt 0 1 250 50 100 150 200 250 _ Show error bars STD _ Export lateral profile as ASCII amp EPS Show normalized in one graph Display lateral profiles Figure 17 GUI for ploting lateral profiles of masses or ratios the profile width is greater than 1 the standard deviation of the values derived from the pixel values
13. 1 nk x 100 4 PE rf r rf 100 5 from Poisson errors represent a theoretical precision of the mean derived from the fact that the count of ions detected during dwelling time is a random variable with Poisson distribution Cameca WinImage User s Guide v 2 0 8 p 82 The depth profile output additionally contains the result of a test for a significant trend with depth Fig 14A This is obtained by fitting the values of mj Eq 2 and rj Eq 3 with a polynomial of order POL and testing whether the variance of fit residuals is significantly lower than the variance of the original values Maximal tested value of POL is 5 The significance testing is done using Levene s test default significance level a 0 05 POL gt 0 indicates significant trend with depth whereas POL 0 indicates no significant trend with depth 20 A fhome lpolerec nanosims E3T6lightCF319a 8 13c 12c cell MEANI Poiss El MEAN2 Poiss E2 MEANS Poiss E3 MEAN4 plane POL 0 POL 0 POL 0 POL 0 POL 0 POL 0 1 2 319e 02 8 293e 03 2 f32e 02 1 238e 02 1 264e 02 3 836e 05 2 1 587e 02 155e 03 1 630e 02 9 490e 03 9 67 7e 03 3 241e 058 3 1 729e 02 7 120e 03 3 593e 02 1 493e 02 5 302e 03 2 378e 05 4 1 824e 02 7 513e 03 3 500e 02 1 346e 02 6 5929e 03 3 171e 08 B 2 D expdata birgit E3T6lightCF319a 8 12C14N dac i Xi Yi MEAN STD1 MEDIANiI SI4ZE1 PIXELS1 1 18 48 195 02 4 946e 00L 9 641e1000 4 922e1001 2 01 58 2 39 00 112
14. 50 100 150 200 250 Figure 13 Exported ROI outlines together with unique identification numbers First select the external image via File Load external image Fig 13B This can be a bitmap image in any common format such as TIF PNG etc Beforting importing the external image it is recommended to crop it in a third party image processing program such that it is slightly larger but not too large than the field of view captured in the nanosIMS image If necessary the cropped image can also be rotated by 90 or 180 degrees to improve its resemblance to the nanoSIMS image Second select the nanoSIMS image that will be used as template for alignment via File Load nanosims image Fig 13B This can be any accumulated image that has been stored in the Matlab format extension mat stored in the mat sub directory during the previous steps of the analysis see Sec 5 5 Subsequently use the Action menu to define reference points that correspond to each other in both images Fig 13C Proceed by adding a point in the external image followed by the addition of the corresponding point in the nanoSIMS image Use left mouse click to define the point s position and right mouse click to confirm it If the reference points were defined incorrectly remove them before proceeding with another pair of points Use Action Show alignment to visually inspect the alignment quality Fig 13D 19 When satisfied with the alig
15. Look QNanoSIMS User s manual Lubos Polerecky Max Planck Institute for Marine Microbiology Bremen Germany version 24 04 2012 http www microsen wiki net lans Summary Look NanoSIMS is a program for the analysis and processing of nanoSIMS and SIMS data It can be used freely for research purposes It is an open source program and can therefore be modified freely by anyone to meet their specific needs We ask users who make modifications to the program to share them with others by e mailing their improved code to the person in charge of the program maintenance who will then post the updated version on the program s website currently LP If you experience problems with the execution of the program discuss them first with your colleagues that are experienced in using the program If this does not solve your problem contact LP In your e mail describe the problem and include in attachment the dataset for which you experienced the problem as well as a screenshot of your LookQ NanosIMSs session especially the Matlab s command window with the error message If you wish to have new features added and don t know how to do that contact LP describing the main idea Since this program is at present being actively developed there is a good chance that your request will be rapidly fulfilled Citation L Polerecky B Adam J Milucka N Musat T Vagner M M M Kuypers 2012 Look NanoSIMS a to
16. OI to select it for removal and the right mouse click to remove it Select Remove multiple ROIs from the menu to remove several ROIs at once whereby the identification 17 numbers of the unwanted ROIs should be entered in the Matlab s command window Select Combine multiple ROIs into one from the menu and enter ROI identification numbers in the Matlab s command window to combine specific ROIs into one If the most recently defined ROI overlaps with one or more of the ROIs defined previously the overlapping pixels will belong to the most recently defined ROI Thus to draw a ROI with a hole draw the bigger ROI first followed by the drawing and removal of the smaller ROI If the ROI classification file see section 5 9 was defined and selected before ROI definition it is automatically updated upon every ROI addition or removal provided that the option Automatically update classification file is checked Select Display ROIs to display currently defined ROIs and Save ROIs to save them to disk It is recommended to use the default file name cells mat This will facilitate rapid post processing of different datasets in a later stage of the analysis see Sec 5 11 Interactive ROI definition is completed by quitting the GUI and exporting the ROI outlines via Export ROIs image from the ROIs menu This step will display and ex port ROI outlines together with automatically assigned identification numbers Fig 12 These numb
17. QSA correction parameters Usually these corrections are not necessary and this step can be skipped Load raw data from disk Click on Ask for range of planes before loading if you want to select specific planes when loading the raw data from disk Autoscale and display plane images for all masses Decide which mass will be used as a base mass for plane alignment during accumulation Display alignment mass While viewing deselect planes that should be excluded from accumulation At the end define the alignment region Accumulate drift corrected images The alignment information will be stored in a file xyalign mat Autoscale and display the accumulated images to verify the alignment quality If not satisfied with the alignment delete the xyalign mat file choose a different alignment mass or alignment region and repeat steps 4 5 Display accumulated masses via Output Display masses Define expressions for ratio images Display ratio images via Output Display ratios 10 11 ie 13 14 l5 16 VE 4 2 Define ROIs based on a template image mass ratio RGB overlay external If an external image is chosen as template align it first with the nanoSIMS image Save the ROI file to disk recommended file name cells mat Export the image of ROI outlines This will automatically assign a unique identi fication number to each ROI Display depth profiles of ratios in ROIs Check whethe
18. ale manually as min max The scale is not changed when a new dataset is loaded Select Display plane images for all masses Fig 1B to view single planes for the detected masses Fig 5 The displayed value in each pixel and for each plane represents the counts of the corresponding mass in that pixel and plane accumulated during dwelling time 10 Mass display oog gt E dsj lt i gt d gt l Close gird 12C14N 12C15N Figure 5 GUI for displaying all detected masses and planes 5 3 Drift corrected accumulation of planes The drift corrected accumulation of planes is controlled by settings specified in the Accumulation options box and in the images fields Fig 1A These include the name of the base mass the number of the base plane the alignment region within the base mass and the identification numbers of planes that should be included in the accumulation Specify the name of the base mass in the Base mass for alignment field It is recom mended to select the mass that exhibits high contrast visual features e g well separated cells or filaments and is characterized by the highest signal to noise ratio among the image pixels Usually the secondary electron SE image is selected When working with cells on a polycarbonate filter the image of C 4N or 3 S is also a good alternative Select Display alignment mass from the Input menu In the provided GUI click arrows to view the images of the
19. are classes Export images for all masses and ratios as PDF Z Include cell outline color w _ Zero outside cells Export images for each variable as PDF MWPlot2D WPlot3D Display error bars Export plots amp tests as PDF Figure 21 GUI for the definition A and processing B of metafile instructions C Types of metafile processing functions offered by the Look NanoSIMS program Metafile processing is done in a dedicated GUI accessed via Post processing Process metafile from the main GUI Fig 21B First select the base directory where all single processed datasets are stored Second select the file with metafile instructions Third specify the names of the files that contain information about ROI outlines cells mat by default and ROI classes cells dat by default Finally specify parameters for controlling the appearance of the graphs and images that will be generated by metafile processing such as the title scaling color and symbol schemes etc Select Plot and export data from the Tools menu Fig 21C to extract and merge all results that have been generated for the datasets listed in the metafile These results will be plotted in scatter graphs Fig 18 and exported in a single text file that can be used for further processing by a third party software Select Compare ROIs and Compare ROI classes from the Tools menu Fig 21C to perform statistical comparison of ROIs and ROI classes for all datasets in the metaf
20. aset from disk Before loading data from disk select Dead time Dead time correction settings Bim g _ Apply dead time correction Ref 1 amp QSA correction settings from the Input menu N eorr 1 Y N_ meas bkg 1 N meas bka dt le 3 _ Apply OSA correction Ref 2 N_corr N_meas 1 K 2 QSA corrections Fig 4 Y detector yield N_meas measured counts per second Select Ask for range of planes before loading 9 lt ksfound counts per second dt dwelling time in nanoseconds Fig 1B to enable the possibility to interactively N_corr dead time corrected counts per second Fig 1B to specify parameters for dead time and define planes that will be loaded from disk O nanan n Hog Po Select Load RAW dataset Fig 1B to load 7 eo a o nanoSIMS dataset from disk This will auto i 2 EI matically fill the mass fields in the Detected masses j L 5 box with names of the detected masses as well as Dwelling timeums 1 fill the images fields with which refers to all de OK Cancel tected planes Figure 4 GUI for the definition of dead time and QSA corrections 5 2 Display of mass images Select Autoscale plane images Fig 1B to update the scale fields with the optimum scale for displaying the raw mass images For each detected mass the optimum scale is calculated as the 0 1 and 99 9 percentile of ion counts in all pixels and planes Alterna tively specify the sc
21. ccumulations of the same dataset if reopened in the program Remove the xyalign mat file from disk prior to accumulation if you wish to generate a new alignment 5 4 Display of accumulated mass images Select Autoscale accumulated images to update the scale fields with the optimum scale for displaying the accumulated mass images For each detected mass the optimum scale is calculated as the 0 1 and 99 9 percentile of the accumulated ion counts in all pixels 12 Alternatively specify the scale manually as min max The scale is not changed when a new dataset is loaded Select Display accumulated images for all masses to view images of accumulated ion counts for the detected masses Fig 7 Verify the alignment quality by visually inspecting the sharpness of distinct features in the images Ly Figure 31 206 HSas s AAV9RL B 08 an S IEE 13C 19F 12C14N Esi 12C15N Figure 7 Display of accumulated mass images 5 5 Graphical output I 5 5 1 Accumulated mass images To display accumulated masses with publishing quality Fig 8 deselect all options in the Output options box except the Display images option and select Display masses from the Output menu A particular mass is displayed if its corresponding checkbox is checked 5 5 2 Ratio images To display ratio images with publishing quality Fig 9 deselect all options in the Output options box except the Display images option and select Display ratios fr
22. dentification numbers in the corresponding fields for the R G and B col ors When pixel by pixel is selected the images are combined without modification whereas values averaged over each ROI are used when the option Averaged over ROIs is selected The contrast and saturation of each individual color can be modified by changing the scale of the corresponding image A specific color is not used if the corre sponding field is left empty If you want to combine mass image with ratio images enter the mass name into one of the expression fields and enter the corresponding identification number in one of the color fields Figure 36 aaka BAGH NSUKA IE GO0e a0 JOSU aago annm 3 R 13C 12C G 12C15N 12C14N B 19F 12C R 13C 12C G 12C15N 12C14N B 19F 12C D expdata birgit E3T6lightCF319a_6 D expdata birgit EST6lightCF319a_6 Figure 10 Display of ratios as composite RGB images 15 5 5 4 EPS and PDF output Select Export EPS PDF graphics while displaying the mass or ratio images to export the images as EPS and PDF graphics Note that XIX installation is required to enable PDF export 5 6 ROI definition Type the name of the image that should be used as template for the definition of regions of interest ROIs in the ROI definition template field This can be any of the accumulated mass images e g 12C14N 19F a ratio image derived from the mass images e g 13C 12C 19F 12C 12C an RGB composite image constructed from the i
23. e ratio s depth profile see Sec 5 8 2 If r exhibit a significant trend with depth it is likely that they originate from more than one type of object in the sample e g a cell lying on top of a filter or of another cell with different isotopic composition and are therefore not justified as a basis for ROI comparison For each comparison level homogeneity of variance is tested using Levene s test whereas significant differences between means are tested using Kruskal Wallis and ANOVA tests All tests employ the default significance level a 0 05 To identify which ROIs ROI classes or treatments are significantly different from the others the Kruskal Wallis and ANOVA tests are coupled to a multiple comparison procedure based on Tukey tests This analysis assigns to each tested ROI ROI class or treatment 29 a pair of ranks that allow its comparison with the other ROIs ROI classes or treatments Fig 20D For example assuming that ROI classes c and c3 were assigned pairs of ranks Ro Re and R2 Re min max min max inequality RO lt R max min respectively their comparison is interpreted as follows the means that the mean ratio r for ROIs in class c is significantly greater than the mean ratio r for ROIs in class c2 whereas the mean ratios r and r2 do not significantly differ within the instrumental precision if R gt Rn max A B Statistical tool ROI classes Al
24. ers will be used throughout the rest of the analysis for identifica tion of each ROI Previously defined and saved ROIs can at any time be loaded via Load ROIs from disk To translate defined ROIs in x and y direction specify the corresponding number of pixels in the x and y fields below the Translate ROIs option check the Translate ROIs checkbox and select Translate ROIs by x and y from the ROIs menu Figure 51 Sele __ Figure 50 jo 7 ata birgit E3T6lightCF319a_6 cells c pdata birgit E3T6lightCF319a_6 cells GS udp he os BS y 2 pet CE Ahea O18 G22 21 h16 T Ta d 5 5 N a 1 ri GJ a 13 qd a i oO me RE Cao e i E 4 i f H j 01 amo TO 02 7 a te 1 Ee z Figure 12 Exported ROI outlines together with unique identification numbers 5 6 1 ROI definition based on an external image Type ext in the ROI definition template field to allow ROI definition based on an external image as template Before the ROI definition can proceed the external image must 18 be aligned which is done in a dedicated GUI Fig 13A accessed via ROIs gt Align external template image Fig 1C File Action it E External 100 200 300 400 D B AGE Action Load external image Ctri E Load nanosims image Ctri N Load point list Save point list c Add point Ctri A Remove point Ctri R Update points Ctri U Show alignment Ctri H Export aligned external Ctri E
25. formats Fig 2B A base directory B Directory with processed NanoSIMS dataset Location home lpolerec nanosims Name 5 a2 gt Pal dat lt _ _ directory with text based data output D all data b eps 4 directory with vector graphical output b E3T6lightCF319a_ 3 gt a mat 4 directory with matlab output b E3T6lightCF319a_5 b pdf directory with vector graphical output E EsT6lightcF319a 6 gt B tests lt directory with text based statistical output p ESTONGhICF319a 6a b tex directory with tex output p E3T6lightCF319a_7 gt G tif directory with bitmap graphical output EST6lightCF319a 7a cells dat 4 ROIs classification file r EST6lightCF319a 8 cells mat ROIs definition file 8 TERR area E outputG pdf PDF summary output La z outputG tex E3T6lightCF319a_3 chk_im outputT pdf E3T6lightCF319a_3 im outputT tex clans prefs mat _ calculation and display preferences Ep aie a xyalign mat q alignment file E3T6lightCF319a_6 chk_im E3T6lightCF319a_6 im Figure 2 Organization of the input and output data in Look NanoSIMS Input data format Each dataset must comprise two files i the raw image stack file with extension im and ii the information file extension chk_im The first file contains the detected ion counts stored in a binary format whereas the second file contains information about the structure of the binary file names and
26. hat should be performed and the ratio based on which the selected treatments should be compared and click on Run test Fig 20C The rest of the analysis proceeds as described for the comparison of ROI classes above 5 11 Metafile processing The above described routines allow processing of data and export of results for one dataset The purpose of metafile processing is to rapidly combine and statistically analyze results from multiple datasets Note that each of the datasets must have been processed before it can be included in metafile processing First choose Generate processing metafile from the Post processing menu of the main GUI to open a GUI dedicated to the definition of a metafile with processing instruc tion Fig 21A In this GUI define the dataset name by selecting the corresponding directory that contains the processed information generated by steps described above Subsequently specify the treatment identification number for this dataset this can cor respond e g to a time point in the incubation experiment ROI classes single character class names or for all classes and click Add data set Do this for all datasets that you want to be combined and processed together Subsequently define variables that should be processed These can be mass ratios e g 13C 12C or 12C15N 12C14N or any other value calculated and stored in the dac files e g ROI size x and y position see Sec 5 7 Finally save the metafi
27. ile The types of analysis are the same as those available during the statistical analysis of results obtained for a single dataset see Sec 5 10 Fig 20 Note that because this comparison relies on the results generated during processing of single datasets e g depth profiles of ratios ratios derived from accumulated ion counts ROI classification this information must exist for each dataset listed in the metafile Choose one of the Export PDF functions in the Tools menu Fig 21C to generate a PDF file with an overview of results from multiple datasets Mass and ratio images from all datasets listed in the metafile will be combined in a single PDF file by 28 selecting Export images for all masses and ratios as PDF whereas separate PDF outputs will be generated for the processed variables see metafile definition above if Export images for each variable as PDF is selected By selecting Export plots amp tests as PDF all 2D and 3D graphs as well as graphical representations of the statistical tests that were generated by metafile processing will be combined in a single PDF file Note that installation of BTFX is required to enable this feature 29
28. l j None j Ee ab ae ee ae If Ig Mk _In Treatments AIl None ROI classes All None Wa Mb Mc Md e Ef Mg Ek Mn Treatments All None Means significantly differ ANOVA p 491 Pal 05 Mi _ 2 M1 _ 2 6 ma AN Compare Classes X Test type 12C15N 12C14N _jLog10 transform _ Show multiple comparison graph ANOVA _jlog10 transform _ Show multiple comparison graph 0 01 0 012 0 014 0 016 0 018 0 02 0 022 0 024 0 026 0 028 1307120 Statistical tool Generated by Look NanoSIMS version 09 07 2011 on 19 Jul 2011 15 29 08 ROI classes All d None 2 Means significantly differ ANOVA p 3 19e 05 Analysis of 12C15N 12C14N averaged over ROIs from classes abcdefgk treatment 1 Sta ee ue gie aje mean std devi class N c t Jf Mg ik _In 4 04e 03 6 71e 04 73 a 1 3 89e 03 4 39e 04 86 b 1 3 07e 02 6 06e 03 139 c 1 7 82e 03 2 20e 03 8 f 1 1 89e 02 2 58e 03 27 d 1 1 11e 02 7 80e 03 3 e 1 Bore 4 06e 03 5 97e 04 31 k 1 Treatments All None 8 44e 03 2 84e 03 23 g 1 A fe HRB aaa w ANOVA test 2 Source SS df MS F Prob gt F Compare i Groups 5 92e 02 7 8 46e 03 573 506 2 7le 198 Error 5 63e 03 382 1 47e 05 Treatments X Total 6 48e 02 389 Means significantly differ amongst classes ANOVA p 2 7le 198 Test type ANOVA Ranking based of multiple comparison of the means G bf Let Patio i a A e eS 12C15N 12C14N M c 0 03070 0 02968 0 03172 0 00051 1 1 d
29. le Depending on your operating system and where you installed the program see above this can be done by clicking on the browse for folder button and browsing through the file system to the correct folder or by typing gt gt cd c programs LANS matlab or gt gt cd home user programs LANS matlab in the Matlab s command window Subsequently type gt gt lookatnanosims in the Matlab s command window This will open a main graphical user interface GUI through which all functions of the program are executed Fig 1 Important messages generated during program execution including warnings and error messages are displayed in the Matlab s command window If an error occurs and you cannot fix it please proceed as described in the Summary on the first page of this document Look NanoSIMS version 03 11 2011 B Input ROIs Output Post processing About Input ROIs Output Post processing About Quit Ask for range of planes before loading Dead time amp QSA correction settings home polerec nanosimsdata LANS FISH Load RAW dataset Autoscale plane images home polerec nanosimsdata LANS FISH Ecoli_Azo_Gam42a_5 Display plane images for all masses Display alignment mass wl ic 13 45 P 0 1249 m M 13C 12 10 007 0 7 Nia Accumulate plane images Autoscale accummulated images v 1 2 45 0307 Bim By 19F 12C 13 0 0 1 Display accumulated images for all masses mM 19 91 245 0 17 m Ej 12C15N 12C 0 0037 0 05 gay Load
30. le from the directory corresponding to your platform which can be done using the Matlab s GUI editor menu File gt New gt GUI 1 6 Adjusting graphical output appearance The appearance of the graphical output exported in the EPS and PDF format may also depend on the platform or the Matlab version used In particular the size of the text labels e g axes labels title text relative to the size of the graph image itself may appear too large or too small This can be adjusted by modifying the PaperPosition property of the exported figures in the file fnc print_figure n 1 7 The total number of scanned planes 0 problem The mass counts on some newer systems are stored as ushort 16 bit unsigned inte ger whereas on some older systems they are stored as uint8 8 bit unsigned integers Perhaps this is because of the differences between the 32 bit and 64 bit operating systems that run the Cameca instrument but it is not sure Therefore if you experience problems with loading a file e g the program tells you that there are O planes in your file it may well be that this is the cause In that case open the file read_im_file m in the fnc directory and change if 1 to if O0 on line 12 or so Then save it and continue as before If this does not help please contact LP 2 Running LookQ NanoSIMS To run Look NanosIMS start Matlab and change the current working directory to the location of the lookatnanosims m fi
31. le instructions in a text file Although metafile definition is more intuitive using the dedicated GUI the metafile can also be generated in any text editor 2 A i mada ceaitizen Visa f Process multiple datasets from metafile m i Data set Treatment Cell classes for all settings Tools E3TOCF319a_2 2 i Select from disk Add data set Remove data set Base directory where all the processed datasets home polerec nanosims Metafile containing processing 6lightCF319a_5 aai T6lightCF319a_6 home polerec nanosims meta LP1 txt T lightCF319a_7 RO options E RO definition file mat cells mat Select ROI classification file dat cells dat Select Variable on X1 axis Variable on Yl axis Variable on Z1 X2 axis e g 13C 12C e g 19F 12C e g 1L2C15N 12C14N 13C 12C 12C15N 12C14N 19F 12C Variable on Y2 axis Variable on X3 axis Variable on Y3 axis e g size e g x e g y Size X y Load metafile Save metafile Graph Image options Til X1 scale _jlog Yi seale slog ZI1 X2 scale _ log 0 WO 0 E 0 Y2 scale llog X3 scale llog Y3 scale _ Slog o0 WO 0 E QQ Color scheme Symbol scheme Cell classes e g a bc Treatments e g 1 2 3 Plot and export data Colors rg bme Symbols ox gt lt V Compare ROIs rgbmckolwdpny bs xAveodp Comp
32. m may have been already assigned to the first class etc ROIs that have not been assigned to a class by any of the specified conditions are by default assigned to class otherwise Save the automatically generated ROI classification file to disk under the recommended name cells dat Fig 19C Classification can be verified in the GUI for manual classification see above 5 10 Statistical comparison of ROIs and ROI classes Check the Compare ROIs option and select Display ratios from the Output menu in the main GUI to perform statistical comparison of ROIs for a currently analyzed dataset Similarly select Compare ROIs or Compare ROI classes from the Tools menu during metafile processing see Sec 5 11 to compare ROIs and ROI classes from multiple datasets A dedicated GUI offers statistical tests that can be performed for three levels individual ROIs ROI classes and treatments Fig 20A C Comparison of ROI classes and treatments takes the mean ratios rg calculated from the total ion counts accumulated over ROI pixels over all selected planes Eq 7 as a basis for comparison In contrast comparison of individual ROIs is done based on the per plane ratios rf calculated from ion counts accumulated over ROI pixels in each plane Eq 3 The latter is based on the assumption that r represent independent replicate measurements of the ratio in the object that the ROI represents This assumption can be verified by plotting th
33. nment save the list of reference points via File gt Save point list and export the aligned external image via Action Export aligned external This will generate a TIF file of the aligned external image as well as of its overlay with the nanoSIMS image Both of these images can subsequently be used as template for ROI definition which proceeds as described above Sec 5 6 5 7 Text output Export of ion counts and ion ratios in ROIs in a tabulated format is done by selecting Display masses and Display ratios in the Output menu respectively and selecting the option Export ASCII data in the Output options box With these settings ion counts accumulated over all ROI pixels and over all selected planes as well as ratios derived from these accumulated ion counts see below are saved in files with extension dac in the dat sub directory If the Display depth profiles in ROIs option is selected the results are additionally displayed as depth profiles and exported in files with extension z dat Fig 2B When exporting depth profiles ion counts in plane l and ROI k are calculated as Px m Mi 2 i 1 where Ma is the ion count per dwelling time for mass M in pixel i and plane l and P is the number of pixels in ROI k The mean ratio Fig 14A of masses M and N in plane l and ROI k is calculated as k k Jk rp mi ni 3 while the Poisson percentage and standard errors of the mass ratio are calculated O r7 y 1 m
34. ol for the analysis of nanoSIMS data in environmental microbiology Environ mental Microbiology 14 4 1009 1023 doi 10 1111 j 1462 2920 2011 02681 x lpolerec mpi bremen de Contents Installation instructions 1 1 Installation under Microsoft Windows 008 4 1 2 Installation under Linux 0 0 0 0 00 000000 1 3 Installation under MacOSX 2 2 ee 1 4 Updating Look NanoSIMS 0 000000 eee 1 5 Adjusting program appearance obsolete from version 24 04 2012 1 6 Adjusting graphical output appearance 0 1 7 The total number of scanned planes 0 problem 2 2 Running LookQ NanoSIMS Organization of the input and output data A typical data processing session 4 1 Analysis of a single dataset from scratch 4 2 Analysis of a single dataset continuation or modification of previous 4 3 Analysis of multiple datasets 2 0 aa Data processing functions of Look NanoSIMS 5 1 Loading nanoSIMS dataset from disk 04 5 2 Display of mass images 1 a a a a a 5 3 Drift corrected accumulation of planes 08 5 4 Display of accumulated mass images 0 000000 Do Graphical output I sses wk wR RARE DRE ESHER OS 5 5 1 Accumulated mass images 0 00000 2G 5 5 2 Ratio images hw caw be ee eae a 5 5 3 Composite RGB images 0 0 0000000 2G 5 5 4 EPS and PDF outp t haw 6
35. om the Output menu A particular ratio is displayed if its corresponding checkbox is checked The formula for calculation of the ratio image is specified in the expression box It can be any valid arithmetic expression e g 13C 12C to calculate a simple mass ratio 13 DER MBR Figure 4 12 14N Cf Figure 6 tog 12c15N data birgit E3T6lightCF319a_6 12C14N birgit EST6lightCF319a_6 log 12C15N L E E 0 1000 2000 19F 12C14N 0 10 20 30 40 50 60 70 0 500 1000 1500 2000 2500 3000 0 0 5 1 1 5 2 Figure 8 Display of masses in publishing quality 13C 12C 13C to calculate the natural abundance of C 1000 13C 12C 13C 0 01 to calculate the C enrichment relative to a standard here the natural abundance of 0 01 in per mil 32S size to calculate the concentration of a specific mass in the ROI i e total counts normalized to the ROI size The scale is specified by entering min max in the corresponding scale field When the scale is changed and enter is pressed the image is displayed immediately i e without selecting Display ratios from the Output menu using the given scale allowing rapid optimization Use the following additional options to customize the display of accumulated ion images and ratio images By checking the corresponding B W checkbox the image is exported as a black and white TIF image By checking the Log10 transform checkbox the image is displayed
36. on rgb1 or ratio rgb2 images or an external image ext The external image can be any bitmap image stored in a common graphics format e g TIF or PNG Alignment between the external and ion images is done as described in Sec 5 6 1 Define cells using image rgb2 Select interactive channel Zoom Display ROIs SaveROIs Quit v Automatically update dassification file Interactive thresholding Cri A E Draw ellipse Ciri A y a d es Draw rectangle Ctri4 T aie Top P E Split ROIs by line Cirip elon R E Take the coarse drawn line Draw ROI freehand Ctrl D Take droumscribing ellipse Remove ROI interactively Ctri R v Always ask Remove multiple ROIs yy Duplicate ROI interactively Combine multiple ROIs into one Figure 11 GUI for interactive ROI definition In this case a composite RGB image is used as template For all alternatives the selected template image can optionally be smoothed before it is used for ROI definition Smoothing is done using a median filter with the two dimensional kernel size specified in the Smoothing kernel field in pixels This will result in generally smoother ROI outlines when defined using the interactive thresholding method for ROI definition see below If you do not wish to do image smoothing before ROI definition use 1 1 as the smoothing kernel 16 Select INTERACTIVE ROIs definition tool from the ROIs menu to start a GUI dedicated to ROI definition Fig 11 Fir
37. over the profile width can also be displayed The profiles can optionally be exported in a text and graphical format by checking the Export lateral profile as ASCII amp EPS checkbox 5 8 4 Scatter plots To display scatter plots of ion ratios in ROIs Eq 7 check the Plot x y z graph option and select Display ratios from the Output menu The mean ratios in ROIs are displayed together with error bars that correspond to the Poisson standard error Eq 9 The ratios that should be displayed are specified by entering their identification numbers in the corresponding x y and z fields in the Output options box If ROIs have been classified the different classes are displayed with different symbols and colors Fig 18 5 8 5 PDF output Select Generate LaTeX PDF output from the Output menu to generate a summary of the results obtained during the analysis of a single nanoSIMS dataset The graph ical and text based outputs are stored under default file names outputG pdf and out putT pdf respectively The types of images and graphs included in the graphical out put are specified by selecting the corresponding Output options Note that installation of BTFX is required to enable this feature An example of a PDF file generated by this function can be downloaded from http www microsen wiki net doku php id lans 23 Figure 39 Figure 39 Se DS agsSs F ARV9EA B Oso OSG as F ARO Pe a l 0H a 0 x D expdata birgit E3T6lightCF319a_6 D
38. r ratios exhibit significant trends with depth Display ratios as images with ROI outlines histograms lateral profiles scatter plots or RGB images Classify ROIs Save the ROI classification file to disk recommended file name cells dat Export ratios in ROIs in graphical and text formats Perform statistical comparison of ROIs and ROI classes for the currently analyzed dataset Export processing results in a PDF file Store settings of the current data processing session via Output Store preferences recommended file name prefs dat Analysis of a single dataset continuation or modification of previous analysis Do steps 1 2 described in Sec 4 1 Load previously stored settings from disk e g from the file prefs mat via Input Load stored preferences Accumulate drift corrected images Alignment information stored in the xyalign mat file will be automatically applied Load ROIs from disk e g from the file cells mat Modify ROIs and ROI classification if required Continue with steps 10 17 listed in Sec 4 1 4 3 Analysis of multiple datasets 1 Ensure that all processed data are in the same directory see Fig 2 2 Generate processing metafile 3 Process metafile plot data from multiple datasets in scatter plots compare ROIs ROI classes or treatments generate PDF output of the results 5 Data processing functions of LookQ NanoSIMS 5 1 Loading nanoSIMS dat
39. s as the ROI The shape is expressed as the ratio between the long and short axes of the ellipse circumscribing the ROI 5 8 Graphical output II 5 8 1 Histograms To display histograms of ion counts and ion ratios in pixels that belong to ROIs Fig 15 left check the Display histograms option and select Display masses and Display ratios from the Output menu respectively If ROIs have been classified see section 5 9 the histograms are constructed separately for each ROI class Fig 15 right 21 5 8 2 Depth profiles To display depth profiles of ion counts Eq 2 and ratios Eq 3 for each defined ROT check the Display depth profiles in ROIs option and select Display masses and Display ratios from the Output menu The depth profiles are plotted together with error bars that correspond to the Poisson standard error Eq 5 Additionally fitting poly nomials are also displayed when a significant trend with depth is detected Depth profiles in multiple ROIs can be displayed and exported in a dedicated GUI Fig 16 _ Figure 24 pixel count OSes AQQe9ed 4 Oj ao 1000 800 D O O 400 200 aan Figure 23 wisi EEEO Er aare E x D expdata birgit E3T6lightCF319a_6 D expdata birgit EST6lightCF319a_6 a 5 67e 003 1 63e 003 gt b 5 21e 003 1 28e 003 c 2 41e 002 7 76e 003 o d 1 66e 002 5 89e 003 e 7 84e 003 3 32e 003 o f 8 61e 003 5 64e 003 o g 8 02e 0
40. st select the ROI file and ROI classification file if these have been previously created or press Cancel if these files do not exist Subsequently use the Action menu to define ROIs The most basic way to define ROIs is to draw them freehand This is done by selecting Draw ROIs freehand from the Action menu Draw the ROI outline with the cursor while keeping the left mouse button pressed Release the button when finished drawing Optionally move the ROI outline in x and y directions using mouse Double click on the ROI to confirm its final position Afterwards specify whether the ROI outline should be defined as the coarse polygon drawn or as an ellipse that circumscribes this polygon To enable or disable questions about the type of ROI outline polygon versus circumscribed ellipse check the relevant option in the Freehand ROI options menu An alternative to free hand ROI drawing is to draw them as ellipses or rectangles which is done by selecting the corresponding item from the Action menu After each drawing the ROI can be moved and resized before its final position is confirmed by double clicking When the contrast between the background and the signal used for ROI recognition is high interactive thresholding is the preferred method for ROI definition due to its speed and reproducibility After selecting Interactive thresholding from the menu click with the left mouse button on the image to automatically select a ROI that e
41. tall them on your computer Purchase Matlab together with the Image Processing and Statistical Toolboxes from Mathworks www mathworks com and install them on your computer Download the LookQ NanoSIMS distribution file from http www microsen wiki net doku php id lans and extract its content in a directory with read write access rights e g c programs LANS matlab Installation under Linux Install the TeX ETRX distribution for your Linux system Install Ghostscript and one of the common viewers of EPS and PDF files e g gv acroread evince Purchase Matlab together with the Image Processing and Statistical Toolboxes from Mathworks www mathworks com and install them on your computer Download the LookQ NanoSIMS distribution file from http www microsen wiki net doku php id lans and extract its content in a directory with read write access rights e g home user programs LANS matlab 1 3 Installation under Mac OS X 1 Install the TEX BTFX distribution www tug org mactex 2 Install Ghostscript and one of the common viewers of EPS and PDF files note that these might be installed by default on your Mac 3 Purchase Matlab together with the Image Processing and Statistical Toolboxes from Mathworks www mathworks com and install them on your computer 4 Download the LookQ NanoSIMS distribution file from http www microsen wiki net doku php id lans and extract its content
42. xtends over pixels whose values do not fall below the value in the selected clicked pixel multi plied by a threshold value The threshold value is 0 5 by default but can be increased or decreased interactively by pressing the up or down arrow key respectively If a new pixel is selected the ROI is automatically redrawn according to the current threshold allowing rapid optimization This procedure leads to a ROI in the shape of a polygon By pressing the left or right arrow key an ellipse circumscribing this polygon is drawn in stead and its axes can be decreased or increased by further pressing the respective arrow key Press Esc to cancel the current interactive ROI definition or Enter to confirm ROI position Once confirmed the ROI is defined as pixels that are inside the drawn polygon ellipse including the edge Note that every ROI drawing by interactive thresholding must be terminated by pressing Esc or Enter otherwise the program will proceed in an unexpected way When using an RGB image as template for ROI definition the color channel used for automatic ROI drawing can be changed to red green or blue through the Select interactive channel menu Any previously defined ROI can be split by a line that is drawn through the ROI which is done via Split ROIs by line in the Action menu Any previously defined ROI can be removed by selecting Remove ROI interactively Use a left mouse click on the R
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