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1. coordinates of a red labelled cell ALU MAP A text file containing the coordinates of the map outline The file comprises a number of sections each starting with a single number which is the number of lines to read in the next section These lines have two numbers each the x and y coordinates of a vertex of the map outline References 1 Sterratt DC Lyngholm D Willshaw DJ Thompson ID 2013 Standard anatomical and visual space for the mouse retina computational reconstruction and transformation of flattened retinae with the Retistruct package PLoS Comp Biol 92. doubly XGREEN x coordinate if cell labelled green but not doubly YGREEN y coordinate if cell labelled green but not doubly XDOUBLE _x coordinate if cell labelled doubly YDOUBLE y coordinate if cell labelled doubly XGRID sample box cell is in YGRID sample box cell is in PERIM perimeter of cell AREA area of cell ONE PER GRID BOX GRIDX grid location of centre of sample box GRIDY grid location of centre of sample box XGRIDCOO x coordinate of centre of sample box YGRIDCOO y coordinate of centre of sample box BOXSIZEX size half width of sample box in x direction BOXSIZEY size half width of sample box in y direction COMPLETE whether counting of sample has been completed TOTALCEL total number of cells in this box TOTALRED total number of red only cells in this box TOTALGRE total number of green only cells in this box TOTALDOU total number of double cells in this box MEANPERI average perimeter of all cells MEANAREA average area of all cells Table 2 Column headings of the SYS SYS file Retistruct version 0 5 9 Date 2014 12 01 9 A The IDT Data format The data for each retina is stored in a separate directory Within each directory there are two files SYS SYS A table in SYSTAT format containing the coordinates of the red green and doubly labelled cell bodies and counts of labelled cell bodies within each grid box The column headings shown in Table Each row of the table contains information only on a subset of the data e g the
3. up the outline image png in IMAGEJ 2 Threshold the image Image Adjust Threshold so that the labelled points are visible 3 Select Analyze Set Measurements and make sure that Centroid is checked 4 Select Analyze Analyze Particles Make sure that Display results and Clear results are checked Click on OK 5 A window entitled Results should appear This contains the X and Y coordinates of the detected points In this window select File Save as and save the file as datapoints csv 6 Open this file with either EXCEL or OPENOFFICE CALC and remove all the columns apart from the X and Y columns Replace the X with the name of the data set and the Y with the colour it should be displayed in RETISTRUCT 7 Save datapoints csv in the retina s directory along with outline roi and image png if an image is desired lhttp rsbweb nih gov ij docs tools html Retistruct version 0 5 9 Date 2014 12 01 4 2 1 2 CSV format This is the same as the IMAGEJ ROI format except that the outline is contained in the two columns of a file called outline csv Each column should have a heading e g uK uyn 1 5 10 76 The optic disc outline can be supplied in a similar file called od csv 2 1 3 IDT format This is the format used in Ian Thompson s lab see Appendix A These files are contained in a directory To open the files corresponding to a retina click on the open file icon and navigate to the directory cont
4. Retistruct version 0 5 9 Date 2014 12 01 1 RETISTRUCT manual David C Sterratt 2nd December 2014 1 Installation 1 1 Install the necessary system packages 1 1 1 Fedora Linux As root yum install R core yum install R devel yum install gtk2 yum install gtk2 devel yum install mesa libGL devel yum install mesa libGLU devel 1 1 2 Ubuntu Linux At the command line enter sudo apt get install r base r cran rgl libgtk2 0 dev 1 1 3 Mac OS X First install R for Mac OS X available from http www r project org Under Mac OS X Leopard and Lion this should be sufficient Under Mac OS X 10 8 Snow Leopard it may be necessary to install the following external packages e Xcode free from App Store e Xquartz http xquartz macosforge org http r research att com libs GTK_2 18 5 X11 pkg 1 1 4 MS Windows First install R for MS Windows available from http www r project org 1 2 Install the core Retistruct package 1 Start R 2 Type source http retistruct r forge r project org install R The first time this runs it should create a personal directory for R packages and it will take a few minutes to install some required packages 3 Test that it is working as follows from within R library retistruct demo retistruct method Retistruct version 0 5 9 Date 2014 12 01 2 If all is well you should be prompted to press Return a few times and Figure 1 from the paper I will appea
5. a and the markup To import this data into MATLAB cd into that directory and type clear load r mat This puts a number of variables into the workspace as shown in Table To produce a polar plot of data points try the following code polar Dss green 2 Dss green 1 180 pi 90 g hold on polar Dss red 2 Dss red 1 180 pi 90 r hold off The radial axis indicates the latitude in degrees measured from the retinal pole In the matlab subdirectory of the distribution there are some scripts to produce polar polar plots including the locations of tears and landmarks To create PDF plots of all the retinae in a directory try makefigures retinae output_directory There are also some embryonic scripts to create polar plots plot_datapoints_polararea m and polararea m 2 10 Running a batch of reconstructions The RETISTRUCT library can be used to reconstruct a batch of retinae which have been marked up Suppose that the directory retinae contains a directory tree in which there are data directories con taining the raw outline datapoint and image files and the saved markup files In order to perform the reconstructions we create a new directory retinae reconstructions and run the following sequence of commands in R R gt library retistruct gt retistruct batch tldir retinae outputdir retinae reconstructions This command will go through the retinae directory looking for vali
6. aining the SYS and MAP files On opening this directory the retinal outline should appear in the RETISTRUCT window 2 2 The retinal display In the Show section at the bottom left of the screen there are checkboxes that allow you to show various types of information Markup Locations of tears and the dorsal or nasal pole Stitch Locations of how the algorithm has stitched tears only visible after the reconstruction step Grid Lines of latitude and longitude projected back onto the flattened retina only visible after the reconstruction step Datapoints Locations of data points such as the locations of beads of dye Landmarks Landmarks such as the optic disc Strain This shows information about how the retina has been reconstructed after the reconstruction step has taken place Preserve area When this is selected the polar plot displays an area preserving projection Contours Display Kernel Density Estimate contours of the data points By default contours at 5 25 50 75 and 95 of the maximum density are shown This can be changed by a command like this at the R prompt options contour levels c 5 25 50 This command would cause the 5 25 and 50 contour lines to be displayed 2 3 Marking up the retina Add tear To add a tear click on this button then click on three points in turn which define a tear The order in which the points are added does not matter Tears contained within a tear can be marked up but tear
7. d data directories If it finds one it sets about trying to reconstruct the retina As it reconstructing each retina it writes to log file in retinae reconstructions Once the reconstruction is complete it saves a number of plots in this directory in PDF format It also adds a line to a summary log file in retinae reconstructions called retistruct batch csv This file contains a number of columns Dataset The directory of the data set Return The return value from the process Result A summary of the result including if any errors were returned E The total error of the optimised reconstruction El The error due to purely to the lengths of links in the optimised reconstruction nflip The number of flipped triangles EOD The distance of the Optic Disc from the inferred centre of the retina in degrees If the OD has not been marked up this is NA To export the reconstruction data in a directory hierarchy in which retistruct batch has been run run the following sequence of commands in R R gt library retistruct gt retistruct batch export matlab tldir retinae Retistruct version 0 5 9 Date 2014 12 01 FOR EACH BOUNDARY MAPNUM id number of boundary MINLAT min latitude MAXLAT max latitude MINLON min longitude MAXLON max longitude LABLAT latitude of label LABLON longitude of label FOR EACH CELL XRED x coordinate if cell labelled red but not doubly YRED y coordinate if cell labelled red but not
8. de of each point measured in radians Location of Karcher mean of green labelled cell bodies in spherical coordinates The first column contains the latitude of each point measured in radians The second column contains the longitude of each point measured in radians Area of convex hull of green red points on sphere The convex hull is essen tially a polygon drawn around data points A structure in which each element contains spherical coordinates in the same latitude longitude format as above of a tear A structure in which each element contains spherical coordinates in the same latitude longitude format as above of a landmark An object containing information about Kernel Density Estimates of the loca tions of cell bodies green_flevels Contour heights determined by finding heights that exclude a certain fraction of the probability For example the 95 contour is excludes 95 of the probability mass and it should enclose about 5 of the points Contour labels These give the label e g 5 25 50 where these are the percentages above of each contour Note that there may be more than one contour at the same level so this vector may con tain more elements than flevels The first ele ment of green_labels labels the contour whose coordinates are specified in green_contourst the second element of green_labels relates to green_contours2 and so on The total area in square degrees enclosed by each contour This is a matrix with the f
9. he retina using a batch process Section 2 10 2 6 Reconstructing the retina To reconstruct the retina click on the Reconstruct retina button This causes a lengthy sequence of operations to be performed Stitching Links between corresponding points on parts of the retinal outline contained in tears are made Triangulation A triangular mesh is placed over the flattened retina Initial projection to sphere The mesh is projected roughly onto a sphere Optimisation The locations of the mesh points on the hemisphere is adjusted so as to minimise a weighted sum of the squared differences between the lengths of links in the mesh on the hemisphere and on the flattened retina whilst ensuring that as few triangles as possible are flipped At the end of the reconstruction process a polar plot appears next to the flattened retina When Landmarks are shown the location of the cuts and tears in the polar coordinates can be seen When Strain is shown the polar plot is replaced by a scatter plot of the length of links in the reconstructed object versus the length on the flattened object The colours of the points indicate the degree of expansion or compression from the flattened object to the reconstructed object 2 7 Saving the reconstruction To save the markup click on the Save button in the toolbar This saves various files to the directory containing the data files When the files in the directory are opened again al
10. irst column giving the contour label see above and the next column giving the area green_labels green_tot_contour_areas green_kappa green_h green _maxs_phi green_maxs_lambda green g xs greeng ys green_g_f green_gpa_xs green_gpa_ys green_gpa_f green_contours1 green_contours2 green_contour_areasi green_contour_areas2 The concentration parameter of the Fisher density determined by the kernel fitting algorithm A pseudo bandwidth parameter the inverse of the square root of kappa Units of degrees Lattitude of maximum point of kernel estimate Longitude of maximum point of kernel estimate Kernel density estimates on standard polar grid This can be plotted in MATLAB using the command contour KDE green_g_ xs KDE green_g_ys KDE green_g f Kernel density estimates on area preserving polar grid Plotted in MATLAB as above Coordinates of contours See green_labels above for more explanation Area contained within each individual contour See green_labels above for more explanation An object containing Kernel Regression estimates of the density of points de rived from the grouped data points All fields correspond to KDE above Table 1 Variables exported in the r mat file Retistruct version 0 5 9 Date 2014 12 01 7 2 9 Reading reconstruction data into Matlab By default when a reconstruction is saved a subset of the data is stored in a file called r mat in the same directory as the raw dat
11. l the markup information cut locations location of nasal point and rim angle is loaded and the reconstruction will also appear unless there has been a major upgrade of the software in which case the retina will need to be reconstructed using the Reconstruct retina button 2 8 Reading reconstruction data into R It can be useful to have access to the data underlying a reconstruction directly for example to allow statistical analysis To load saved date into R type the following into R r lt retistruct read recdata path to reconstruction directory The resulting object r contains various fields which can be accessed using the R operator e g r Dss green gives a list of the spherical co ordinates of green labelled datapoints See Table I for a list of the most useful quantities Other quantities for example the locations of the mesh points in the flat and spherical retina are also available The documentation for ReconstructedQutline ReconstructedDataset getDss and getSss in the user manual has more information about this Retistruct version 0 5 9 Date 2014 12 01 phid Dss green Dss red DssMean green DssMean red DssHullarea green DssHullarea red Tss Sss KDE KR The latitude of the rim expressed in radians Locations of green red labelled cell bodies in spherical coordinates The first column contains the latitude of each point measured in radians The second column contains the longitu
12. ove information as will be described below At present there are three formats of directory that RETISTRUCT can read Most users will probably find the the IMAGEJ ROI format most convenient 2 1 1 ImageJ ROI format This format allows you to load images of retinae whose outlines have been marked up in IMAGEJ To create the image and outline files for this format 1 Create a directory to save the files created to 2 Open up IMAGEJ or FIJI 3 Use File Open to open the image Retistruct version 0 5 9 Date 2014 12 01 3 4 Use Image Scale to down sample so that the resolution is less than 1000x1000 This is not crucial but will speed up things later 5 Save the down sampled version of the image to the file name image png in the directory created in Step 1 6 Use the Polygon Tool 2 to mark the edge of the retina According to the IMAGEJ manual To create the selection click repeatedly with the mouse to create line segments When finished click in the small box at the starting point or double click and ImageJ automatically draws the last segment The points that define a polygon selection can be moved or deleted and new points can be added To delete a point click on it with the alt key down To add a point click on an existing point with the shift key down 7 Open the ROI manager by selecting Analyze Tools ROI Manager 8 Click on the Add t button 9 Click on the More Save button In the Save
13. r drawing the 3D renditions takes some time There will be reports of warnings they are to do with printing the figures to files You can view them by typing warnings 4 A further test produces Figure 2 demo low high 5 A further quite long running test produces Figure 6 demo figure6 1 3 Install the Retistruct GUI If the previous step works 1 Type source http retistruct r forge r project org install gui R 2 Under MS Windows there may be an error message saying that libatk 1 0 0 dll is missing from your computer Click on OK Another message will appear asking if you want to install GTK Agree to this After this has finished quit R without saving the workspace image and restart 3 If all works an interface window should appear A number of sets of demonstration data are available from the Demo menu item 2 Running Retistruct To start the program start R At the R prompt type gt library retistructgui gt retistruct A window should appear 2 1 Opening the files for a retina There are a number of types of information associated with a flat mount retina that RETISTRUCT can process e The coordinates of the outline of the flattened retina e An image of the flat mount retina optional e The coordinates of labelled data points within the flat mount retina optional To import this information into RETISTRUCT for each retina a directory should be created containing files with the ab
14. s cannot cross over one another Move Point To move one of the points defining a tear click on this button then click on the point which you desire to move then click on the point to which it should be moved Remove tear To remove a tear click on this button then click on the apex of the tear marked in cyan on the plot Mark nasal To mark the nasal pole click on this button then click on the point which is the nasal pole If the nasal or dorsal pole has already been marked the marker is removed from the existing location The nasal pole should not be in a tear If the nasal tear is placed within a tear no error is reported at this stage but it will be reported later Retistruct version 0 5 9 Date 2014 12 01 5 Mark dorsal As above except for the dorsal pole Mark OD To mark the optic disc click on the structure marked in orange which you think is the OD Once clicked on the structure should become blue PhiO This determines the latitude of the rim of the reconstructed hemisphere It depends on the age of the animal 2 4 Metadata Data Flip DV Flip the DV axis to compensate for microscope orientation Affects display of retinae Eye Specify whether the eye is Right or Left Affects display of retinae 2 5 Saving the markup and metadata To save the markup click on the Save button in the toolbar This saves various markup files to the directory containing the data files This saved data can be used to reconstruct t
15. selection box that appears enter the file name outline roi and make sure this file is saved to the same directory as the image png file i e the directory created in Step 1 10 Optionally mark up the optic disc in the same way as the outline Save this ROI to a file called od roi in the same directory as image png 11 Open RETISTRUCT 12 Click on the Open File icon and select the directory containing image png and outline roi The retinal image should now appear with the outline shown By default the outline is in black If this isn t visible against the image press the Properties button in the interface or select Edit Properties and change the Outline colour Specifying the scale Optionally the scale of the image can be specified by providing a file scale csv in the same directory as the image and ROI files This file should contain two lines the first with the heading Scale and the second with the length of the side of a pixel in micrometres For example Scale 1 5 Reading in data points In general the coordinates of data points are read in from a csv file called datapoints csv The two cells of the first line of the file contain the name of the group of data points and the colour that these should be displayed in RETISTRUCT Marking up the points from the image in IMAGEJ will ensure that the coordinates are in the same system as those used for the image and the outline To do this 1 Open the image used for marking
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