User Guide
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User Guide for the streamlined, less biased, but still manual registration of fluorescent tracks to the Allen mouse brain Reference Atlas Software by Nick Steinmetz & Philip Shamash in the laboratory of Kenneth Harris & Matteo Carandini at UCL Table of Contents 1. Data & code to have on hand (2) 2. Pre-processing (3) 3. Navigate to the correct slice in the reference atlas (6) 4. Transform and overlay histology to match reference (8) 5. Select dye track locations (11) 6. Visualize Probe Track (13) 7. FAQ (18) 1 1. Data & code to have on hand Imaged slides of brain slices with fluorescent track(s) Microns per pixel of these images Prior knowledge of the number, entry point, and insertion depths of all probes is highly recommended Have MATLAB installed (R2017 used for all testing), preferably on a Windows computer Have a (computer) mouse with a scroll wheel Get the code at github.com/cortex-lab/allenCCF and add this folder with subfolders to your MATLAB Path Additionally, download the reference atlas data at http://data.cortexlab.net/allenCCF/ and suporting code at https://github.com/kwikteam/npy-matlab 2 2. Pre-processing Go from an unprocessed image of many brain slices, to a folder of nice-looking, properly oriented, and ordered brain slice images (skip if you already have this) Open ProcessHistology.m Enter the folder containing the histology slide images, the folder the processed slice images should be saved to, the name of the histology slide images in anatomical order, and a few other parameters o If your images are downsampled to the correct size (10 microns per pixel), set use_already_downsampled_image = true Run this cell to initialize parameters Run the following cell to downsample and adjust the large histology images (downsampling very large images may take a minute); Instructions should appear on adjusting the image o Scrolling shifts the max or min pixel intensity saturation points; space bar shifts between control of the max vs. the min; ‘c’ shifts to the next color channel; arrows move to the next image; etc. 3 Before (above) and After (below) preprocessing When done with all slides, run the following cell (‘CROP AND SAVE SLICES’). More instructions will appear in the console. o Select a slice image by clicking and dragging around it; try to go in order, either anterior to posterior or posterior to anterior o The slices needn’t be perfect; they will be after the next, and last, pre-processing step. Cropping too big is ok. 4 o Do so for each desired slice on the slide – for now, the most important is to crop the entire slice (and press ‘space’ to move to the next histology slide) Finally, run the last cell in ProcessHistology.m o Instructions appear. Most important: scroll to get the slice oriented straight, press ‘f’ to reflect the slice horizontally (so all slices are in the same orientation – a necessary step for the following steps), press ‘c’ to crop the image further (necessary if the image is larger than 800 x 1140), and left/right arrows to save and navigate between slices Before (left) After (right) Done! Now you should have the large images saved in your designated ‘save_folder’, as well as a subfolder in there called ‘processed’ containing the processed slices, in order. 5 3. Navigate to the appropriate slice in the Allen reference atlas With the folder of processed images, find each corresponding slice in the reference atlas; or search the atlas without the slice images in a simpler GUI Open the file AlignHistologyToProbe.m and enter the folder with the ordered, processed slices. o if you did not process the images through the above process, you may still need a similar naming convention (Name of Animal_slide number.slice number.tif) – e.g. ‘Richards_2.12.tif’ o You will need to first download the reference atlas structural volume, annotation volume, and annotation data. This can be done on http://data.cortexlab.net/allenCCF/ Download the structure tree ending in ‘2017.csv’ o You must also download and add to your path the following repository: https://github.com/kwikteam/npy-matlab o Enter the file paths of the annotation volume in the line starting ‘av = …’; of the structural volume in the line starting ‘tv = …’; and of the annotation data in the line starting ‘st = …’ o Run the file. The first time, it will take a while to load the reference brain data. Note that the simpler function allenAtlasBroswer(…) can be used to find slices in the reference atlas, without any accompanying GUI that uses the histology images (Slice Viewer). It is not compatible, however, with sections 4 -6. Basic instructions appear. o On the Slice Viewer, press ‘left’ or ‘right’ to switch slices. o On the Atlas Viewer, scroll to move through coronal slices. Press ‘up’ to make scrolling now adjust the angle of the slice, along the dorsal-ventral axis or press ‘right’ to make scrolling now adjust the angle of the slice, along the medial-lateral axis. Using these features, navigate to the approximately correct slice as seen in the Slice Viewer 6 7 4. Transform and overlay histology to match reference We’ve found a similar slice in the reference atlas, but room for error remains – transform and overlay the slice with the reference to get even more exact When you’re done exploring the brain regions on this slice with the mouse pointer, or by pressing ‘o’ to highlight regions (and pressing ‘o’ again to turn that off for this next bit), press ‘t’ on the Atlas Viewer to initialize a transform Also press ‘t’ with the Slice Viewer figure selected, so that both figures are in transform mode. Select salient points on one figure, and then the corresponding point on the other. 10-20 points should do, depending on the slice. o You can always add more points later o Press ‘d’ to erase the current transform and start over 8 Press ‘h’ to transform and overlay the histology, and thereafter switch between 3 viewing modes. This also saves the transform. o Press ‘a’ to toggle region annotation outlines. Now you can slightly alter the reference outlines position (using the scroll bar and arrow keys as before) and/or alter the transformation by clicking more points in both figures and pressing ‘h’ again Press ‘h’ 3 times to return to present view While pressing ‘h’ saves the transform, after changing the atlas position or angle, you can press ‘x’ to save that change After pressing ‘h’ and ‘a’; Arrow shows a minor improvement made by adding 2 points to the transform and modifying the angle of the slice in the reference atlas After improving the fit 9 To move on to the next slice, use the left/right arrows on the Slice Viewer. Then, you can press ‘h’ to get to reference move, scroll to the slice in the atlas corresponding to your new histology, and repeat the process. However, there is a more streamlined way to move between slices on the Atlas Viewer: press ‘left’, and then you can scroll between the slices you’ve transformed! If a slice is not yet transformed, it will take you to the reference view so that you can do so. o It is not necessary to transform every slice, if for example two slices show redundant information about the probe Or, to bring up a particular transform that has been saved, navigate to that slice in the Slice Viewer and press ‘l’ after selecting the Atlas Viewer 10 5. Select dye track locations Localize the dye track(s) in the histology Navigate through transformed slices on the Atlas Viewer by pressing ‘left’ and then scrolling. Press ‘a’ to see the region boundaries Press ‘p’ to toggle probe point mode. Now you can click points on the figure corresponding to the probe track. Press ‘n’ to add a probe (up to 11 probes), and press ‘p’ to toggle between them (check the console to see which probe has been selected) Press ‘d’ to delete the most recent probe point from the currently active probe. Press ‘s’ to save the probe points for probe visualization Press ‘l’ twice (once loads a transform) to load saved probe points (or ‘w’ to launch probe view mode – see below) 11 Pressing ‘p’ will also bring up a third window: the Transformed Slice & Probe Point Viewer. Pressing left and right will move through the slice images (transformed, if applicable) o This allows you to see surrounding slices with their probe points, to help determine which probe a particular fluorescent track belongs to 12 6. Visualize Probe Track See the fruits of your labour Load saved probe points by pressing ‘l’ twice, and toggle between the probe of interest by pressing ‘p’ Press ‘w’ to toggle probe viewer mode for the currently selected probe (will load probe 1 if none is selected or loaded). o This will show a best fit line, the slice that this line lies along, all the clicked probe points (with proximity from the best fit indicated by brightness) o Feel free to press ‘o’ to see the probe intersects, or to scroll and explore nearby slices … o Clicking on or around the probe track will find the point in the histology images closest to the clicked point, and display it in the Transformed Slice & Probe Point Viewer window 13 Open displayProbeTrack.m and enter some parameters o processed_images_folder and probe_save_name_suffix should be the same is those used in AlignHistologyToProbe o probes_to_analyze: a list of the probes you would like to analyze found in the file set by the two aforementioned variables. ‘all’ will analyze all saved probes; [1, 5] will analyze the 1stand 5th probes o probe_lengths is a list of the known insertion depths of all probes saved in this file (if you are not analyzing some probes, you can set their depths to 0). Setting it as one number sets all probes to that depth. o active_probe_lengths is the distance from the bottom tip of the electrode that contains recording sites o probe_radius is the range in microns around the best fit line corresponding to the probe that will be queried to estimate a confidence metric (distance from best fit line to nearest different brain region – see plot below) o show_parent_category will also include a confidence metric for 2nd-order parent regions (e.g. prelimbic area vs. prelimbic area layer 6a) – this feature is slow. o probe_past_tip_to_plot: how far past the estimated tip of the probe should be plotted? (see plot below) o scaling_factor: set the scaling between the probe length that you know from the insertion depth and the depth in the reference atlas brain. 1.0-1.2 is a reasonable range. Aligning the electrophysiology to known landmarks (e.g. white matter) can also help to estimate this value. Set this variable to ‘false’ to use the most ventral clicked point as the expected electrode tip, and to then derive the scaling factor automatically. o show_region_table: output and show a borders_table with data on the regions displayed in the plot (see below): their upper and lower borders in microns from the surface, acronym, name, area index as represented in the downloaded annotation volume see all abbreviations and hierarchical region structure is the downloaded csv file ‘structure_tree_safe_2017.csv’ 14 example parameters for a brain with 11 probe tracks Example table output from probe 4 (the dark green probe below) 15 All probes overview output (Brain View), using scaling_factor = 1.1 Same data, using scaling_factor = false (get probe scaling from histology) 16 Probe 4 (dark green track in Brain View above) Region abbreviations, as defined in borders_table and the downloaded .csv file Start of active recording sites on the probe Plot starts at brain surface Confidence in parent region (here, somatosensory cortex) shown in dark gray show_parent_category set to false for this plot Here, probe tip was calculated by the insertion depth and scaling factor Distance to the nearest brain region in um along the plane 17 orthogonal to the probe, as a measure of confidence Here, 300 um past probe tip plotted (distances are in reference atlas space) 7. FAQ Frequently asked questions There’s a bug or error – what should I do? o Close all the figures and re-start the file That doesn’t work – what should I do? o Feel free to raise an issue at github.com/philshams/allenCCF Is there a tool to know how I should orient my probe, before I do the recording? o Andrew Peters crafted one called allen_atlas_probe.m, using the same reference data as above (arrays called tv, av, and st). This file can be found in the repository. 18
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