Before we start the script to automatically process the images, we want to manually review the images. Manually reviewing the images allows us to make sure that the images we use don't have any problems.

First, create a new folder in the folder containing the original images called "excluded". We will put any images we want to exclude from further analysis here.

Next, open each .lsm image file in ImageJ. Use Image --> Stacks --> Orthogonal Views (or shortcut Shift-Command-H) to get the orthogonal views of the stack. If the image has ANY of the following problems, put it in the "excluded" folder.

I find it helpful to scroll through the stack (in all 3 dimensions) to check for the following problems.

• Organoid is flat on the bottom. I believe this happens when the fixation destroys the Matrigel and the organoid gets flattened against the bottom of the glass. Scroll over for example

• The upper part of the organoid appears stretched. I believe this happens when part of the organoid was beyond the working distance of the objective and moving the objective upwards doesn't bring the objective closer to the sample, but rather lifts the sample off the stage. Scroll over for example

• At least half of the organoid has not been captured. You can tell because the approximate diameter of the organoid never appears to decrease. Scroll over for example

next, you want to make sure you did not image the same organoid twice. Open all image in ImageJ

Now that the original folder(s) contains the .lsm files you want to analyze (and the "excluded" subfolder), you are ready to prepare the images in MATLAB.

Make the folder containing the raw images MATLAB's working directory.

#!matlab

cd /Volumes/LAUREN_05/Data_Microscopy/Scope_CDB_Core_710/18_09_18_Organoids_Round_144/plate2/well01_normal/

Run the image preparation script.

#!matlab

organoids.prepare_images

When prompted, enter the names of each channel. The names you should use are the same names Raj Lab microscopes use for fluorophores (gpf, dapi, alexa, etc). Note that you will only enter this information once, so it should be the same for all images in the folder.

When prompted, select "Yes" for orthogonal slices.

Next, you will be asked how much to stretch the XZ images. I usually use 6. Close the window when you have entered the number.

Next, you will be shown the XY slices of the first image stack. Scroll through the stack and decide which slices to include. The next window will ask you what the starting and ending slices should be.

Generally, I exclude any bottom slices that do not have nuclei in them. For the top, I exclude slices AFTER I am sure that the organoid diameter is decreasing (the goal is to make sure I have analysis for the bottom half of the organoid, plus a little more to be safe).

Next, you will be shown the XZ slices of the first image stack. Scroll through the stack and decide which slices to include. The next window will ask you what the starting and ending slices should be.

Generally, I include slices where nuclei are visible.

The past two steps will repeat for each image in the folder.

Then, repeat this entire process for any other folders you want to include in the same Quantius job.