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These instructions explain how to go from raw .lsm images of organoids to morphology measurements.
The images must be organized into folders. These folders are called data folders and the code is built to run on a single data folder at a time.
One data folder should contain images of organoids from a single perturbation imaged on a single day. The name of the folder is always wellXX_Y, where XX is 01 - 08 (to represent what well of the 8-well chamber I was imaging) and Y is the name of the perturbation (normal, 1.6uMSU668, day04, etc). Note, I sometimes have folders titled wellXX_Y_Z where Z denotes some other meaning NOT used by the code.
All images in a data folder are named posZZZ.lsm where ZZZ is a unique position number (001, 002, etc).
The code will make the following assumptions about the images:
- All the images in the data folder have the same order of channels (for example, the first channel is always DAPI, etc).
- There is only 1 organoid to analyze in each image. There can be more than 1 organoid in the image, but only 1 will be analyzed.
You can track the progress of each data folder through the pipeline here. Each row represents one data folder.
I also use this document to record wet lab details about each sample. Each row represents a single sample (an 8-well chamber where each well has a different perturbation).
The organoids2 repository (this repository) and rajlabimagetools repository must be on MATLAB's path for each step.
The steps of the pipeline must be executed in order (see below). For example, if you have completed step 4 (measured the features), and you change the segmentations (in step 2), you must re-run steps 3 and 4. The only exception to this are the different parts of step 2. Each section of step 2 can be run independently and in any order.
This also means that if you re-run a particular step (or piece of code) you will write over the files generated in the last run.
Step 2A: Segment Each Structure
Step 2B: Identify Each Cell Type