This project contains three scripts used to process images for the Microtechnology, Medicine and Biology Lab. Details involving the purpose of this code can be found in PAPER.
First, download Eclipse IDE or another IDE or your choice. To download this project, click Code → Download ZIP.
Extract the downloaded zipped file somewhere.
Copy the "repository" folder into your own repository, which is located in C://Users/MMB/.m2. This will make the libraries needed for the project available to use.
In Eclipse, go to File → Import → Maven → Existing Maven Projects.
Choose the unzipped Code and check the Project. Finish the dialog.
Now, there should be a Neutrophil-Project project in the explorer. Dependencies should all be resolved.
Motility analysis involves thresholding.LocalThresholding and MotilityAnalysis.java. They contain code for analyzing the cell motility. Bright-field videos (.avi, 30X magnification, ～8 FPS, 10 s) were recorded on a Nikon Ti. The videos were analyzed using a customized code in Java.
Create a LocalThresholding object:
LocalThresholding thresholder = new LocalThresholding();
To change the parameters, use the other constructor:
LocalThresholding thresholder = new LocalThresholding(3, 15, 0.1, 0.4, 5);
Create a RunAnalysis object with desired parameters:
RunAnalysis ra = new RunAnalysis(thresholder, srcFolder, destFile, 5, 10);
Run the analysis using:
ra.run();
The resulting csv will be saved to the destFile path.
The explanation of these parameters can be found in the methods section of the paper.
Navigate to src/main/java/analysis/RunAnalysis.java and start coding in the main method.
Full documentation is available in the doc folder at doc/analysis/RunAnalsis.html and doc/thresholding/LocalThresholding.html.
Particle analysis uses the ImageJ particle analyzer to measure the areas of particles. There is a multiplier to convert the result from pixels to square microns. It includes a GUI that allows users to choose between two different thresholding methods. There are many variables that can be changed.
resultColumn is the name of the column which will be calculated and saved into the resulting csv file. Examples include "Area", "Circularity", and "Perimeter", and any measurement that's an option in ImageJ.
automatic determines whether the GUI is displayed or not. If it is set to true, the script will be run using Default for all images. If it is set to fault, the GUI will pop up when run.
series is an array with the number of series you want in each replicate. This is intended for use with replicates. For example, say you have 3 conditions each with 3 replicates in a single nd2 file. If you set series to {3,3,3}, the first three series will be put into the first column, the next three in the second column, and the last three in the third column. This results in each condition taking up a single column instead of three. Make sure that the sum of the numbers in the array equals the total number of images.
seriesTitles builds off of series so that each column can be named instead of just Condition 0, Condition 1, Condition 2. Make sure that the length of seriesTitles matches series.
channel determines which channel of the nd2 file the script will use. Note that the first channel is channel 0.
MICRONS_PER_PIXEL is a multiplier to the final result to convert it from pixels to microns squared.
Set the variables as desired above, then run. A file chooser window will pop up. Select the folder containing all the nd2 files you want to process. You should have each timepoint in a separate nd2 file. The nd2 files must have the format "NAME1-123h.nd2" where the hour can be one to three digits. Note that the script does a deep search for nd2 files, so make sure only the nd2 files you want to process are present in the folder and its subfolders.
The output will be divided by 0.518 to normalize to the size of the well and multiplied by 100 to convert it to a percentage.
Clumping analysis uses a novel algorithm to determine how much clumping there is in an image.
Many of the options are also part of Particle Analysis and are described above. These are the unique ones:
gridSizes is an array of different grid sizes to test. Each grid size will be saved in a separate csv.
crop is an ROI. The algorithm firsts crops the image to this ROI. The format is (top left X coordinate, top left Y coordinate, width of ROI, height of ROI).
Set the options as desired, then run.