'''

OrgM: Organoid Size Measurer - ImageJ Macro written in Python

Input - Directory containing Organoid bright field images

Output - CSV file containing size and shape descriptors for each organoid image

Written by: Eddie Cai and Rhalena Thomas - NeuroEDDU

'''

# Import required packages

import os, sys, math, datetime

from ij import IJ, ImagePlus, ImageStack

from ij.io import DirectoryChooser

from ij.measure import ResultsTable

from ij.measure import Measurements

from ij.process import ImageProcessor

from ij.process import ImageConverter

from ij.gui import WaitForUserDialog

from ij.gui import GenericDialog

from ij.plugin.frame import RoiManager

from ij.plugin.filter import ParticleAnalyzer

# Set Threshold mode

thresholdMode = False

gd = GenericDialog("Set Threshold Mode")

gd.addChoice("Would you like to enable thresholding mode?", ["No, run the normal macro", "Yes, enable thresholding mode"], "No")

gd.addMessage("Watershed will NOT be run in threshold mode and WILL BE RUN in macro mode.")

gd.showDialog()

if gd.getNextChoice() == "Yes, enable thresholding mode":

thresholdMode = True

# Set default thresholds:

# round_threshold is the minimum roundness a roi must have to be considered an organoid for the isOrganoid column

# area_threshold is the minimum area a roi must have to be considered an organoid for the isorganoid column

# minimum_size is the minimum area to be considered an ROI

gd = GenericDialog("Other Thresholds.")

gd.addMessage("Ajust after you have determined if new thresholds are needed.")

gd.addStringField("Round threshold", "0.62")

gd.addStringField("Area Threshold", "50000")

gd.addStringField("Minimum Size", "3000")

gd.showDialog()

round_threshold = float(gd.getNextString())

area_threshold = float(gd.getNextString())

minimum_size = float(gd.getNextString())

#set pix_width and pix_height to real dimensions per pixel

gd = GenericDialog("Dimension Options")

gd.addMessage("Conversion from pixles to uM :Evos 10X pixle width/height = 0.8777017 uM")

gd.addMessage("Conversion from pixles to uM :Evos 4X pixle width/height = 2.1546047 uM")

gd.addMessage("Conversion from pixles to uM :Calculate for your objective and enter below")

gd.addStringField("Pixel Width:", "0.8777017")

gd.addStringField("Pixel Height:", "0.8777017")

gd.showDialog()

pix_width = gd.getNextString()

pix_height = gd.getNextString()

# Get input and output directories with GUI

dc = DirectoryChooser("Choose an input directory")

inputDirectory = dc.getDirectory()

dc = DirectoryChooser("Choose an output directory")

outputDirectory = dc.getDirectory()

with open(outputDirectory + "output_"+datetime.datetime.now().strftime("%Y-%m-%d-%H-%M")+".csv", "w") as output:

# set the output column names for the csv sheet

output.write("Subfolder, File Name,Feret,MinFeret,Average Feret,Area,Equivalent Circle Diameter, Ellipse Major, Ellipse Minor, Circularity,Roundness,Solidity, MeetsCriteria \n")

subfolders = []

# Finds subfolders in input directory

for subfolder in os.listdir(inputDirectory):

if os.path.isdir(inputDirectory + subfolder):

subfolders.append(subfolder)

# If there are no subfolders, runs on images in input directory instead

if len(subfolders) == 0:

subfolders = [""]

for subfolder in subfolders:

#Opens each image

for filename in os.listdir(inputDirectory + subfolder):

imp = IJ.openImage(inputDirectory + subfolder + '/' + filename)

if imp:

# 10X objective

IJ.run(imp, "Properties...", "channels=1 slices=1 frames=1 unit=um pixel_width=" +str(pix_width)+ " pixel_height=" +str(pix_height)+" voxel_depth=25400.0508001") # Change to a GUI option later?

# Threshold, fills hole and watershed

ic = ImageConverter(imp);

ic.convertToGray8();

IJ.setAutoThreshold(imp, "Default dark")

if thresholdMode:

imp.show()

IJ.run("Threshold...")

WaitForUserDialog("Title", "Adjust threshold").show()

IJ.run(imp, "Convert to Mask", "")

IJ.run(imp, "Invert", "")

IJ.run(imp, "Fill Holes", "")

if not thresholdMode:

IJ.run(imp, "Watershed", "")

#Measure particles

table = ResultsTable()

pa = ParticleAnalyzer(ParticleAnalyzer.EXCLUDE_EDGE_PARTICLES, Measurements.AREA | Measurements.FERET | Measurements.CIRCULARITY | Measurements.SHAPE_DESCRIPTORS | Measurements.CENTROID | Measurements.ELLIPSE, table, minimum_size, 9999999999999999, 0.1, 1.0)

pa.setHideOutputImage(True)

pa.analyze(imp)

index = -1

maxArea = -1

# Check if Column even exists (in case it didn't measure anything)

if table.getColumnIndex("Area") != -1:

# Find the ROI with the largest area

for i, area in enumerate(table.getColumn(table.getColumnIndex("Area"))):

if area > maxArea:

index = i

if thresholdMode:

imp.show()

# Writes everything in the output file

if index != -1:

diameter = 2* math.sqrt( float(table.getValue("Area", index)) / (2* math.pi))

isOrganoid = table.getValue("Area", index) > area_threshold and table.getValue("Area", index) > round_threshold

output.write(str(subfolder) + ',' + filename + ',' + str(table.getValue("Feret", index)) + ',' + str(table.getValue("MinFeret", index)) + ',' + str((table.getValue("MinFeret", index)+table.getValue("Feret", index))/2) + ',' + str(table.getValue("Area", index)) + ',' + str(diameter) + ',' + str(table.getValue("Major", index)) + ','+ str(table.getValue("Minor", index)) + ','+ str(table.getValue("Circ.", index)) + ',' +str(table.getValue("Round", index)) + ',' + str(table.getValue("Solidity", index)) + ',' + str(isOrganoid))

else:

output.write(str(subfolder) + ',' + filename + ",NA,NA,NA,NA,NA,NA,NA,NA,NA,NA,NA")

output.write('\n')

imp.changes = False

imp.close()

# End of macro

cat = """

\(__)|"""

print(cat)