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perimeter_meter.py
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perimeter_meter.py
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#@ String (visibility=MESSAGE, value="<html>Perimeter Meter 0.3 <br></html>") docmsg
#@ Float(label="Depth of cortex (um)", required=true, value=10, stepSize=0.1) band_thickness
#@ Boolean(label="Do Gaussian blur background subtraction?", value=true) blurFlag
#@ Float(label="radius of blur (µm)", value=5) blurSigma
#@ Boolean(label="Show blurred image", value=false) showBlurFlag
"""
A plugin that measures the intenisies of equally thick bands around organoids.
Count frames represented in RM
Make empty imw with correct dimensions
Extract frames from ROIS
duplicate -> Gaussian blur 5 um
subtract blur from frames
run analysis
"""
from ij import IJ, ImageStack, ImagePlus
from ij.gui import Roi, Overlay
from ij.plugin.frame import RoiManager
from ij.plugin.filter import Analyzer, GaussianBlur
from ij.plugin import HyperStackConverter, ImageCalculator
from ij.measure import ResultsTable
from java.awt import Color
import math
def getCurrentRoiMean(imp, channel):
#Returns the mean of the rurrent roi active in imp for channel
imp.setC(channel)
return imp.getStatistics().mean
def getBandMean(imp, roi, band_thickness, channel):
"""
Returns the mean of a band_thickness thick band
around the supplied roi, positive value for growing
the band, negative for shrinking.
"""
imp.setRoi(roi)
strt = getCurrentRoiMean(imp, channel)
IJ.run("Enlarge...", "enlarge="+str(band_thickness))
roi_o = imp.getRoi()
out = getCurrentRoiMean(imp, channel)
#Are we growing or shrinking the ROI?
if band_thikness > 0:
return(out-strt)
else:
return(strt-out)
def getOutsideBand(imp, contourRoi, band_thickness, label, addToRm = False):
"""
Returns a ROI consisting of controurRoi expanded by band_thickness
minus (XOR) contourRoi. Roi is named: "Outside band frame NN".
addToRm specifies if ROI is to be added to the RoiManager.
"""
_rm = RoiManager(True) #secret RoiManager
_rm.addRoi(contourRoi)
roi_idx = _rm.getIndexes()[-1] #record index for XOR
imp.setRoi(contourRoi)
#enlarge contourRoi
IJ.run("Enlarge...", "enlarge="+str(band_thickness))
roi_o = imp.getRoi()
_rm.addRoi(roi_o)
roi_o_idx = _rm.getIndexes()[-1]
_rm.setSelectedIndexes([roi_o_idx, roi_idx])
_rm.runCommand('XOR')
roi_o = imp.getRoi()
roi_o.setName("Outside band frame "+label)
roi_o.setPosition(imp)
roi_o.setFillColor(Color(0, 0, 255, 32)) #12,5% transparent red
if addToRm:
RoiManager.getRoiManager().addRoi(roi_o)
return roi_o
def getCortexBand(imp, contourRoi, band_thickness, label, addToRm = False):
"""
Returns a ROI consisting of controurRoi minus contourRoi shrunk
by band_thicness. addToRm specifies if ROI is to be added to the
RoiManager.
"""
_rm = RoiManager(True) #secret RoiManager
_rm.addRoi(contourRoi)
roi_idx = _rm.getIndexes()[-1] #record index for XOR
imp.setRoi(contourRoi)
#shrink contourRoi
IJ.run("Enlarge...", "enlarge="+str(-band_thickness))
roi_o = imp.getRoi()
_rm.addRoi(roi_o)
roi_o_idx = _rm.getIndexes()[-1]
_rm.setSelectedIndexes([roi_o_idx, roi_idx])
_rm.runCommand('XOR')
roi_o = imp.getRoi()
roi_o.setName("Cortex band frame "+label)
roi_o.setPosition(imp)
if addToRm:
RoiManager.getRoiManager().addRoi(roi_o)
return roi_o
def getInside(imp, contourRoi, band_thickness, label, addToRm=False):
"""
Returns a ROI consisting of controurRoi shrunk
by band_thickness. addToRm specifies if ROI is to be added to the
RoiManager.
"""
imp.setRoi(contourRoi)
#shrink contourRoi
IJ.run("Enlarge...", "enlarge="+str(-band_thickness))
roi_o = imp.getRoi()
roi_o.setName("Inside frame "+label)
roi_o.setPosition(imp)
roi_o.setStrokeColor(Color.red)
roi_o.setFillColor(Color(255, 0, 0, 32)) #12,5% transparent red
if addToRm:
RoiManager.getRoiManager().addRoi(roi_o)
return roi_o
def listTimepointsInRoiManager(rm):
"""
Returns a list of the unique time points represented in a
RoiManager
"""
out = []
for idx in range(rm.getCount()):
t = rm.getRoi(idx).getTPosition()
if (t not in out):
out.append(t)
return out
def extractFrame(frameNumber, imp, z=1):
"""
Extracts and returns all channels from one single frame of imp as an ImageStack
frameNumber is one based
z is slice, defaults to 1
"""
inStack = imp.getStack()
outStack = ImageStack(imp.width, imp.height)
for c in range(imp.getNChannels()):
stk_idx = imp.getStackIndex(c+1, z, frameNumber)
ip = inStack.getProcessor(stk_idx)
ip = ip.duplicate()
slice_lab = inStack.getSliceLabel(stk_idx)
outStack.addSlice(slice_lab, ip)
#outStack.update(ip) #Sets stack attributes to match ip
return outStack
def extractFrames(frameList, imp):
"""
Extracts and returns all channels corresponding to frame numbers in frameList
returns imp as HyperStack
frameNumber is one based
"""
outStack = ImageStack(imp.width, imp.height)
n_chan = imp.getNChannels()
for fn in frameList:
stk = extractFrame(fn, imp)
for stk_idx in range(1, stk.getSize()+1):
ip = stk.getProcessor(stk_idx)
outStack.addSlice(ip)
imp_o = ImagePlus("extracted", outStack)
imp_o = HyperStackConverter().toHyperStack(imp_o, n_chan, 1, len(frameList))
return imp_o
def blurStack(inStack, sigma):
"""
Returns an gaussian blurred version of ImageStack
with sigma (int) applied
"""
blur = GaussianBlur()
outStack = ImageStack()
inStack = inStack.duplicate()
for stk_idx in range(1, inStack.getSize()+1):
ip = inStack.getProcessor(stk_idx)
#ip = ip.duplicate() #don't mess with the original
blur.blurGaussian(ip, float(sigma))
outStack.addSlice(ip)
return outStack
def blurImp(imp, sigma):
"""
runs blurStack on imp
returns imp
"""
n_chan = imp.getNChannels()
n_frames = imp.getNFrames()
inStack = imp.getStack()
outStack = blurStack(inStack, sigma)
imp_o = ImagePlus("blurred_"+str(sigma), outStack)
imp_o = HyperStackConverter().toHyperStack(imp_o, n_chan, 1, n_frames)
return imp_o
def subtractImps(imp1, imp2):
"""
Subtracts the pixels in imp2 from imp1
Returns imp
"""
ic = ImageCalculator()
#imp_o = ic.run("Subtract create stack", imp2, imp1)
imp_o = ic.run("Subtract create stack", imp2, imp1)
return imp_o
def _runAnalysis(imp):
imp.setActivated()
imp.show()
rt_ch2 = ResultsTable()
rt_ch3 = ResultsTable()
anal_2 = Analyzer(imp, rt_ch2)
anal_3 = Analyzer(imp, rt_ch3)
olay = Overlay()
for idx in range(rm.getCount()):
imp.setT(idx+1)
contourRoi = rm.getRoi(idx).clone()
contourRoi.setPosition(imp)
imp.setRoi(contourRoi)
label = str(rm.getRoi(idx).getTPosition())
contourRoi.setName("Contour Frame " + label)
o_band_roi = getOutsideBand(imp, contourRoi, band_thickness, label, False)
c_band_roi = getCortexBand(imp, contourRoi, band_thickness, label, False)
in_roi = getInside(imp, contourRoi, band_thickness, label, False)
roiz = [contourRoi, o_band_roi, c_band_roi, in_roi]
for c in [2,3]:
if c == 2:
anal = anal_2
rt = rt_ch2
else:
anal = anal_3
rt = rt_ch3
for roi in roiz:
imp.setRoi(roi)
imp.setC(c)
anal.measure()
rt.addValue("Channel", c)
rt.addValue("Frame", rm.getRoi(idx).getTPosition())
for roi in roiz:
imp.setRoi(roi)
stack_idx = imp.getStackIndex(1, 1, roi.getTPosition())
#roi.setPositon(stack_idx)
olay.add(roi)
imp.setOverlay(olay)
rt_ch2.show(title+"_Ch2")
rt_ch3.show(title+"_Ch3")
# Get current ImagePlus & set up variables
imp = IJ.getImage()
title = imp.getTitle()
cal = imp.getCalibration()
luts = imp.getLuts()
sigma_px = blurSigma/cal.pixelHeight
rm = RoiManager.getRoiManager() #user facing RoiManager
frameList = listTimepointsInRoiManager(rm)
imp1 = extractFrames(frameList, imp)
imp1.setCalibration(cal)
if blurFlag:
imp2 = blurImp(imp1, sigma_px)
imp2.setCalibration(cal)
imp3 = subtractImps(imp2, imp1)
imp3.setTitle(title+" subtracted")
_runAnalysis(imp3)
if showBlurFlag:
imp2.show()
else:
_runAnalysis(imp3)