Problems with Thresholding



Hello There!

I have a problem with thresholding. I want to calculate the ratio between euchromatin (light pixels) and heterochromatin (dark pixels) in an 8 bit grey scal image stack.
This is the macro I use:

// Clean up the results table
run(“Clear Results”);

setAutoThreshold(“Default dark”);
setThreshold(90, 255);
setOption(“BlackBackground”, false);
run(“Convert to Mask”, “method=Default background=Dark calculate”);

// Fill holes is not necessary for nuclear lamina meassurements
// Fill holes to create a complete mask of the cell body
//run(“Fill Holes”, “stack”);

// Set the scale to how much pxs equal 1 µm
// has to be adjusted for each image stack!!!
run(“Set Scale…”, “distance=180 known=1 pixel=1 unit=”);

// Measure Stack

// This macro measure all the slices in a stack.

macro “Measure Stack” {
setOption(“Stack position”, true);
for (n=1; n<=nSlices; n++) {
run(“Create Selection”);

// von

So if I change the grey values within the same threshold method (eg. Default) I always get the same meassurements. It doesn’t matter if the threshold is from 80-240 or 111-240 …I always get the same surface meassurement values!

Does anyone know how to fix this?

Thanks in advance!!!