Count live, apoptotic or necrotic cells with IMAGE background subtraction

Parameters:

Name	#	Type	Description
Channel of nuclear marker	1	integer	The first linked image window with matching channel number will be invoked.
Channel of apoptosis probe	2	integer	The first linked image window with matching channel number will be invoked.
Channel of necrosis probe	3	integer	The first linked image window with matching channel number will be invoked.
Apoptosis probe threshold (%)	4	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Necrosis probe threshold (%)	5	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Positive control scaling (percentile, both probes)	6	real	Adjust this value depending on the number of positive cells in the positive control. If the positive control has very few dead cells, a larger (closer to 100 percentile) value is required.
Number of tiles in x	7	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	8	integer	The image consists of this number of equal sized tiles in y dimension.
Approximate cell diameter	9	integer	Diameter of the nucleus in pixels
Debris cutoff (percentile)	10	real	This percentile of the image histogram sets the intensity value where the maximum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below ant "Max value".
Minimum cell fluorescence (%)	11	real	Cells dimmer than this in filtered and 1-1000 rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Cell boundaries (% of max fluorescence)	12	real	Cells dimmer than this in filtered rescaled TMRM+FLIPR projection images will be rejected. Increase this value if debris dimmer than the cells is detected.
Weld segments into round objects	13	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.

Description:

Counts cells positive or negative for an apoptosis and a necrosis marker.
Segments an image of nuclear stains into single nuclei. Then scales cell death marker fluorescence based on Histogram Reference Images and counts nuclei that are brighter than a percent value of the reference image.
Generate Histogram Reference Images using the " Count live apoptotic or necrotic cells with IMAGE background subtraction POZ. CTRL. REFERENCE " pipeline
This pipeline also uses Background Reference Images that must be generated before running this pipeline. Please generate background reference image by setting it in an Image Window by right-click/Set as Reference Image/Background, or use the “Create reference image for multiwell plate using median” pipeline to automatically generate background from multi position image sets.
Output:
Column Total n : all detected cells

Apoptotic X
Necrotic O = apoptotic cells

Apoptotic
Necrotic X = necrotic cells

live cells: Total n - the sum of the last three columns (double negatives)
Tiled images: the background tiling pattern is efficiently removed by spatial filtering if the recording was performed without overlap and image registration. Provide the number of tiles in x and y direction.

V2
Segment welding has been enabled and separation of segments has been disabled.
No clipping during intensity scaling for seed calculation.
Intensity classifiers are applied to the seeds, as well as to the secondary segmentation.
V3
Fixed association of “Minimum cell fluorescence (%)” with intensity classifiers and “Cell boundaries (% of max fluorescence)” with Watershed "Determine boundaries at" function parameters.