Whole well nuclear count time lapse 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.
Number of tiles in x	2	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	3	integer	The image consists of this number of equal sized tiles in y dimension.
Debris cutoff (percentile)	4	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".
Approximate cell diameter	5	integer	Diameter of the nucleus in pixels
Minimum cell fluorescence (%)	6	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)	7	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	8	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Minimum shape factor	9	real	1 for disc, smaller for irregular shapes. 0 for not checking

Description:

Measures cell death as a fraction of all cells using a nuclear and a cell death marker.
Segments an image of a nuclear stain into single nuclei. Then scales cell death marker fluorescence based on a reference image and counts nuclei that are brighter than a percent value of the reference image.
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.
Generate reference image using the "fraction of cell death time lapse reference.ips"
The Excel Data Window output is set to place the time courses into consecutive columns.

*If single nuclei are detected as multiple segments, increase the approximate cell diameter. In addition, you may turn segment welding on.
*If multiple nuclei are detected as single segments, decrease the approximate cell diameter. In addition, you may turn segment welding off.
*If debris is detected a nuclei, increase background cutoff or minimum cell fluorescence.
*If dimmer cells are missed, decrease background cutoff or minimum cell fluorescence.
*If bright debris outshines cells, decrease debris cutoff percentile.
If segmentation goes as it is desired, process the whole microplate using the double blue arrowhead button and ‘Run Pipeline … on All Stage Positions’.

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.
Minimum shape factor parameter has been added.