Fraction of cell death time lapse

Parameters:

Name	#	Type	Description
Channel of nuclear marker	1	integer	The first linked image window with matching channel number will be invoked.
Channel of cell death probe	2	integer	The first linked image window with matching channel number will be invoked.
Background Level, (percentile)	3	real	Background is calculated as percentile for each frame.
Cell death probe threshold (% of pos. control)	4	real	Percent of the brightness of the positive control, marked as the Histogram Reference Image
Use inhomogeneous background removal with spatial filtering	5	boolean	Yes executes output #1, No executes output #2.
Spatial filtering: Number of tiles in x	6	integer	The image consists of this number of equal sized tiles in x dimension.
Spatial filtering: Number of tiles in y	7	integer	The image consists of this number of equal sized tiles in y dimension.
Spatial filtering: Largest object size (pixels times number of tiles)	8	real	Cut on of the band pass Butterworth filter
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:

Measures cell death as a fraction of all cells using a nuclear and a cell death marker.
Segments an image of nuclear stains into single nuclei. Then scales cell death marker fluorescence based on a Histogram Reference Image that serves as a positive control. Counts nuclei that are brighter than a percent value of the reference image, and expresses as the fraction of all cells. This is performed for all frames of a timelapse.
The reference image is a positive control, with a strong fluorescence signal. Using Histogram Reference Images reduces the well-to-well variability of the readout.
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.

*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 minimum cell fluorescence.
*If dimmer cells are missed, decrease minimum cell fluorescence.
*If bright debris outshines cells, decrease debris cutoff percentile.

Generate reference image using the "fraction of cell death time lapse reference.ips"

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
DFT background removial is optional
Cell counts are also plotted