Whole well strip cell count

Parameters:

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

Description:

Whole well cell count calculated from recording using a strip of tiles across the well, and extrapolation of cell numbers to the whole well.
Set approximate cell diameter. To this end load an image, zoom in using the magnifier glass Main Toolbar button, and then using the linear ROI button draw a line across a nucleus. Double-click the status bar of the Image Window to see the length of the ROI (Size of active ROI).
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.
Run the pipeline on a single well and observe the results. In the overlay Image Window the gray fluorescence image should be well matched by the colored segments:

*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’.
The “Strip to Well Cell Count” function takes radial inhomogeneity into account.

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.