Seahorse well cell count with nuclear stain

Parameters:

Name	#	Type	Description
Number of tiles in x	1	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	2	integer	The image consists of this number of equal sized tiles in y dimension.
Mask ROI File	3	string	*.roi file
Approximate nucleus diameter	4	integer	Diameter of the nucleus in pixels
Background cutoff (%)	5	real	Details dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Debris cutoff (percentile)	6	real	Brighter details of the image than this will be ignored. Decrease this value if bright debris results wrong scaling of nuclei.
Minimum nucleus fluorescence (%)	7	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Nucleus boundaries (% of peak fluorescence)	8	real	For "Bound locally", the boundary of each object is determined at this % of the maximal intensity of the object relative to its neighborhood. For "Bound uniformly" this is a pixel intensity value.
Weld segments into round objects	9	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.

Description:

Microplate whole-well cell count, also used for normalization of Seahorse respirometry data.
Paraformaldehyde fix and stain respirometry microplates with Hoechst 33342 or DAPI. Tiled image the bottom of the well at low magnification, e.g. 10x (~1.6um/pixel resolution). This tiled image is the input for this pipeline.
First set the well ROI. To this end load an image, and load a previously used ROI with the “Seahorse well cell count with nuclear stain LOAD well ROI” pipeline. Move or erase and redraw the ROI. This has to be an area-type ROI, encircling the well. Then save the ROI using the “Seahorse well cell count with nuclear stain SAVE well ROI” pipeline.
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).
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’.
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.
Internal version: V2.1

Version history
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 updated description