Measure intensities using segmentation of the same image

Parameters:

Name	#	Type	Description
Subtract reference image background	1	boolean	Use I/O Set Reference Image or the Context Menu of the Image Window to mark Background Reference Images. The Background Reference Image with the corresponding channel number will be subtracted here. Adjust channel numbers with Edit/Rename if needed.
Local background subtraction by median rolling ball	2	boolean	Rolling ball-style background subtraction will be performed: the median of the image is subtracted from the image
Median rolling ball size	3	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Background Level (Percentile)	4	real	Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording. This has no effect if rolling ball is used.
Image stabilizer period (frames)	5	integer	1 if comparing to the first frame. Use more frames if not comparing to the first frame and the view field drifts slowly.
Image stabilizer compares to first frame	6	boolean	“Yes” is the fastest way of image stabilization. Turn it off if the image changes a lot during the time lapse, and it is not possible to compare later frames to the first frame any more
ROI drawing: approximate cell diameter	7	integer	Diameter of the cells in pixels
ROI drawing: debris cutoff (percentile)	8	real	Brighter details of the image than this will be ignored
ROI drawing: minimum cell fluorescence (%)	9	real	Cells dimmer than this will be rejected. Increase this value if debris dimmer than the cells is detected.
ROI drawing: cell boundaries (% of peak fluorescence)	10	real	The boundary of each object is determined as this % of the maximal intensity of the object relative to its neighborhood.
ROI drawing: weld segments into round objects	11	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Plot normalization	12	string	This option supersedes ΔF/F0 normalization.
Show results in microplate format	13	boolean	Organizes data in table according to microplate well associations in additional worksheets.

Description:

This pipeline segments an image of cytosolic or nuclear stain into single cells or nuclei, and then measures intensities in the same channel, after performing background subtraction.

To adjust segmentation:
If single cells are detected as multiple segments, increase the approximate cell diameter.
If multiple cells are detected as single segments, decrease the approximate cell diameter.
If debris is detected as cells, increase minimum cell fluorescence.
If dimmer cells are missed, decrease minimum cell fluorescence.
If only bright debris is detected, decrease debris cutoff percentile.
If brightest areas are clipped or fused, Increase debris cutoff percentile.
If ROIs are smaller than cells, decrease cell boundaries (% of peak fluorescence).
If ROIs are too large or spilling over to background, increase cell boundaries (% of peak fluorescence).

Version history
V2: local and reference image background subtraction options were added to the pipeline.
Improved segmentation.