Measure fluorescence ratio in single-cells using segmentation-based ROIs

Parameters:

Name	#	Type	Description
Channel Number (Numerator)	1	integer	The first linked image window with matching channel number will be invoked.
Channel Number (Denominator)	2	integer	The first linked image window with matching channel number will be invoked.
Subtract reference image background	3	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	4	boolean	Rolling ball-style background subtraction will be performed: the median of the image is subtracted from the image
Rolling ball: Median filter size (pixels)	5	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Background Level (percentile)	6	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 compares to first frame	7	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
Image stabilizer period (frames)	8	integer	1 if comparing to the first frame. Use more frames if not comparing to the first frame and the view field drifts slowly.
ROI drawing: approximate cell diameter	9	integer	Diameter of the nucleus in pixels
ROI drawing: 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".
ROI drawing: 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.
ROI drawing: cell boundaries (% of peak fluorescence)	12	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	13	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Plot normalization	14	string	This option supersedes ΔF/F0 normalization.
Show results in microplate format	15	boolean	Organizes data in table according to microplate well associations in additional worksheets.

Description:

This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of the time lapse, creates ROIs from the segments and copies ROIs to the original image. Finally ROI means are plotted.
Applicable to Ca2+-imaging (e.g. Fura-2), ratiometric GFP-based sensors (e.g. cameleons, ATeam, HyPer) imaging.

Version history:
V2
Minimum cell fluorescence classifier is also applied for the seeds, speeding up segmentation.
Segment welding is used preventing oversegmentation.
Local background subtraction option.
V3
Changed plotting property: Place channels into columns
V4
Changed plotting property: Expect only a single channel - in order to tidy up output
V5
Added reference image option