Count senescent cells with SA-BGAL staining densitometry

Parameters:

Name	#	Type	Description
Show histogram of staining OD	1	boolean	Yes executes output #1, No executes output #2.
Show overlay image	2	boolean	Yes executes output #1, No executes output #2.
Export overlay image	3	boolean	Yes executes output #1, No executes output #2.
Export with scalebar	4	boolean	Turns on scale bar.
Channel of nuclear marker	5	integer	The first linked image window with matching channel number will be invoked.
Channel of SA-BGAL	6	integer	The first linked image window with matching channel number will be invoked.
Positive staining threshold (OD)	7	real	Segment considered to be positive for the given channel if mean intensity is greater or equal to the threshold.
Number of tiles in x	8	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	9	integer	The image consists of this number of equal sized tiles in y dimension.
Nuclei: Positive control reference image operation for nuclei detection	10	string	The positive control is an image with many visible cells. Using a positive control avoids amplifying background noise in wells with little or no cells.
Nuclei: Nucleus diameter (pixels)	11	integer	Diameter of the nucleus in pixels. Nuclei ranging around this size will be selected.
Nuclei: Clip bright foci (e.g. nucleoli)	12	boolean	Bottoms at zero and ceilings at range value. When gamma<>1 value is used, zero clipping always occur.
Nuclei: Debris cutoff for ICC and nuclear stain (percentile)	13	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 at "Max value".
Nuclei: Minimum cell fluorescence (%)	14	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.
Nuclei: Shape factor (minimum; 0-1)	15	real	1 for disc, smaller for irregular shapes. 0 for not checking
Nuclei: Weld segments into round objects	16	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Nuclei: Discard segments at edges of the image	17	boolean	Any segment that has at least 10% of its boundary at the edge of the image will be discarded.
Width of perinuclear ring to measure OD	18	integer	Diameter of the structuring element in pixels
Thin process suppression (30-80)	19	real	Cut off of the band pass Butterworth filter
Dark level (pixel intensity, if no dark current reference image)	20	real	This value will be used as dark current, if no dark current (unilluminated) image was provided.
Export filename	21	string
Export format	22	string	Available formats: "AVI", "MOV", "MP4", "JPG", "TIF"...
Scale bar size (um)	23	real	Length of the scale bar in the units given above.

Description:

Counts senescent cells based on fluorescence labeling of nuclei and senescence associated beta-galactosidase (SA-BGAL) staining absorbance.
Segments an image of nuclear stains into single nuclei, and optical density (OD) images are calculated using blank and dark current images.
SA-BGAL Image: Bright field, grayscale image of SA-BGAL staining. For the classical stain use a red (e.g. Cy5) emission filter to record absorbance.
Blank image: using the same optical setting an empty (specimen-free) bright field image is recorded.
Dark current image: using the same optical setting an image with no illumination is recorded. This is optional, a fixed intensity value can be given as 'Dark Level'.
Set Blank and Dark current images by loading them and using the right-clicking the image / Set Reference Image. The reference images must have the same channel number as the SA-BGAL image. Use Edit/Rename to change the channel number if needed.
If multiple blank or dark current images are available in a multi-position recording, generate dark current and blank images using the “Create background reference image(s) for multiwell plate using median of wells” pipeline with “Background / Dark Current” as type for dark current and “Blank for OD / Normalization” as type for blank.

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.
V4
Added missing OD threshold option
Added options to show histograms and overlay images and to save overlay images
Added options to fine tune suppression of OD from refraction caused by thin cellular processes.
Added using optional fixed value instead of dark current image.
V5
Nuclear segmentation was replaced with one matching that of used in the histometry pipelines, including a switch for reference images and bright foci clipping.