Image processing pipelines in Image Analyst MKII
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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.
Width of perinuclear ring to measure OD 10 integer Diameter of the structuring element in pixels
Approximate nucleus diameter 11 integer Diameter of the nucleus in pixels
Debris cutoff (percentile) 12 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 nucleus fluorescence (%) 13 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.
Nucleus boundaries (% of max 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.
Weld segments into round objects 15 boolean Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Thin process suppression (30-80) 16 real Cut off of the band pass Butterworth filter
Dark level (pixel intensity, if no dark current reference image) 17 real This value will be used as dark current, if no dark current (unilluminated) image was provided.
Export filename 18 string
Export format 19 string Available formats: "AVI", "MOV", "MP4", "JPG", "TIF"...
Scale bar size (um) 20 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 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.