Seahorse well cell count with nuclear stain with marker (immunocytochemistry)

Microplate whole-well cell count, also used for normalization of Seahorse respirometry data. Total cell count and marker positive nuclei are cells with marker positive perinuclear area are calculated.
Images are either independently auto-scaled for cell and positive cell detection (self-referenced) or based on a positive control reference (make and use reference image), that is an arbitrary other image. This pipeline also detects and removes fluorescent debris from images an allows compensation for the area not looked because of debris.

Reference image operation: by default, as the previous versions of this pipeline, the calculation of the marker positivity is self-referenced. Thus, it is independently calculated for each image. This is set by the “Reference image operation” = ”Self-referenced” option. This works well if none of the samples have very few, e.g. <5% of positive cells. If cell density or marker positivity greatly varies between samples, or if some samples have very few cells or few positive cells, use positive control reference. The positive control is one of the images from the data set with about maximum number of cells and positive cells. To make reference image set “Reference image operation” = ”Make reference image”. Process a positive control sample. The reference images will be minimized, and reused at the following operations. Then set “Reference image operation” = ”Use reference image” and process all data. To process a different data set, close first reference images by “File/Close All Reference Images” and make new reference images.

To adjust the sensitivity of marker detection: to increase sensitivity, decrease “Positive counts threshold: Marker reference top percentile” and increase “Positive counts threshold: Minimum Marker fluorescence (%)”. To decrease sensitivity, do the opposite. If you adjust these values, the reference image does not have to be recalculated.

Debris avoidance: to increase sensitivity, increase “Large debris removal sensitivity (%)” and decrease “Large debris minimum size (length in pixels)”. To decrease sensitivity, do the opposite. Debris avoidance may avoid cells or positive cells, if the cell count or positive cell count is too low. In this case set a small (non-zero) %, e.g. 1 as sensitivity.


Paraformaldehyde fix and stain respirometry microplates with Hoechst 33342 or DAPI, and a single immunofluorescence or other stain. 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: "Debris filtered v4"
This pipeline was used in Gerencser AA. Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells, BBA - Bioenerg. 2018

Version history
the "with marker" version of "Seahorse well cell count with nuclear stain" is based on V2 of the original pipeline.
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 debris filtering was added to remove large fluorescent debris from the images, that prevent cell detection underneath. The residual area of the well is also saved into the output, so cell count may be corrected for it.
V4 added perinuclear measuement, updated labels and description
V5 added reference images and options for adjusting debris avoidance
V6 fixes to make and use reference images.
V7 changed location of roi file, improved hints