Microplate whole well cell count (automatic well positioning, with positive control)

Parameters:

Name	#	Type	Description
Channel Number, nuclear stain	1	integer	Channel number associated with the image, as appears in the Multi-Dimensional Open Dialog. Use Edit/Rename to view or change channel number.
Positive control reference image operation	2	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.
Suppress debris and outside of well areas	3	boolean	Brighter areas corresponding to the bottom of microplate outside the well area will be masked.
Debris masking: minimum diameter of continuous bright spot	4	real	Farthest points of the maximal cross section. 0 for not checking
Debris and well edge detection sensitivity (%)	5	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Cells: Nucleus diameter (pixels)	6	integer	Diameter of the nucleus in pixels. Nuclei ranging around this size will be selected.
Cells: Debris cutoff for ICC and nuclear stain (percentile)	7	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".
Cells: Minimum cell fluorescence (%)	8	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.
Cells: Shape factor (minimum; 0-1)	9	real	1 for disc, smaller for irregular shapes. 0 for not checking
Cells: Weld segments into round objects	10	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Cells: Discard segments at edges	11	boolean	Any segment that has at least 10% of its boundary at the edge of the image will be discarded.
Estimate area covered by cells (empty area detection)	12	boolean	Yes executes output #1, No executes output #2.
Empty area detection: minimum diameter	13	integer	Diameter of the structuring element in pixels
Empty area detection: copy the above value here	14	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Plate table output	15	boolean	Organizes data in table according to microplate well associations in additional worksheets.
Local background subtraction method	16	string	The Nth output will be executed when the Nth value is selected here from a ;-separated list given below at "Possible selector values".
Local background: Rolling ball: median filter size	17	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Local background: Spatial filtering: Number of tiles in x	18	integer	The image consists of this number of equal sized tiles in x dimension.
Local background: Spatial filtering: Number of tiles in y	19	integer	The image consists of this number of equal sized tiles in y dimension.
Local background: Spatial filtering: Largest object size (for background removal, pixels times number of tiles)	20	real	Size of the object to be passed by the filtering. The pixel size must be multiplied by the number if tiles (in x or y). Objects larger than this will be removed as background. Cut on of the band pass Butterworth filter.

Description:

Robust microplate whole-well cell count with auto detection of well and debris avoidance. No well ROI is used for this pipeline. Images are either independently auto-scaled for cell detection (self-referenced) or based on a positive control reference (make and use reference image), that is an arbitrary other image.

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.

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.

Output:
Col1: cell count
Col3: well area excluding debris where cell were counted (use this to normalize counts for whole well)
Col4: (optional): estimated area that is covered by cells, excluding empty areas (see Empty area options; use this to alternatively normalize counts for whole well, if cells got part washed off before counting)

Sample:
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. The area outside of the well must be bright in the fluorescence image for auto detection of the well.

Adjustments:
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: "washoff v7"

Version history:
V2:
Changed shape factor to follow changes in core calculations
Added option to weld segments to round objects and discard segments touching the edges of the image