Measure MULTIPLE shape parameters of cells or nuclei in tiled images

Parameters:

Name	#	Type	Description
Number of parameters measured (1-3)	1	real	Select how many parameters do you want to measure, and enter what do you want to measure below.
Morphological parameter 1	2	string	Plots and saves time course of selected morphological data
Morphological parameter 2	3	string	Plots and saves time course of selected morphological data
Morphological parameter 3	4	string	Plots and saves time course of selected morphological data
Plot type (statistics to plot)	5	string	Available plot types: "Mean", "Each", "Sum", "Variance", "Variance/Mean", "SD/Mean". These statistics represent the population of segments in the image or bounded by constraining ROIs.
Plot in separate rows (set Yes if Plot type is Each)	6	boolean	When "Place channels into columns" is set to yes repeated plotting in a Multi-dimensional open position will enter data in the same row to make data from different channels appear next to each other. Set this to yes to override this behavior and force each plot command to create a new row in the Excel Data Window.
Constrain to ROIs	7	boolean	Calculates only segments overlapping (any small intersection) with the active ROI in the segmented image.
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.
Minimum nucleus diameter	10	integer	Explicit minimum diameter of objects in pixels.
Approximate nucleus diameter	11	integer	Diameter of the nucleus in pixels. Objects varying around this size will be detected.
Background cutoff (%)	12	real	Details dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Debris cutoff (percentile)	13	real	Brighter details of the image than this will be ignored. Decrease this value if bright debris results wrong scaling of nuclei.
Minimum nucleus fluorescence (%)	14	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Nucleus boundaries (% of peak fluorescence)	15	real	For "Bound locally", the boundary of each object is determined at this % of the maximal intensity of the object relative to its neighborhood. For "Bound uniformly" this is a pixel intensity value.
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.

Description:

Cell or nucleus count or morphology measurement for tiled images, plotting up to 3 morphological parameters into consecutive columns.

Input: Tiled (optionally) image at low magnification, e.g. 10x-40x (~1.6-0.4 um/pixel resolution).
Output: data column Ch1 is for “Morphological parameter 1”, Ch2 for 2, Ch3 for 3.

The pipeline removes uneven background and tiling pattern. Then cells or nuclei are identified using watershed segmentation. Optionally, up to 3 morphological measurements are performed on the segmented image and entered into columns in the Excel Data Window. If using “Each” for plot type to record data for each object, set “Plot in separate rows” to Yes to obtain objects organized to columns and measurements into rows.

Paraformaldehyde fix and stain samples with Hoechst 33342 or DAPI.
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).
Set minimum size threshold.
No other shape classifiers are applied here, e.g. no minimum shape factor in contrast to other cell counting pipelines.
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.

Based on "Seahorse well cell count with nuclear stain" V2