Count DAB positive nuclei in HE stained sections

Parameters:

Name	#	Type	Description
Channel Number : Red	1	integer	Red channel of an RGB bright field image
Channel Number : Blue	2	integer	Blue channel of an RGB bright field image
Approximate cell diameter	3	integer	Diameter of the nucleus in pixels
Debris cutoff (percentile)	4	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 staining intensity (%)	5	real	Cells dimmer than this in filtered and 1-100 rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Cell boundaries (% of max fluorescence)	6	real	Cells dimmer than this in filtered rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
Weld segments into round objects	7	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Minimum Shape factor	8	real	A larger value will remove irregular objects.
Intensity threshold for DAB staining	9	real	Intensity threshold for DAB positivity. Use the histogram to determine desired threshold.
Coefficient Matrix	10	string	Matrix for separation of HE and DAB.

Description:

This pipeline measures DAB intensity over nuclei and counts positive ones in HE stained histological sections, imaged at low 10-20x magnification.
Input: Brightfield, RGB image.
Output: counts of all nuclei, DAB-positive nuclei, and a histogram of DAB. Intensity is defined here as inverted brightfield pixel value, and not optical density. In the output images all nuclei are shown in green with the positive ones appearing in yellow (red overlay). Click the histogram to show cells with a particular DAB staining intensity.
Adjust “Approximate cell diameter” to the visible size of nuclei in pixels. Adjust “Minimum nucleus staining intensity (%)” for the desired sensitivity, where a lower value will detect more cells and debris. Increase “Minimum Shape factor” if nuclei are all round and background is recognized as cells, or decrease it if irregular shaped nuclei are missed.
Tune the sensitivity of the detection by entering a value in “Intensity threshold for DAB staining” corresponding to the histogram x-axis.
Draw an ROI before analysis on the image to constrain measurements to a particular area of the image.