Measure DAB staining intensity in 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
Histogram calculation	3	string	Enable histogram calculartion. Use "None" to record staining intensity for each cell.
Histogram: number of bins	4	integer	When the output is histogram, the histogram is calculated using this number of equal sized bins, where the lowest bin starts at the smallest data point and the highest bin ends at the largest data point. If explicit range definition is used below, the bins will bound the given range. Use zero bin size for automatic binning.
Approximate cell diameter	5	integer	Diameter of the nucleus in pixels
Debris cutoff (percentile)	6	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 (%)	7	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)	8	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	9	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Minimum Shape factor	10	real	A larger value will remove irregular objects.
Coefficient Matrix	11	string	Matrix for separation of HE and DAB.

Description:

This pipeline measures DAB intensity over nuclei in HE stained histological sections, imaged at low 10-20x magnification.
Input: Brightfield, RGB image.
Output: List of DAB staining intensities, one per nucleus. 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.
Draw an ROI before analysis on the image to constrain measurements to a particular area of the image.