Count cells with aggregates using nuclear and whole cell markers

Parameters:

Name	#	Type	Description
Channel Number: Nucleus marker	1	integer	The first linked image window with matching channel number will be invoked.
Channel Number: Cell marker	2	integer	The first linked image window with matching channel number will be invoked.
Channel Number: Foci	3	integer	The first linked image window with matching channel number will be invoked.
Number of tiles in x	4	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	5	integer	The image consists of this number of equal sized tiles in y dimension.
Positive control reference image operation for nuclei detection	6	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.
Local background: Spatial filtering: Largest object size (for background removal, pixels times number of tiles)	7	real	Size of the cellular objects 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.
Nuclei: Nucleus diameter (pixels)	8	integer	Diameter of the nucleus in pixels. Nuclei ranging around this size will be selected.
Nuclei: Debris cutoff for ICC and nuclear stain (percentile)	9	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".
Nuclei: Minimum cell fluorescence (%)	10	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.
Nuclei: Shape factor (minimum; 0-1)	11	real	1 for disc, smaller for irregular shapes. 0 for not checking
Nuclei: Weld segments into round objects	12	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Nuclei: Discard segments at edges of the image	13	boolean	Any segment that has at least 10% of its boundary at the edge of the image will be discarded.
Cell marker gating: maximum area (pixels)	14	integer	Volume in 3D, area in 2D. 0 for not checking
Cell marker gating: minimum area (pixels)	15	integer	Volume in 3D, area in 2D. 0 for not checking
Foci: Spatial filtering: Largest object size (for foci detection, pixels times number of tiles)	16	real	Size of the foci 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.
Foci gating: maximum area (pixels)	17	integer	Volume in 3D, area in 2D. 0 for not checking
Foci gating: minimum shape factor (0-1)	18	real	1 for disc, smaller for irregular shapes. 0 for not checking
Foci gating: minimum fluorescence intensity (%)	19	real	Mean of pixel intensities in Image B. Use 0 for not checking

Description:

This pipeline counts cells that have diffuse, punctate/foci or both fluorescence in the cytosol. The analysis relies on a nuclear, a cellular and the foci fluorescence marker. Foci are detected by a combination of spatial filtering and image segmentation.
Input: a three-channel image of nuclei (e.g. Hoechst 33342 or DAPI), cells (e.g. CellTrackers), and an aggregating label, e.g. a GFP-tagged protein. Images may be tiled. If no tiling is used, set tiling x and y to 1.
Output: counts for total all possible combinations of cells with diffuse and/or aggregated labeling.
Adjustments:
Set approximate nucleus 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 too few of the foci are detected, decrease the “Foci gating: minimum fluorescence intensity (%)”.
*if too many foci are detected, increase the “Foci gating: minimum fluorescence intensity (%)”.

If segmentation goes as it is desired, process the whole microplate using the double blue arrowhead button and ‘Run Pipeline … on All Stage Positions’.

A basic version of this pipeline has been used in:
1. Peters TW, Nelson CS, Gerencser AA, Dumas KJ, Tavshanjian B, Chang KC, Lithgow GJ, Hughes RE. Natural Genetic Variation in Yeast Reveals That NEDD4 Is a Conserved Modifier of Mutant Polyglutamine Aggregation. G3 (Bethesda) [Internet]. 2018;8(11):3421–3431. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30194090 PMID: 30194090