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Count cells with foci vs diffuse fluorescence

Parameters:
Name # Type Description
Channel Number, Label 1 integer The first linked image window with matching channel number will be invoked.
Channel Number, DIC 2 integer The first linked image window with matching channel number will be invoked.
Positive control reference image operation for nuclei detection 3 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.
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.
Image scaling maximum for debris suppression (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 at "Max value".
DIC: DFT high pass cut on ω (pixels) 7 real Cut on of the band pass Butterworth filter
DIC: minimum cell area 8 integer Volume in 3D, area in 2D. 0 for not checking
DIC: maximum cell area 9 integer Volume in 3D, area in 2D. 0 for not checking
DIC: minimum shape factor 10 real 1 for disc, smaller for irregular shapes. 0 for not checking
Foci: DFT high pass cut on ω (pixels) 11 real Cut on of the band pass Butterworth filter
Foci: maximum area 12 integer Volume in 3D, area in 2D. 0 for not checking
Foci: minimum intensity (%, scaled by DFT) 13 real Minimal intensity of diffuse fluorescence expressed as % of maximal intensity of unfiltered images. The DFT filtering lowers intensities, therefore a lower % value needs to be used than without filtering.
Diffuse: DTF band pass cut on ω (pixels) 14 real Cut on of the band pass Butterworth filter
Diffuse: DTF band pass cut off ω (pixels) 15 real Cut off of the band pass Butterworth filter
Diffuse: minimum intensity (%, scaled by DFT) 16 real Minimal intensity of foci expressed as % of maximal intensity of unfiltered images. The DFT filtering lowers intensities, therefore a lower % value needs to be used than without filtering.
Background level (percentile) 17 real This percentile of the image histogram sets the intensity value where the minimum of the Look Up Table (LUT) is scaled. Use -1 to override this with fixed value set below at "Min value".
Description:
Counting cells with and without fluorescence aggregates (foci). The original use of this pipeline was to detect aggregates of a GFP-tagged protein in yeast. Yeast cells are detected in an off-focus DIC image, where cells look brighter than the background. Mammalian cells and a fluorescence label to identified also may be used with this pipeline. The algorithm separates background, aggregates and diffuse fluorescence using band-pass and high-pass filtering in frequency domain using DFT. Then images are segmented based on local maxima and objects are gated by size classifiers.

Input: 2 channels one with the aggregating fluorescent label, and the other is off-focus DIC or a fluorescence marker for all cells. May use tiling. Use medium-high magnification to resolve aggregates (e.g. 40x).

Output: number of cells with diffuse or aggregated fluorescence is counted. Histograms of fluorescence intensity of all cells, foci, and diffuse fluorescence is calculated. Image intensities are scaled to the “Image scaling maximum for debris suppression (percentile) “ percentile of a positive control sample (reference image, see below). This top percentile will be 100% image intensity.

Reference image operation: First load a positive control image with high amounts of aggregates and run this pipeline with the “Positive control reference image operation for nuclei detection”=”Make reference image” parameter to set up the positive control reference image. Then set this parameter to ”Use reference image” to process other images. To use the pipeline in self-referencing mode, leave this parameter as ”=”Make reference image”.
Adjustment:
DFT cut on or cut off ω values are in pixels, where larger values correspond to higher frequencies, thus smaller objects.
To make back round subtraction stricter in cell detection, increase DIC: DFT high pass cut on ? (pixels). If cells break up or develop holes, decrease this value. Adjust “DIC:” shape parameters as desired.
To make foci detection stricter:
*Increase Foci: DFT high pass cut on ω (pixels)
*Decrease Foci: maximum area
*Increase Foci: minimum intensity (%, scaled by DFT)
To make diffuse fluorescence detection stricter:
*To improve background suppression, increase Diffuse: DTF band pass cut on ? (pixels)
*To improve separation from foci, decrease Diffuse: DTF band pass cut off ? (pixels)
*Increase Diffuse: minimum intensity (%, scaled by DFT)

This is a gregarized version of the pipeline used in “Peters TW, Nelson CS, Gerencser AA, Dumas KJ, Tavshanjian B, Chang KC, Lithgow GJ, Hughes RE. 2018 Natural Genetic Variation in Yeast Reveals That NEDD4 Is a Conserved Modifier of Mutant Polyglutamine Aggregation. G3 (Bethesda). 2018 Nov 6;8(11):3421-3431. doi: 10.1534/g3.118.200289.”