Count cellular foci with secondary cell segmentation

Parameters:

Name	#	Type	Description
Number of labels to measure (1-4)	1	integer	Select how many label channels tom measure. Each channel can be selected multiple times.
Histogram or distribution calculation	2	string	Select Histogram to show output as histogram, or None to show individual cell values.
Channel Number, nuclear stain	3	integer	Channel number of the nuclear marker used for nucleus (primary) segmentation.
Background subtraction method, nuclear stain	4	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
Morphological parameter measurement for nuclei	5	string	Plots and saves time course of selected morphological data
Channel Number, cell area marker	6	integer	Channel number of the whole cell marker used for secondary segmentation.
Background subtraction method, cell area marker	7	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
Morphological parameter measurement for cells	8	string	Plots and saves time course of selected morphological data
Foci: Channel Number, for foci (label #1)	9	integer	Channel number of label #1 to be measured. Each channel can be selected multiple times for different measurements.
Foci: Channel type, label #1	10	string	Switch between fluorescence intensity and optical density measurement. Optical density calculation needs already set up blank reference image (cell-free image) for the given channel number.
Foci: Measure label #1 over	11	string	Intensity measurement is performed either over the nucleus or in a perinuclear ring or in the whole cell, marked by secondary segmentation of the cell area marker.
Foci: Background subtraction method, label #1	12	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
Foci: Positive control reference image operation	13	string	The positive control is an image with many visible foci. Using a positive control avoids amplifying background noise in samples with little or no foci. Alternatively enter fixed threshold value here.
Foci: Sensitivity (0-1, less is more sensitive) or threshold	14	real	Sensitivity factor for Otsu's threshold determination. Smaller value will result a smaller threshold for foci brightness compared to local background.
Foci: maximum size (area, pixels)	15	integer	Maximal pixel area counted as foci.
Foci: Perinuclear ring width (pixels), label #1	16	integer	Width of the ring around the nucleus to measure perinuclear area, in pixels. This is used only if "Perinuclear ring" is selected above.
#2: Channel Number, label #2	17	integer	Channel number of label #2 to be measured. Each channel can be selected multiple times for different measurements.
#2: Channel type, label #2	18	string	Switch between fluorescence intensity and optical density measurement. Optical density calculation needs already set up blank reference image (cell-free image) for the given channel number.
#2: Measure label #2 over	19	string	Intensity measurement is performed either over the nucleus or in a perinuclear ring or in the whole cell, marked by secondary segmentation of the cell area marker.
#2: ackground subtraction method, label #2	20	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
#2: Intensity measurement type of label #2	21	string	Available plot types: "Mean", "Sum", "Variance", "PDI variance", "PDI sd", "Line Scan". These statistics describe pixels within each segment. To calculate statistics between segments use the "Mean of all traces" option.
#2: Perinuclear ring width (pixels), label #2	22	integer	Width of the ring around the nucleus to measure perinuclear area, in pixels. This is used only if "Perinuclear ring" is selected above.
#3: Channel Number, label #3	23	integer	Channel number of label #3 to be measured. Each channel can be selected multiple times for different measurements.
#3: Channel type, label #3	24	string	Switch between fluorescence intensity and optical density measurement. Optical density calculation needs already set up blank reference image (cell-free image) for the given channel number.
#3: Measure label #3 over	25	string	Intensity measurement is performed either over the nucleus or in a perinuclear ring or in the whole cell, marked by secondary segmentation of the cell area marker.
#3: Background subtraction method, label #3	26	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
#3: Intensity measurement type of label #3	27	string	Available plot types: "Mean", "Sum", "Variance", "PDI variance", "PDI sd", "Line Scan". These statistics describe pixels within each segment. To calculate statistics between segments use the "Mean of all traces" option.
#3: Perinuclear ring width (pixels), label #3	28	integer	Width of the ring around the nucleus to measure perinuclear area, in pixels. This is used only if "Perinuclear ring" is selected above.
#4: Channel Number, label #4	29	integer	Channel number of label #4 to be measured. Each channel can be selected multiple times for different measurements.
#4: Channel type, label #4	30	string	Switch between fluorescence intensity and optical density measurement. Optical density calculation needs already set up blank reference image (cell-free image) for the given channel number.
#4: Measure label #4 over	31	string	Intensity measurement is performed either over the nucleus or in a perinuclear ring or in the whole cell, marked by secondary segmentation of the cell area marker.
#4: Background subtraction method, label #4	32	string	Select background subtraction method: Percentile, Rolling ball, Spatial filtering, None. See settings below. Background subtraction is performed in the same way for all channels.
#4: Intensity measurement type of label #4	33	string	Available plot types: "Mean", "Sum", "Variance", "PDI variance", "PDI sd", "Line Scan". These statistics describe pixels within each segment. To calculate statistics between segments use the "Mean of all traces" option.
#4: Perinuclear ring width (pixels), label #4	34	integer	Width of the ring around the nucleus to measure perinuclear area, in pixels. This is used only if "Perinuclear ring" is selected above.
Histogram: number of bins	35	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.
Positive control reference image operation for nuclei detection	36	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.
Suppress debris and outside of well areas	37	boolean	Brighter areas corresponding to the bottom of microplate outside the well area will be masked.
Debris masking: minimum diameter of continuous bright spot	38	real	Farthest points of the maximal cross section. 0 for not checking
Debris and well edge detection sensitivity (%)	39	real	Pixel value, percentile, or factor times Otsu optimal threshold value (typically 1)
Nuclei: Nucleus diameter (pixels)	40	integer	Diameter of the nucleus in pixels. Nuclei ranging around this size will be selected.
Nuclei: Debris cutoff for ICC and nuclear stain (percentile)	41	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 fluorescence (%)	42	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)	43	real	1 for disc, smaller for irregular shapes. 0 for not checking
Nuclei: Weld segments into round objects	44	boolean	Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Cells: Minimum area (pixels)	45	integer	Volume in 3D, area in 2D. 0 for not checking
Cells: Maximum area (pixels)	46	integer	Volume in 3D, area in 2D. 0 for not checking
Cells: Minimum fluorescence (%)	47	real	Mean of pixel intensities in Image B. Use 0 for not checking
Cells: Boundaries are at (%) of peak fluorescenece	48	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.
Nuclei and Cells: Discard segments at edges of the image	49	boolean	Any segment that has at least 10% of its boundary at the edge of the image will be discarded.
Background percentile	50	real	If background subtraction method is percentile, this percentile value will be used in all channels.
Local background: Rolling ball: median filter size	51	integer	The median is calculated over a width x width rectangular area around each pixel of the image.
Local background: Spatial filtering: Number of tiles in x	52	integer	The image consists of this number of equal sized tiles in x dimension.
Local background: Spatial filtering: Number of tiles in y	53	integer	The image consists of this number of equal sized tiles in y dimension.
Local background: Spatial filtering: Largest object size (for background removal, pixels times number of tiles)	54	real	Size of the object 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.
Optical density: optional dark current intensity value	55	real	Enter average pixel intensity measured in an unilluminated, dark image, if not using a dark current reference image when calculating optical density.

Description:

This pipeline counts bright foci (aggregates, spots) per cell and measure fluorescence intensities or optical densities over whole cells areas, nuclei and perinuclear areas using a nuclear and cellular marker and 1-4 label channels. Use the "Count nuclear or cellular foci" pipeline instead if foci in the nucleus are to be counted, and a whole cell label is not available. The pipeline can run in single or tiled images and in whole wells with automatic suppression of outside of the well areas and debris (see “Suppress debris and outside of well areas”). Different measurements may be done in the same label channel, and each label can be defined as fluorescence or optical density (“Channel type”). One channel is used for foci count and 0-3 channels for intensity measurements.

Background subtraction:
The background subtraction technique can be specified for each channel. Choose percentile-based global or local background subtraction methods. All channels use the same method, and the parameters are at the end of the pipeline parameter list. For median or special filtering, the object size must be bigger than the cell size.
Tiled images: the background tiling pattern or vignetting 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 in the “Local background: Spatial filtering: Number of tiles in x” and “…y” parameters.

Sample: fixed or live cells stained for nuclei e.g. with Hoechst 33342 or DAPI, and showing other fluorescence of interest. Whole cells are marked, e.g. with phalloidin. Medium or high magnification (40x or above).

Input: 3-4 channels fluorescence image showing a nuclear and cellular markers and immuno- or other fluorescence visible over the nuclei or in perinuclear areas. Low or medium magnification 10-40x. May be tiled and whole well image. The area outside of the well must be bright in the fluorescence image for auto detection of the well.

Output: 1 morphological parameters of choice of nuclei and cells, the count of foci per nucleus (perinuclear area or cell) and 0-3 intensity or optical density measurements in the same or different label channels. For intensity measurement the “Intensity measurement type of label #” statistics (Mean, Sum, Variance, Punctate over diffuse ratio) is calculated over nuclei or perinuclear area see (“Measure label # fluorescence over” and “Perinuclear ring width”). Output data are single cell values or histogram (see “Histogram or distribution calculation”). Note that the order of labels measured in the output worksheet may vary due to parallel processing.

Reference image operation: by default, the calculation of nuclei is self-referenced. Thus, it is independently calculated for each image. There are two separate reference image options, one for the nucleus counting and one or the foci counting. This is set by the “Positive control reference image operation for nuclei detection” = ”Self-referenced” option. This works well if none of the samples have very few cells (foci) compared to others. If cell density varies between samples, or if some samples have very few cells (foci), use positive control reference. The positive control is one of the images from the data set with about maximum number of cells and positive cells. To make reference image set “Positive control reference image operation for nuclei detection” = ”Make reference image”. Process a positive control sample. The reference images will be minimized, and reused at the following operations. Then set “Positive control reference image operation for nuclei detection” = ”Use reference image” and process all data. The foci detection alternatively allows a fixed threshold that is a pixel intensity for the peak brightness of foci compared to the local background.

Debris avoidance:
To increase sensitivity, increase “Large debris removal sensitivity (%)” and decrease “Large debris minimum size (length in pixels)”. To decrease sensitivity, do the opposite. Debris avoidance may avoid cells or positive cells, if the cell count or positive cell count is too low. In this case set a small (non-zero) %, e.g. 1 as sensitivity.
Background subtraction:
Choose percentile-based global or local background subtraction methods. For tiled images: the background tiling pattern or vignetting 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 in the “Local background: Spatial filtering: Number of tiles in x” and “…y” parameters.

Adjustments:
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).
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 threshold in “Foci: Sensitivity (0-1, less is more sensitive) or threshold” and may increase the “Foci: maximum size (area, pixels)”.
*if too many foci are detected, increase the threshold in “Foci: Sensitivity (0-1, less is more sensitive) or threshold” and may decrease the “Foci: maximum size (area, pixels)”.

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

Version history
V1: based on “Fluorescence and absorbance histometry using nuclear and secondary whole cell segmentation (1-4 labels - advanced background options)”.