Image processing pipelines in Image Analyst MKII
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Measure intensities in single-cells using ROIs drawn by segmentation

Name # Type Description
Subtract reference image background 1 boolean Use I/O Set Reference Image or the Context Menu of the Image Window to mark Background Reference Images. The Background Reference Image with the corresponding channel number will be subtracted here. Adjust channel numbers with Edit/Rename if needed.
Local background subtraction by median rolling ball 2 boolean Rolling ball-style background subtraction will be performed: the median of the image is subtracted from the image
Rolling ball: median filter size 3 integer The median is calculated over a width x width rectangular area around each pixel of the image.
Background Level (percentile) 4 real Background is calculated as frame-by-frame median of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording. This has no effect if rolling ball is used.
Image stabilizer compares to first frame 5 boolean “Yes” is the fastest way of image stabilization. Turn it off if the image changes a lot during the time lapse, and it is not possible to compare later frames to the first frame any more
Image stabilizer period (frames) 6 integer 1 if comparing to the first frame. Use more frames if not comparing to the first frame and the view field drifts slowly.
ROI drawing: approximate cell diameter 7 integer Diameter of the cell in pixels
ROI drawing: debris cutoff (percentile) 8 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".
ROI drawing: minimum cell fluorescence (%) 9 real Cells dimmer than this in filtered and 1-1000 rescaled images will be rejected. Increase this value if debris dimmer than the cells is detected.
ROI drawing: cell boundaries (% of peak fluorescence) 10 real The boundary of each object is determined as this % of the maximal intensity of the object relative to its neighborhood.
ROI drawing: weld segments into round objects 11 boolean Weld touching segments if they form a rounder object together. Use this to avoid objects fragmenting into multiple segments.
Plot normalization 12 string
Show results in microplate format 13 boolean Organizes data in table according to microplate well associations in additional worksheets.
This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of the time lapse, creates ROIs from the segments and copies ROIs to the original image. Finally ROI means are plotted.
Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

Version history:
Minimum cell fluorescence classifier is also applied for the seeds, speeding up segmentation.
Segment welding is used preventing oversegmentation.