Image processing pipelines in Image Analyst MKII
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Measure intensity of whole view field with background masking

Parameters:
Name # Type Description
Background level, percentile 1 real Background is calculated as frame-by-frame mean of those pixels that are the darker than this percentile of image histogram for the duration of the entire recording.
Mask level factor (0.2-1.5) 2 real If the colored area in the resultant image is less than what is expected to be cells decrease mask level factor. If the colored area in the resultant image appears to cover background increase the decrease mask level factor.
Plot normalization 3 string
Show results in microplate format 4 boolean Organizes data in table according to microplate well associations in additional worksheets.
Description:
The pipeline calculates average fluorescence intensity for the whole view field after stabilization of the image time series, background subtraction and masking of background regions. The mask is calculated from maximum intensity projection of the time series therefore accounts for cellular movement during the timelapse. This pipeline is ideal for obtaining fluorescence intensities from low-light level single-channel recordings, where cellular movement occurs. Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

If cells are very sparse use background level of 50 percentile.
If the culture is subconfluent use background level of 20 percentile.
If the culture is confluent use background level of 5 percentile.
If the colored area in the resultant image is less than what is expected to be cells decrease mask level factor.
If the colored area in the resultant image appears to cover background increase the decrease mask level factor.


Version history
V2: updated description, adjusted "Align Series" parameters