Count Division and Cell Death ( IACountDivAndDeath )

Parameters:

Name	Short Name	Type	Description
Type of Algorithm	type	string
Death (intensity) Threshold	thresholdIntensity	real	Dead cells stain brighter than live ones with nuclear stains, this threshold value distinguishes dead cells form live ones.
Division (derivative) Threshold	thresholdDerivative	real	Mitotic cells stain brighter than live ones with nuclear stains, this threshold value distinguishes mitotic cells form live ones.
Width of differentiation window	derivwidth	integer
Width of observation window	transientwidth	integer	How long does a change persists (in frames)
Maximal parent-daughter distance	divdist	real	Distance of parent and daughter in pixels when first detected.
Maximal parent cell size	maxparentsize	integer	Area in pixels, applied to observation window. 0 = disabled
Maximal daughter cell size	maxdaughtersize	integer	Area in pixels, applied to the observation window. 0 = disabled
Minimal cell size	mincellsize	integer	Area in pixels, minimal size to be counted as live cell. 0 = disabled
Data Table Output	dataoutput	boolean	Shows results in text window or Excel Data Window.

Description:

Counts Cell Division and Cell Death in image series of nuclear staining.
Image A has to be segmented and tracked first.
Image B is the original (or high pass filtered) nuclear staining image.
Division is defined by an increase followed by a decrease in the fluorescence intensity of the same object in time. The increase and the decrease each has to happen in a time window given by "Width of observation window" after each other. The increase and the decrease has to be over a threshold "Division (derivative) Threshold" given in intensity change per frame.
Daughter cell is defined by a decrease in intensity without previous increase.
Dead cell is defined by a permanent increase over the "Death (intensity) Threshold". Cells above the threshold from the beginning are excluded from the analysis. Daughter cells appearing after a division event within "Maximal parent-daughter distance" are assumed to belong to the same event. Pairs of daughter cells appearing together within "Maximal parent-daughter distance" or alone are assumed to be also a division event. Thus the total number of divisions is divisions + pairs of daughter cells + solo daughter cells.
Events are not detected in the last "Width of observation window" frames because the fate of the cell cannot be determined here. These frames are excluded from the calculation of the time span of the experiment.
The "Width of differentiation window" is typically 5, should be less than the "Width of observation window". Larger values suppress noise.
The analysis if further constrained by the following rules for detection of division or daughter events:
- Maximal size criteria
- Increasing mean fluorescence cannot be associated by decreasing summed fluorescence
- Decreasing mean fluorescence cannot be associated by increasing summed fluorescence
- The size of daughter cell cannot decrease
- The intensity has to increase (or decrease) slower than "Death (intensity) Threshold" per frame
Manual touchup:
Use the segment selector in the toolbar to add or remove events, and the "ReCount Cell Division and Cell Death" function to plot data.
Use the Excel Data Window to handle the results.