Mitochondria:cell volume fractionator (original)

Parameters:

Name	#	Type	Description
Channel number for cytoplasmic stain	1	integer	This is the channel number shown in the Multi-Dimensional Open dialog.
Channel number for mitochondrial stain	2	integer	This is the channel number shown in the Multi-Dimensional Open dialog.
Minimal mean intensity cutoff for the cytoplasmic stain	3	real	Frames with smaller mean intensity are discarded.
Sensitivity scaling for the cytosolic stain (percentile)	4	real	A lower value (slightly below 100) increases sensitivity. Cells aggregating the probe in lysosomes need a lower percentile value to select cells and not lysosomes.
Detector bit-depth for detection of saturation	5	real	This value if used to prevent spectral unmixing artifact resulting from saturated pixels. Set this value based on the detector setting for data recording.

Description:

Analysis of Mitochondria:cell volume fraction from confocal microscopic recordings of a cytosolic or cellular stain (e.g. Calcein-AM) and a mitochondrial stain (MitoTracker Red).
The algorithm has been calibrated to electron microscopy, if using a red fluorescent probe for mitochondria (e.g. MitoTracker Red CMX), the pixel size is ~44nm and the pinhole was set to ~ 1 Airy unit for recording the images. Smaller resolution will result overestimation of the volume fraction. For image acquisition please follow protocols given at http://help.imageanalyst.net/protocols.html.
The volume fraction is given by the 2/3 times the ratio of the sum of mitochondrial pixels over the sum of all cellular (including mitochondrial) pixels, where the sum is calculated for all recorded images. The sum and ratio calculation takes place in an Excel template.
Note: The "Sensitivity scaling for the cytosolic stain (percentile)" may need to be adjusted. A lower value (slightly below 100) increases sensitivity, and estimates bigger cell size. Cells aggregating the probe in lysosomes need a lower percentile value to select cells and not lysosomes. The default value 80 is set for INS-1E cells with bright vacuoles in the calcein staining. Other specimens may need higher values around 98-99 percentile.
The algorithm is as it was set up for J Physiol. 2012 590:12 2845-71, with some automation elements added.

Keywords: volume fraction, volume density, mitochondrial biogenesis, mitochondrial abundance, mitochondrial mass, amounts of mitochondria, confocal microscopic stereology, mitochondrial membrane potential measurement

Version history:
V2: An Erase All ROIs command was added to the start to ensure that no user-drawn ROIs interfere with the analysis.