Mitochondria:cell volume fractionator (basic - remove out of focus)

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 image 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.
Hole filling: maximal hole area in the cytosol	5	integer	Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
QC: Minimum cell size	6	integer	Volume in 3D, area in 2D. 0 for not checking
QC: Maximal edge roughness (%)	7	real	Mean of pixel intensities in Image B. Use 0 for not checking

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.
The algorithm is a simplified version of the one described in J Physiol. 2012 590:12 2845-71. Handling of saturation and spectral unmixing has been removed compared to the original version (Mitochondria:cell volume fractionator (original)).
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.

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.