Mitochondria:cell volume fractionator (multiplexed)

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.
Channel number for qualifier stain	3	integer	This is the channel number shown in the Multi-Dimensional Open dialog.
Minimal mean image intensity cutoff for the cytoplasmic stain	4	real	Frames with smaller mean intensity are discarded.
Sensitivity scaling for the cytosolic stain (percentile)	5	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.
Cell tracker threshold (%)	6	real	Mean of pixel intensities if Image B. Use 0 for not checking
Spectral Unmix Coefficient Matrix for channel 2 and 3	7	string	To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.

Description:

Analysis of Mitochondria:cell volume fraction from confocal microscopic recordings of a cytosolic or cellular stain and a mitochondrial stain plus a qualifier stain. The qualifier stain is present only in a subpopulation of the cells, and qualifier stain positive and negative cells are collected in separate columns in the output Excel Data Window.
The algorithm allows for spectral unmixing of the mitochondrial and the qualifier stain. To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
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 based on the simplified version of the one described in J Physiol. 2012 590:12 2845-71. Handling of saturation and spectral unmixing between the mitochondrial and cytosolic stain has been removed compared to the original version (Mitochondria:cell volume fractionator (original)). Then processing of a qualifier stain and segmentation classifiers were added to separate cells positive and negative to the qualifier stain.
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.