Mitochondria:cell volume fractionator (basic with post-hoc staining and hole filling)

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 post-hoc stain 1	3	integer	This is the channel number shown in the Multi-Dimensional Open dialog.
Channel number for post-hoc stain 2	4	integer	This is the channel number shown in the Multi-Dimensional Open dialog.
Post-hoc stain 1 color	5	string	Look Up Table: "Grayscale", "Red", "Green", "Blue", "Pseudocolor", "Intensity Gated Pseudocolor", "Fire", "ROI color", "Off"
Post-hoc stain 2 color	6	string	Look Up Table: "Grayscale", "Red", "Green", "Blue", "Pseudocolor", "Intensity Gated Pseudocolor", "Fire", "ROI color", "Off"
Minimal mean intensity cutoff for the cytoplasmic stain	7	real	Frames with smaller mean intensity are discarded.
Sensitivity scaling for the cytosolic stain (percentile)	8	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	9	integer	Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
Hole filling: maximal hole area in mitochondria	10	integer	Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
Hole filling: close this size of gaps before filling holes	11	integer	Diameter of the structuring element in pixels

Description:

Analysis of Mitochondria:cell volume fraction from confocal microscopic recordings of a cytosolic or cellular stain and a mitochondrial stain, using additional post-hoc immunocytochemistry images. This pipeline is specifically applicable for cells with swollen mitochondria. Image processing in the original volume fractionator captures swollen mitochondria with a hole in the middle. This pipeline has additional components to fill in these holes. In addition, hole filling can repair holes introduced by saturation of images. Holes in the cytosolic stain, e.g. vacuoles are also filled in, thus estimated as cell area. Post-hoc images are recordings of the same view fields as the live cell recording, but after fixation and immunofluorescence staining. To load live plus post-hoc image set, add both files to the “Multi-Dimensional Open Dialog” (use the “File Order” tab), and check “Merge as Channel”.
The pipeline only shows the corresponding immunofluorescence image (use the green double arrow Main Tool Bar button to synchronize Image Window sliders), and the output needs to be manually gated based on these images, using the Editing/Mask ROIs with a fill value of 0 on the binarized “Cell” and “Mitochondria” images. When done, plot data to the Excel Data Window with Plotting/Plot, Plot type=Sum.
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 calculations take 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)), and processing of additional channels was added.
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.
V3: The first version of the "hole filling" flavor of the volume fractionator is based on V2 basic volume fractionator.