Mitochondrial membrane potential assay - measurement of spectral crossbleed

This is the standard pipeline to calculate spectral crossbleed coefficients for the unbiased absolute mitochondrial membrane potential assay.
Recordings with tetramethylrhodamine methyl ester (TMRM) and FLIPR (aka PMPI, plasma membrane potential indicator; FLIPR Membrane Potential Probe) are contaminated with fluorescence crossbleed, more or less, depending on the actual optical setup of the microscope. Mostly FLIPR fluorescence contaminates the TMRM signal. This pipeline (Version 2) allows calculation of spectral crossbleed form a standard TMRM/FLIPR recording for absolute calibrated mitochondrial membrane potential assay. However, spectral crossbleed can be calculated most precisely using single-fluorophore stained samples.

1) Determining spectral crossbleed from TMRM/FLIPR time lapse recording (both stains present):

The crossbleed from TMRM to FLIPR is determined at the baseline of the recording, where TMRM is punctate, and FLIPR is less bright and more diffuse, using high pass filtering. Then the FLIPR to TMRM crossbleed is determined in the ending frames of the recording, where TMRM does not/minimally stains cells.
• Load time course, duplicate each image with no linking and link the duplicates with each other.
• Use “Cut or Truncate Frames” to shorten the first pair of images series to the duration of the baseline, and the second one to the duration of the zero calibration at the end of the run.
• In the pipeline parameters set up channel numbers as background subtraction level in the same way as during the potentiometric analysis.
• Open Excel Data Window.
• First, run the pipeline in the baseline section with these settings:
o Direction of crossbleed (1st:TMRM, 2nd: FLIPR): first->second
o TMRM->FLIPR corssbleed with highpass filtering: Yes
o Size of mitochondria (pixels): 4
• Second, run the pipeline in the baseline section with these settings:
o Direction of crossbleed (1st:TMRM, 2nd: FLIPR): second->first
o TMRM->FLIPR corssbleed with highpass filtering: No
• Average results from multiple recordings.
• Enter the spectral crossbleed matrix as ={{1,second->first},{first->second,1}} in the Mitochondrial membrane potential measurement (TMRM/FLIPR).
• Save the data by File/Save Excel Data if needed.


2) Determining spectral crossbleed from single stained samples:

Two samples, one incubated with TMRM in basal condition, and an other incubated with FLIPR and then with CDC (or PM-PFA; see the ΔψM assay). It is also possible to calculate it from an actual potentiometric recording because the two fluorophores behave differently in time, but this method is less robust and typically allows only calculation of the stronger FLIPR to TMRM crossbleed. This pipeline is supported by an interactive protocol in the Primer.

The input is a two channel recording acquired with the same microscopy settings as the potentiometric recording, so a TMRM and a FLIPR channel. Single or multiple frames. The potentiometric medium is prepared either only with TMRM or on with FLIPR added. For FLIPR also a PM-PFA is prepared and added to the sample ~30 min before recording.

• Adjust channel numbers and the percentile level of background subtraction pipeline parameters to match the settings as you analyze the potentiometric time lapse recordings.
o If the cells are very sparse Use background level of 50 percentile.
o If the culture is sub confluent Use background level of 20 percentile.
o If the culture is confluent Use background level of 5 percentile.
• Select the direction of crossbleed:
o For TMRM-loaded sample: first->second
o For FLIPR-loaded sample: second->first
• Run the pipeline, and observe the resultant Excel worksheet.
o The crossbleed coefficient for "first->second" channel appears in the bottom left corner of the matrix.
o The crossbleed coefficient for "second->first" channel appears in the top right corner of the matrix.
• Run the pipeline in all relevant positions (e.g. use the pull-down menu of the button to toggle mode for partial plate operation) and calculate the median of the coefficients in Excel independently for the two ways of crossbleed.
• Enter the spectral crossbleed matrix as ={{1,second->first},{first->second,1}} in the Mitochondrial membrane potential measurement (TMRM/FLIPR).
• Save the data by File/Save Excel Data if needed.


Version History:
V2: Two-stains operation using high pass filtering was added