Measurement of matrix to cytosol apparent activity coefficient ratio for TMRM

This protocol describes how to measure the apparent affinity coefficient ratio of tetramethylrhodamine methyl ester (TMRM), that is a required for the calculation of the absolute magnitudes and time courses of mitochondrial and plasma membrane potentials in intact cells in adherent cultures. The apparent affinity coefficient ratio describes the binding of TMRM to mitochondrial membranes and the dilution of fluorescence by otherwise light microscopically invisible ultrastructural details of mitochondria (e.g. crista density). This is a confocal microscopic assay. To calculate the apparent affinity coefficient ratio, confocal microscopic fluorescence time-lapses are recorded to follow the leakage of TMRM from the cells as mitochondria are being completely depolarized immediately before the start of the recording. Then the apparent affinity coefficient ratio is calculated by a built-in pipeline in Image Analyst MKII.

Sample preparation, reagents

• Experimental buffer (EB): 120 NaCl, 3.5 KCl, 1.3 CaCl₂, 1 MgCl₂, 0.4 KH2PO4, 5 NaHCO₃, 1.2 Na₂SO₄, 20 TES, 15 glucose , pH7.4 at 37°C
• Mitochondrial depolarization cocktail with FCCP (MDCF):

  Drug	stock (mM)	Final concentration (mM)	Volume to mix (ml)
  Valinomycin	10	1	10
  Oligomycin	10	2	20
  Myxothiazol	20	1	5
  FCCP	10	1	10
  Add EtOH=			55

• TMRM 100mM stock in ethanol or methanol
• Load cells with TMRM (50nM) for >90 min in EB (no TPB or PMPI here).
• The experiment is performed without replacement of the above medium at 37°C
• Immediately before starting the time lapse add 1:1000 MDCF to the cultures.

Configuration of image acquisition with Zeiss LSM Multi Time Series module

The aim is to record a time lapse of 10 frames of decaying TMRM fluorescence after mitochondria have been completely depolarized by the addition of MDCF. The images are acquired with identical scanning settings as during mitochondria to cell volume fraction measurement. Here it is somewhat important to avoid photo-bleaching.

The image acquisition protocol is given for a Zeiss LSM 510 / AIM system here. Use the updated volume fractionator protocol as a guideline for setting the assay up on an LSM780 / Zen / Definite Focus system. The essence of the configuration detailed below is to record a time lapse of TMRM fluorescence at 1024x1024 single plane frames at ~0.044um/pixel resolution, 10 frames at about 1 min/frame interval.

• Microscope Settings (for Zeiss LSM 510):
• Microscope and Configuration Control:
  • Lens: Plan-Apochromat 100x/1.4 Oil DIC
  • Multitrack (containing 1 track):
    • Mitotracker Red: 543->560LP (Ch1)
  • Autofocus track (multitrack with 1 track)
    • Use reflected light of HeNe633 laser in Ch1
• Scan Control:
  • Optical Zoom: 2x
  • Pixel size: ~0.044um (oversampled, but not that much as for deconvolution)
    Dimensions, scan: 1024x1024, Single plane, 2xLine Average, Scan Speed 6
  • Pinholes: 1 Airy
  • Gain: ~500V
  • Data depth: 12 bit
  • Laser power:  HeNe543:50% (set gain or laser power before starting time lapse)
• Multi Time Series module:
  1. Switch to Fixed Location and set up experiment parameters:
     • Experiment Repetitions: 10
     • Autofocus (XY) checked
     • Define Autofocus:
       • Use Cnf (XY), set the saved multitrack configuration for autofocus
       • Correct All Loc
       • Z Range: 30
       • Scan speed 4
     • Define configuration
       • set the saved multitrack configuration for TMRM
       • No of Scans: 1
       • Delay 0
  2. Switch the microscope to eyepiece / transmitted light and search for a good position (cell with well visible nucleus and perinuclear area).
  3. Switch the Multi Time Series module to Multiple Locations. This adds the current position to the list.
  4. Search for 2 more positions and press Insert XYZ after each.
  5. Set up autofocus offsets
     1. In the Define Configuration tab press Load Conf.
     2. In the Scan Control dialog decrease the 543nm intensity to ~15%
     3. Focus and reposition the sample using Fast XY scan. Focus in the middle of the cell to have a nuclear region where no out-of-focus mitochondria appear.
     4. In the Multi Time Series module press Replace XYZ and the Find Z Offset.
     5. Press Next Loc and repeat from point 5.1. Do it for all positions.
  6. Select Image DB to save results.

Assay protocol

1. Add MDCF at 1:1000 to the culture and mix the medium well. Points 2-4 serve to adjust illumination parameters in a Zeiss AIM system.
2. In the Define Configuration tab press Load Conf.
3. In the Scan Control dialog press single.
4. If intensities look fine stop it. If there is saturation decrease the laser intensity and in the Configuration Control store the changed configuration
5. Start recording by pressing Start Time in in the Multi Time Series module

Autofocus settings
TMRM scan settings	These settings (except for the scan speed, averaging) should be similar to the one used for the volume ratio measurement.	Set the Transmission% of the 543nm before starting time lapse
Definition of autofocus	Definition of the TMRM scanning configuration. The number of frames to record are set at the experiment repetitions. Use 3 positions and record without delay between the cycles.

Analysis in Image Analyst MKII (from version 3.0.0)

Use Image Analyst MKII to determine mitochondria to cytosol activity coefficient.

Analysis Protocol

• Open the lsm file.
• Activate the  the "Mitochondrial membrane potential assay - measurement of the apparent activity coefficient ratio - 1 - process" pipeline in the Pipelines/Intensity Measurements/Applications main menu point. The pipeline is pre-configured to work with images recorded using the above acquisition configuration.
• Run the pipeline using the button (or use for automatic loading and processing from a Multi-Dimensional Open dialog)
• Mark mitochondrion free nuclear areas and mitochondrial areas in the respectively named images (see figure below).
• Activate the  the "Mitochondrial membrane potential assay - measurement of the apparent activity coefficient ratio - 2 - plot" pipeline in the Pipelines/Intensity Measurements/Applications main menu point, and run by pressing the button.
• The aR' value will appear in the automatically loaded spreadsheet template
• Save contents of the Excel Data Window form the File main menu.

"Draw nucleus ROIs here" Image	"Draw mitochondrial ROIs here" Image
Nuclear fluorescence	Mitochondrial fluorescence
Contents of the Excel Data Window

Protocol by Akos A. Gerencser 02/11/2010    V2.0 updated 07/29/2015    

Who to cite? This technique has been published here:

1. Gerencser AA, Chinopoulos C, Birket MJ, Jastroch M, Vitelli C, Nicholls DG, Brand MD. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J Physiol. 2012 Jun 15;590(Pt 12):2845-71.