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Assay of absolute magnitude of mitochondrial membrane potential in cells

This protocol describes how to measure the absolute magnitudes and time courses of mitochondrial and plasma membrane potentials in intact cells in adherent cultures. The assay uses fluorescence time-lapse microscopy (epi, confocal or two-photon) to record a time course of tertamethylrhodamine methyl ester (TMRM) and the Molecular Devices FLIPR Plasma Membrane Potential Assay Kit probe (=plasma membrane potential indicator, PMPI) fluorescence. The intact cells are exposed to a calibration paradigm using the reagents described below, and fluorescence responses measured during this challenge are converted into millivolt values using the Membrane Potential Calibration Wizard in Image Analyst MKII.

Equipment

Microscopy requirement:

The assay works with wide field (epifluorescence), confocal and two-photon microscopy.  The epifluorescence microscope has to be capable of low-light level time lapse imaging, e.g. equipped with a fast shutter and a sensitive monochromatic camera. Confocal and two-photon microscopes trivially work in low-light level mode. The image acquisition software must record accurate time stamps for each (pairs of TMRM and PMPI) frames.

Wide field microscopy:

  • Inverted microscope with 10x or 20x fluorescence-optimized, high NA (0.5-0.75) lens
  • Cooled CCD camera
  • Motorized filter turret or filter wheels
  • Fast electronic shutter
  • Optionally: real time autofocus, such as the Nikon's Perfect Focus System or the Zeiss Definite Focus.

Filter sets: a customized filter set achieves better signal to noise ratio because of less correction required for spectral crossbleed. However fluorescence can be captured by standard green (PMPI) and red (TMRM) emission channels. Ideally TMRM emission is collected above 600nm to decrease crossbleed of PMPI.

Filter Spectrum (center/bandwidth nm) Supplier & Cat. # Note
PMPI excit 500/24 Semrock FF01-500/24-25 Standard YFP cube component
PMPI dichroic 520 Semrock FF520-Di02-25x36 Standard YFP cube component
PMPI emitter 542/27 Semrock FF01-542/27-25 Standard YFP cube component
TMRM excit 580/14 Semrock FF01-580/14-25 individual stock filter
TMRM dichroic 594 Semrock Di02-R594-25x36 individual stock dichroic
TMRM emitter 641/75 Semrock FF02-641/75-25 individual stock filter

Alternatively use the PMPI exciter and dichroic for both TMRM and PMPI channels to record only using an emission filter wheel. Note: the emission bands of PMPI and TMRM are wide, and other similar filters may work just as well.

Confocal microscopy:

  • Inverted stage with 10x or 20x fluorescence-optimized, high NA (0.5-0.75) lens
  • 488 or 514 nm lines for PMPI excitation
  • 543 or 561 lines for TMRM excitation
  • Spectral detector set to capture fluorescence similarly to the above specified filters, or filter based detector configured similarly to the above filters

Two-photon microscopy:

  • 10x or 20x two-photon microscopy optimized, high NA (0.5-0.75 or 20x NA 1.0 WI) lens
  • 930 nm excitation
  • Non-descanned detectors with beamsplitter to detect two emission channels simultaneously.
  • The above listed emission filters, or alternatively use a 610 nm long pass filter for TMRM.

Small equipment requirement:

  • 37C waterbath
  • 37C dry or humidified air incubator with no CO2, optionally with a rocker.
  • scale, pH meter, pipettes
Reagents and consumables
Reagents stocks:

 

Reagent Stock Concentration Solvent Storage Supplier & Cat. # Notes
NaCl 240 mM H2O RT Sigma sterile filter
KCl 240 mM H2O RT Sigma sterile filter
KCl 2 M H2O RT Sigma  
CaCl2 1 M H2O RT Sigma sterile filter
Glucose 1 M H2O RT Sigma sterile filter
Glutamine 200mM (cell culturing) H2O -20C Gibco / Life Technologies  
TMRM 50 µM DMSO -20C Life Technologies  
PMPI (plasma membrane potential indicator) (explorer kit vial dissolved in 10ml H2O) H2O -20C Molecular Devices #R8042 FLIPR Membrane Potential Assay Kit (blue) or #R7291 (red)   The bulk kit is 10x more concentrated. Do not filter.
Zosuquidar 25 mM DMSO -20C MedKoo  or Sigma  
           

IAA-94

200 mM DMSO -20C Enzo ALX-550-248-M025 optional component
DIOA 100 mM DMSO -20C Santa Cruz sc-203230A optional component
Bumetanide 100 mM DMSO -20C Sigma optional component
TTX (tetrodotoxin) 1 mM MES/H2O -20C Sigma optional component, dissolve in 10mM MES
Valinomycin 1 mM Ethanol -20C Sigma  
Oligomycin 10 mg/ml Ethanol -20C Sigma  
Antimycin A 20 mM Ethanol -20C Sigma Alternatively use myxothiazol
Gramicidin 10 mg/ml Ethanol -20C Sigma  
Paraformaldehyde 8%, see below H2O -20C   warm up of filter for precipitates
           
2xPM (potentiometeric medium) see composition below H2O RT or 4C   sterile filter

An abbreviations list is at the end of the document.

2xPM (potentiometric medium)

The 2xPM is a Na-free double concentrated stock solution for composition of the assay medium (potentiometric medium). This is required for a series of mixing steps that ensure constant probe concentrations. The full potentiometric medium is composed in the Assay Protocol section below by mixing the 2xPM with equal amount of NaCl or KCl solutions.

Simple 2xPM:

Substance mM (2x of final)
KCl 7
MgCl2 2
KH2PO4 0.8
TES 40
NaHCO3 10
Na2SO4 2.4

Set the pH to 7.6 at room temperature using NaOH. Mix an aliquot of 2xPM with 240mM NaCl 1:1, warm up to 37C and adjust the pH with measured amount of HCl or NaOH. Add the required amount to the rest of the 2xPM. Sterile filter and aliquot into 50ml conicals. Store at RT. Note: Don't include CaCl2 into the 2xPM, because calcium phosphate may precipitate.

Cell culture medium-based 2xPM

Custom-order (e.g. from Gibco / Life Technologies) powdered culture medium omitting the following components: NaCl, phenol red, NaHCO3, riboflavin, folic acid, glucose, glutamine and  Ca2+ (in any form). Note: mind the final phosphate concentration in light of the final concentration of added CaCl2!

Substance mM (2x of final)
Custom culture medium powder (w/o NaCl!) 2x
TES 40 mM
NaHCO3 10 mM

Set the pH to 7.6 at room temperature using NaOH or HCl. Mix an aliquot of 2xPM with 240mM NaCl 1:1, warm up to 37C and adjust the pH with measured amount of HCl or NaOH. Add the required amount to the rest of the 2xPM. Steril filter and aliquot into 50ml conicals. Store at RT. Note: Don't include CaCl2 into the 2xPM, because calcium phosphate may precipitate.

Calibration cocktails:

Anti-swelling cocktail (ASC) - optional

Drug stock (mM) Final (uM) final dilution= 1000 volume= 20
IAA-94 200 100 10
DIOA 100 10 2
Bumetanide 200 80 8
Add DMSO= 0

Mitochondrial depolarization cocktail (MDC)

Drug stock (mM) Final (uM) final dilution= 1000 volume= 100
Valinomycin  1 0.50 50
Oligomycin 10 1 10
Antimycin A 20 1 5
Add EtOH= 35


8% Paraformaldehyde solution:
  1. 8g paraformaldehyde for 100 ml distilled water

  2. Heat to 60 degrees while stirring. Add drops of 10N NaOH until the solution is transparent (usually 1-4 drops per 100ml sometimes more).

  3. Cool to room temperature.

  4. Adjust to pH 7.4

  5. Filter into aliquot tubes. Store frozen at -20C.

  6. Thaw before use in 65C water bath.

Consumables (for working in LabTek coverglass-bottomed chamber or up to 15 wells of a 96-well plate)

  • 15 ml conicals 2x
  • 1.5 ml  and 2 ml tubes
  • Reservoir plate, if using microplate format: polypropylene, 300ul / well volume, round or V bottom, with lid.

Assay protocol:

Generic guidelines for fluorescence microscopic recording of mitochondrial membrane potential:

  • Avoid photo-effects: a stable baseline will indicate no photo-toxicity. Use attenuated and shuttered illumination and not too frequent frame captures.
  • Acquisition interval: the maximum interval is dictated by how fast will TMRM leak out of the cells after MDC addition - the decay curve must be resolved. In beta-cells 35s cycle time for complete calibration is OK. Higher temporal resolution may exert photo-effects. If using complete (known kt) or relative to baseline calibrations, the acquisition interval can be longer.
  • The image acquisition software must record accurate time stamps for each (pairs of TMRM and PMPI) frames.
  • The sample needs to be thermostated to 37C, ideally by a heated environment chamber
  • Pre-warm all added liquids and pipette tips used for addition
  • The sample holder has to be sturdy enough to withstand several liquid additions or removals during the experiment.

Configuration guidelines for Wide field microscopy::

  • Exposure time: 50-200ms, balance exposure times to have similar baseline TMRM intensity to end point PMPI intensity
  • Illumination intensity: attenuate until image looks grainy - and baseline recordings are stable
  • Bin the camera to have ~1-2 um pixels

Configuration guidelines for Confocal microscopy::

  • Open pinhole to improve signal intensity
  • Laser intensity: attenuate until image looks grainy - and baseline recordings are stable
  • Set low image resolution resulting 1-2 um pixels
  • Stick to the same gain settings during experiments, keep intensities in the lower part of the detector range, mind that PMPI will brighten during calibration.

Configuration guidelines for Two-photon microscopy :

  • Stick to the same gain settings during experiments, keep intensities in the lower part of the detector range, mind that PMPI will brighten during calibration.
  • May record z-stacks and set "Mean Intensity" projection in Image Analyst MKII Multi-Dimensional Open dialog Settings tab when opening the recording for analysis.
  • Scan fast with frame averaging for better distribution of heat.

Prepare potentiometric medium (PM) working solutions (right before the assay, use it on the same day):

In a 15ml conical prepare supplemented 2xPM with the probes:

Reagent (stock concentration) amount or concentration in the 2x medium
2xPM 10 ml
CaCl2 (1 M) 1.5 mM (30 ul)
Glucose (1 M) 6 mM (60 ul)
Glutamine (200 mM) 4 mM (200ul)
Zosuquidar (25 mM) 2 uM (0.8ul)
TMRM (50 uM) 10 nM (4ul)
PMPI (explorer kit) 1:200 (100ul)
TPB (1 mM) 1 uM (20 ul)

Mix with vortexing.

  1. Save 1ml of this supplemented  2xPM in an 1.5ml tube. Note: a modified culture medium-based 2xPM may form precipitation by time with Ca2+ added. In this case save this 2xPM without Ca2+ added and supplement it before use.

  2. Prepare PMNa: in an other 15 ml conical mix 7ml NaCl (240 mM) and 7ml of the above supplemented 2xPM.

  3. Prepare PMK: in a 2ml tube mix 1ml KCl (240 mM) and 1ml of the above supplemented 2xPM.

Handling of cultures:

  1. Wash cell cultures with PMNa twice. Use 400-500 ul for LabTek 8-well chambered coverglass or 150ul for reduced-area 96-well plate wells.

  2. Incubate in a CO2-free warm air incubator, optionally on rocker for 90min

  3. Replace medium for fresh PMNa and transfer the culture onto the microscope stage and incubate further 30min. Note: keep the culture warm while transferring to the microscope.

Note: All tubes and reservoirs used for handling PMNa need to be pre-soaked in PMNa, because probes may get depleted by absorption on the walls.

Note: The dilution of 2x media stocks was designed to ensure constant probe concentrations around the sample.

Preparations before time-lapse recording:

While the cell culture dish is preincubating on the microscope stage prepare:

  1. Set up acquisition parameters based on the above recommendations (Generic guidelines).

  2. Prepare PMNa-MDC by pipetting 500ul PMNa into a pre-soaked tube and add 1.5ul ASC + 1.5 ul MDC + 1.5ul TTX

  3. Supplement the PMK tube with 2ul ASC + 2ul MDC + 2ul TTX

  4. Mix challenge treatments at 2x concentration with PMNa. If using larger aqueous volumes, e.g. for glucose addition, mix 1M glucose with the supplemented 2xPM at 1:1 resulting a 0.5M glucose stock with identical probe concentration to PMNa. Use this stock to prepare the 2x challenge treatment.

  5. Set the volume in the well to support 1:1 additions, e.g. 200ul in a LabTek 8-well chamber or 100ul in a 96-well plate well.

Time-lapse recording:

  1. Record a baseline of 30 frames. Longer baselines allow better precision and also a test for non-phototoxic conditions. Stop recording after the baseline. Individual recordings will be merged in Image Analyst MKII.

  2. Make the challenge addition by adding the 2x concentrated challenge prepared in PMNa at 1:1, stir well and remove the added volume.

  3. Record challenge for an arbitrary length of time

  4. Add PMNa-MDC at 2:1 volume to the well and stir well.

  5. Quickly start a 20 min recording.

  6. In 3-5 steps add (or replace medium) by increasing amounts of supplemented PMK, e.g. 50,100,150, 200 ul for a Labtek 8-well chamber or 10,20,40,75 for reduced are 96-well plate. The volumes are arbitrary, but note down the initial volume and the additions and removals, these values will be required for the calibration. After each addition wait 1 minute, then record 5 frames.

  7. Prepare PMK-PFA by mixing 440ul 2xPM 60ul 2M KCl and 500ul 8%PFA supplemented with 1ul of 10mg/ml gramicidin

  8. Add PMK-PFA at 1:1

  9. Record for another 20-30 min until PMPI fluorescence stabilizes at a maximum level.

The live-cell time lapse experiment ends here. PMK-PFA fixed samples can be processed for immunocytochemistry with any-colored fluorescence labeling as PMPI and TMRM washes out during premeabilization. If the microscope is equipped with appropriate stage motorization, the same view fields can be re-imaged after staining, to pick specific cell types during analysis.

Data analysis in Image Analyst MKII

The analysis of TMRM/PMPI recordings requires spectral unmixing. See related "Calculation of spectral unmixing coefficients for mitochondrial membrane potential measurement" protocol. The spectral unmixing coefficient matrix qualifies the configuration of the microscope, so it has to be re-measured if the relevant microscope configuration changes, but not for different specimens. The ratio of the two (TMRM and PMPI) exposure times affects the coefficients, but a change exposure time can be accounted for by changing exposure correction parameters in the Spectral Unmixing function by editing the pipeline.

Analysis of the recording:

  1. Open the recording using the File/”Open Image Series/Measurement” or the  toolbar button. In the Open Image/Measurement dialog select all recorded files comprising the time lapse.  Alternatively use multiple file selection in the Windows Explorer and drag-and-drop on the Image Analyst MKII. Note: check the correct order in the Files tab of the Multi-Dimensional Open dialog.

  2. Select the “Mitochondrial membrane potential measurement (TMRM/PMPI)” pipeline in the Pipelines/”Intensity Measurements”/Applications main menu point.

  3. Configure the pipeline as follows:

    1. Set channel number associations for TMRM and PMPI

    2. Use a smaller background level (10-30 percentile) for confluent cultures and ~50 percentile for sparse cultures. Percentiles below 5-10 may increase noise.

    3. Enter the coefficient matrix as above calculated.

    4. Set cell diameter in pixels. To measure it open the recording, draw linear ROI across a typical cell using the   toolbar, and then reading out its length by double-clicking the status bar of the image to bring up an Image Property window. (Alternatively use the context menu of the Image Window).

    5. If you have previously saved the calibration configuration, enter the file file name into the corresponding pipeline parameter or leave this field blank.

  4. Press the  button on the main toolbar or in the bottom of the Multi-Dimensional Open dialog.

  5. To calibrate PMPI and fluorescence traces to millivolts, either follow the step-by-step configuration of the “Membrane Potential Calibration Wizard” (below) or open the “calibration configuration.ips” configuration file accompanying the image data files.

  6. Press the  button to perform the calibration or the  button to transfer calibrated potentials to Excel.

Configuration of the Membrane Potential Calibration Wizard:

Optionally first reset the Membrane Potential Calibration Wizard by pressing the  button.

1.       Calibration Method tab:

1.1.     Choose the plasma membrane potential calibration method: “Complete with known kP (K-steps)”

1.2.     Choose the mitochondrial membrane potential calibration method: “Complete”

2.       Input/Output tab: the input images have been already selected by the pipeline.

3.       Wizard – Data Ranges tab:

3.1.     Select baseline by pointing the range in one of the graphs on the left, and press the “Select baseline button”

3.2.     Select the recording after addition of PMNa-MDC, thus the complete mitochondrial depolarization, and press the “Select MDC (K-eq)” button.

3.3.     Select last segment of the recording where PMK-PFA was added, thus the final complete depolarization, and press the “Select CDC (zero)” button.

3.4.     Select the K+-steps, the end of the PMNa-MDC addition and the number of steps made by supplemented PMK addition or replacement. Press the “Select a K+ step(s)” button.

3.5.     To calculate [K+] during K-steps, enter the K+ concentration in the potentiometric medium (PM), in the K-based potentiometric medium (PMK), and the medium volume in the well before starting K+-steps. From the second line of the table, in each line of the table enter values for the arbitrarily made medium removals and PMK additions into the “PM Removal” and “PMK Addition” columns, repsectively. Press the “Calculate [K+]ec button”.

4.       Wizard – Assumed Parameters tab: No action is required, use the default rate constant for PMPI redistribution kP=0.38 s-1, and assume no significant non-K-permeability of the plasma membrane during K-steps by using the default PN=0.

5.       Wizard – Constants tab: set the cell specific parameters here:

5.1.  VF (mitochondrion:cell volume fraction): use the Mitochondrion:cell volume fractionator” protocol to measure this value using confocal microscopy, or assume it based on literature. Typically between 4 (fibroblasts) - 8 (primary beta cells, neurons) % for cultured mammalian cells.

5.2.  VFM (matrix:cell volume fraction): this value affects the results only little (because aR’ –see below- largely cancels its effects). The default is 0.8

5.3.  aR’ (apparent activity coefficient ratio): use the “Mitochondrial membrane potential assay - measurement of the apparent activity coefficient ratio” pipeline to calculate this value using confocal microscopy. See protocol here. Typically 0.36 (human beta-cells) - 0.41 (rat neurons).

5.4.  Leave all other constants at their default values.

6.    Press the Calibrate button to perform the calibration. Note: not all cells in the view field can be calibrated, therefore error messages will appear. Press cancel to see no more messages, or check “Suppress messages” in the bottom of the Calibration Wizard.

7.    To save results press  or right-click / ”Copy Plot Data” in the graphs on the graphs on the right.

8.    To explore the results:

8.1.  Use the  and  buttons to see the regression analysis used to calculate calibration parameters

8.2.  Use the  button to see only the time course preceding the calibration steps.

8.3.  Use the  button to see only those plasma membrane traces that also were successfully calibrated for mitochondrial membrane potential. Press  again to refresh the results.

8.4.  Right-click / “Calculate Mean” to see mean±SE of all calibrated traces. To undo mean calculation, press  again. Note: if the mean calculation is performed on the fluorescence traces on the left, then the mean data will be calibrated.

9.    Quality control of the calibration:

9.1.  Check “Calculate the error of calibration” in the Data ranges tab within the Wizard tab and press . Now the predicted error of the calibration is shown. Note: optionally, check the “Suppress messages” on the bottom.

9.2.  Check “Edit all parameters”. Detailed lists of all parameters for PMPI and TMRM calibration, common calibration constants, and error propagation parameters are shown in tabs appearing on the right of the Wizard tab on the top.

9.3.  In the PMPI parameters, set Quality control by propagated error of baseline to “Yes” and press . Traces with larger predicted error than set at the parameter below disappear now.

9.4.  In the TMRM parameters, set Quality control by propagated error of baseline to “Yes” and press . Traces with larger predicted error than set at the parameter below disappear now.

10. Using the Expert Mode: When the Edit all parameters checkbox is checked in the Data ranges tab within the Wizard tab, all parameters of the calibration algorithms can be directly accessed in the tabs appearing on the right of the Wizard tab. These can be used alternatively to the Wizards tab to enter any of the parameters. Use the “Fill in Range(s)” button to automatically enter a range from the selection made in the right graphs.

11. Fine tuning the calibration

11.1. Switch to the “Constants” tab, for this the “Edit all parameters” needs to be checked in the “Wizard” tab.

11.2. To accommodate to the temporal resolution and noise of the recording, adjust the “Differentiation kernel width” to 11 frames. Larger width suppresses noise by providing more smoothing, but also smears fast changes.

11.3. Median filter for baseline, fft0 and fp0: if using the “Take maximum of CDC Range” in the “PMPI Parameters” tab or “Take minimum of CDC Range” in the “TMRM Parameters” use this option to suppress noise affecting minimum and maximum calculations.

12. Automation

12.1. Save an arbitrary calibration configuration using the  button in the Membrane Potential Calibration Wizard.

12.2. In the Pipeline Parameters (Main Window Parameter Bar; the “Mitochondrial membrane potential measurement (TMRM/PMPI)” pipeline is activated) click the “Calibration configuration file name (*.ips)” parameter, and click the button appearing at the end of the line. Select the saved calibration file.

12.3. To automate saving the results give a filename in the “Output Excel Data save file name (*.xlsx)” using the string parser, such as =%LoadBaseName%%LoadStageNumber:2%.xlsx. See more about the string parser in the Main menu Help/”Help on Expression Evaluation”. Optionally give a path, or set the default path to be used in the Main menu Files/”Set Folder Locations”.

12.4. Use the pull down menu of the  button on the main toolbar or in the bottom of the Multi-Dimensional Open dialog to select “Run pipeline … on all stage positions” or “Run pipeline … on partial plate”.


Abbreviations:
PMPI, plasma membrane potential indicator
TMRM, tertamethylrhodamine methyl ester (mitochondrial membrane potential indicator)
PM, PMK, PMNa : potentiometric medium, K-based potentiometric medium, Na-based potentiometric medium
PMP, plasma membrane potential
MMP, mitochondrial membrane potential
MDC, mitochondrial depolarization cocktail
CDC, complete depolarization cocktail
Keq, K+-equilibrium (potential)
PN and PD, sum of the concentration*permeability values of ions other than K+ in the Goldman (GHK) equation; using relative permeabilities to K+; N: numerator, D:denominator.
DTE,FTE : rate of TMRM decay, normalized with PMP and TMRM fluorescence normalized with PMP, used for calculation of calibration parameters using linear regression.
VF : mitochondria:cell volume fraction
VFM : matrix:mitochondria volume fraction
aR` : apparent activity coefficient ratio, expresses probe binding to mitochondrial membranes, differences in chemical activities between the cytosol and the mitochondrial matrix and the dilution of TMRM fluorescence by the intermembrane space.
RAV : a parameter calculated from VF, VFM and aR`
fP0 and fT0 : fluorescence at zero potentials of PMPI and TMRM, respectively
fPX and fTX : fluorescence background and high-affinity binding of PMPI and TMRM, respectively
kP and kT : rate constant of PMPI and TMRM equilibration through the plasma membrane at zero potential, respectively.
r^2, kPr2, fpxr2, TMRMr2 : r square values of the respective linear regressions
zTM,zT,zP : the apparent charge of TMRM in the mitochondrial inner membrane, in the plasma membrane and of PMPI in the plasma membrane, respectively.
sP,sT : structural parameters of the electrostatic barrier model for PMPI and TMRM.

Additional keywords: tertamethylrhodamine ethyl ester, TMRE, JC-1, mitochondrial assay, oxidative phosphorylation assay, mitochondrial membrane potential measurement, proton motive force, phosphorylation potential, ATP/ADP

 


Protocol by Akos A. Gerencser 12/13/2015 V1.2        

minor update (V1.2): 12/13/2015: typos, added links
original date (V1.1): 07/29/2015

Who to cite? The theory of the 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.

The beta-cell specific application and the Membrane Potential Calibration Wizard was used for the first time here:

  1. Gerencser AA. Bioenergetic Analysis of Single Pancreatic Beta-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects. Endocrinology 2015 Oct;156(10):3496-503 

We used this technology also in the following paper:

  1. Birket MJ, Orr AL, Gerencser AA, Madden DT, Vitelli C, Swistowski A, Brand MD, and Zeng X. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. Journal of Cell Science, 2011 24:348-58.