Measurement of mitochondria to cell volume fraction (V3.1)

This protocol describes how to measure mitochondria:cell volume fractions using a confocal microscope and image analysis in Image Analyst MKII. The assay was tuned to result similar volume fractions to electron microscopic stereological measurements when the below specified optical configuration is used for image acquisition. The technology is based on segmentation and binarization of high-resolution confocal micrographs of a cytosolic and a mitochondrial stain.

Sample preparation, reagents

Experimental buffer (EB) in mM: 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
Alternatively use full culture medium and CO₂ control during microscopy.
Calcein-AM 2mM stock in DMSO
MitoTracker Red CMX 100mM stock in DMSO
Load cells with calcein-AM 0.5-1 µM plus MitoTracker Red 25-50 nM for 30 min in EB. Certain cell types need higher dye concentrations, up to 2-µM calcein-AM plus 100-nM MitoTracker Red
Replace medium over the cultures with dye-free EB. Image at 37°C or alternatively at RT to mitigate mitochondrial movement and dye leakage. If calcein excessively leaks out of the cell, add 500 µM - 2mM Na-sulfipyrazone.

Image acquisition with Zeiss LSM780 ZEN 2011, 2012 and with or without Zeiss LSM Multi Time Series PLUS version 24 or 35 module with Definite Focus

See links to previous versions of this protocol for older systems on the bottom of the page.

The aim is to record best possible quality, slightly oversampled images at evenly spaced z-coordinates from the bottom to the top of the culture. Due to strong photo-toxicity and photo-bleaching it is not possible to use z-stacking, but different x,y-coordinates of the culture are scanned systematically at incrementing the z-focus. To automate this, the Zeiss LSM Multi Time Series dialogue is used with overriding Z-offset values with an Microsoft Excel / Windows regedit trick. See also recording of volume fraction data without using the Multi Time Lapse module. Microscope Settings are given below for Zeiss LSM 780, ZEN2011 software with Multi Time Series PLUS, ZEN 2011-24 module equipped with Definite Focus. Here the protocol is given for an LSM780 equipped with a spectral detector and 561nm diode laser. See 'Manual image acquisition' below if using other microscope or not having the Multi Time Lapse module.

Start ZEN 2011 software and load the Multi Time Series macro. If this macro is not configured to be accessed from the menu, Press Macro/Macro, go to Assign Macro tab, press '...' browse to C:\ZEN\Macros\MultiTime\ MultitimeZen2011.lvb (or similar). Wait until control comes back to the dialog (can take a while). Select an entry position and provide a caption in the Text field, and select the first field in 'Macros'. Press Apply. Wait until control comes back to the dialog then close it.
Go to the acquisition tab. Check Show all tools and the check Show all in each panel. Expand each panel.
Define the following acquisition configuration and save in the Experiment Manager as 'calcein-MTR'. If you have previously defined it load it.:
Use 'Smart Setup' for Calcein and Rhodamine. Choose the fastest, one track configuration. Alternatively configure it from scratch:

Light Path

Channel/Frame/One track mode
Ch1: calcein: 493-556nm
Ch2: Mitotracker Red: 566-690nm
Appropriate dichroic (488/561/633) for dual 488/ 561 nm excitation. Check out the 488 and 561nm lasers (use 543 excitation if that line is available, then spectral unmixing may be needed)
Optionally use transmitted light detector - only for presentation purposes, this is not needed for analysis

Acquisition mode:

Lens: Plan-Apochromat 63x/1.4 Oil DIC (you will use Zoom 3) or Plan-Apochromat 100x/1.4 Oil DIC (you will use Zoom 2)
Scab mode: Frame
Frame Size: 1024x1024
Line Step 1
Speed 6
Averaging:1
Bit depth:16 [Important: some versions of the volume fraction processing pipeline below need to be adjusted for the bit depth!]
Direction ---->
Scan area: 0,0,0 Zoom 3 (for 63x lens or 2 for 100x lens)
[Pixel size: ~0.044um (oversampled, but not that much as for deconvolution)] [Important:smaller resolution(larger um/pixels value) will casuse overestimation of the volume fraction]

Channels

Lasers:

488 selected, 4-10%
561 selected, 10-30%

Pinhole: press 1AU
Gain ~450-550, Integration mode
Digital Offset: 0
Digital Gain 1.0

Setup of the Multi Time Series PLUS, ZEN 2011-24 or 35 Dialogue:

Saving tab

Enter file name
Select Image Folder
Keep Final Image Open : checked
Save Final Image: checked
Single File Output: checked
Use the Store/Apply Recipe to save and load settings here
Recall Locations List: checked

Acquisition tab

Scan Configuration: 'calcein-MTR': this need to be set after selecting positions, And Apply to All Locations pressed
No of Time Points within Block 1
Interval 0
Press 'Select Parameters to Apply to All Locations and/or All Blocks' and check everything

Timing Tab

Experiment Repetitions:1
Group repetitions 1

Location tab

Multiple Locations
Press 'Clear All' before starting to mark positions

Autofocus

Definite Focus checked.

Save the above settings in the Saving tab Store/Apply Recipe under the 'volumeratio' name. This name will be used later on to override Z-offsets in the system registry.

Saving tab

Acquisition tab

Grid tab (described below)

Autofocus

Timing Tab (missing)

Manual image acquisition (without Multi Time Series PLUS Module):

Set up laser powers and gains to result no saturation of images. Saturated pixels prevent accurate analysis.
Identify a homogeneous region of the culture towards the middle of the well under eyepiece. In the steps below, using Live scan center cells into the scan area, if cells are smaller than scan area. If cells are larger, do not center them, because they will not be evenly sampled. In this case advance between fields use arbitrary number of turns of the fine stage control knob and do not align cells to the view field. This will avoid biasing the amount of nucleosol/cytosol present in the images.
Using Live scan focus to the bottom of the cells, then acquire and save one frame. Always acquire only once one specific view field, the applied laser intensity damages the cells.
Acquire 2-4 more bottom plane frames in different areas.
Using Live scan focus to the bottom of the cells, then raise the focal plane by 1 mm.
Acquire 3-5 different view fields at this plane.
Repeat point 3 until getting to the top of the cell culture. Different cell types need different z-step size, look for the cell layer thickness, and record 5-7 planes.
Laser intensities and gains may be increased by advancing in z. If image becomes noisy decrease scan speed.
Use the range indicator. DO NOT SATURATE IMAGES! (when having multiple cell types, non-interesting details can be saturated)

Setting up the Multi Time Series PLUS module of the Zeiss ZEN software, for automated z-focusing:
1. Focus an area in the sample that is not going to be recorded.
2. Using Live scan, estimate the apparent thickness of the cells. For a 10-plane cycle, divide thickness by 9, this will be the step size.
3. In Microsoft Excel, prepare the ‘volumeratioDefiniteFocus.xls’, by entering the following formulae, entering the actual numerical step size in microns instead of ‘stepsize’:
4. Cell A1="""BlockZOffsetDF"&TEXT(ROW(A121),"0")&"_1""="
5. Cell B1=""""&TEXT(-MOD(ROW(B121)-1,10)*stepsize-0,"0.00E+000")&""""
6. Adjust the number of rows identical to the number of positions that will be acquired below.
7. In Microsoft Notepad create a text file named volumeratio.reg by copying the following rows:

Windows Registry Editor Version 5.00

[HKEY_CURRENT_USER\Software\Carl Zeiss Jena GmbH\UI\AutoTime\VolumeRatio]

[HKEY_CURRENT_USER\Software\Carl Zeiss Jena GmbH\UI\AutoTime\VolumeRatio\Block1]

Copy the two columns from Excel below these lines and save volumeratio.reg. (In the save dialog set file type to All files to prevent edition of txt extension)
These file names are arbitrary.

Automated image acquisition at manually selected coordinates for sparse and small cells (requires the Multi Time Series PLUS module of the Zeiss ZEN software)

1. Focus an area in the sample that is not going to be recorded.

2. Set up laser powers and gains to result no saturation of images. Saturated pixels prevent accurate analysis.

3. Save the adjusted ‘calcein-MTR’ configuration.

4. In the ZEN press Locate and switch to eyepiece / transmitted light

5. If doing post-hoc immunocytochemistry, set the top edge of the well into the center of the view field, go to the Acquisition tab and zero the focus by pressing 'Manually' in the Focus panel, and zero the stage by pressing 'Set zero' in the Stage panel

6. Go back to the Multi Time Series

7. Search for a 50-100 cells under eyepiece, center each in the view field and press 'Add NEXT'.

8. Optionally cycle around all the selected cells to see that they are centered, and there are no cells imaged more than once.

9. Move to the first position

10. In the ZEN turn off 488 and set 561 laser to 5% power and using live scan focus the lowest plane where mitochondria are sharply visible. Stop scanning.

11. In the Multi Time Series, Autofocus tab, press ‘Find Z Offset’. [The offset will show zero, but the focusing is now initialized.]

12. Save the above settings in the Saving tab Store/Apply Recipe under the ‘VolumeRatio’ name. Wait until done.

13. In the Windows Explorer double click volumeratio.reg and OK to enter its contents to the system Registry.

14. Use the Store/Apply button to Apply parameters into registry under the name of “VolumeRatio”.

15. In the Acquisition tab set Scan Configuration to 'calcein-MTR': and press Apply to All Locations.

16. Verify other settings as above given in the Multi Time Series, including file name and folder.

17. Press Start.

18. If doing post-hoc immunocytochemistry, after finished imaging in the Saving tab Store the recipe under a unique name. This will store the image coordinates for later revisiting.

Automated image acquisition along a grid in each well of a multiwell plate for confluent cultures or large cells (requires the Multi Time Series PLUS module of the Zeiss ZEN software)

1. Focus an area in the sample that is not going to be recorded.

2. Set up laser powers and detector gains to result no saturation of images. Saturated pixels prevent accurate analysis.

3. Save the adjusted ‘calcein-MTR’ configuration.

4. In the ZEN press Locate and switch to eyepiece / transmitted light

6. Go back to the Multi Time Series

7. Select a homogeneous area of in each well to be imaged and press 'Add NEXT'. Note: you may press the clear all locations button before this step.

8. Optionally cycle around all the selected wells to see that they are still in focus.

9. Optionally save the settings.

10. In the Grid tab set 10 for X and 5 for Y and 100 microns for both Grid steps. [Note that these numbers have to be entered even if they are already there by default, because the dialogue tends to take zero grid steps otherwise (this was seen at version 24).]

11. Check Meander mode and press Create Multi Grids.

12. Verify that you have now 50 times the number of wells selected locations.

13. Move to the first position (just press Next Loc)

14. In the ZEN turn off 488 and set 561 laser to 5% power and using live scan focus the lowest plane where mitochondria are sharply visible. Stop scanning.

15. In the Multi Time Series, Autofocus tab, press ‘Find Z Offset’. [The offset will show zero, but the focusing is now initialized.]

16. Save the above settings in the Saving tab Store/Apply Recipe under the ‘VolumeRatio’ name. Wait until done. [Note: It is important to used this configuration name for the procedure below.]

17. Adjust the number of rows in the ‘volumeratioDefiniteFocus.xls’ to match (or exceed) the number of total positions, and copy columns to the ‘volumeratio.reg’ as above described.

18. In the Windows Explorer double click ‘volumeratio.reg’ and OK to enter its contents to the system Registry.

19. Use the Store/Apply button to Apply parameters into registry under the name of “VolumeRatio”.

20. In the Acquisition tab set Scan Configuration to 'calcein-MTR': and press Apply to All Locations.

21. Verify other settings as above given in the Multi Time Series, including file name and folder.

22. Press Start.

23. Keep an eye on the microscope, checking whether the Definite Focus correctly engages as the acquisition moves from well to well. If fails reload the configuration saved in #9 and repeat from #10 using the remaining well positions.

24. If doing post-hoc immunocytochemistry, after finished imaging in the Saving tab Store the recipe under a unique name. This will store the image coordinates for later revisiting.

5. Recording post-hoc immunocytochemistry (requiring the Multi Time Series PLUS module of the Zeiss ZEN software)

1. In the ZEN load the previously defined ‘counterstain’ configuration. If not yet defined do the following:

1. load ‘calcein-MTR’ configuration, and change the Light Path and Channels to match the fluorescence of the label. Usually calcein and Mitotracker Red are washed out by this time or outshined by the staining.

2. Keep the same Acquisition mode settings, however Faster speed and Bi-directional scanning may be used.

3. Save the new configuration as ‘counterstain’ (arbitrary name).

2. Press Locate and switch to eyepiece / transmitted light and set the top edge of the well into the center of the viewfield, go to the Acquisition tab and zero the focus by pressing 'Manually' in the Focus panel, and zero the stage by pressing 'Set zero' in the Stage panel

3. Go back to the Multi Time Series and under Saving tab Store/Apply Recipe load the previously stored coordinates. [Note, that stored coordinates need to be loaded after pressing ‘Set zero’ above.]

4. In the ZEN load the live cell calcein-MTR recording. You may split the screen to see live image and the original recording in the same time.

5. In the Multi Time Series select any position and using Live scan in the ZEN, try to find the same cell compared to the live recording, by dragging the scan area in the Acquisition mode panel. [Note, that tiled acquisition is also an option to find the same cell]

6. If not finding the cell, the zero position may need to be adjusted, and coordinates re-loaded.

7. If the Live scan image matches the previously recorded calcein-MTR image, stop scanning and save the ‘counterstain’ configuration.

8. Move to the first position

9. Using live scan focus the lowest plane where the cell is sharply visible. Stop scanning.

10. In the Multi Time Series, Autofocus tab, press ‘Find Z Offset’. [The offset will show zero, but the focusing is now initialized.]

11. Save the above settings in the Saving tab Store/Apply Recipe under the ‘VolumeRatio’ name. Wait until done. [Note: It is important to use this configuration name for the procedure below.]

12. In the Windows Explorer double click the previously used ‘volumeratio.reg’ and OK to enter its contents to the System Registry.

13. Use the Store/Apply button to Apply parameters into registry under the name of “VolumeRatio”.

14. In the Acquisition tab set Scan Configuration to ‘counterstain’ and press Apply to All Locations.

15. Verify other settings as above given in the Multi Time Series, including file name and folder.

16. Press Start. [Note, if split-screen was used, you can side-be-side scroll the live-cell recording to verify the match.]

Analysis in Image Analyst MKII (Version 3.0.0 and above)

Use Image Analyst MKII to determine areas corresponding to mitochondria and the whole cell by using adaptive thresholding.

Four pipelines are available under the Pipelines/Morphological Measurements/Applications main menu point, with slightly different functionalities:

Mitochondria:cell volume fractionator (basic)
- generic use, no crossbleed correction
Mitochondria:cell volume fractionator (original)
- automatic crossbleed correction and saturation handling. Note: for 488/561 excitation no spectral unmixing is required, but for 488/543 is recommended.
Mitochondria:cell volume fractionator (multiplexed)
- the basic pipeline plus evaluation of a marker channel to distinguish two population of cells in the same view fields based on fluorescence intensity and calculation of volume fractions separtately
Mitochondria:cell volume fractionator (basic with post-hoc staining)
- the basic pipeline plus handling of post-hoc immunofluorescence recordings

Adjustments necessary to run the pipeline

Select the pipeline in the main menu, and look the parameter bar (below). Adjustment may need to be done if using a different microscope configuration for recording than the above specified. The "Sensitivity scaling for the cytosolic stain (percentile)" parameter may need to be adjusted if cell density is very different or the cells have bright vacuoles.

$Volume fractionator parameters$

Parameters of the basic volume fractionator:

Channel number for ...: set the corresponding channel number as shown in the Multi Dimensional Open dialog.
Minimal mean image intensity cutoff for the cytoplasmic stain: The pipeline discards blank images to prevent biasing intensity rescaling of the image series. Such blank frames may occur in high z-planes, or if an automatically spread grid misses any cells. The mean intensity of an image containing minimal useful information needs to be set. To find this value create a whole-image ROI and plot mean intensities. Look typical values for empty frames, and set a slightly larger value.
Sensitivity scaling for the cytosolic stain (percentile): 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.

Additional parameters for the original volume fractionator:

Detector bit-depth for detection of saturation : provide the detector bit depth set before acquisition e,g, 8,12 or 16.

Additional parameters for the multiplexed volume fractionator:

Cell tracker threshold (%) : this threshold will be used to distinguish the two populations (percent of maximal fluorescence)
Spectral Unmix Coefficient Matrix for channel 2 and 3 : Spectral unmix matrix between Mitotracker Red and presumably deep red fluorescence multiplexer stain

Additional parameters for the basic with post-hoc staining volume fractionator:

Set channel numbers and lookup tables for the post-hoc staining. These images will appear next to the processed images helping manual masking of desired cell types.

Performing the analysis

Open the recording (up to 100 frames for basic processing and up to 50 frames for multiplexed or with post-hoc stain processing with 4GB or more RAM). If data was recorded in separate files use multiple file selection to open.
Run the pipeline . Alternatively use the button to load and process.
The pipeline will automatically load a template spreadsheet, fill in the data and calculate the volume fraction.
Save the contents of the Excel Data Window using the File/Save Excel Data main menu.

Analysis protocol with post-hoc immunostaining

Frames corresponding a sample (well) need to be in single files, one file of live recording, one file of immunocytochemistry.
Set USE BIO–FORMATS as file type and select both files to load. Alternatively load one file, and add the second in the Multi-Dimensional Open dialog File Order tab.
In the Multi-Dimensional Open dialog File Order tab verify that the order is: first the live recording, and second is the immunocytochemistry recording.
If the recorded set is too large to fit into the memory, checkmark and entering Settings/Frame of the Time Lapse e.g. 1-50 in the Multi-Dimensional Open.
Switch to the Open tab and Check merge as Channels.
Uncheck any transmitted light channel.
Press Open
Run the pipeline . Alternatively use the button to load and process. The pipeline will automatically load a template spreadsheet, fill in the data and calculate the volume fraction.
Close graph windows and arrange the following windows on the screen for good visibility: The overlay image, the binarized cytosol and mitochondria image and the overlay ICC image.
Optional: for faster erasing frames disable auto-scaling in all non-binarized images by dragging either end of the intensity scale bar and therefore setting scaling into fixed-value mode.
Press on the main toolbar to synchronize frame-to-frame scrolling of the windows.
Identify and eliminate frames that contain no positively stained cells using the cut tool on the main toolbar, and pressing Cut current.
On each frame mask non-positive cells and debris. For this:

Erase all ROIs
Compare the immunocytochemistry and the live overlay images. Draw ROIs as given below in the live overlay or the "Mitochondria" image.
Draw ROIs around positive cells and set Editing/Mask Active ROI parameters to: Outside,0,Yes,Yes
Or alternatively draw ROIs around negative cells and debris and set Editing/Mask Active ROI parameters to: Inside,0,Yes,Yes
Right click and Process This With Mask ROIs for both "Mitochondria" and "Cell" images.

Erase all ROIs (right-click, context menu on the image)
Create an image-sized ROI using the Create ROI sized as the image pipeline from the Pipelines menu.
Re-plot pixel sums using Plotting/Plot parameters:No,No,Sum,No,No,None,"",No,Yes,Yes,No,No,No for both "Mitochondria" and "Cell" images. This updates data in the Excel Data Window.

Plot ROIs

Note: the parameters not visible above are set to "No".

Save the contents of the Excel Data Window using the File/Save Excel Data main menu.

Protocol by Akos A. Gerencser 07/29/2015 V3.1

Who to cite? This technique has been published here:

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.

We used this technology in the following papers:

Choi SW, Gerencser AA, Lee DW, Rajagopalan S, Nicholls DG, Andersen JK & Brand MD. Intrinsic bioenergetic properties and stress-sensitivity of dopaminergic synaptosomes. J. Neurosci. 2011 Mar 23;31(12):4524-34
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.
Birket MJ, Casini S, Kosmidis G, Elliott DA, Gerencser AA, Baartscheer A, Schumacher C, Mastroberardino PG, Elefanty AG, Stanley EG, Mummery CL. PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function. Stem Cell Reports. 2013 Dec 12;1(6):560-74.

Using post-hoc immunostaining:

Shaltouki A, Sivapatham R, Pei Y, Gerencser AA, Momčilović O, Rao MS, Zeng X. Mitochondrial alterations by PARKIN in dopaminergic neurons using PARK2 patient-specific and PARK2 knockout isogenic iPSC lines. Stem Cell Reports. 2015 May 12;4(5):847-59.
Gerencser AA. Bioenergetic Analysis of Single Pancreatic Beta-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects. Endocrinology 2015 in press doi:10.1210/en.2015-1552

Previous versions of the protocol:

Analyze Time Lapse Recordings with Image Analyst MKII

Video Tutorials

Publications using Image Analyst MKII

Try Image Analyst MKII for free

Measurement of mitochondria to cell volume fraction (V3.1)