Measurment of mitochondria to cell volume fraction

Saving tab	Acquisition tab	Grid tab (described below)
Autofocus	Timing Tab (missing)

1. Manual image acquisition (without Multi Time Series PLUS Module):
1. Set up laser powers and gains to result no saturation of images. Saturated pixels prevent accurate analysis.
2. 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.
3. 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.
4. Acquire 2-4 more bottom plane frames in different areas.
5. Using Live scan focus to the bottom of the cells, then raise the focal plane by 1 mm.
6. Acquire 3-5 different view fields at this plane.
7. 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.
8. Laser intensities and gains may be increased by advancing in z. If image becomes noisy decrease scan speed.
9. Use the range indicator. DO NOT SATURATE IMAGES! (when having multiple cell types, non-interesting details can be saturated)

 

2. 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]

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

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.    Select a homogeneous area of in each well to be imaged  and press 'Add NEXT'.

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.]

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

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

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

This protocol assumes that the user is familiar with the following sections of the online manual:

• Spatial filtering in Fourier domain
• Fixing imaging artifacts (Image Stabilizer, Channel Alignment and Spectral Unmixing)
• Using masking to constrain areas to plot or process
• Binarization, Thresholding, Segmentation
• Pipeline

Pipelines with and without spectral unmixing

Note: for 488/561 excitation no spectral unmixing is required. For 488/543 excitation use the ‘unmix’ versions of the pipeline. This uses an adaptive spectral unmixing, based on the differential staining of mitochondria and the cytosol.

Adjustments necessary to run the pipeline

This adjustment may need to be done if using a different microscope configuration for recording than the above specified. The following steps are dependent on the bit depth of the recording:

·         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.

·         If spectral unmixing is used, the pipeline contains an algorithm to protect saturated pixels.

To adjust bit depth in the pipeline, do the following:

1.    Load the pipeline

2.    If this is with spectral unmixing, in the first Threshold command (captioned as Threshold: saturation) set as value: 65535 if using 16 bit or 4095 of using 12 bit recording.

3.    To set value for ‘Remove blank frames’ command, perform the following procedure, if there are missed blank frames:

a.    Create a 1024x1024 ROI using the Plotting/Create ROI command.

b.    Press plot.

c.    Find an intensity that clearly distinguishes missed, blank frames.

d.    Enter this intensity value in the Value field of the ‘Remove blank frames’ command of the pipeline.

4.    Save the pipeline.

Adjustment of the number of immunocytochemistry channels:

1.    Load the pipeline

2.    In the top right corner of the pipeline diagram add or remove channels as follows:

a.    Unlock editing by clicking …

b.    To remove a channel right click and delete unnecessary channel number and corresponding ‘Set scaling/LUT’ and ‘Window command’ commands below.

c.    To add a channel, right click and copy-paste a channel number and corresponding ‘Set scaling/LUT’ and ‘Window command’ commands below.

                                          i.    Drop new channel number (‘Get Linked Channel’ command) on ‘Remove blank frames’  and set channel number as appears in the Multi-Dimensiona lOpen dialog

                                        ii.    Drop ‘Set scaling/LUT’  on channel number

                                       iii.    Drop ‘Window command’  on ‘Set scaling/LUT’ 

                                       iv.    Drop ‘Attach Overlay Image’ on ‘Set scaling/LUT’ 

3.    Save the pipeline.

 

Protocol without post-hoc immunostaining

• Load the pipeline ‘volume fraction.ips’  using Pipeline/New Pipeline Window and then pressing the open button on the toolbar.
• If data was recorded in separate files use multiple file selection to open. Load the lsm file(s) corresponding to the serial sections of one sample by sorting in Windows Explorer according to Time, multiple selecting and dropping on the Image Analyst. If the recorded set is a single file, and too too large to fit into the memory, use the Bio-Formats load and then open only a range of frames by checkmarking and entering Settings/Frame of the Time Lapse e.g. 1-50 in the Multi-Dimensiona lOpen. Press Open.
• Optionally open Excel Data Window
• Switch to the pipeline window (Pipeline/Show) and modify the channel assignment in the pipeline if required.
• Run pipeline by pressing …
• Save Excel Data by File Save Excel Data

Protocol with post-hoc immunostaining

• Load the pipeline ‘volume fraction with ICC.ips’  using Pipeline/New Pipeline Window and then pressing the open button on the toolbar.
• 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.

1. Alternatively load one file, and add the second in the Multi-Dimensiona lOpen dialog File Order tab.

• In the Multi-Dimensiona lOpen 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-Dimensiona lOpen.
• Switch to the Open tab and Check merge as Channels.
• Uncheck any transmitted light channel.
• Press Open
• Switch to the pipeline window (Pipeline/Show) and modify the channel assignment in the pipeline if required.
• Run pipeline by pressing …
• 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:

1. Erase all ROIs
2. Compare the immunocytochemistry and the live overlay images. Draw ROIs as given below in the live overlay image.
3. Draw ROIs around positive cells and set Editing/Mask Active ROI  parameters to: Outside,0,Yes,Yes
4. Or alternatively  draw ROIs around negative cells and debris  and set Editing/Mask Active ROI parameters to: Inside,0,Yes,Yes
5. Right click and Process This With Mask ROIs for both mitochondrial and cell pixels images.

• Measure number of positive pixels per frame using Plotting/Plot parameters:No,No,Sum,No,….anything in both images
• Copy values to Excel (outside of Image Analyst) by right clicking the graphs and selecting Copy Y data only.  [alternatively Excel data window can be used instead, for this set the MD output parameter of Plot to Yes.].
• Calculate the sum of both columns of pixel numbers. Divide mitochondria by the cell. Multiply this ratio by 0.667. This is the mitochondria:cytosol volume fraction.

 

 

"volumeratio preprocess confocal mask and unmix.ips"

Tuning the pipeline

The pipeline has been originally tuned to produce similar volume fractions to electron microscopy stereology on INS-1E cells. Normally the detection of mitochondrial profiles does not have to be adjusted. However, the cytosolic staining can be quite different between cell types because of differences in confluence and presence or absence of intracellular vacuoles with very bright staining. Therefore the rescaling percentages before thresholding of the calcein image may have to be adjusted.

•  
• For cortical neurons pre-process as follows ("volumeratio preprocess confocal mask and unmix.ips", the steps of the pipeline are detailed below, use the to run the pipeline and proceed to the next step):

1. Threshold: Mask above Pixel value = 4095 (Threshold from local max/min=None) both images before unmixing, if there were saturated areas.
2. Subtract Background: at 5 percentile per frame
3. Blind Spectral Unmixing with NMF: perform automatic unmixing. The UnmixNFM matrix have to be defined in the Preferences/Convolution kernels first as {{1,0.2},{0.01,1}} meaning that we expect mostly calcein crossbleed into the Mitotracker channel.
4. Inprint Mask: holes created by masking are filled in with this function.
5. When the pipeline finished running close the copied calcein image, and keep working with the unmixed Mitotracker and the non-unmixed calcein image.

•  
• If the culture is uniform, proceed to next step. If the the mitochondrial volume ratio in a certain cell type is to be measured in a mixed culture:

1. Work with the calcein image performing the processing steps below by hand:
2. Subtract Background: at 5 percentile per frame
3. Threshold: Bottom at Pixel value = 0 (Threshold from local max/min=None).
4. For each frame of the calcein image draw ROI and perform Apply ROI mask by Outside/0/Current frame only=Yes. Mask only the calcein image.
5. Save ROIs for later use.
6. If previously drawn ROIs are loaded the image set can be masked by advancing frames by -> and ROIs by > and repeating the Apply ROI mask for each frame

• Process the unmixed Mitotracker and masked or original calcein image with the Pipeline “volumeratio process confocal cortical-masked.ips” (the steps of the pipeline are detailed below, use the to run the pipeline and proceed to the next step):

1. The Calcein image is processed as follows:
2.  Wiener Filter Smooth: adaptive filter for noise removal. Mask width=5, Noise level=0.01. Increase noise level for greater noise suppression or decrease if details are smudged.
3. Set scaling/color and Write back scaled values: values between 1-90 percentile of each frame is mapped between 0 and 1000 for each frame, so the new intensities in each of the frames will be similar.
4. Threshold: Mask below Pixel value = 1 (Threshold from local max/min=None): this re-masks the area originally masked when selecting for the cells of interest, so these areas will not contribute into the threshold calculation in the next step:
5.  Threshold: Above Otsu by frames at 1 (Threshold from local max/min=None): this is an adaptive, but uniform threshold for each frame. 
6. The Mitotracker Red image is processed as follows:
7. Set scaling/color and Write back scaled values: values between 0-99.99 percentile of each frame is mapped between 0 and 4095 for each frame, so the new intensities in each of the frames will be similar.
8. 2D DFT Butterworth BP filter: 80/1.5/2000/100/pixels/.../absolute=No : this is a highpass filter with a cuton at 80 pixels spatial frequency to show mitochondria only. Note that if the resolution of image acquisition is changed a different cuton frequency will be required. See more here and here.
9. Threshold: Bottom at Pixel value = 0 (Threshold from local max/min=None): this removes negative values created by the high pass filter.
10. Wiener Filter Smooth: adaptive filter for noise removal. Mask width=3, Noise level=0.01. Increase noise level for greater noise suppression or decrease if details are smudged.
11. Image Arithmetic: the filtered Mitotracker image is masked with the cytosolic area
12. Set scaling/color and Write back scaled values: values between 0-99.5 percentile of the whole image series resulting brighter images and saturation of the brightest pixels. The 99.5 is an important empirical value which sets the sensitivity of the method.
13. Threshold: Mask below Pixel value = 100 (Threshold from local max/min=None): this suppresses background. Increase this value if mitochondria look overflowing.
14. Threshold: Above Otsu by frames at 1, Threshold from local max/min=Bound Maxima Locally, Determine boundaries at 10(%). This performs the actual locally adaptive thresholding outlining close to the zero crossings of the highpass filtered mitochondrial profiles, therefore reflecting the width of the original fluorescence objects at half maximal intensities, thus the physical size of the mitochondria.
15. Finally the area of the white (1) pixels is measured by drawing or loading a big ROI encircling the whole the whole image Plot ROIs/ Plot type=Sum.

• Stereologic correction factor

The factor K_V=0.667 derives from the stereologic correction formula for spheres with equal thickness to the section and truncated at half maximal intensity. Since mitochondria are thinner than the optical thickness, they are blurred by definition to the size of optical thickness, therefore the thickness of mitochondria equals to the section thickness. The correction formula  (Weibel and Paumgartner 1978), where g is the relative section thickness, which is 1 (see above). r is the relative smallest visible cap section, which is 1 where objects are clipped at half maximal intensity. Therefore K_V=2/3. Cells are a lot thicker than the focal plane therefore no correction factor applies for the calcein channel.

Protocol by Akos A. Gerencser 03/03/2014 V2.0        

Who to cite? We used this technology in the following papers:

1. 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
2. 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.

Other references:

1. Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates.
   Weibel ER, Paumgartner D. J Cell Biol. 1978 May;77(2):584-97.