Our research program focuses on structural and functional aspects of mitochondria, particularly as they relate to aging-related dysfunction. To this end, we use a broad array of biophysical, biochemical, and structural techniques to explore the basic mechanisms of mitochondrial aging and to develop therapeutic interventions to preserve and restore mitochondrial health. Mitochondrial function and structure are inextricably related.
Standard light microscopy techniques are suitable for measuring large-scale mitochondrial features, like basic morphology and network structures. Analyzing mitochondrial ultrastructure or features that require higher resolution than afforded by traditional optical microscopy is typically done using electron microscopy or tomography on fixed samples. Super resolution microscopy is a fluorescence-based imaging technique that measures features beyond the diffraction limit.
For mitochondria, this includes measurements of nanoscale protein distribution and cristae architecture. The STED imaging protocol described here allows researchers to measure the detailed structure of the inner membrane in living cells. Live-cell STED imaging allows us to observe and quantitatively analyze complex features of cristae under physiologically relevant conditions without the need for sample fixation.
This will give researchers novel insights into the dynamic changes that occur in ultrastructural features and their temporal responses to stressors and pharmacological compounds. To begin, rapidly thaw a frozen stock of SH-SY5Y cells in FBS supplemented with 10%DMSO and dilute tenfold with prewarmed media. Then centrifuge the cells and remove the supernatant.
Resuspend the cell pellet in five milliliters of prewarmed media and seed cells in a T25 flask. To prepare cover slips coated with PDL, apply 600 microliters of 50 micrograms per milliliter PDL solution to each well of sterile chambered cover slips. The volume of PDL added to each well varies based on well size.
Incubate the cover slips at room temperature for one hour. After incubation, remove the PDL solution and rinse the cover slip thrice with 1.8 milliliters of distilled water. Allow the coated chamber to air dry for two hours.
Once the SH-SY5Y cells reach 80 to 90%co-fluency, dissociate them by adding trypsin solution. Neutralize the trypsin by adding a prewarmed DMEM medium. Centrifuge the cell suspension, and resuspend the pellet in DMEM.
Count the cells on an automated cell counter. Next, seed the cells at a density of 1.5 times 10 to the 4th cells per square centimeter onto the PDL-coated chambered cover glass. On day one and day three, replace the medium with DMEM supplemented with either 5 or 2%FBS respectively, 1%antibiotic antimycotic, and either 10 micromolar RA or 95%ethanol of the same additive volume to serve as the vehicle control for differentiation.
Prepare a PKMO stock in pre-warmed phenol red free DMEM supplemented with 2 or 10%FBS, dependent on differentiation state, 1%antibiotic antimycotic, and 20 millimolar HEPES. On day six, rinse the cells twice with imaging media and incubate cells with the PKMO solution at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. After staining, rinse the cells thrice with prewarmed imaging media.
For the final wash, incubate the cells for 30 minutes at 37 degrees Celsius and 5%carbon dioxide. After incubation, replace the final wash with fresh prewarmed imaging media containing 20 millimolar HEPES. Open the Lightbox Software for image acquisition.
To select the laser and filter sets, use parameters for an orange dye by selecting the dye used in the staining from the dye list. Using an overview, click on LIVE to focus the cells. Once the cells are in focus, adjust the confocal acquisition settings.
Next, select the rectangular ROI button and create a region of interest around a mitochondria of interest by clicking and dragging to shape the region. To adjust detector gating for stimulated emission depletion or STED acquisition, next to the GENERAL menu, select the GATING menu or click and hold to add the menu to the view. For STED acquisition, set the excitation laser to 15 to 20%and the STEP depletion laser to 20 to 25%with 10 line accumulations.
Use a pixel dwell time of four microseconds and a pixel size of 20 to 25 nanometers. Perform time lapse by selecting the Time dropdown menu, then setting the number of iterations to five and a time interval of 25 or 30 seconds. A Z-stack can be taken using the Volume option and adjusting the desired Z-volume range and step size.
Open STED image deconvolution software to deconvolute raw STED images with the software algorithm. After ensuring microscopic parameters are correct, select the Express button and set the deconvolution type to Fast, Standard, Aggressive, or Conservative for varying degrees of deconvolution power. Execute the deconvolution.
Save the images in ICS2 format In image J, click on File, then Open to open the ics2 files from the deconvolution software. Select Plugins, then segmentation, followed by Trainable Weka Segmentation to open the deconvoluted STED images in the Trainable Weka Segmentation plugin. In the Segmentation Settings, select the Gaussian blur, Membrane projections, and Sobel filter features.
Label one class as Cristae and the other as Background. Next, draw a line over the structure to assign to either class. Then select the Add button on the right-hand side for either Cristae or Background.
Then select the Train classifier button on the left-hand side to generate a map based on the information provided to the plugin. Click the Save classifier button to save the classifier settings for future segmentation. Use the Cristae Probability Map to threshold the image in image J to generate a binary mask, and then go to Analyze, then Analyze Particles.
Finally, draw a multi-point line, adjust the line thickness to several pixels wide, and spline the line to fit the mitochondria. In this study, imaging of mitochondria in undifferentiated, retinoic acid differentiated SH-SY5Y cells with time-lapse imaging is shown. The deconvoluted STED images can be used to determine cristae periodicity in a given area and cristae size and shape measurements.
