Determination of Mitochondrial Morphology in Live Cells Using Confocal Microscopy

Our research focuses on basic neuropharmacology. Specifically, we study how drug molecules work. We aim to understand the relationship between potential drug compounds and mitochondrial function in Parkinson's disease.

Prior studies review longstanding lab cell analysis challenges even with manufacturer guidelines. To solve this, we outlined a detailed step-by-step protocol for assessing mitochondrial morphology in lab cells. Our protocol uses a simple mitochondrial stain, fluorescence imaging and open source software.

This reduces cost, increases accessibility, and makes it easier to use than complex alternatives like electron microscopy. To begin, obtain cultures of SH-SY5Y cells grown in flasks containing DMEM/F12, supplemented with 10%FBS. Detach the cells for one minute using a 0.05%trypsin solution.

Then centrifuge the cells at 100 G for three minutes before passaging. Next, suspend the SH-SY5Y cells in DMEM/F12 medium supplemented with 1%FBS. Then seed the cells onto confocal dishes with glass bottoms and maintain them overnight.

For MPP+stimulation, replace the culture medium with fresh medium with or without 1-methyl-4-phenylpyridinium for 24 hours. For MitoTracker staining, dilute MitoTracker stock solution with pre-warmed DMEM/F12 medium to obtain a 50 nanomolar working concentration. Keep the staining solution protected from light throughout the procedure.

Remove the culture medium from the confocal dishes. Wash the cells twice with fresh DMEM/F12. Then pipette one milliliter of the MitoTracker working solution into the dish.

Incubate for 15 minutes at 37 degrees Celsius in the dark. Now remove the MitoTracker working solution and wash the dishes twice with DMEM/F12. After the last wash, add one milliliter of DMEM/F12 to each dish.

Incubate in the cell incubator before imaging. To pre-equilibrate the confocal microscope for imaging, under the acquisition panel, adjust the light path parameters by setting the excitation and emission wavelengths. Set the pinhole size to 1.2 airy units, pixel scan size to 1024 by 1024, pixel dwell time to 2.4 microseconds with averaging of two.

Now use the halogen light to focus on the cells using a plan apochromatic 60X objective lens with a numerical aperture of 1.4, minimizing phototoxicity. Adjust the laser power and high voltage just below the saturation level. Then set the zoom factor to three and capture the images.

Move the region of interest to focus on other cells under the microscope. Then capture images without changing any settings. To enhance image quality prior to binary transformation and skeletonization, first open the image.

Select an area of a single cell using the ROI tool. Now, sequentially click on process, filters, unsharp mask function to enhance sharpness. Then use process and enhance contrast to reduce noise amplification.

Click on process, filters, and the median option to remove salt and pepper noise. To convert the image to binary, click on process followed by binary and choose the make binary option. Skeletonize the binary image by clicking process, binary, and skeletonize.

Then group all skeleton pixels by selecting analyze, followed by skeleton and analyze skeleton 2D 3D function. Simplify the analysis by using the MiNA tool to calculate nine descriptive parameters in the skeletonized image. The fluorescence intensity of mitochondrial staining increased progressively with MitoTracker red CMXRos concentration from 25 nanomolar to 200 nanomolar.

But at 200 nanomolar, substantial cytoplasmic background staining was observed. Mitochondrial morphology was preserved better in DMEM/F12 medium compared to PBS at one hour post staining, although DMEM/F12 also showed higher background fluorescence. Dual frame averaging significantly improved the signal to noise ratio in mitochondrial imaging compared to single scan mode.

And increasing pixel dwell time from 2.4 to 10.8 microseconds resulted in brighter images, but caused photobleaching. Increasing high voltage intensified background noise, while increased laser power led to photobleaching. The MiNA tool produced consistent skeletonization outputs for both dim and bright images, indicating robust segmentation across different fluorescence intensities, with the bright image exhibiting 5.5 fold higher intensity than the dim image.

MPP+treatment at 500 micromolar disrupted mitochondrial morphology in SH-SY5Y cells compared to the untreated control, resulting in a significantly reduced mitochondrial footprint.