The overall goal of this procedure is to monitor single mitochondrial events such as superoxide flashes and membrane potential fluctuations in situ or in vivo to achieve real-time evaluation of mitochondrial function in a physiologically relevant context. This is accomplished by first exposing the skeletal muscle in the mouse's hind limb. Next time-lapse confocal images of skeletal muscles are obtained in vivo.
Then the mouse heart is perfused and imaged using confocal microscopy. Finally, the images are processed and data is analyzed. Ultimately, results can be obtained that show single mitochondrial superoxide flashes and membrane potential fluctuations in skeletal muscles, in vivo, or in perfused heart through time-lapse and two dimensional confocal images.
The main advantage of this technique over existing muscles like evaluating mitochondrial function in isolating mitochondria is that this technique allows the real time evaluation of single mitochondrial function in a physiologically relevant condition. We demonstration of this method is critical as a knife, animal, or knife tissue Confocal imaging are difficult to learn because it's more steep and complicated procedure. After preparing isotonic balanced salt solution or IBSS, Krebs HENSEL Light Buffer, or KHB and surgical instruments according to the text protocol, anesthetize a mouse with an intraperitoneal injection of Pentobarbital and perform a toe pinch to confirm that the animal is sedated.
Using a razor, remove the hair on one of the hind limbs before disinfecting the site with 70%ethanol. Next, make an incision on the skin along the outer side of the limb to expose the gastric anemia muscles. Then using sharp forceps, gently pick up the epimysium and use scissors to make an incision through it.
Further dissect to remove the epimysium and expose the muscle fibers beneath it. Add 500 nanomolar tetraethyl, rod domine methyl ester, or TMRM to the IBSS and immerse the muscle in the solution for 30 minutes. After loading TMRM into the muscle, wash the muscle with TMRM free IBSS using a 40 x oil objective.
Place the mouse on its side on the confocal microscope stage and restrain the rear limb so that the exposed skeletal muscle is in a custom made chamber against the cover slip. Then gently press down on the leg to make tight contact between the tissue and the cover slip and immerse the exposed muscles in IBSS using an eight bit pixel depth and a sampling rate of one second per frame record a two dimensional serial scan of the muscle. Collect sequential images by first exciting at 405 nanometers and collecting at greater than 505 nanometers.
Then exciting at 488 nanometers while collecting at greater than 505 nanometers. For dual wavelength excitation of MT.CCP YFP, use sequential excitation at 405 488 and 543 nanometers and collect emission at 505 to 5 45.505 to 5 45 and greater than 560 nanometers respectively. For triple wavelength excitation imaging of M-T-C-P-Y-F-P and TMRM to image the mouse heart, inject the animal with 200 units of heparin.
After waiting 10 minutes and euthanizing the mouse by thoracotomy quickly remove the heart and lungs and thymus attached to it. Quickly transfer the implanted tissue to ice cold IBSS. Then identify the lobes of the thymus and gently peel them back.
To expose the ascending aorta, remove the lung and thymus and isolate the aorta by carefully removing any surrounding tissue. Next, make a cut at the upper end of ascending aorta before the first branch of the aortic arch. Then with two micro suturing forceps, gently hold the wall of the aorta to expose the lumen and carefully place it onto a cannula.
Use a micro vessels clamp to hold the aorta in place and quickly tie sutures around it. Remove the clamp and use forceps to carefully check that the tip of the cannula is above the aortic root. Secure additional ties if necessary to hold the heart in place.
Turn on the peristaltic pump and perfuse the heart. At one milliliter per minute, the heart will resume beating upon perfusion. After 10 minutes of stabilization, perfuse the heart with 10 micromolar BLEs statin and 100 to 500 nanomolar TMRM.
The heartbeat will slow down after 10 minutes. Next, adjust the position of the perfusion system and put the heart on the microscope stage equipped with an adapter to heat the chamber to 37 degrees Celsius. Add one milliliter of KHB perfusion solution in the chamber to partially submerge the heart.
Use a peristaltic pump to remove the effluent from the chamber. Gradually increasing the speed of the pump to provide sufficient flow to the heart to ensure tight contact of the heart with the cover slip and to further suppress the heartbeat, apply gentle pressure on the heart. Image the organ as described for the muscle earlier in this video, adjusting the focal plane to reveal the clearest image possible.
Using the physiological analysis module of the confocal microscope software. Begin by opening the automatically generated database and then the 2D image file to be analyzed. Click region of interest or ROI mean.
To switch to the mean of ROIs mode. Turn off the display of channels except for CP YFP at 488 nanometers for selecting the flashes. Zoom in on the image and manually move the slide bar to play the serial 2D images.
Next, identify single mitochondrial superoxide flashes by locating the site where the fluorescent signal increases transiently. Use the appropriate ROI tool to mark the flashes the trace showing time dependent fluorescence change of each ROI will show up beside the image. After selecting all the flashes, turn on the display of other channels.
Select an ROI on the image outside of the cell for background signal subtraction. Then output the average fluorescence of each ROI together with the time labels. Record the number of flashes in each of the serial scanning image files together with the scan time and area of the cell.
Use excel to calculate flash frequency as number of flashes per 100 seconds and per 1000 square micron cell area. To calculate the amplitude time to peak and decay time for each flash. Use a custom developed program for flash parameter analysis that's part of a custom developed program written in interactive data language.
Using this protocol in vivo imaging of single mitochondrial events can be performed in skeletal muscles of anesthetized mice, followed by insi imaging in the perfused heart. As shown here. TMRM, which is a mitochondrial membrane potential indicator, is often used to verify the location of M-T-C-P-Y-F-P and should show a complete overlapping pattern with the M-T-C-P-Y-F-P signal.
Its spectra are distinguishable from that of CP YFP, and by using the sequential excitation method, the emission signals of TMRM and M-T-C-P-Y-F-P will not interfere with each other. These images demonstrate that single mitochondrial superoxide flash accompanied by membrane depolarization can be identified in the serial 2D scanning images, which showed a transient fluorescence increase over the background signals in both skeletal muscle tissues and the myocardium. Besides high resolution, adequate fluorescence intensity is also required.
This can be achieved by modulating the laser intensity and the gain in the collecting channels. In general, the basal fluorescence signal from the cell is set at one third to one fourth of the maximal intensity of the channel. Since the expression level of M-T-C-P-Y-F-P and the loading of TMRM can vary among animals, fine tuning of the imaging conditions should be done for each experiment, both physiological and pathological perturbations, such as metabolic substrates.
Electrical stimulation as indicated here, and ischemic reperfusion have been used to show that superoxide flash activity responds to changes in cellular metabolic status. While attempting this procedure, it's important to remember to maintain appropriate anesthesia of the mouth and the correct condition of the perfused heart. Following this procedure, other methods like ischemia and fusion can be performed in order to answer additional questions like mitochondrial dysfunction during myocardial leukemia and reperfusion injury.
