Mitochondria dysfunction is associated with many disease, particularly neurodegenerative and metabolic disorders. Therefore, if we can understand how mitochondria is regulated by lysosomes, it helps to develop new therapeutic strategies. In this protocol, we demonstrated a dual-color CLEM method to visualize mitochondrial lysosome contact.
Correlative light and electron microscopy is an imaging technique that combines the advantages of light microscopy and electron microscopy. In this protocol, we maximize the advantage of CLEM using dual-color probes to visualize mitochondrial lysosome contact. It is challenging to clearly distinguish mitochondria degrading within lysosomes using traditional electron microscopy sampling methods.
This study presents high-resolution CLEM result that clearly showed the effector domain of lysosomes and mitochondria. The dual-color taking method was used to accurately distinguish mitochondria effectors within lysosomes. Our research has revealed the mitochondria interact with the lysosomes.
Our research has revealed that mitochondria interacted with lysosomes in different ways in response to different external stresses. By studying the causes and regulators of these phenomena, we will gain insight into understanding intracellular mitochondrial quality control. To begin, culture HeLa cells at a density of one times 10 to the power of five in 35-millimeter glass grid-bottomed culture dishes.
On the next day, co-transfect the cells at 30%to 40%confluency with plasmids using the transfection reagent. After transfection, treat the cells with dimethyl sulfoxide or U18666A and bafilomycin A1 for 18 hours. For confocal imaging, remove the culture media and immediately add 250 microliters of warm fixative solution at 30 to 37 degrees Celsius.
Immediately remove the fixative. Replace it with 1.5 milliliters of fresh fixative and incubate it on ice for 30 minutes. Then wash the cells three times for 10 minutes each with one milliliter of ice-cold 0.1 molar sodium cacodylate buffer.
Next, add one milliliter of cold 20-millimolar glycine solution to quench the aldehyde. After incubating the cells on ice for 10 minutes, wash them three times for 10 minutes each in cold 0.1 molar sodium cacodylate buffer. Then add 500 microliters of Amplex red solution to the sample dish and incubate for five minutes on ice.
Afterward, remove the Amplex red solution and rinse the cells three times for 10 minutes each with 0.1 molar sodium cacodylate buffer. Use a confocal microscope to image the area around the cells of interest in three-by-three tile scan mode. Capture both differential interference contrast and fluorescent signals to identify and record the grid and letters on the grid dish using a 40-times objective.
Image the target cells at high magnification using a 40-times objective and acquire a Z-stack image. After imaging the Amplex red-stained cells using a confocal microscope, add one milliliter of 1%reduced osmium tetroxide at four degrees Celsius and incubate for one hour. Then rinse the sample three times for 10 minutes each with distilled water at four degrees Celsius.
Dehydrate in a graded ethanol series for 15 minutes each time at room temperature. Now mix ethanol with epoxy resin in volume ratios of three to one, one to one, and one to three, preparing a total of 300 microliters of each mixture. Pour each epoxy resin mixture into the dish and incubate at room temperature for one hour.
Next, prepare the embedding capsule by filling it with fresh epoxy resin. Turn the tube or capsule upside down in the region of interest and place it in an oven at 60 degrees Celsius for 48 hours to polymerize. Then immerse the bottom glass and the polymer block in liquid nitrogen to separate them.
Place the specimen block in the sample holder of the ultra microtome and set it in the trimming block. Use a razor blade to trim the resin into a rectangular or trapezoidal shape around the object of interest. Place the trimmed specimen block in the sample holder and adjust the diamond blade.
Using an ultra microtome, cut ultra-thin sections approximately 60 to 70 nanometers from the flat embedded cells. Collect the serial sections on a formvar-coated slot grid before drying them on a hot plate. Afterward, stain the sections with contrast stain for two minutes and lead citrate for one minute.
Then place the grid in the transmission electron microscope, or TEM, sample holder for observation. Based on the differential interference contrast image from the light microscope, identify the target cell of interest and image the entire cell area using tiled overlapping images at 1, 700 times magnification To obtain the correlative light and electron microscopy images of HeLa cells using ImageJ Fiji, start ImageJ Fiji and click on file, new, TrakEM2 blank, and select storage folder to create a new TrakEM2 project. Drag and drop the raw tile image data into the workspace to open the tile image dataset.
Click the right mouse button and select the line, montage all images in this layer and elastic non-linear block correspondences. Then click okay to accept the default values for the remaining parameters. Next, click the right mouse button and select export, followed by make flat image to create a stitched image.
Then click file and save as to save the image. To resize the fluorescence image, open the photo enlargement software. Click on file, open and select the image.
Enter the width and height. To match the size of the electron microscope image. Select the algorithm to be applied in the resize method.
Click on file and save as to save the resized image. Open ImageJ Fiji and drag and drop the resized fluorescence and the stitched electron micrograph into the workspace. Click on plugins big data viewer and big warp to start the registration.
Then click on the dropdown menu to select the fluorescence micrograph image as the moving image and the electron micrograph image as the target image. Using functions such as zoom, image rotation, and move, compare the fluorescence and electron micrograph images. Press the space bar on the keyboard to switch to landmark mode and press the left mouse button to mark at least three landmarks identified in both images.
After marking all the identified landmarks, go to file, export moving image, and click okay. A new pop-up window with the transformed fluorescence micrograph image will appear. Click on file and save as to save the transformed fluorescence micrograph image.
To overlay the CLEM images, drag and drop the transformed fluorescence micrograph image and the stitched electron micrograph image into the ImageJ workspace. Click on image, type, and eight-bit color. To set the same image type for both the fluorescence and electron micrograph images.
Click on image, color, and merge channels. To combine the images. Click on file and save as to save the CLEM image.
Mitochondria trapped within lysosomes were observed in bafilomycin A1-treated cells indicating inhibited autophagy and mitochondrial quality reduction, while control cells showed mitochondria interacting with but not engulfed by lysosomes, the mitochondrial disruption observed in control cells showed that mitochondria surrounded lysosomes rather than being engulfed by them. U18666A-treated cells showed increased interactions between mitochondria and lysosomes, with some lysosomes engulfed by mitochondria.
