This protocol provides a standardized approach to image and quantify the changes in mitochondrial morphology across multiple tissues. In C elegance, mitochondrial morphology is often associated with its functionality. Changes in the mitochondrial morphology can be observed under various disease conditions and with the progression of aging, which often correlate with dysregulation in mitochondrial function, Different tissues, and see elegance.
Exhibit unique mitochondrial structures that require distinct imaging strategies. Therefore, selecting an appropriate microscope and optimizing sample preparation is essential for successful tissue specific imaging of mitochondria and C Elegance. Here we chose to use the transgenic cele strains expressing mitochondria localized GFP instead of conventional mitochondrial dyes.
This allows us to avoid off target effects, onal bi penetrance, and the complex sample preparation steps necessary to visualize mitochondria in different tissues using a dye. Our lab's primary focus is studying mitochondrial homeostasis during stress and aging. Standardizing a mitochondrial imaging protocol is essential to minimize extramental errors and generate reproducible results regarding changes in mitochondrial morphology under various biological conditions.
To begin, obtain clean glass slides. For sample preparation, add 10 to 20 microliters of M nine solution onto the glass slide and place the desired number of adult worms at specific ages in the solution. On the slide, cover the worms in the M nine solution with a cover glass to image muscle.
Mitochondria, nudge the cover slip to roll the worms. Then with a nail polish seal the sides of the cover glass to prevent the evaporation of the M nine solution. Use a standard wide field microscope to image muscle and epidermal mitochondria expressing the fluorescent tags.
Use a confocal microscope for imaging intestinal mitochondria and optimize the imaging settings to suit the specific experimental setup. To begin the analysis, open the Fiji application after installation to download and install the mito mapper macro. First, download the source code one.
Copy the code listed under A IJM code for MIT mapper 1.0. Open Fiji, and go to plugins, followed by new and macro paste the copied code into the macro window. Then save the macro for future use via file and save as.
Navigate to file, and then open for opening a 3D microscopic image with Zs.In Fiji, create a max projection under image, followed by stacks and Z project. Select the range of slices within in-focus images for max projection and choose max intensity as the projection type to save the max projected image. As a tiff, go to file and click save as followed by tiff.
Crop the regions of interest from the full image with the rectangle tool and navigate to image and crop. Go to file followed by save as and tiff to save the crop image in Tiff format. Drag and drop the previously saved mito map or macro file onto the Fiji toolbar.
Click run. When prompted, select the folder containing the TIFF file saved in the previous step. When prompted to select an area, create a rectangle in the image using the rectangle tool and press okay to confirm.
Finally, go to file and save the data with all the values related to mitochondrial morphology as a dot CSV file. Mitochondrial morphology was tissue specific with tubular mitochondria aligning with muscle fibers in muscles interconnected web-like structures in the intestine, and more rounded or oval. Mitochondria in the hypodermis imaging with a confocal microscope improved the visualization of intestinal mitochondria by reducing out of focus light compared to compound microscopy.
On slides, mitochondria displayed fragmentation after 30 minutes in the M nine buffer with epidermal mitochondria showing the most prominent fragmentation. Aging resulted in mitochondrial fragmentation in all tissues appearing as truncated and spherical structures. MIT mapper quantification indicated that the object length changed in muscles and the junction point changed in the hypodermis.
