The overall goal of this video is to determine the application of serial-section electron tomography to study the mitochondrial ultrastructure of Drosophila indirect flight muscle. This method can help answer key questions in the field of structure cell biology, such as the structure and the functional relationship to mitochondria. The main advantage to this technique is that the cellular ultrastructure is revealed in three dimensions.
To begin the experiment, anesthetize five Drosophola specimens on ice and immerse each fly in one milliliter of 4%low melting agarose in phosphate buffer. Allow the agarose to solidify on ice. Then, use a vibrating blade microtome to section agarose gel-embedded Drosophola into slices with 100 micrometer thickness.
Immerse the slices in fixative solution containing 2.5%glutaraldehyde in zero point one molar phosphate buffer. Wash the tissue sections in three drops of phosphate buffer then follow with a wash with two drops of phosphate buffer with 20%BSA. Place the sections in gold carriers for high-pressure freezing, or HPF, filled with buffer and 20%BSA.
Next, load the sample containing the carriers into a high-pressure freezer according to the user manual. After freezing, release the carriers from the holder under liquid nitrogen and transfer them to a free substitution device cooled to negative 140 degrees Celsius. Perform the free substitution protocol with the FS cocktail containing 2%glutaraldehyde, 2%osmium tetroxide, 0.1%uranyl acetate in acetone.
Embed specimens in resin at room temperature. Then, polymerize the resin at 65 degrees Celsius for 16 hours. Trim the specimen blocks to expose the desired block face that contains tissue.
Next, pretreat 10 nanometer gold particles with 1%BSA for 30 minutes. Wash and suspend the gold particles in phosphate buffered saline, or PBS. Overlay the gold particles on copper slide grids coated with carbon film to create fiducial markers.
Cut serial sections 200 to 250 nanometers in thickness using an ultramicrotome. Then, collect the serial sections on slot grids using a perfect loop for thin sections. Stain the sections with Reynolds lead citrate for 10 minutes.
Overlay a second layer of fiducial gold particles on the top of the sections. Load the grid onto a dual-axis tomography holder and insert into the transmission electron microscope operating at 200 kilovolts. Align the microscope at the eucentric focus.
Set up the automatic data collection software, adjust and align the electron beam at the multiscale imaging setting, acquire camera dark and bright references in an empty area without carbon film under the tomography collection setting. Next, collect a grid atlas at low magnification. Select mitochondria on serial sections as targets for tomography collection, acquire a tilt series from negative 60 degrees to positive 60 degrees with two degree increments on axis A for each target.
Finally, collect the second tilt series. Rotate the specimen holder 90 degrees. Acquire a new atlas.
Select corresponding positions and acquire the tilt series on axis B for each target. Using this method, 2-D micrographs and 3-D tomographs from serial sections covering the entire volume of a mitochondrion were generated. The joined serial-section tomograms are projected to create a longitudinal section with the z-axis shown vertically.
Serial-section electron tomography was applied to analyze structural feautures of mitochondrial cristae that reflects its energetic state and aging. Slices of mitochondrial electron tomographic reconstructions reveal switches between lamellar membranes through the z-axis. Tomographic segmentation illustrating a left-handed spiral in 3-D.
Cristae switching patterns were analyzed and color rendered on the segmentation model. A tomographic slice shows the lateral matrix confluency across cristae membranes. A segmentation model was generated of the tomogram in showing lateral matrix confluency and representative cristae.
After watching this video, you should have a good understanding of how to perform serial-section electron tomography, which may be adapt to study any cellular structure in three dimensions.
