This volume electron microscopy technique is used to perform biological studies that require the observation of a 3-D structure. Offering a rod, fielder view, and sample storage before image retakes. Fx2 Asan engineered the path stages that catalyzes the deabilation to increase the electron test of target structures and can be used to locate a specific target of interest.
These particles combines the use of correlative light and 3-D electron microscopy technique to investigate the interactions between membranous organelles and host cells. 16 to 24 hours after transfection, gently wash the culture with 250 microliters of 30 to 37 degree Celsius fixation solution. Quickly replacing the wash with 1.5 milliliters of fresh fixation solution, and placing the culture on ice for 30 minutes.
At the end of the incubation, rinse the cells with three ten minute washes in one milliliter of ice-cold 0.1 molar sodium cacodylate buffer per wash. After the last wash, add one milliliter of ice-cold 0 to 4 degrees Celsius 20 millimolar glycine solution to the dish for a ten-minute incubation on ice followed by three five minute washes with one milliliter of fresh, cold 0.1 molar sodium cacodylate buffer per wash. Next, label the cells with 500 microliters of freshly prepared DAB solution.
And incubate the cells on ice for approximately five to forty-five minutes. When a light brown stain is visible under an inverted light microscope, rinse the cells three times with one milliliter of fresh, cold 0.1 molar sodium cacodylate buffer for ten minutes per wash. Then, use a phase contrast inverted microscope to visualize the DAB staining at a 100 times magnification or higher and mark the bottom of the glass where the region of interest is located.
For electron microscope block sample preparation, first post-fix the cells with one milliliter of 2%reduced osmium tetroxide for one hour at four degrees Celsius. At the end of the fixation, rinse the cells with three five-minute washes and one milliliter of fresh distilled water per wash. Before treating the cells with one milliliter of filtered TCH solution for 20 minutes.
At the end of the incubation, wash the cells three times in distilled water as just demonstrated and fix the cells with a 30-minute incubation in 2%reduced osmium tetroxide. At the end of the second fixation, wash the cells three times with distilled water and cover the culture with one milliliter of aqueous 1%uranyl acetate overnight at 4 degrees Celsius protected from light. The next morning, wash the cells three times with distilled water before staining with one milliliter of 60 degree Celsius warmed Walton's lead aspartate solution for 30 minutes in a 60 degrees Celsius oven.
At the end of the incubation, wash the cells three times in distilled water. And incubate the culture in a graded series of 20-minute 2 milliliter ethanol aliquots. After the last 100%ethanol exposure, incubate the cells for 30 minutes in one milliliter of three to one ethanol to low viscosity embedding mixture medium at room temperature.
At the end of the incubation, replace the medium with one milliliter of one-to-one ethanol to low viscosity embedding mixture medium for another 30 minute incubation at room temperature. Next, replace the medium with one millilter of one-to-three ethanol to low viscosity embedding mixture medium for an additional 30 minutes at room temperature. Then, replace the medium with one milliliter of 100%low viscosity embedding medium overnight, followed by embedding of the sample in 100%low viscosity embedding mixture for 24 hours at 60 degrees Celsius.
For transmission electron microscopy analysis, the next day, use an ultra microtome to obtain 90 nanometer thick sections and observe the grid under transmission electron microscopy at 200 kilovolts. Before beginning the sample preparation, clean Indium Tin Oxide coated glass coverslips with gentle agitation in isopropanol for 30 to 60 seconds. At the end of the agitation, drain off the excess alcohol and place the coverslips in a dust-free environment.
When the coverslips are dry, use a plasma coater to glow discharge the coverslips for one minute. Then insert the coverslips into the substrate holder and place the substrate holder into the knife boat. To obtain samples for scanning electron microscopy, insert the embedded sample block into the sample holder of the ultramicrotome.
And set the holder into the trimming block. Use a razor blade to trim away the excess resin around the target position so that the face of the block acquires a trapezoid or rectangular shape. The leading and trailing edges should be strictly parallel to each other.
It is not easy to acquire a large number of serial sections. However, you have to have the leading edges and trailing edges completely parallel. Next, insert the sample holder into the arm of the ultramicrotome.
Place the diamond knife in the knife holder, and fill the knife boat with filtered distilled water. Then, carefully push the handle of the holder to adjust the carrier position so that the edge of the coverslips is close to the knife. After obtaining the sample, stop the sectioning process, slowly open the clamping screw of the tube and drain the water boat.
When the ribbon collection process is complete, remove the substrate with the handle of the clamping device and dry the ribbon. The use of genetically tagged plasmids expressing proteins of interest, allows the identification of the transfected target cells after staining for the tagged proteins. Correlative light electron microscopy can then be used to further visualize the labeled cell culture.
For example, the dark stain generated by SCO1-APEX2 is observed exclusively in the mitochondrial intermembrane space and not in the matrix space of the mitochondria. Code transfected cells with both SCO1-APEX2 and HRP-KDEL plasmids express a highly dense electron signal in the mitochondrial intermembrane space and the endoplasmic reticulum. However, no endoplasmic reticulum staining is observed in cells that are transfected with SCO1-APEX2 only.
For serial imaging by scanning electron microscopy, an overview of the whole array image is created using a backscatter electron detector over a large area. Next, the region of interest is placed in the first section and propagated to all the other sections. Finally, the region of interest containing five target cells is imaged with five nanometer imaged pixels.
Magnification reveals detailed subcellular structures such as the Golgi apparatus, mitochondria, and endpolasmic reticulum. Serial imaging clearly reveals that endoplasmic reticulum-mitochondria contacts occur on different z-planes. You should have a good understanding of how to locate a specific target structure using correlative light and electron microscopy to investigate the effects of a specific identity modification.
These staining techniques combined with volume electron microscopy could be used for larger scale EM to investigate cellular interactions particularly in eurovirus. Remember the glutaraldehyde, also enteterocide and TCH are very toxic, so be sure to wear protective clothing and to work in a fume hood when using these materials.
