The overall goal of the following experiment is to get the three dimensional structure of the exosome complex using the random conical tilt method. This is achieved by first depositing a thin layer of carbon film onto the holy carbon grid to make the support substrate for the protein complex and the stain as a second step. The specimen and the stain are applied onto the grid, which buries the specimen into heavy metal salts.
Electron microscopy is then carried out to get tilt pairs of micrographs of the specimen. Finally, results are obtained that show the three dimensional structure of the exosome complex based on image analysis using the random conical tilt method. Generally, individuals new to this method will struggle because sometimes good preparation of specimen for random conical tilt method is difficult as well as the procedure for the image processing that follows View.
Demonstration of this method is critical as the grade and specimen preparation steps are difficult to learn without an illustration of sample assessment. The the principle of the random conical tilt method requires taking a pair of micrographs of the same region of a specimen inside the electron microscope. One picture is taken of the specimen in an un tilted position, and the other picture is taken of the specimen tilted at an angle between 50 and 70 degrees Using the computer, the digitized micrograph pair is put side by side and images from the same particles are selected in this illustration.
These images are marked by numbers in three dimensional coordinates. The images of un tilted particles and their tilted partners are correlated to each other by the direction of the tilt axis and the tilt angle. The alignment of the un tilted particle images brings the images of tilted particles to their corresponding as aMule locations using multiple images of tilted particles filling the azimuthal space, the three dimensional structure of the molecule can be reconstructed using a back projection algorithm to fabricate the grids first, prepare a 0.5%form VAR solution by adding 0.45 grams of poly vinyl formal resin, and 90 milliliters of chloroform to a 100 milliliter glass beaker.
In a fume hood, cover the beaker with aluminum foil and dissolve the form var resin. With the eight of a small stir bar and a magnetic stir, the resin takes about 15 minutes to dissolve. Meanwhile, clean pre-cleaned microscopy glass slides in methanol and with Kim wipes.
After the form var resin is fully dissolved. In chloroform, add one milliliter of 50%glycerol to the surface of the solution. Adjusting the volume of the added glycerol affects the density of the holes in the holy carbon.
Tip the tip of an ultrasonic into the solution. At about one inch depth, use maximum power to sonicate for one minute, which will make an emulsion of glycerol droplets in the form of our solution. Longer sonication will cause smaller size of holes in the holy carbon.
The solution becomes milky after this step. Immediately after the sonication dip, the clean glass slides vertically into the emulsion. For one second, take them out and blot the bottom of the slides using filter paper to form a thin plastic film over the surface of the slides.
After the chloroform evaporates, check the density and size of the holes in the film under a light microscope. The preparation condition described here should produce 10 to 20 holes in each square of a 400 mesh grid with diameters between three and four micrometers. Once the glass slides are dry, cut the edge of the plastic film on the slide surface.
Float the film off on the surface of distilled water by vertically dipping the slide into water. The thin film on the water surface can be observed at a glancing angle against the light reflection. Place 400 mesh copper grids on the film, one by one with the grid smooth surface facing down.
Pick up the plastic film with grids on it using a piece of paper. Flip the paper and let it dry. In a Petri dish, soak the paper in methanol to remove the residual glycerol in the holes and allow the paper to air dry.
Use a carbon evaporator to coat the grids with a layer of carbon to a thickness of approximately 20 nanometers. Using the evaporation time to control for the desired thickness. The thickness can be determined by the gray color of the carbon as compared with the gray color of an known thickness carbon layer.
Soak the carbon layered grids with chloroform in a glass petri dish for half an hour to remove the form var form var removal, followed by drying of the grids results in homemade holy carbon grids. Next, evaporate a thin layer of carbon with about a five nanometer thickness onto a freshly cleaved mica surface. Then carefully put the holy carbon grids on a piece of filter paper underneath distilled water.
In a homemade apparatus, float the thin carbon from the mica surface onto the water surface and slowly deposit it onto the holy carbon grids. Pick up the filter paper with grids on it and let it dry. In a fume hood, begin negative staining of the exosome complex by making a fresh 2%urinal formate solution.
As described in the written procedure, cover the solution tube with a piece of aluminum foil To prevent light exposure, the solution must be used the same. Day glow discharge one thin carbon over wholly carbon grid using a glow discharge apparatus. Next place a piece of clean parfum on the bench pipette three 50 microliter droplets of urinal formate stain solution on top of the perfil, dilute the exosome complex to a concentration of a approximately 50 nanomolar Using dilution buffer pipette four microliters of the diluted protein until the glow discharged grid.
Let the sample stay on the grid for one minute, then blot the residual solution with a piece of filter paper using tweezers. Flip the grid immediately on top of the first stain droplet and rinse the grid by moving it back and forth several times for about 10 seconds. Repeat this process for each of the three stain droplets after the last rinse, let the stain stay on the grid for an additional one minute.
Then blot the stain away with a piece of filter paper. Keep a thin layer of stain solution on the grid surface and allow the grid to dry in a fume hood. Place the sample grid in the sample holder and then put the holder into an FEI TE nine 12 electron microscope.
Check the sample grid at low magnification to find the best stained squares. A good square appears to have a dozen holes with dimensions of about one to two micrometers and dark stained areas in them. Turn on the low dose mode of the FEI user interface and align the search focus and expose position in low dose mode.
Set the search into fraction mode with 1.5 meter camera length. Set the focus to 150 K magnification and set the exposed to 50 K magnification with one second measured exposure time. Normally, a recognizable feature on the grid at both search and exposure mode is used to perform the alignment.
Find the holes with good staining in search mode and save the locations. Take a CCD picture of the square. Tilt the specimen to 55 degrees and take another picture.
Compare the two pictures and identify the paired holes in the two micrographs. Tilt the stage back to zero degrees. Use the low dose kit to take pictures of each hole identified in the search mode at high magnification with a defocus of about minus 0.7 micrometers.
After pictures have been taken of all the holes, tilt the stage to 55 degrees using the same magnification. Take micrographs of the tilted specimen with a defocus of about minus 1.2 micrometers. Identify the corresponding tilt pair of micrographs based on the patterns in the low magnification micrographs using the irregular pattern of the homemade holy carbon grids.
To help the correlation choose micrograph tilt pairs with even distribution particles and without halos around the particles. Those micrographs with obvious stain halos around them should be discarded to process the data. Start with program setup In the procedure.
Image processing will be demonstrated using process 2D in the Eman package. To change the image format the iMagic five package to do the 2D alignment and the spider package. To perform the 3D reconstruction and refinement, convert the DM three Gatan digital image to spider image format.
Using the process 2D command, pick the particle pairs. Using the web program distributed in the spider program package, the program automatically saves the coordinates of the particles box out all the selected particles using the spider script. The script saves the tilted and un tilted particle stacks.
Convert the UN tilted particles into IMAG five format. Using the EM two EM program band pass, filter and mask the UN tilted particles using the InCorp prep command in IMAG five and center the band pass masked particles using the center command. In imag five, align and classify the particles iteratively into homogeneous classes using a batch script automatically executing IMAG five programs.
Next, use the MSA names in class command in IMAG five to generate a class's lookup table of the particles, generate a plot file for translation and rotation values of alignment for each particle using the header command. In iMagic five, convert the aligned particles to spider format using the EM two EM program. Then change the classes lookup table into spider document files.
The translation and rotation values of alignment for each particle are also converted into a spider document file band pass filter, mask and center the tilted particles just as for the un tilted particles in IMAG five, generate a new data set for center tilted particles. Create angular document files from the multi reference alignment documents generated from IMAG five and the DCB files generated by the web program. Use back projection script in spider to get one initial model for each class average.
Finally, perform projection refinement of the initial volume using the spider script against all the un tilted particles to get the final volume using RCT. About 50 reconstructions of the exosome have been obtained. The volumes are generated from tilted particles corresponding to each particle in the 50 classes of un tilted particles.
Using back projection implemented in the spider package, the projection matching refinement was then performed Using these volumes as initial models and the displayed 3D volume of the exosome was generated at about 18 ang extra resolution, we can then duck in the atomic models of different parts of the complex in the 3D map. After watching this video, you should have a good understanding of how to use RCT to get three dimensional reconstructions from generating a good narrative standing grid of specimen to peer-to-peer micrographs, and finally to obtaining the reconstruction using image analysis techniques. Besides the protocol we described here, there are several methods to get the final reconstructions in image analysis.
The most critical step for getting a reliable correct reconstruction is to work with a well prepared specimen.
