The overall goal of the following experiment is to localize proteins and electron micrographs using correlated fluorescence and electron microscopy.Here. This method known as nano FEM, is used to localize proteins in C elgan. First, a transgenic worm expressing histone TD eos is embedded in plastic and sectioned.
Each section is imaged by both photo activated localization microscopy or palm and scanning electron microscopy. The two micrographs for each section are aligned using fiducial markers using Photoshop. The correlative image shows the localization of histone molecules in the nucleus.
So the main advantage to the technique that we are going to describe today is that instead of using immuno em, we are using genetically encoded floors so that every protein is associated with a fluorescent protein. When dealing with immuno em, many of the antibodies can't act with plastics. It's just not compatible, and if it is, the antibodies are not able to penetrate deep into the section where the antigens are.
By using fluorescent proteins, every protein is tagged with a Fluor. So today shige wbi will be demonstrating this technique. The following procedure will be demonstrated using transgenic nematodes that express histones tagged with the fluorescent protein TDE os.
For this demonstration, we will use bacteria as a cry protectant, but you can also use 20%BSA for freezing sea elegance. One hour before freeze substitution, fill the automated freeze substitution or a FS device with liquid nitrogen. Also in advance, prepare the needed fixatives according to the instructions in the accompanying document, store the fixatives in one milliliter, aliquots in cryo vials, and place them in liquid nitrogen until they're used.
Use a paintbrush to scoop animals from a lawn of bacteria and transfer them to fill a 100 micron specimen carrier Slightly overfill the cup to ensure that there are no airable. When kept, place the specimen cup in the armature of a high pressure freezer to freeze it. Then immediately transfer the sample to a liquid nitrogen bath next to the freezing chamber.
Next, using an all and a pair of forceps, transfer the specimen into a cryo vial containing fixatives. Make sure the specimen stays under the liquid nitrogen at all times. Repeat this process for each specimen, then transfer all the cryo vials into the a FS unit.
Once all of the cryo vials are placed in the a FS unit, start the program. The freezing and processing protocol is shown in this diagram. During the initial minus 90 degree Celsius incubation, the vitreous ice is replaced with the fixative in acetone.
Then as the sample temperature rises, the fixative in the liquid acetone cross links the sample to preserve structure. When the temperature in the a FS unit reaches minus 60 degrees Celsius place scintillation vials containing 20 milliliters of 95%acetone in the a FS to cool them. Then when the temperature reaches minus 50 degrees Celsius, use a Pasteur pipette with a P 200 tip to remove the fixative and add about one milliliter of the pre-cool 95%acetone to each sample.Bile.
Repeat this process every 20 minutes over two hours. Also, during the minus 50 degrees Celsius incubation place a scintillation vial containing 20 milliliters of 0.1%urinal acetate in 95%acetone into the a FS chamber to pre-cool it to minus 50 degrees Celsius. Then after the final acetone wash, add about one milliliter of urinal acetate solution to each vial to stain the sample to enhance membrane contrast.
If the program has been paused, restart it. When the temperature reaches minus 40 degrees Celsius place, scintillation vials containing 95%ethanol in the a FS chamber. When the temperature reaches minus 30 degrees Celsius, use a pasture pipette to replace the urinal acetate with 95%ethanol every 20 minutes for two hours.
It is important to use ethanol in this step since the resin will not polymerize in the presence of acetone. In the next part of the protocol, the specimens are embedded into plastic while the specimens are at minus 30 degrees Celsius. In the a FS, replace the 95%ethanol with pre-cool 30%glycol methacrylate resin or GMA in 95%ethanol after three to five hours replace the 30%GMA with 70%GMA after four to six hours, remove the 70%GMA and incubate the specimens in 97%GMA overnight.
The stepwise increase of GMA concentration allows penetration of the media into the tissues. Note that since 3%water is added to the GMA resin, 97%GMA is not diluted. With ethanol the next day, transfer the samples to an embedding mold in the A FS using a Pasteur pipette place the samples in the embedding mold then exchange the 97%GMA for freshly prepared GMA three times over six hours at minus 30 degrees celsius.
After the third exchange, tap on the bacteria matrix to separate the specimen from the cryoprotectant. Add the initiator nn dimethyl udine to the 97%GMA in a ventilation vial. The amount of initiator added is 1.5 microliters per one milliliter GMA immediately add the GMA solution to each embedding mold and position the animals in the mold so that the animals are evenly spaced.
Restart the program so that the temperature increases to minus 20 degrees Celsius. Although the resin polymerizes quickly within one hour, allow the plastic to cure overnight at minus 20 degrees Celsius. To be sure it is thoroughly polymerized.
Once the plastic has cured, store the specimen in a nitrogen gas filled vacuum bag at minus 20 degrees Celsius. Sectioning of GMA embedded specimen should then be performed as described in the accompanying document for photo activated localization microscopy or palm imaging. The microscope system should be set up with the E-M-C-C-D camera set to minus 70 degrees Celsius or below.
Using a pipette, apply about 50 microliters of 100 nanometer gold particles at one times 10 to the 10th particles per milliliter in solution to the sample to be imaged. Place a black cover case over the samples and allow the solution to sit for 30 seconds on the cover slip. To remove the gold solution, blow it to the edge of the cover slip and absorb the solution with a kim wipe.
Then apply vacuum grease to a circular cover slip holder and insert the cover slip. Apply immersion oil to the underside of the cover. Slip directly below the sample.
Insert the sample holder into the slot on the stage of the microscope and adjust it so that as tight and centers the sections above the objective. Take special care not to touch the objectives. Next, using brightfield illumination, locate the sections using a 10 x objective lens.
Then change to the 100 x objective lens and focus on the specimen. If necessary, switch to fluorescence illumination using the 488 nanometer laser with the intensity set to 10%Allow five minutes to elapse for the sample temperature to equilibrate. Then identify the regions with the brightest fluorescence and bring them into focus.
Switch the lasers to 561 nanometers and increase the intensity to 100%To bleach out the background autofluorescence bleach the sample for approximately two minutes. If the focus changes during bleaching, allow five minutes to elapse before adjusting the focus and capturing images. When bleaching is complete, activate the 405 nanometer laser and set it to the lowest intensity.
Start collecting the images at 20 frames per second. Collect enough frames to achieve the goal of the experiment here. 5, 000 to 6, 000 frames are taken to determine the localization of 584 histone proteins within a nucleus.
Once 5, 000 frames are collected, all the molecules are identified and mapped by the imaging software. The final fluorescence image or so-called some turf image is a composite of all of the fluorescence detected on the camera. In contrast, the palm image maps the OID of each fluorescent spot and only indicates where each protein is located.
Prior to SEM imaging stain the sections using 2.5%urinal acetate in water for four minutes. Wash off the urinal, acetate thoroughly with filtered milli QE water. After the sections are dried, use a sputter to carbon coat the cover slip until it becomes fairly dark.
Apply carbon conductive tape at the edge of the cover slip on one end and to the metal stub on the other end so that the electrons that accumulate on the surface of the cover slip are grounded. Mount the cover slip in the SEM chamber and locate the specimen on the microscope. Take a low magnification image of the specimen, then zoom into the region of interest and obtain high magnification images.
Repeat this process with a new section until all sections have been imaged. Next, the palm and EM images must be aligned. To do this first, open the acquired SEM images in Photoshop.
Create a new window with dimensions of 5, 000 by 5, 000 pixels and 300 pixels per inch resolution. Copy the load magnification SEM image into the new window. Use the free transform command found under the edit menu to scale the image so that it fills up the entire window.
Then copy the higher magnification SEM images and scale and transform as necessary. Select image rotation from the image dropdown menu bar and flip the sum turf image horizontally. Copy and paste the sum turf image into a new layer.
Use the free transform command found under the edit menu to scale the image so that the image is roughly the same size as the low magnification electron micrograph. Then rotate the image as necessary to match the fluorescence of the gold particles with the corresponding structures visualized in SEM. Next, copy the palm image into a new layer and apply the same transformation to extract a higher magnification image.
Zoom into the region of interest and copy the palm image from the region. Paste the image into a higher resolution SEM image for presentation. A gradient transparency should be applied to the palm image so that only the black background, but not the palm signals become transparent.
Copy the transform palm image into a new layer. Select the palm signals but not the background by using color range in the select dropdown menu. Then select a background pixel as a reference and turn on the invert option.
Cut the desired palm signals and paste them to a new layer. Apply transparency to the background layer and set it to 10%This allows for visualization of the palm signals by making the background transparent without compromising the intensity. Histone protein.
His 11 tagged TD eos was stably expressed in the nematode c elgan, and the transgenic animals were processed and imaged using the protocol described in this video. Palm and electron micrographs were acquired from the same section. The gold nanoparticles, which appear in both the fluorescence and electron micrographs were then used to align the two images.
Here we zoom in on a region near the top end of the micrograph. As can be seen here. Subcellular details such as a nucleus.
A nucleus, nu nuclear pores, and endoplasmic reticulum can be observed. However, the tagged histone molecules are exclusively localized to the nucleus, but not the nucleus As expected, the correlative palm and electron microscopy thus allows for protein localization at the highest resolution. After performing this procedure, one can take these sections with the protein distribution and stack them using array tomography so that you can generate a three dimensional structure, which shows where the proteins are distributed in the cell.
And remember that when using osmium tetroxide that it's a very danger, dangerous chemical, and you should always wear gloves.
