The overall goal of this microscopy method is to precisely localize protein expression in relation to different subcellular organelles in the larval zebrafish retina by correlating super-resolution and scanning electron microscopy images. This method can help answer key questions in the retinal development and cellular pathway fields, such as the study of protein mislocalization in mutants. The main advantage of this technique is that it combines super-resolution with scanning electron microscopy to attain highly accurate protein localization data within the structural context of the sample.
The collection of Tokuyasu sections on silicone wafers substantially facilitates handling and the use of platinum shadowing for contrast provides a good topography of the sample. So, this method can provide insight into larva zebrafish retina studies. One of its major advantages is that it can also be applied to many other biological systems, such as identification of protein expression in mouse tissue.
After dissecting the eyes from fixed zebrafish larvae according to the text protocol, warm up 15 milliliters of 12%local food brand gelatin in PBS at 40 degrees Celsius. Then aspirate the PBS from the tubes of eyes and add the gelatin solution. Gently tap the tube to ensure the gelatin infiltrates the sample, and incubate it for 10 to 30 minutes at 40 degrees Celsius in a thermoblock with gentle shaking or in a water bath.
In a 40 degree Celsius water bath, fill 12 by five by three millimeter silicone or polyethylene flat embedding molds with warm gelatin. Then using a pipette add two eyes per mold and under a binocular use a dissection needle to properly align them. After allowing the gelatin to cool down at room temperature for one minute, place it at four degrees Celsius for 20 minutes to harden.
Under the binocular, use a razor blade to re-trim the gelatin block to fit one eye per block. Transfer the gelatin embedded eyes to 2.3 molar sucrose in PBS on ice. Incubate the samples at four degrees Celsius overnight.
Then exchange the samples to fresh 2.3 molar sucrose solution. And store the tissue at four degrees Celsius, or minus 20 degrees Celsius for storage up to several months. Re-trim the gelatin block to almost the size of the eye before transferring it to a cryo-pin.
Then freeze the block in liquid nitrogen, and transfer it to a cryo-ultramicrotome. Using a diamond knife in the cryo-ultramicrotome cut 110 nanometer thick sections at minus 120 degrees Celsius. Pick the sections with a wired loop containing a droplet of 2%methylcellulose and 2.3 molar sucrose solution.
Then transfer the sections to a seven by seven millimeter silicone wafer. To wash the wafers, pipette four drops, and place the wafers upside down on the drops. Incubate the wafers on the drops at zero degrees Celsius for 20 minutes.
Then wash the wafers two times in PBS at room temperature for two minutes each. Incubate the samples three times in 0.15%glycine in PBS for one minute each. Then use PBS to wash the samples three times for one minute per wash.
Pre-incubate the tissue with PBG for five minutes. Next, add rabbit anti-Tom20 in PBG to the sections. And incubate the wafers at room temperature for 30 minutes.
With PBG, wash the tissue six times for one minute per wash. Then pre-incubate the samples with PBG at room temperature for five minutes. Replace the PBG with Alexa 647 anti-rabbit divalent Fab fragments in PBG, and incubate for 30 minutes.
Wash the samples in PBG six times for one minute per wash. Then use PBS to wash the wafers three times for two minutes. Add DAPI and PBS to the wafers and incubate for 10 seconds.
Then use PBS to wash the samples two times for two minutes each. Place the wafer on a droplet of a one to one solution of 80%glycerol and imaging buffer containing an oxygen scavenging system and briefly incubate it. Transfer the wafers, tissue side down, onto a fresh drop of the one to one mixture of 80%glycerol and imaging buffer on a glass bottom Petri dish.
Use a pipette from all sides to remove most of the liquid underneath the wafer. Then use silicone stripes to fix the wafer to the bottom of the Petri dish. Place the mounted sections on an inverted microscope with a high numerical aperture oil immersion objective.
Prior to imaging, let the sample equilibrate to the microscope temperature in order to minimize or reduce lateral and axial drift. Center the area of interest and acquire wide field epifluorescence reference images. Change to the super-resolution operation mode.
Adjust the exposure time of the camera to 15 milliseconds and set the electron multiplying gain to the maximum of 300. Next, illuminate the sample in epifluorescence mode with the 642 nanometer laser at maximum laser power. As soon as the single molecule blinks are well separated in each frame, so that the probability is low that individual signals overlap, reduce the laser power to almost 1/3.
Record the raw image in epifluorescence by acquiring a minimum of 30, 000 frames. From the raw data, under Tools, t-Series Analysis use a detection threshold of 30 photons. Click Evaluate to generate a localization event list.
Under Tools, in the Eventlist Processing panel click Create Image to visualize the super-resolution image by Gaussian fitting, applying a rendering pixel size of four nanometers. Remove the silicone stripes, and add a drop of PBS close to the edges of the wafer to lift it up from the Petri dish. After using PBS to wash the wafer two times for two minutes each, use 0.1%glutaraldehyde in PBS to post-fix it for five minutes.
Then wash the sample again two times for two minutes per wash in PBS. Incubate the wafer in one drop of 2%methylcellulose in water on ice two times for five minutes per incubation. Then insert the wafer in a centrifuge tube, and centrifuge at 14, 100 times g for 90 seconds.
Following the spin, use conducting carbon cement to mount the wafer on an SEM aluminum stub. Using an electron beam evaporation device, add a layer of two to 10 nanometers of platinum carbon on the sample by rotary shadowing with the following settings. Image sections with a scanning electron microscope at 1.5 kilovolts, two millimeter working distance, and with an in lens secondary electron detector.
Use Fiji to open both light and electron microscopy images by clicking File, Open. Adjust the canvas size by clicking Image, Adjust, Canvas Size. Then bring both images to a stack by clicking Image, Stacks, Images to Stack.
In Fiji, open a new track EM2 interface by clicking File, New, Track EM2 new. Import the stack with both images by right clicking on the black window and selecting Import stack. Align the light microscopy image to the electron microscopy image manually with landmarks by using a right mouse click on the image and selecting Align, Align layer manually with landmarks.
Pick the Select tool to add landmarks. Use the shape of the nuclei as a reference to select the same edges in both images and to add several points. Apply alignment with an affine model by right clicking and select Apply transform, Affine Model.
Finally, change the layer transparency to assess the quality of the alignment. This panel shows a low magnification wide field image of a five day post-fertilization zebrafish retinal section. The same area is shown here by scanning electron microscopy.
These higher magnification images show Tom20 staining in red with clusters at the mitochondria. The expression of Tom20 is shown here as detected by GSDIM microscopy. In this image, the same section combines correlative super-resolution and scanning electron microscopy.
Tom20 staining appears within the mitochondrial cluster at the outer membranes of mitochondria. The fluorescence DAPI signal in the nuclei corresponds with the topography of the SEM image. This SEM image provides context to the Tom20 staining in the GSDIM image.
Mitochondrial cristae are clearly visible, and the Tom20 staining is localized to the outer membranes of mitochondria. The membranes of the outer segment of the photo receptors are clearly resolved. While attempting this procedure, it is important to remember to optimize as best as possible your labeling and imaging parameters.
Only optimally labeled samples are suitable for super-resolution. Samples must never dry at any time during the labeling procedure. This procedure could be extended to double labeling immunofluorescence, and thus allow the localization of two different proteins within a specific structure.
In case of more complex samples, the use of fiducial markers is recommended to obtain a precise alignment of the images. After watching this video, you should have a good understanding of how to perform correlative studies for localizing proteins in relation to the different organelles of the cell in a complex tissue sample with super-resolution precision.
