The overall goal of this large-scale electron microscopy or a nanotomy experiment is to define alternations from the macromolecular level to the tissue level, in today's case, in the degenerating zebrafish brain. Nanotomy can help answer key questions in life science. For example, in neurodegeneration and in type 1 diabetes research.
The main advantage of nanotomy is that unbiased information is acquired, ranging from macromolecules, organelles, cells, to tissues. This allows quantitation and open-access data sharing via nanotomy.org. Although nanotomy provides insight into immune maintenance and microglia in the zebrafish neurodegenerative brain, it also is applied to other disease models, including cell culture, mouse, and even human tissues.
Generally, scientists new to this technique struggle because the amount of data is overwhelming. This can be a thousand times more than obtained with traditional EM.After fixing zebrafish larvae according to the text protocol, cut the larval heads rostrally to the hindbrain to facilitate penetration of the osmium. Place the larvae in 1%osmium tetroxide, 1.5%potassium ferrocyanide, in 0.1 molar sodium cacodylate and incubate on ice for two hours.
Then use double distilled water to rinse the embryos three times for five minutes each time. Prior to embedding, the samples need to be dehydrated in a series of ethanol. To embed the embryos, after incubating in diluted epoxy resin overnight according to the text protocol, remove the diluted resin and use pure resin to replace it.
Incubate for 30 minutes, then replace the resin a second time and incubate for another 30 minutes. After refreshing the resin a third time, incubate at room temperature for three hours. Then, incubate at 58 degrees Celsius for 15 minutes.
Finally, incubate under low pressure at room temperature for one hour. Next, under a dissection microscope, use a needle or toothpick to orient the heads in commercially available silicon flat embedding molds. Then polymerize the epoxy resin at 58 degrees Celsius overnight.
When the specimen is fully hardened, use razor blades to trim away excess resin from the stub. To detect the right positioning, use a glass knife or a diamond histoknife on an ultramicrotome to cut semithin larvae sections for toluidine blue/basic fuchsin. Transfer the semithin sections onto microscopic slides by picking them up with a glass Pasteur pipet whose tip has been closed by melting it in a flame.
Dry on a hot plate until no water is left. Next, place the samples in 1%toluidine blue in water and incubate the sections on a hot plate for 10 seconds to stain them. Then, after using water to rinse the sections, use 05%basic fuchsin in 1%sodium tetraborate to stain the samples for 10 additional seconds.
With a normal light microscope at 10X to 40X, examine the samples. When the proper site or orientation is reached, use easily identifiable anatomic structures, including olfactory pits, eyes, or gray-white matter boundaries, to identify the brain region of interest during ultrathin sectioning and to adjust the sectioning angle when the sample is tilted. Continue sectioning the epoxy resin block with a diamond knife to cut ultrathin 70 nanometer sections.
Mount each section on a single slot L2 by 1 form barcoded copper grid to allow acquisition uninterrupted by grid bars. A square millimeter might seem small. It will just fit in the opening.
In this way, we prevent grid bars blocking our sample. Realize every artifacts introduced in our sample will be recorded compared to traditional EM.Contrast the samples with heavy metals according to the text protocol and store in a grid box. To mount the sample in the scanning electron microscope, or SEM, place the grid from the transfer box with the section in the multiple grid sample holder and transfer it into the chamber of the SEM.
After aligning the detector according to the text protocol, pre-irradiate the sample by zooming out so the complete area to be scanned fits the image window. Once the aperture has been changed to 120 micrometers and the image has been defocused, use the reduced area scan option to make the scanned area as tight as possible. Next, set the frame rate to scan the frame in approximately one to two seconds.
Then zoom in at least 100X and scan a small area for 10 seconds. If the brightness in the area still changes, continue pre-irradiation. When this area does not change brightness compared to its surroundings, pre-irradiation is sufficient.
While in focus, select the lightest area or feature and set the scan speed so that details are visible. In the microscope software, adjust the brightness and contrast by carefully watching the histogram to keep all the pixels in the dynamic range. Do the same for the darkest areas and features.
Go back to the bright area and check again so that there is some space on both sides of the histogram. For steps four six to four eight, adjustment of the brightness and contrast has to be done very carefully so that the whole area is within the dynamic range. Zoom out so the complete area to be scanned fits the imagining window and start the large area acquisition program.
Then use the wizard option to set up a mosaic by selecting an area from the screen. Use a pixel size of two to five nanometers, depending on what details are necessary, and set a dwell time of three microseconds for scanning transmission electron microscopy, or STEM. Press optimize to check microscope settings and the time needed will be displayed.
Then switch on the external scan generator again and press continue. To analyze the STEM data, open a large scale EM file viewer program and open the file called mosaic info, which will open the tiled tif files. Choose the option Auto Stitch Entire Mosaic and choose the following parameters:overlap mode equals half, stitching threshold equals 0.90, and noise reduction equals automatic.
If stitching criteria cannot be fulfilled, zoom in and manually place the tile in position and click Continue As Is.Export the data as an HTML file or as a single tif. Include the final pixel size and the file name to enable measurements later, then analyze the data according to the text protocol. As shown here, nanotomy of control brain sections reveals typical ultrastructural features of neural tissue of the rostral forebrain including olfactory fiber bundles, neuronal nuclei and neuronal sub-compartments including synapses.
Here, subcellular structures include different nuclear morphologies and foci in different cell types and organelles including Golgi apparatus, endoplasmic reticulum, mitochondria, synaptic vesicles, and post-synaptic densities. Unexpectedly, the nuclei of cells lining the ventricle are very electron dense compared to other cells dispersed more laterally in the brain. In microglia, the nuclei also show dark foci, possibly heterochromatin, that are absent in other cells in the brain.
This image from a large scale EM of a coronal section in a zebrafish larva undergoing neuronal ablation reveals phagocytic microglia. Features characteristic of microglia in mammalian cells were also found in these cells, including prominent Golgi apparatus and numerous inclusions such as lysosomes. Once mastered, acquisition can be done within a day, where traditional EM will at least cost you a week.
While attempting this procedure, it's important that a microscope operator plays a crucial role in obtaining high quality images. This is not just a push a button technique. We now applied nanotomy not only to the zebrafish model of neural degeneration, but we also used it for the immunolabeling of cells and to study tissues of flies, human tissues, and more.
This technique paves the way for any researchers in any field. They can revisit the data sets online on nanotomy.org. They can then explore putative alterations, ranging from the nanometer to the millimeter scale and all models studied.
After watching this video, you should have a good understanding of how to apply nanotomy to your research question and the cellular or tissue system you are investigating. Don't forget, only a professional should do sample preparation because of the toxic reagents and the ethical consideration, but everybody can try the website nanotomy. org at home.
