Investigating a Zebrafish Larval Brain Injury Using Large-Scale Scanning Transmission Electron Microscopy

Take resin-embedded, chemically fixed ultrathin brain sections from healthy and brain-injured zebrafish larvae mounted on a one-hole grid. 

The sections are stained with heavy metals to enhance image contrast during microscopic imaging.

Place the grid in a sample holder and transfer the holder to the microscopy chamber.

Align the grid under the electron gun, which generates an electron beam.

An array of electromagnetic lenses and coils focuses the electron beam onto the sections.

Move the sample holder to scan the sections in a raster pattern.

As the electron beam passes through the tissue sections, the stained electron-dense regions scatter more electrons, while the unstained regions transmit the electrons.

The detector collects these electron signals, creating high-resolution brain images.

Compared to the control, the injured brain shows phagocytic microglia containing numerous intracellular inclusions, a characteristic feature of injury-induced neurodegeneration.

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 a frame in approximately 1 to 2 seconds. Then, zoom in at least 100x and scan a small area for ten 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 the 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 4-6 to 4-8, 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 imaging 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 2 to 5 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.