ssSEM and FIB-SEM imaging

The serial ultrathin sections produced from ultramicrotomy were imaged under a high-throughput scanning electron microscope (Merlin, Carl Zeiss Microscopy GmbH, Oberkochen, Germany; located at ScopeM). The sections together with the holder was fixed onto the sample stage and loaded into the microscope chamber.

The approximate locations of the serial sections were first determined with 5 kV of extra-high tension (EHT, the voltage applied to the electron gun) and an in-lens detector. Ten intact consecutive sections near the end of the ribbon, which were closer to the block face of the sample, were selected, and the locations of the HVC region were determined on the sections (Figure 2. 3, c).

The stage was slowly moved upwards so that the working distance (WD, the distance between the sample surface and the electron gun) was 3.5 mm. The EHT was then reduced to 1.6 ± 0.1 kV, and the view mode was switched to the energy selective backscattered electron detector (EsB), with the EsB Grid voltage set to 550 V and the detector probe current set to 550 pA. In my preparation, the EsB detector provided more membrane contrast and dynamic signal ranges. Therefore, all of the high-resolution ssSEM datasets were acquired with the EsB detector in the pixel-averaged noise-reducing scanning mode. After fine adjustments of the imaging parameters, the focus and astigmatism correction were optimized. All of these adjustments were set manually with SmartSEM software (Carl Zeiss Microscopy GmbH).

The parameters of image acquisition were then set with ATLAS 3D software (Fibics Incorporated, Ottawa, Canada). The brightness and contrast of the images were fine-tuned to maximize the dynamic range of the electron micrograph. Imaging sites of approximately 80 × 80 µm on the tissue sections that were at the center of HVC were selected, located, and stored in ATLAS 3D. The relocation on each section was precisely defined to ensure repeat imaging of the same region on the selected serial sections. The detection limit of the EsB detector in the current setting was around 50 µm. Beyond this limit, focus distortion and brightness aberrations began to appear.

Therefore, for each 80 × 80 µm imaging site, 2 × 2 square-shaped tiles were automatically generated by the software to cover the entire imaging area of the ROI.

Relocation of sequential imaging of the four tiles was done by automatic stage

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movement. Auto-focus and auto-stigmator were implemented on each tile prior to the acquisition. For all of the ssSEM datasets in these studies, the imaging pixel size was set to 4 nm, and the electron beam dwell time was set to 7 µs. Once all of the parameters were set, the scanning and image acquisition were performed automatically with the ATLAS 3D software. Fluctuations in brightness and contrast occurred due to instability of the electron source. Therefore, the quality of the acquired images was monitored frequently with remote desktop control (VNC Viewer, RealVNC Ltd, Cambridge, UK). For any quality problems, the current session was stopped, reconfigured, and then restarted from the end of the last acceptable acquisition point. The acquired ssSEM datasets, stored in 8-bit grayscale tiff formats, served to quantify the synapse density with the dissector method (see Figure 2. 5 and Method section 2.2.5).

On the other hand, the carbon-coated brain sample blocks were imaged with a FIB-SEM microscope (Auriga 40, Carl Zeiss Microscopy GmbH; located at ZMB).

The sample holder was placed on the staged and aligned with the electron gun as was done with the Merlin microscope. The EHT was turned on and set to 5 kV, and the secondary electron detector was used to obtain live images of the block surface.

The WD was at least 10 mm. Focus, astigmatism, brightness, and contrast were adjusted to visualize the sample block face.

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Figure 2. 4: FIB-SEM imaging of the block-face of the brain sample. (a) Photograph of the chamber during the imaging session. The blue dashed line indicates the electron beam path, the green dashed line indicates the gallium ion beam path, and the red dashed line indicates the plane wherein the sample surface lies. Note that the angle between the electron and ion beams is fixed in the instrument at 54° and that the sample surface should be perpendicular to the ion beam during milling and imaging. (b) Overview of the sample surface before the FIB imaging session. The red dashed circle indicates the location of HVC. (c) Overview of the same sample surface after the FIB imaging session.

The red square indicates the imaged area. (d) A magnified image of the red square in (c).

The imaged sample was already milled away by the ion beam. The yellow square here indicates the approximate X-Y location of the imaged stack.

The center of HVC was again located under the FIB-SEM microscope (Figure 2. 4, b). The stage was then tilted 54° in order to facing perpendicular to the gallium ion beam (Figure 2. 4, a). The WD was then reduced to 4.8 mm, and the EHT was reduced to 1.7 ± 0.2 kV. This procedure was performed to ensure that the ROI on the sample surface was located precisely at the coincident point of the cross-beam system (the crossing point of the blue and green lines in Figure 2. 4, a). The X-Y axis and focus were monitored and adjusted throughout this process in order to maintain a clear image of the center of HVC. The FIB gun was then switched on.

Using the same magnification and the common features on the tissue sample surface, the SEM and FIB views were coupled. The imaging mode was switched back and forth between the SEM and FIB views. The Z-axis was moved only in the FIB view, while the beam shift in the X-Y axis was only used in the SEM view. Once the SEM and FIB views were well aligned, a 40 × 40 µm ROI was chosen near the center of HVC while avoiding large synapse-free structures, such as blood vessels and cell bodies. A coarse trench was then milled into the tissue at the ROI location with a 16-nA ion beam current to expose the cross-sectional surface (Figure 2. 4, c and d).

A fine polish was then performed with 600 pA ion current. Fine tuning of the electron beam and the imaging parameters were then performed on the exposed cross-section with SmartSEM software, as was done during the setup of the Merlin microscope. Similarly, the same imaging parameters were used and fine-tuned in the ATLAS 4D software (Fibics Incorporated). The EsB detector signals were obtained while the EsB Grid voltage was set at 1.3 kV. An angle correction was applied in the acquired image to compensate for the tilting of the sample surface. The image pixel size was set to 5 nm for all of the FIB-SEM datasets acquired, and the electron dwell time was set at 13 µs. 10-nm-thick layers were then serially milled away with a 600-pA ion current, and the exposed block surfaces were serially imaged with a 8 × 8-µm square-shaped ROI window (Figure 2. 4, d, red square) defined in the ATLAS 4D software. The milling and imaging progresses were monitored. If quality problems occur, or hidden (which means contained inside the tissue block and subsequently exposed from advanced tissue milling) large synapse-free structures appear in the ROI, the current session would be stopped and discarded. After reconfiguration, a new acquisition would be started. Auto-focus and auto-stigmator

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were set to perform every 90 min to counter the physical milling-induced focus drift in the Z-axis.

The final FIB-SEM image stacks for all of the samples were 8 × 8 µm with 800 serial slices, which physically corresponded to an 8 × 8 × 8-µm cube. Thus, the acquired FIB-SEM datasets, stored as 8-bit grayscale multipage tiff image sequences, served to perform the synapse subtype classifications and segmentations (see Method section 2.2.6 and 2.2.7).