Dataset DOI: 10.5061/dryad.dz08kpsd2

Description of the data and file structure

The SBEM volume of the D. melanogaster antenna was generated following the CryoChem protocol (Tsang et al., 2018). Briefly, dissected antennae were immediately subject to high-pressure freezing in a solution of 0.15 M sodium cacodylate and 20% BSA using a high-pressure freezing machine (Bal-Tec HPM 010). The frozen samples were then transferred to a freeze-substitution solution containing 0.2% glutaraldehyde (#18426, Ted Pella, CA, USA), 0.1% uranyl acetate (Electron Microscopy Sciences, USA), and 1% water in acetone (#AC326800010, ACROS Organics, USA) in a liquid nitrogen bath. Freeze substitution was performed in a Leica EM AFS2 device held at –90°C for 58 hr, from –90°C to –60°C for 15 hr, at –60°C for 15 hr, from –60°C to –30°C for 15 hr, and then at –30°C for 15 hr. During the final hour at –30°C, samples were washed three times (20 min each) in an acetone solution containing 0.2% glutaraldehyde and 1% water, before being transferred to ice for 1 hr prior to rehydration (see below).

The cryofixed samples were gradually rehydrated in a series of nine rehydration solutions, each for 10 min on ice.

1. 5% water, 0.2% glutaraldehyde in acetone
2. 10% water, 0.2% glutaraldehyde in acetone
3. 20% water, 0.2% glutaraldehyde in acetone
4. 30% water, 0.2% glutaraldehyde in acetone
5. 50% 0.1 M HEPES (Gibco, Taiwan), 0.2% glutaraldehyde in acetone
6. 70%, 0.1 M HEPES, 0.2% glutaraldehyde in acetone
7. 0.1 M HEPES
8. 0.1 M sodium cacodylate with 100 mM glycine
9. 0.1 M sodium cacodylate

After rehydration, the samples were subject to en bloc heavy metal staining in a solution of 2% OsO₄, 1.5% potassium ferrocyanide, and 2 mM CaCl₂ in 0.1 M sodium cacodylate for 1 hr at room temperature. Then, samples were washed five times with water (5 min per wash) before being incubated in 0.5% thiocarbohydrazide (Electron Microscopy Sciences, USA) for 30 min at room temperature. Following another series of water washes, the samples were incubated in 2% OsO₄ for 30 min at room temperature. After a final rinse with water, the samples were transferred to 2% aqueous uranyl acetate (filtered with 0.22 µm filter) at 4°C overnight. The samples were then washed with water and then subjected to the dehydration steps described below.

Dehydration was performed in six consecutive 10 min steps: 70% ethanol, 90% ethanol, 100% ethanol (twice), followed by 100% acetone (twice). All ethanol steps were conducted on ice. The first acetone step used ice-cold acetone, while the second was performed with acetone at room temperature.

Resin infiltration was carried out in a 1:1 solution of Durcupan ACM resin and acetone and incubated overnight on a shaker. The samples were then transferred in fresh 100% Durcupan ACM resin twice, with a 6–7 hr interval between transfers. During incubation in 100% resin, the samples were placed in a vacuum chamber on a rocker to facilitate the evaporation of residual acetone. After an overnight incubation in 100% resin, the samples were embedded in fresh resin and polymerized at 60°C for at least 2 days. The composition of Durcupan ACM resin (Sigma-Aldrich) was 11.4 g component A, 10 g component B, 0.3 g component C, and 0.1 g component D.

Microcomputed X-ray tomography was used to determine the position and proper orientation of the resin-embedded specimens. Samples were mounted on aluminum pins with conductive silver epoxy (Ted Pella) and sputter coated with gold-palladium for SBEM imaging with a Gemini SEM 300 (Zeiss) equipped with a Gatan 3View 2XP microtome system and the OnPoint backscatter detector.

The antennal SBEM volume was acquired at 2.5 kV using a 30 μm aperture, with the electron gun set to analytic mode and the beam operating in high-current mode. Nitrogen gas was used for focal charge compensation to reduce charging artifacts. Imaging was performed with a dwell time of 1 μs, a pixel size of 5 nm, and a Z-step of 40 nm. The X and Y pixel numbers were 1909 and 2061, respectively, and there were a total of 2751 Z slices. After data collection, the images were converted to MRC format, and rigid alignment of the image slices was performed using cross-correlation in the IMOD image processing package.

Files and variables

File: SBEM_Volume.mrc

Description: Drosophila melanogaster antenna serial block-face electron microscopy volume. The X and Y pixel numbers were 1909 and 2061, respectively, and there were a total of 2751 Z slices.

Code/software

Option 1. IMOD

We used the IMOD image processing package (https://bio3d.colorado.edu/imod/) to view these volumes. Download the appropriate version that is compatible with your operating system.

1. Open the 3dmod application (the main viewing component of IMOD).
2. In the startup window, click on "Select..." next to "Image file(s)" and browse to select the downloaded file.
3. In case of low system memory: Before clicking "Open", look for the "Cache" option in that same startup window. Set the cache to 5 sections. This reduces the memory used so the file can load smoothly.
4. Click Open.

Option 2: Fiji / ImageJ

If you do not have specialized electron microscopy software, you can open the file in Fiji using the Bio-Formats plugin to bypass memory limits. However, this option results in poorer image quality.

1. Open Fiji/ImageJ.
2. Go to File > Import > Bio-Formats.
3. Select the .mrc file.
4. In the Bio-Formats Import Options window that pops up, look under the "Dataset Organization" section.
5. Check the box for "Use virtual stack".
6. Click OK. The file will stream directly from your storage without overloading your computer's RAM.

System Requirements

Because these volumes are large, the primary bottleneck is system memory (RAM).

• Minimum RAM: 8 GB (but the file must be opened with cache turned on, as described above).
• Recommended RAM: 16 GB or higher to load the volume at full resolution.
• Operating System: Windows, macOS, or Linux.

Access information

Other publicly accessible locations of the data:

• Cell Image Library (https://www.cellimagelibrary.org/) with the accession numbers CIL:57519.