Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local authorities.

1. Preparation of Ultrathin Sections on Silicon Wafers

1. Sample fixation
   1. Prepare the fixative solution containing 0.1% glutaraldehyde, 4% formaldehyde in 0.1 M cacodylate buffer.
      NOTE: CAUTION! Use caution when working with glutaraldehyde, formaldehyde and cacodylate buffer, wear appropriate personal protective equipment and work in a fume hood.
   2. Euthanize 5 days post fertilization (5 dpf) zebrafish larvae with tricaine (ethyl 3-aminobenzoate methanesulfonate, 0.4% w/v in PBS (phosphate buffer saline, pH 7)) as previously described¹⁵.
   3. Fix the larvae by immersion in pre-chilled fixative solution on ice. Remove the head and incubate overnight at 4 °C with gentle rocking.
   4. Dissect the eyes by trimming the tissue around the eye using fine forceps and a microdissection scalpel (under a binocular) in chilled fixative solution on a 1% agarose-bedded plate. Transfer the eyes to a tube with fresh pre-chilled fixative.
   5. Wash twice in PBS (phosphate buffer saline, pH 7.4) for 5 min each wash at room temperature (RT).
2. Gelatin infiltration and mounting
   1. Warm up 15 mL local food brand gelatin 12% w/v in PB (phosphate buffer, 0.1 M pH 7.4) at 40 °C. Remove the PBS and add gelatin solution to the tubes containing the eyes. Tap the tube gently to ensure the gelatin infiltrates the sample and incubate for 10 – 30 min at 40 °C in a thermoblock with gentle shaking or in a water bath.
   2. Fill 12 mm x 5 mm x 3 mm silicon or polyethylene flat embedding molds with warm gelatin in a 40 °C water bath. Add two eyes per mold using a pipette, align them properly under a binocular using a dissection needle and let the gelatin cool down at room temperature for 1 min and harden at 4 °C for 20 min.
   3. Re-trim the gelatin block under the binocular to fit one eye per block using a razor blade.
   4. Transfer the gelatin embedded eyes to 2.3 M sucrose in PB on ice. Incubate at 4 °C overnight.
   5. Exchange to new 2.3 M sucrose solution and store at 4 °C or -20 °C; after this step, samples are ready for sectioning or can be stored at -20 °C for several weeks to months.
   6. Re-trim the gelatin block to almost the size of the eye before transferring to a cryo-pin. Freeze in liquid nitrogen and transfer to a cryo-ultramicrotome.
3. Cryosectioning
   1. Cut 110 nm thick-sections at -120 °C with a diamond knife in the cryo-ultramicrotome.
   2. Pick the sections with a wired loop containing a droplet of 2% methylcellulose (in water) and 2.3 M sucrose solution (1:1). Transfer sections to a 7 mm x 7 mm silicon wafer. Store sections at 4 °C until further processing.

2. Immunolabelling

1. Wash wafers with PBS at 0 °C for 20 min on four drops by placing the wafers upside down on the drops. Wash in PBS 2x 2 min at room temperature.
2. Incubate 3 times in 0.15% glycine in PBS, for 1 min each. Wash 3x for 1 min/wash with PBS. Pre-incubate with PBG (PBS with 0.5 % Bovine Serum Albumin BSA and 0.2 % Gelatin type B) for 5 min.
3. Incubate with rabbit anti-Tom20 (see the Table of Materials) in PBG (4 µg/mL) for 30 min at room temperature.
4. Wash 6x for 1 min/wash in PBG. Pre-incubate with PBG for 5 min at RT. Incubate with anti-rabbit Alexa 647 F(ab')₂ (see the Table of Materials) in PBG (7.5 µg/mL) for 30 min.
5. Wash 6x for 1 min/wash in PBG. Wash 3x 2 min in PBS. Incubate with DAPI (4 µg/mL) in PBS for 10 s. Wash 2x 2 min in PBS.

3. Super-resolution Microscopy

1. Incubate the wafer briefly (10 s) by placing it on a droplet of a 1:1 solution of glycerol (80%) and imaging buffer containing an oxygen scavenging system (200 mM Phosphate buffer containing 10% glucose, 0.5 mg/mL glucoseoxidase, 40 µg/mL catalase, 15 mM beta-mercaptoethylamine hydrochloride (MEA HCL), pH 8.0).
2. Transfer the wafers (section facing down) to a glass bottom (thickness 170 ± 5 µm) Petri dish onto a fresh drop of the 1:1 mixture of glycerol (80%) and imaging buffer (as in step 3.1).
3. Remove from all sides, with a pipette, most of the liquid underneath the wafer. Use silicone stripes to fix the wafer to the bottom of the Petri dish.
   NOTE: Silicone stripes are made out of two-component silicone-glue (3 mm x 12 mm).
4. Image sections on an inverted microscope using a high numerical aperture (e.g. 160X / NA 1.43; see the Table of Materials) oil immersion super resolution dedicated objective. Prior to imaging, let the sample equilibrate to microscope temperature in order to minimize/reduce lateral and axial drift.
5. Center the area of interest and acquire first widefield epifluorescence reference images. Change to the super resolution operation mode. Adjust the exposure time of the camera to 15 ms and set the electron multiplying (EM) gain to the maximum of 300.
6. Illuminate sample with the 642 nm continuous wave laser at maximum laser power (corresponding to ~2.8 kW/cm²) in epifluorescence mode. As soon as the single molecule blinks are well separated in each frame, so that the probability is low that individual signals overlap, set the laser power to ~0.7 kW/cm². Record the raw image in epifluorescence mode by acquiring a minimum of 30,000 frames.
   NOTE: All these parameter can vary depending on different specimens and labeling densities.
7. From the raw data, generate a reconstruction event list (localizations of each single molecule blink in the raw image) using a detection threshold of 30 photons (needs to be adjusted according to the sample) by clicking "Evaluate" in the 't-series analysis' under 'Tools'.
8. Visualize the super resolution image by Gaussian fitting¹⁶ applying a rendering pixel size of 4 nm by clicking "Create Image" in the 'event list processing panel' under 'Tools.'
   NOTE: For generating the super resolved image the integrated software tools of the system used in this study were employed. However, the described super resolution imaging could be performed on any other single molecule localization based system, and the data could be processed with open source software tools as thunderSTORM¹⁷.

4. Platinum Shadowing

1. Remove silicon stripes and add a drop of PBS close to the edges of the wafer to lift it up from the Petri dish. Wash the wafer 2x 2 min in PBS, then post fix it with 0.1% glutaraldehyde in PBS for 5 min. Wash 2x for 2 min/wash in PBS.
2. Incubate the wafer twice for 5 min/incubation in 1 drop of methylcellulose 2 % in water on ice. Insert the wafer in a centrifuge tube and centrifuge at 14,100 x g for 90 s. Mount it on a SEM aluminum stub with conducting carbon cement.
3. Add a layer of 2 to 10 nm of platinum/carbon on the sample by rotary shadowing at 8° using an electron beam evaporation device settings: 1.55 kV, 55 mA, at 0.3 nm/s, and an angle of 8°, rotation level 4.

5. Scanning Electron Microscopy

1. Image sections with a scanning electron microscope at 1.5 kV, 2 mm working distance and with an In lens secondary electron detector.

6. Alignment of Light and Electron Microscopy Images

1. Open both types of images with Fiji¹⁸ by clicking "File|Open". Adjust canvas size by clicking "Image| Adjust| Canvas size" and bring both images to a stack by clicking "Image| Stacks| Images to stack". Save the stack as tiff file type.
2. In Fiji, open a new TrackEM2¹⁹ interface by clicking "File|new|TrackEM2 (new)". Import the stack with both images by right clicking on the black window and selecting "Import stack".
3. Align light microscopy image to electron microscopy image manually with landmarks by using a right mouse click on the image and selecting "Align|Align layer manually with landmarks".
   1. Pick the select tool (the black arrow) to add landmarks. Use the shape of nuclei as reference to select the same edges in both images (Supplementary Figure 1). Add several points (minimum three points need to be selected). Apply alignment with an affine model by right mouse clicking, and selecting "Apply transform| Affine model".
4. Change layer transparency (see Supplementary Figure 1) to assess the quality of the alignment.