A protocol for Epon-embedding-based correlative super-resolution light and electron microscopy

This protocol describes a detailed step-by-step sample preparation procedure for Epon based post-embedding correlative super-resolution light and electron microscopy (SR-CLEM). The newly developed �uorescent protein, mEosEM, is the core in this protocol. The advantage of this method is to simultaneously obtain high-quality LM and EM images from the same ultrathin section of Epon-embedded sample after conventional chemical �xation.


Introduction
Combining the nanoscale localization information from super-resolution light microscopy with ultrastructure context from EM, SR-CLEM is a versatile imaging tool.Fluorescent probe and labeling strategy that can balance the LM uorescence, EM contrast and structural preservation are in great demand.Genetically encoded uorescent proteins (FPs) are widely used in SR-CLEM for its speci city and biocompatibility.However, the strong oxidizer osmic acid (OsO 4 ) and the hydrophobic embedding resin Epon will destroy the uorescence of existing FPs.With mEosEM, a newly developed PCFP that has OsO 4 and Epon embedding resistance, the same-section SR-CLEM under conventional chemical xation condition with FP is enabled.The procedure described in this Protocol Exchange chapter focuses on detailing the SR-CLEM process of same-section Epon-embedding sample using mEosEM, and is related to the Nature Methods manuscript NMETH-BC38466B titled: "An Epon embedding-and osmium acidresistant uorescent protein for superresolution CLEM".
6. Wash the cell pellet with ice-cold H 2 O three times for 5 min on ice, blocking light.7. Cells were stained en bloc with 2% UA for 1 h on ice, blocking light.
8. Wash the cells with ice-cold H 2 O three times for 5 min on ice, blocking light.9. Dehydrate cells in increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 100%) for 6 min each on ice and further dehydrate cells in 100% acetone one time for 6 min at room temperature, blocking light.
11.In ltrate the cell pellet with 25% Epon in acetone at room temperature for 1 h on shaker, blocking light.
12. In ltrate the cell pellet with 50% Epon in acetone at room temperature for 2 h on shaker, blocking light.
13.In ltrate the cell pellet with 75% Epon in acetone at room temperature for 3 h on shaker, blocking light.
14.In ltrate the cell pellet with 100% Epon at room temperature two times for 12 h on shaker, blocking light.
15. Embed the cell pellet in 100% Epon at 60 °C for 12 h, blocking light.
16. Coverslip cleaning.Coverslips were washed in a mixture of 250 ml of H 2 O, 50 ml of ammonium hydroxide, and 50 ml of hydrogen peroxide in a 500 ml beaker for 2 h at 142 °C.After being rinsed 6 times in H 2 O, the coverslips were carefully transferred to a beaker containing anhydrous ethyl alcohol overnight and dried on a clean bench.17.Pioloform coating of coverslips (Figure 1).To coat a coverslip with Pioloform, we placed a glass slide on the desktop centrifuge using double-sided adhesive tape and then mounted a coverslip on the glass slide.Then, 75 µl of 1% Pioloform in chloroform was applied to the coverslip before spinning for 3 min.This treatment produced a thin layer of Pioloform on top of the coverslip.The coverslip was then coated with 250 µl of 0.1% fresh poly-L-lysine for 1 h.Then, 80-nm gold nanoparticles were diluted with H 2 O to 25% and sonicated for 15 min.The poly-L-lysine was rinsed off with H 2 O, and the coverslip was blowdried.Finally, the diluted gold nanoparticles were placed on the coverslip for 1 h, and the coverslip was rinsed with H 2 O and blow-dried 1 .
18. Cut sections to 100 nm using Ultra-microtome (Leica, EM UC6).Place a section on the center of the pioloform coating of coverslip.
33. Add a drop of 2% UA on a piece of Para lm on the desk.Put the slot grid on the top of the 2% UA droplet, section side down, for 15 min, blocking light.34.Pass the slot grid over nine deionized water droplets for washing.
35.Dry the slot grid with lter paper.36.Place a piece of Para lm in a Petri dish.To absorb excess carbon dioxide from the air in the Petri dish, add a few NaOH pellets inside the dish and cover the Petri dish for 5 min.Add a drop of Sato's triple lead ( ltered through a 0.2-µm lter) onto the Para lm.Put the slot grid on the top of the lead droplet, section side down.Put the cover back on the Petri dish and stain for 5 min.37. Pass the slot grid over nine deionized water droplets for washing.
38. Dry the slot grid with lter paper.The slot grid is now ready for TEM imaging.39.TEM imaging.Find the same cell using different magni cation DIC images (160 X, 100 X, 40 X 16 X, 10 X).TEM images were taken using MORADA G3 camera on FEI Spirit TEM operated at 100 kV with 1 s exposure time.40.SMLM analysis was performed using custom routines in MATLAB as described previously 2 , with a modi cation to remove the background and the noise.The reconstruction parameters were shown in Figure 2. 41. eC-CLEM was used to register the LM image with EM image using gold nanoparticles as ducial makers using non rigid (2D or 3D) mode 3 .The whole predicted error map given by eC-CLEM could be used to evaluate the registration accuracy.

Troubleshooting Time Taken
It takes four days to nish the whole process.