Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

1. Preparation of Patterned Sapphire Discs for Cell Culture

1. Transfer the patterned sapphire discs (see Table of Materials), with an alphanumeric pattern etched onto one of their surfaces, into a 15 mL conical centrifugation tube.
   NOTE: Alternatively, conventional 0.05 mm thick sapphire discs without alphanumeric pattern can be used in which a reference pattern is created by carbon coating them with a finder grid on top. To this aim, they should be cleaned with ethanol before the carbon pattern is applied.
2. Wash them thoroughly with ethanol. Spread them out in a Petri dish with filter paper and let them dry. Place them on a glass slide separate from each other using slim long tweezers.
3. Check with an inverted microscope (4X magnification) whether the pattern of coordinates is readable on every sapphire disc.
   NOTE: If this is not the case, flip the sapphire discs with the help of the tweezers.
4. Store the sapphire discs in a Petri dish.
   NOTE: The protocol can be paused here.

2. Cell Culture with Patterned Sapphire Discs

1. Sterilize the patterned sapphire discs with an ultraviolet (UV) crosslinker for 5 min.
2. Take the sapphire discs to the cell culture biosafety cabinet. Sterilize the long tweezers with ethanol in the biosafety cabinet.
3. Add half of the cell culture medium (see Table of Materials) to a cell culture dish. Transfer the sapphire discs carefully with the long tweezers into the cell culture dish.
4. Seed 1 x 10⁵ Huh7-Lunet cells, stably expressing the T7 polymerase resuspended in the other half of the cell culture medium, onto the culture dish.
   NOTE: Calculate the number of cells to be seeded in such a way that at the end point of the experiment the cell confluency should be not over 20–30%. High cell density will hinder the relocation of the cells at later stages by EM. Furthermore, over-confluency might result in the detachment of cells from the sapphire discs.
5. Check that the sapphire discs remain at the bottom of the culture dish and that the alphanumeric coordinates are still readable with the inverted microscope.
   NOTE: If the discs float in the cell culture medium, use the long tweezers to push the discs to the bottom and eventually to flip them back to the correct orientation.
6. Transfect the cells the next day as previously reported in references^21,23 following the transfection agent's manufacturer instructions.

3. LM Imaging of Cells Growing on Patterned Sapphire Discs

1. Transfer the sapphire discs at the end point of the experiment to a metal imaging plate containing 30–50 µL/ position of pH indicator-free medium to reduce nonspecific background fluorescence.
   NOTE: The metal plate described here (Figure 2) has been designed at the European Molecular Biology Laboratory (EMBL) workshop. In the absence of this plate or a similar one, glass-bottom cell culture dishes could be also used instead for LM. It is important to change the normal cell culture medium for medium without pH indicator. Note also that long exposure times should be avoided because they might cause photobleaching, leading to reactive oxygen species (ROS) accumulation and physiological damage of the cells^24,25.
2. Image the cells under an inverted widefield fluorescence microscope.
   NOTE: When using a green fluorescent protein (GFP)-tagged protein, as shown in the representative results, 450–490 nm and 500–550 nm excitation and emission filters, respectively, should be used.
3. Acquire first a low magnification image (10–20X) of the cells to allocate cells expressing FPs. Record the coordinates of the patterned sapphire discs where the cells of interest are located by using differential interference contrast (DIC) microscopy.
   NOTE: Stitching several areas might help to have a better overview of the location of the cell/cells of interest. Note also that phase-contrast microscopy could be optionally used here.
4. Acquire high magnification (fluorescence and DIC) images (63–100X, oil-immersion objectives) of the same cell/cells to better discern the subcellular localization of the protein of interest within the intracellular space.
   NOTE: DIC images provide contextual information. This information is often critical to correlate LM images with EM micrographs later on, because the final correlation is more precise with "anatomical" landmarks of the cell. As some sapphire discs might break during cryo-immobilization, it is highly recommended to prepare triplicates per condition. Furthermore, some cells detach from the discs during HPF and the subsequent steps of this protocol, while the ultrastructure of other cells might contain artifacts as a consequence of freezing damage. Therefore, at least two areas of the discs should be imaged via LM to ensure that in the end there will be enough cells to be analyzed. Importantly, these two areas should be on opposite sides of the disc. In this manner the resin block where the cells are embedded can be cut into two halves and sections of these areas can be obtained.

Figure 2: Schema for the LM imaging of cells growing on patterned sapphire discs. (A) The sapphire discs are transferred with help of fine tweezers to an imaging plate. (B-C) This plate has been specially designed at the EMBL workshop in Heidelberg to fit into the light microscope, where the sapphire discs can be imaged with cell culture medium without pH indicator. It consists of a metal slide, 75 mm x 25 mm x 1 mm, with four-five holes milled into it with a 3 mm drill bit to fit the size of the sapphire discs. In addition, small grooves were milled on each side of the holes at a 90° angle. These holes make it easier to access the discs with forceps when manipulating them. To allow for optimal imaging conditions glass coverslips No. 0 (0.085 to 0.13 mm thick) were glued below the holes with UV glue and placed under UV light to harden. (D) High magnification image of the patterned sapphire disc depicting the alphanumeric coordinates that are etched on its surface. The patterned sapphire discs are commercially available (see Table of Materials). Please click here to view a larger version of this figure.

4. Cryo-Immobilization of Cells Growing on Patterned Sapphire Discs via HPF

1. Place the "A" and "B" aluminum carriers for HPF (see Table of Materials) in a Petri dish with filter paper soaked with 1-hexadecene. Immerse the patterned sapphire discs containing the cells in a Petri dish with 1-hexadecene.
   NOTE: Move the discs a bit to wash the cell culture medium away.
2. Assemble the sapphire discs between an "A" and a "B" aluminum carrier in the HPF holder as follows (Figure 3B). On the bottom, place a "B" carrier with its flat side facing upwards. Place the sapphire discs on this flat side with the cells facing upwards. Add an "A" carrier with its 0.1-mm depth facing the cells.
   NOTE: Because the patterned sapphire discs are thicker than the conventional sapphire discs, the commercially available "A" carrier (designed to be used with non-patterned 0.05-mm sapphire discs) had to be cut down to 0.05 mm at this 0.2 mm depth side at the EMBL workshop in order to fit into the HPF holder (Figure 3A). To this aim, the "A" carrier is inserted into the end of a metal tube so that its 0.2 mm side is exposed and remains still while cutting it down. The cut down side is then sanded so that it is flat. Alternatively, a special aluminum carrier can be used on top of the patterned sapphire disc (commercially available, see Table of Materials), being in this case the HPF "sandwich" composed only of the sapphire disc and this carrier.
3. Close the holder of the HPF machine and freeze the cells.
   NOTE: This protocol is specific for a particular HPF machine. Other HPF machines could be alternatively used^6,26. In any case, please be aware of the existence of new generation machines from the suppliers.
   CAUTION: Use safety goggles and hearing protection headset.
4. Place the frozen "sandwich" in a polystyrene box with liquid nitrogen for temporary storage.
   CAUTION: Use goggles and get in contact with the local safety officers to be informed about the potential dangers when handling liquid nitrogen.
   1. To avoid confusion, place each "sandwich" in a separate 0.5 mL microcentrifuge tube (inside the polystyrene box) marked with a number.
      NOTE: The protocol can be paused here. If freeze substitution (FS) cannot be immediately performed, the samples can be stored in cryo-tubes (with a hole punched in the top) inside boxes, that are held in a rack in a cryogenic liquid nitrogen dewar (see Table of Materials). This liquid nitrogen container should be located in a room with oxygen gas sensors, to avoid suffocation in case of an oxygen deficiency.
      CAUTION: All the procedures described below (steps 5 and 6) must be carried out in a biosafety cabinet. Furthermore, most of the reagents used are hazardous. Before using them, it is mandatory to read carefully the Material Safety Data Sheets provided by the manufacturers, as well as ask the safety officers about the local rules to ensure safe handling. For the correct disposal of these used materials, as well as for the proper use of the equipment described below, it is also required to consult the local institute’s health and safety procedures. 

Figure 3: Schema of the assembly of patterned sapphire discs between two aluminum carriers for HPF. (A) Cut-away views of a conventional "A" carrier, that has to be cut on its deeper side from 0.2 mm to 0.05 mm. (B) The 0.16 mm thick sapphire disc with the alphanumeric pattern etched on the surface is assembled into two aluminum carriers for HPF, as follows: The sapphire disc is placed on the flat side of a "B" carrier with the cells facing upwards. A chopped "A" carrier with its 0.1-mm depth facing the cells is placed on top to close the "HPF sandwich". The aluminum carriers and the patterned sapphire discs are commercially available (see Table of Materials). Please click here to view a larger version of this figure.

5. FS of Cryo-Fixed Cells

1. Fill up the FS machine (see Table of Materials) with liquid nitrogen. Set the temperature to -90 °C and wait until the machine reaches this temperature.
2. Prepare the FS medium containing 0.2% osmium tetroxide (OsO₄) (v/v), 0.1% uranyl acetate (UA) (w/v) in glass-distilled acetone while the FS machine is cooling down.
   NOTE: To prepare, (for instance) 12 mL of FS medium, mix 600 µL of 4% OsO₄with 0.012 g of UA in a small glass container. Dissolve this mixture in an ultrasonic bath device for 5 min. Add 11.4 mL of glass-distilled acetone with a pipette and mix well.
3. Fill up 1.5 mL microcentrifuge tubes with 200-500 µL of FS medium, close the lid and transfer them to the FS machine. Wait 10 min for the FS medium to cool down.
4. Transfer the frozen "sandwiches" containing the sapphire discs with cells in liquid nitrogen using cooled long tweezers to the tubes containing the FS medium.
   CAUTION: Use cryo-protective gloves and goggles.
   NOTE: The HPF "sandwiches" could be disassembled spontaneously during their handling. In this case, make sure that the frozen sapphires discs are transferred to the microcentrifuge tubes. The aluminum carriers can be discarded.
5. Run the FS as follows until the next day²⁷: from -90 °C to -80 °C for 8 h; from -80 °C to -50 °C for 8 h; from -50 °C to -20 °C for 2 h; from -20 °C to 0 °C for 2 h.

6. Resin Embedding of Freeze Substituted Samples

CAUTION: All the procedures described below must be carried out in a biosafety cabinet. Furthermore, most of the reagents used are hazardous. Before using them, it is mandatory to read carefully the Material Safety Data Sheets provided by the manufacturers, as well as ask the safety officers about the local rules to ensure safe handling. For the correct disposal of these used materials, as well as for the proper use of the equipment described below, it is also required to consult the local institute's health and safety procedures.

1. Take out the substituted specimens from the FS machine and place them on ice for 20 min.
2. Keep the samples at room temperature for 20 min and wash them thoroughly (3 times, 10 min each) with glass-distilled acetone. Discard the aluminum carriers with long tweezers if they are still in the microcentrifuge tubes after the FS.
3. Infiltrate the cells (on the remaining sapphire discs) in a four-step epoxy resin series using 1 h incubations in 25%, 50%, and 75% resin in glass-distilled acetone, followed by overnight incubation with 100% resin. Exchange the 100% resin the next day for 1h.
4. Carefully place the sapphire discs into flow-through rings mounted in reagent baths filled with 100% resin. These rings are used as polymerization molds for 10 sapphire discs.
   NOTE: The sapphire discs must be completely pushed to the bottom with the coordinates facing upwards in a readable manner. This could be checked with help of a binocular attached to a fume extractor. Alternatively, a binocular inside a biosafety cabinet could be used instead. Importantly, there are numbers (1-10) engraved on the bottom of the reagents that help to identify the samples.  In addition, for identification of the samples a paper tag could be included in the resin block.
5. Polymerize the samples in the oven at 60 °C for 48 h.

7. Removal of Patterned Sapphire Discs from the Polymerized Resin Blocks

1. Remove the hard resin blocks containing the cells from the oven.
   NOTE: The protocol can be paused here. If not, let the blocks cool down to room temperature before cutting them.
2. Cut the embedding molds with a razor blade to have access to the resin blocks. Remove the cylindrical resin blocks using tweezers.
   NOTE: If a paper tag has not been included in the resin block before polymerization, number the blocks with a permanent marker and store them in 1.5 mL microcentrifuge tubes. The protocol can be paused here.
3. Immerse the tip of the resin block containing the sapphire disc in a polystyrene box containing liquid nitrogen until it stops "bubbling". Then, immerse the tip of the resin block in boiling H₂O.
4. Repeat the two previous steps as many times as needed until the sapphire disc falls off of the resin block.
   CAUTION: Use goggles and cryo-protective gloves.
   NOTE: If this does not work, use a razor blade to remove a bit of the polymerized resin around the discs before trying again. The protocol can be paused here.

8. Targeted Trimming and Ultrathin Sectioning for TEM

1. Identify the area on the resin block face where the cells of interest are present by examining the block face with the binocular of an ultramicrotome.
   NOTE: The negative imprint of the coordinate pattern from the patterned sapphire discs is retained on the block face. This allows the identification of the area where the cells of interest are located for targeted trimming. The LM images must be flipped vertically in order to facilitate the finding of the same position of the block face.
2. Trim the embedded cell monolayer containing the cell/cells of interest to a small flat pyramid with the shape of a trapezium (~ 200 µm × 250 µm average size) with a clean razor blade.
3. Obtain 70-nm ultrathin sections of the embedded cells with a 35° diamond knife.
4. Place the sections on EM slot grids with help of ultra-fine tweezers.
   NOTE:: The use of mesh grids should be avoided because the sections could be masked by the grid bars.
5. Store the grids in a grid box.
   NOTE: The protocol can be paused here.

9. TEM Analysis of Ultrathin Sections

1. Place the slot grid into the holder of the transmission electron microscope. Acquire low magnification (overview) images, where the complete cell profile of the cell of interest is visible.
   NOTE:: The cell/cells of interest can be found back by using the cell profiles and the locations of the cells to one another as references, comparing to the DIC views acquired before by LM.
2. Acquire high magnification images of the cell/cells of interest, to try and find, at the ultrastructural level, features that correspond to the fluorescent signals observed previously by LM.
   NOTE: Montage images can be obtained at high magnification to have an overview at high resolution of the cell/cells of interest. Furthermore, it is often necessary to acquire images of the same cell in consecutive serial sections to be able to reconstruct the cell in 3D before comparing to the fluorescence images.