Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

NOTE: This protocol pertains to samples containing cells grown or deposited on transmission electron microscopy (TEM) 3 mm flat gold grids with a holey carbon support film that have been vitrified by plunge freezing or high-pressure freezing. This protocol assumes that samples have already been imaged using a conventional epifluorescence and brightfield microscope to map locations of interest for imaging in cryoSIM. See Figure 1 for an overview of the entire protocol.

1. Preparation of the cryo-stage

1. Prepare ice-free liquid nitrogen (LN₂) by passing LN₂ through a funnel lined with any standard scientific dry paper wipes.
   NOTE: As LN₂ causes burns, wear appropriate personal protective equipment, including cryo-protective gloves and goggles, when handling it. Such liquids may displace oxygen and cause rapid suffocation and should be handled in a properly ventilated area.
2. Remove surface dust from the cryo-stage with pressurized air. Expel liquid from the pressurized air container before using it.
3. Ensure the sample-loading cartridge is in place with the appropriate chambered sample holder (check that there is no grid left in the sample holder from a previous experiment) (Figure 2).
4. Remove the lid from the external dewar of the cryo-stage, and pour filtered LN₂ until approximately 1/4^th full. Wait until initial boiling subsides before pouring more; fill the vessel to about 2/3^rd full. Replace the lid carefully, pointing the nozzle away from the handler and the stage/optics while LN₂ boils out from the outlet.
5. Once LN₂ has stopped coming out of the outlet, place the outlet pipe over the stage dewar on the cryo-stage.
6. Plug-in the power source of the stage, and connect the USB cable to the cryo-stage. Ensure the heated sample chamber lid is plugged in. Plug in the external dewar to the stage.
   NOTE: Do not plug in the external dewar until the outlet pipe has been positioned over the stage dewar on the cryo-stage. If LN₂ overflows (or to prevent it from overflowing), pull out the USB cable for ~10 s, allow it to equilibrate, and reconnect the USB cable to reactivate the sensor.
7. After LN₂ has been delivered into the stage dewar, press the release button on the cryo-stage to allow LN₂ to enter the sample chamber.
   NOTE: Do not leave the system unattended while LN₂ is filling the chamber.
8. Wait for ~30-45 min to allow the system to cool and stabilize before commencing image acquisition. Periodically check the external dewar, and top-up with filtered LN₂ if it is less than a quarter full (approximately every hour).

2. Transfer of the sample storage box into the cryo-stage

NOTE: Immerse the sample storage box, holder, and the tips of any instruments (e.g., forceps) inside filtered LN₂ to cool them before touching any cold surfaces such as the sample or any objects inside the sample chamber. Wear a laboratory coat and gloves when handling biological samples.

1. Ensure vitrified samples are in a cryo-compatible container, and bring them to the microscope. Press the corresponding button on the cryo-stage to turn on the light in the sample chamber.
2. Use the hex key on the cassette tool to open the two plates of the sample transfer cassette. Open the plates wide enough to drop in the grid between the two plates, but avoid opening to the maximum open position to prevent the grid falling through the other side.
3. Use long forceps to lift the sample grid box out of the LN₂, turn it where the notch aligns with the position of the storage position inside the stage, and place it onto the stage. Use the appropriate device (e.g., a screwdriver) to open the storage box lid to the correct sample position.
4. Using inverted forceps (or any fine-tip surgical forceps), remove the TEM grid from the sample holder, immerse it inside the LN₂, and drop it into position in the sample transfer cassette, keeping close to the LN₂ during the transfer process. Ensure the carbon film side is placed so that it will ultimately be facing the objective on the sample bridge.
5. Close the sample cartridge using the hex key on the cassette tool. Close and remove the storage box along with any remaining samples.
6. Use the magnet point on the cassette tool to lift and mount the cartridge containing the grid onto the sample bridge. Keep it immersed/close to the LN₂ during the transfer process. Ensure the orientation is appropriate for the bridge (the two magnets will make contact with the magnets on the bridge). Place the cassette flat within the positioning pins of the bridge, and gently nudge to ensure it is fixed.
   ​NOTE: A sample cassette for clipped grids that have been prepared for subsequent focused ion beam milling is also available at the CryoSIM facility at beamline B24.

3. Stage docking and focusing

1. Move the cryo-stage lid opening to the imaging position, and turn off the sample chamber light. Slide the stage towards the optics to align it under the objective lens. Gently drop the objective into position using the lever, ensuring that it rests within the lid of the cryo-stage, but does not touch it.
   NOTE: Ensure the external Dewar's outlet pipe does not touch the stage dewar on the cryo-stage at any point during data collection.
2. Cover the stage and optics with an opaque black curtain. Start the control software Cockpit on the cryoSIM PC (Figure 3 and Figure 4).
3. Click on the readout mode button for each camera, and set it to CONV 3 MHz. Check that the temperature of each camera is -80 °C, and the camera fan is off.
4. Turn on the reflected camera. Under Lights, choose ambient (exposure 20 ms), and under linkam, click on condenser. Click on the Video mode button.
5. In the Mosaic view window, zoom out (mouse scroll) to see the grid outline. Click on Find stage if it cannot be seen. Center the grid by double-left-clicking in the middle of the circle.
6. Focus the sample until the grid support film (or any other relevant sample feature) is in focus, using the up and down keys to move the cryo-stage up and down, and using the 9 and 3 keys on the numerical pad to change the z-step (set it to 20 µm for initial focusing).
   ​NOTE: If the user runs out of travel in z, the stage can be manually moved up or down using the wheel under the cryo-stage. Turn it by one notch at a time, and check if the sample is within the range of view. Alter the direction again if focusing worsens.

4. Brightfield mosaic acquisition

1. Once the stage is centered, turn off video mode, and collect a visible light mosaic (click on Run mosaic in the Mosaic view to produce tiles of visible light images that spiral outward from the center). If the grid is bent, try different positions on the grid (double left click within Mosaic view), re-focus, and click on Run mosaic again to collect partial mosaics. Alternatively, drop-in a focused image on top of the mosaic (Figure 3).
2. Save the view by clicking on Save mosaic. Give it a short filename containing information on the storage box and respective grid number (a timestamp will be appended automatically to the file name).

5. Identification of areas of interest

1. Inspect the brightfield mosaic alongside any previous fluorescence "map" images for where cells or biological features of interest have previously been located. Check if those areas produce suitable fluorescence.
   1. Turn off the ambient light and condenser as well as the video mode if active. Turn on the required excitation laser (405, 488, 561, or 647), and choose the corresponding camera and filter, initially at 50 mW, for 50 ms exposure time.
      NOTE: Increase/decrease these camera and filter settings depending on the fluorescence signal.
   2. Press 0 to snap an image and * to auto-contrast. Alternatively, manually adjust the contrast by using the slider at the bottom of the image.
      NOTE: Switch on the Laser on sign for the room.
   3. Once biologically interesting cells with suitable fluorescence have been found, mark their positions using the Mark site button in Mosaic view.
      NOTE: These marked sites will appear in the enclosed list and can be accessed by double-clicking on the coordinates. When returning to a marked site, zoom into the area before double-clicking.
   4. Continue marking all potential sites before commencing image acquisition. Re-save the mosaic with the marked sites; click on Save sites to file.
      1. To stitch the mosaic images together using the StitchM software (developed inhouse at beamline B24), drag and drop the .txt mosaic file into the StitchM file with extension .bat and save the combined tiff image of the mosaic tiles in the same folder. To save an image with the marked sites, drag and drop the mosaic.txt file and the markers.txt file into the icon at the same time.
         ​NOTE: Balance data collection against the needs of the project (e.g., if correlative imaging will be done, check how many images can be taken with the partner-imaging modality, and choose the appropriate number of sites for cryoSIM imaging). In addition, if the external dewar requires refilling with LN₂ , the cryo-stage system will change position, and the marked sites will likely not return to the same locations; therefore consider this when choosing the number of sites to be imaged.

6. Data collection strategy

1. Set the laser exposure time based on the counts in the dynamic range in the fluorescence image (at the bottom of the camera views window). Choose which filter to apply, and optimize these settings for each wavelength of excitation light to be used, turning each laser on separately.
   ​NOTE: Although 10,000-20,000 counts are optimal, lower counts are acceptable if there is a good contrast. Ideally, check filter settings before each image stack is acquired because cells at different areas of the grid could have variable fluorescence levels.

7. Data collection

1. Click on both cameras to turn them on. Return to one of the marked sites, and focus on the desired depth again. Once in focus in an area of interest, move out of focus upwards (↑ key) in the XY window in the Macro Stage to choose the height of the z stack to acquire, and click on Save top. Move out of focus downwards (↓ key), click on Save bottom and then on Go to the centre, and check that the image is still in focus.
   NOTE: The sample height will be shown in the window.
2. Right-click on slm (spatial light modulator) in the Cockpit window, and make sure the angle is set at 0.41. In the Cockpit window, select Single-site experiment.
   NOTE: Do not click the slm on; Cockpit will automatically turn it on during image acquisition.
   1. From the dropdown list, select Structured Illumination. Alter the Stack height so that it equals the z-height + 1 µm.
      NOTE: The addition of 1 µm ensures the capture of the entire sample in z and minimizes reconstruction artefacts.
   2. Enter the exposure times (ms) for the 405 nm and 488 nm lasers in the upper row (for the reflected camera) and the exposure times for the 561 nm and 647 nm lasers in the lower row (for the transmitted camera).
      NOTE: The values for exposure time should match the values decided on previously and shown in the main Cockpit window.
   3. Input a file name (naming convention: box number_grid area_filters_FL) (FL (fluorescence) or BF (brightfield) depending on what type of imaging is being done). Click on Update to produce a new file containing the date and time without overwriting previous files. Then, click on Start.
3. Check the Camera view while data is being collected. Re-take the images if there is any xy displacement. If the LN₂ dewar refills the cryo-stage during image acquisition, abort the process by clicking on the Abort button in the Cockpit software.
   1. Once the external dewar has finished refilling the stage dewar, repeat the experiment because the refill displaces the sample vertically. Refocus the image to re-center it in z, and repeat the Single-site experiment. Repeat the Single-site experiment for all combinations of lasers and filters needed.
      NOTE: It takes approximately 30 s-1 min to finish refilling. During the imaging of one grid, refilling will happen ~4-8x.
4. At each position, collect a z stack using visible light.
   1. Switch off the lasers, and switch on Ambient light and condenser. Under Single-site experiment, select Z-stack, set the Ambient light to 20 ms exposure, and keep z height as above.
5. Repeat steps 7.2 to 7.4 for all sites marked on the grid.
6. Before moving to another sample, delete the mosaic by clicking on Delete tiles.
   1. Draw a square around all the tiles to delete them. Delete the markers as well by selecting all of them in the list and then clicking on Delete selected sites.
7. Turn off the ambient light and condenser before proceeding to change the grid. Follow the steps in section 4 in reverse order to undock the stage from under the objective and change the grid.

8. After imaging

1. After imaging is finished, undock the stage, and remove all the samples. Turn off the sample chamber light. Unplug the external dewar, and decant any remaining LN₂ into another cryo-compatible container, allowing the dewar to safely return to a normal temperature.
2. Put the lid plug on the cryo-stage. Wait until the option to bake-out the cryo-stage display becomes available after no more LN₂ remains in the stage dewar. Press the bake-out button to enter the heating mode, and remove any moisture from the cryo-stage to avoid ice formation.

9. Reconstruction

1. Transfer raw SIM data files to the appropriate workstation for reconstruction. Run processing in batches through the Processing Task builder window using channel-specific optical transfer functions (OTFS) (calculated from multi-fluorescent beads point spread functions) and K₀ angles (0.29278, -1.8028, 2.3786) with a constant Wiener filter for all channels of 0.004 and a bias offset of 200.
2. In the Additional options panel, ensure that negative intensities are discarded by keeping other options unchecked. Save the SIR images into a folder named "processed".
   ​NOTE: Commercial SIM reconstruction software usually produces reconstructed SIM data and retains the file name, but appends SIR.dv at the end. A log file is also created that contains the processing protocol, steps, and statistical information on reconstruction success.

10. Chromatic shift correction

1. Download the software Chromagnon⁷ to correct for the chromatic shift.
2. Use the chromatic shift reference matrix that corresponds to the fluorescence wavelengths of the data collected (provided by the beamline).
   ​NOTE: The reference file is a 'chromagnon.csv' file, which contains the alignment parameters and has been obtained from calibration images using multi-fluorescent nanoparticles. It can be used to batch-process multiple data sets at once.
   1. Choose the appropriate reference file which matches the laser wavelength and filter used for imaging the sample, and add it to the reference field. Add the reconstructed SIR data to be aligned in the source field, and click on Run.
   2. Check that the fluorescence signal is now aligned in the images. For batch-processing, place all SIR images to be aligned in the source field and the reference file in the reference field, and press Run all.