Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

This method makes super resolution cryo imaging possible on whole biological cells to precisely identify cellular structures. It can also be used in conjunction with other modalities as part of a correlative imaging workflow. The main advantages of this technique are that super resolution imaging can be rapidly done in cryogenic conditions using conventional fluorophores and relatively low light doses.

CyroSIM is a powerful tool that provides insight towards understanding cellular ultrastructure dynamics in response to internal or external cues. Its application include vaccine production and roll out, quality control, post-market surveillance, antibody engineering and optimization, as well as nano particle characterization. Maneuvering samples within the cryo-stage takes practice to ensure the safety of the grid.

It's also best to familiarize yourself with the controls in the Cockpit window before data collection. To begin, remove the lid from the external Dewar of the cryo-stage and pour filtered liquid nitrogen until it's approximately a quarter full. Wait until initial boiling subsides before pouring more and fill the vessel to about two thirds full.

Replace the lid carefully, pointing the nozzle away from the handler as the liquid nitrogen boils out. Once the liquid nitrogen has stopped coming out of the outlet, place the outlet pipe over the stage Dewar on the cryo-stage. Plug in the power source, connect the USB cable and plug in the external Dewar to the stage.

After delivery of liquid nitrogen, press the release button on the cryo-stage to allow it to enter the sample chamber and wait for 30 to 45 minutes for the system to cool and stabilize before commencing with image acquisition. 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 the grid between the two plates, but do not open to the maximum position.

Use long forceps to lift the sample grid box out of the liquid nitrogen. Turn it so that the notch aligns with the storage position inside the stage and place it onto the stage. Use the appropriate device to open the storage box lid to the correct sample position.

Using inverted forceps, remove the TEM grid from the sample holder, immerse it inside the liquid nitrogen ensuring that the carbon film side is placed so that it will ultimately be facing the objective on the sample bridge and drop it into position in the sample transfer cassette. Close the sample cartridge using the hex key on the cassette tool. Use the magnet point on the cassette tool to lift and mount the cartridge containing the grid onto the sample bridge.

Keep it immersed or close to the liquid nitrogen and in proper orientation. Place the cassette flat within the positioning pins of the bridge and gently nudge it to ensure it is fixed. Close and remove the storage box along with any remaining samples.

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, then 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. Cover the stage and optics with an opaque black curtain, then start the control software Cockpit on the cryoSIM PC.Click on the readout mode button for each camera and set it to CONV3 megahertz.

Check that the temperature of each camera is 80 degrees Celsius and that the camera fan is off. Turn on the reflected camera, under light, choose ambient and under linkam, check on condenser, then click on the video mode button. In the Mosaic view window, zoom out to see the grid outline.

Click on Find Stage if it cannot be seen and center the grid by double left clicking in the middle of the circle. Use the up and down keys to focus the sample until the grid support film or any other relevant feature is in focus. Use the nine and three keys on the numerical pad to change the Z step.

Once the stage is centered, turn off video mode. Collect a visible light mosaic by clicking on Run Mosaic in the mosaic view to produce tiles of visible light images that spiral outward from the center. Save the view by clicking on Save Mosaic.

Inspect the Bright-field mosaic alongside any previous fluorescence map images by turning off the ambient light and condenser as well as the video mode, then turn on the required excitation laser and choose the corresponding camera and filter, initially at 50 milliwatts for a 50 millisecond exposure time. Press zero to snap an image and star to auto contrast. Alternatively, manually adjust the contrast by using the slider at the bottom of the image.

Once biologically interesting cells with suitable fluorescence have been found, mark their positions using the mark site button in Mosaic view. Continue marking all potential sites before beginning image acquisition. Re-save the mosaic with the marked sites by clicking on Save sites to file.

To stitch the mosaic image using the stitchEm software, drag and drop the text mosaic file into the stitchEm file with the extension BAT and save the combined TIF image of the mosaic tiles in the same folder. To save an image with the marked sites, drag and drop the mosaic text file and the markers text file into the icon at the same time. Set the laser exposure time based on the counts and the dynamic range in the fluorescence image at the bottom of the camera view window.

Choose which filter to apply and optimize the settings for each wavelength of excitation light to be used, turning each laser on separately. 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 using the up arrow key in the XY window on the macro stage to choose the height of the Z-stack to acquire and click on Save top. Move out of focus using the down arrow key, click on Save bottom, then on Go to center, verify that the image is still in focus. In the Cockpit window select Single-site experiment.

From the drop-down list select Structured Illumination. Alter the stack height so that it equals the Z height plus one micro meter. Enter the exposure times in milliseconds for the 405 and 488 nanometer lasers in the upper row for the reflected camera and the exposure times for the 561 and 647 nanometer lasers in the lower row for the transmitted camera.

Input a file name and click on update to produce a new file containing the date and time without overriding the previous files, then click on Start. If the liquid nitrogen Dewar refills the cryo-stage during image acquisition, abort the process by clicking on the ABORT button in the Cockpit software. At each position, collect a Z-stack using visible light by switching off the lasers and switching on the ambient light and condenser.

Under Single-site experiment, select Z-stack and set the ambient light to 20 millisecond exposure, maintaining the Z height. Repeat this process for all marked sites. After imaging is finished undock the stage and remove all samples.

Turn off the sample chamber light. Unplug the external Dewar and decant any remaining liquid nitrogen into another cryo compatible container, allowing the Dewar to safely return to a normal temperature. Wait until the option to bake out the cryo-stage display becomes available after no more liquid nitrogen remains in the stage Dewar.

Press the bake out button to enter the heating mode. Put the lid plug on the cryo-stage. The resolution in cryoSIM is significantly higher than that in standard epifluorescence microscopy.

The fluorescence map from a conventional epifluorescence microscope can be used to locate areas of interest for imaging and the corresponding cryoSIM image can be obtained from a location on the grid. A sample containing U2OS cells was stained with a mixture of green microtubule cytoskeleton dye and red mitochondria dye resulting in the staining of the microtubule component of the cytoskeleton and the mitochondria. Subsequent imaging showed the localization of mitochondria within the cell, as well as the arrangement of the microtubules, highlighting the structural framework that they provide to the cell and the assembly of the cytoskeleton around organelles.

The SIM reconstruction process can produce artifacts which can be identified using SIMCheck, a free imageJ plugin. This modulation contrast map generated using SIMCheck shows areas of low modulation contrast within the nucleus area, indicating that this region is going to be more susceptible to reconstruction artifacts. The white arrow indicates a reconstruction artifact.

When attempting this protocol ensure that the sample remains submerged or close to liquid nitrogen at all times to avoid de-vitrification. Also pay attention to exposure times and counts in the dynamic range, since this affects the quality of data and avoids laser damage to the sample. Following cryo imaging, the same samples can be imaged with other modalities such as cryo-soft x-ray tomography, which we have here at the B24 Beamline.

By combining cryoSIM images with images containing structural information about the cell, we can answer key additional questions on cellular ultrastructure and function.