Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

My research develops optical microscopy methods to preserve cellular features in cryogenic lamella during focused ion beam milling, enabling detailed analysis by cryogenic electron tomography to uncover native biological organization. Optical microscopy has been integrated into CryoFIB-SEM systems, but rarely at the same position as the FIB. This necessitates registration-based guidance approaches to preserve fluorescently labeled targets in final lamella.

To begin, manually cool the transfer station using liquid nitrogen once the stage has reached cryogenic temperatures. Load one clipped autogrid into the sample cassette designed for the cryogenic light microscopy system. Ensure that the autogrid is positioned between the glued cover slip and the spacer.

Tighten the ring screw to close the cassette and insert it into the slot on the transfer station. Attach the transfer station to the cryogenic light microscopy system. Use the internal chamber camera to guide the sample cassette onto the sample stage.

In the light microscopy software, move the sample stage from the loading position to the predefined three beam position. In the FIB-SEM software, set the magnification to 100X and click on the top left window. In the alignment tab of the light microscopy software, click on the Z and select the Play function to acquire a SEM atlas of the grid.

Identify a square area with broken carbon film and double click on it to move the sample stage to that location. Return to the software interface. Activate the FIB and acquire an image.

Adjust the magnification using the dropdown menu on the top left corner. Press Play to display the FIB image. Then set the step size using the slide bar.

Now click Z and Z to adjust the stage Z position until the horizontal line present in the ion and the electron images align with the same feature. Select the SEM tab in the light microscopy software. Use the or X and Y buttons to align a common feature between the SEM and ion images.

Now turn on the fluorescence microscope. Select the FLM tab, set reflected light imaging conditions, and select Play. Then align the reflected light image with the SEM image.

Zoom into the image and click the rectangle option to define a milling pattern for the ion beam. Press Start Patterning in Display 2 to mill a small pattern using the focused ion beam. Confirm that the milled pattern is visible in both the focused ion beam and fluorescent microscope views.

In the light microscopy software, move to the Localization tab and activate the appropriate conditions to set up the fluorescent channels. Assign each fluorophore to a separate channel and include an additional channel for brightfield. Adjust the LED power and exposure time for each fluorescent channel to identify the target of interest.

Once the parameters are satisfactory, acquire a pre-milling fluorescent z-stack by stepping through the axial direction. Then mark the target as a region of interest. Switch to the FIB-SEM software and draw a lamella at the region of interest.

Perform rough milling to remove the bulk material while keeping the fluorescence microscope on to monitor signal changes. Open the Python-based interferometric toolkit from the terminal to monitor fluorescent signal during lamella thinning. Thin the ROI to approximately two micrometers using the cleaning cross-section mode.

For a target with an axial extent of over 300 nanometers, in the Python GUI select the target in the fluorescence micrograph. The software will automatically begin plotting brightness as a function of time with each new frame. Monitor fluorescence intensity through the Python toolkit.

Mill the sample using cleaning-cross-section mode to remove any remaining support film from below the region of interest. Observe for sudden drops in fluorescence intensity as cues to switch milling direction. If a sharp decrease is observed when milling from the bottom up, stop and begin milling from the top down.

After completing top-down milling, finalize the lamella by milling from the bottom up in cleaning cross-section mode until the desired thickness is reached. Then capture a post-milling fluorescence image of the lamella. Transfer the sample to a CryoTEM.

Load the post-milling multi-channel fluorescence image and the CryoTEM projection image into the custom projective transformation software for region of interest registration. Now select eight to 10 pairs of reference points that are visible in both fluorescence and electron microscopy images to compute projective transformation. Use the overlaid image as a guide to select an imaging area.

Then select appropriate magnification and acquisition parameters for the tilt series depending on the size of the target structure and desired resolution. Differentiated macrophage cells labeled with SiR-Tubulin displayed a single bright fluorescent punctum, approximately one micrometer in diameter, corresponding to the MTOC, along with fibro-like structures radiating towards the cell periphery consistent with the microtubule network. As the sample was milled to approximately 800 nanometers in thickness, the singular fluorescent MTOC punctum resolved into two distinct speckles, each approximately 700 nanometers in diameter.

Fluorescence intensity increased after removal of non-fluorescent bulk material and the absorptive support film, followed by a sharp decrease as the lamella was further thinned from two micrometers to below 200 nanometers. Correlated cryogenic fluorescence and electron microscopy enabled precise targeting of structures during milling. Reconstructed tomograms and segmented models reveal that the centrioles are composed of microtubule triplets.

In cases where only one fluorescent speckle was retained in the final lamella, a single centriole was visualized in the corresponding tomogram. Accuracy of fluorescence based on millimilling is limited by registration approaches, which suffer a lot from diffraction-limited resolution, refractive index mismatch, and also milling-induced motion. Simultaneous optical microscopy and FIB milling removes registration errors by instead using real-time feedback from fluorescence microscopy to guide the milling process.

This avoids errors due to the diffraction limit and refractive index mismatch, enabling precise milling that preserves fluorescently labeled structures for cryoelectron tomography within the final lamella. Our research makes it possible to see cellular structures that were previously challenging to directly visualize by cryoET, shedding light on molecular mechanisms behind a wide spectrum of key micromolecular machines.