Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus

My research focuses on establishing a standardized cryo-ET workflow to enable in-situ structural analysis of thick tissues like the mouse hippocampus. Existing methods lack reliability for thick tissues, so this protocol integrates optimized steps for standardized analysis. To begin, place a 10 L droplet of 10%sucrose solution on a clean glass surface.

Rapidly dissect the hippocampus from a freshly euthanized mouse. Using a sharp razor blade, excise small tissue pieces of 0.5 to 1 cubic millimeters. Using a toothpick or fine brush, transfer the tissue onto the prepared copper support TEM grids within the high-pressure freezing carrier.

Use 2-methylpentane to keep the tissue moist and adjust its positioning on the grid, while minimizing mechanical stress to avoid grid damage. Then, cover the tissue and grid with a 3-millimeter diameter copper foil disc. To complete the assembly, place the second high-pressure freezing carrier, flat-side down, on top to form a sealed waffle sandwich.

And check the freezing parameters to ensure there is no abnormality in the instrument. Ensure the total time from tissue dissection to freezing does not exceed 90 seconds. Obtain a new 300-mesh gold grid to serve as the final receiver grid for the lift-out procedure.

Under a stereo microscope at room temperature, gently clip the grid into a compatible cryo-FIB AutoGrid ring. Then, store the assembled AutoGrid directly in a dry oven, maintained at approximately 40 degrees Celsius, to minimize frost accumulation before use. While continuously immersing the assembly in liquid nitrogen, carefully separate the vitrified TEM grid from the surrounding waffle assembly components.

Using pre-cooled fine-tipped tweezers, gently pry the two high-pressure freezing carriers apart. Next, on a pre-cooled cryo-loading workstation, mount the isolated tissue grid into a second cryo-FIB AutoGrid ring under the protection of liquid nitrogen. Load both AutoGrids into the designated slots of the cryo-transfer shuttle sample holder.

Orient each AutoGrid so that the milling notch faces outward and is positioned vertically to enable a lower milling angle. Keep the fully loaded shuttle immersed in liquid nitrogen or maintained in a pre-cooled state until transfer into the FIB-SEM. Load the sample stage into the instrument using the sample rod.

After cryo-transfer into the microscope, navigate to the tissue grid at low SEM magnification. Then, sputter coat the grid surface with platinum at 30 milliampere for 15 seconds to provide conductive protection. Deposit a 0.5 to 1.0 micrometer thick organometallic platinum cap using the gas injection system for approximately 60 seconds.

Apply a final sputter coated platinum layer to the grid surface at 30 milliampere for 15 seconds. Using the SEM electron beam image, navigate to the selected region of interest on the tissue grid. To orient the sample for front-side milling, adjust the stage to approximately 110 degrees rotation and 7 degrees tilt to present the sample surface at an angle suitable for ion beam access.

For trench milling, define a rectangular trench surrounding the target tissue block using the ion beam operated at 30 kilovolts. Begin milling at a distance from the block using a relatively high current of 15 nanoampere to achieve rapid material removal. As the trench walls approach the block to within a few micrometers, reduce the ion beam current to 7 nanoampere to achieve more precise milling control.

Leave one side of the block attached to the bulk material to create a temporary holding tab. For tissue blocks with an estimated thickness greater than 20 micrometers, perform an undercut milling step from the reverse side to facilitate the final lift out. Rotate the stage and adjust the tilt to access the bottom of the block.

Mill the underside connection using the focused ion beam at 30 kilovolts and a current of 3 nanoampere. Mill the bottom of the metal block to fit tightly against the sample block. Precision mill the contact surface between the sample and the gold block to make the contact tighter.

To attach the sample to the needle, ensure that the gold block is in direct contact with the target sample. Using the focused ion beam at 30 kilovolts and 0.5 nanoampere, perform 8 to 12 sequential cross-section milling patterns at the interface to weld the gold block to the sample. To lift out the tissue block, sever the remaining tab connecting the block to the bulk sample using the focused ion beam.

Carefully maneuver the micromanipulator needle to lift the isolated tissue block completely clear of the trench and the surrounding grid. Retract the needle and transport the block to the receiver grid. Navigate the micromanipulator needle to a pre-cooled 300-mesh gold receiver grid.

Lower the block onto a chosen grid square. Mill away small ice crystals on the grid to make the tissue block and grid adhere more tightly. Lower the tissue block so that it just contacts the grid.

Mill a 3 to 4 micrometer-thick slice from the block onto the grid film using the ion beam. Next, weld the slice securely by milling, using cleaning cross-section patterns on both sides of the junction, and reapply the conductive and protective coating to the grid surface as demonstrated previously. For lamella thinning, after setting the stage tilt, define the final lamella position within a smooth area of the tissue block.

Perform systematic milling using ion beam currents and over-tilt compensations specified in the presented table. When within 1 to 2 micrometers of the lamella, optionally mill a stress-relief trench approximately 1.5 micrometers away to prevent bending or curling of the lamella. After lamella preparation, acquire images using cryo-electron microscopy.

Tissue blocks processed by high-pressure freezing with the waffle method exhibited a uniform and electron-transparent appearance. Reconstructed tomograms resolved mitochondrial structures within the cellular environment. Macromolecular complexes, such as ribosomes, were clearly resolved in the cytoplasm.

Cytoskeletal and membrane-associated cellular features and nuclear pore complexes were also visible in the reconstructed tomograms. Synaptic structures and features, including calcium stores and myelin sheath structures, were clearly seen in the tomograms. This protocol enables researchers to study native cellular outer structure directly within complex tissue environments.

The most critical challenge in this cryo-ET protocol is achieving uniform, artifact-free vitrification of thick tissue samples. Following this procedure, researchers can localize regions of interest using correlative light and electron microscopy or labeling techniques, then perform sub-tomogram averaging and structure analysis to resolve macromolecular complexes.