High-Pressure Freezing for Organoids: Cryo CLEM & FIB Lift Out

Key learnings — at a glance

• HPF vs plunge—for 3D tissues/organoids: Why HPF (EM ICE) is preferred for larger/thicker specimens—including organoids and tissue slices—and how it preserves native structure for downstream cryo‑ET/FIB workflows.
• 3D targeting inside organoids with Cryo‑CLEM: Use cryo confocal overview imaging to locate ROIs in planchets and carry coordinates into FIB‑SEM for trench placement and lift‑out.
• Contamination/devitrification control during transfers: Cover box (dry N₂), GN₂ “curtain” at the cryogen area, and vacuum transfer help minimize exposure and maintain cryo integrity across instruments.
• Low contrast & charging in HPF samples: What to expect in cryo‑volume EM of organoids (lower contrast, charging) and how lifetime tools/optimized excitation can reduce background and laser power during cryo fluorescence.
• Lift‑out & lamella quality: Step‑by‑step guidance on serial lift‑out, copper redeposition “welding,” receiving‑grid orientation (front face towards FIB), and shallow‑angle polishing to ~200 nm.

Selecting the vitrification method depends on sample size, nature, and downstream workflow. Plunge freezing with the EM GP2 is appropriate for thin suspensions, single particles, and small cells. EM ICE high‑pressure freezing (HPF) is used for larger or thicker specimens—including tissues, monolayers on sapphire, and the waffle method—to maintain native structure for cryo‑ET, cryo‑FIB and related routes. Decision points focus on practical limits (thickness, complexity) and alignment with subsequent steps such as lamella preparation and correlative imaging.

For reproducible ice thickness, the webinar covers single‑side blotting with optional grid rotation, sensor‑blotting (stops upon liquid contact with configurable extra move), and configurable pre/post‑blot delays—plus chamber humidity/temperature readouts and a GN₂ “curtain” to shield the cryogen area during transfers.

Clean handling from preparation to imaging is supported by a coordinated chain: the cover box (dry nitrogen atmosphere, pre‑cooling for tools) for grid loading into the transfer shuttle; EM VCT/VCM vacuum transfer for holders (grids, planchets, pins); and docking to EM ACE600 for cryo coating, then onward to cryo‑SEM. This integrated path aims to limit exposure, reduce handling steps, and maintain cryo conditions during movement between instruments.

Target identification and correlation are supported by STELLARIS Cryo. Overview imaging (widefield/confocal) locates samples on grids or planchets; TauSense/FALCON lifetime tools help separate signal from background (e.g., carbon film). The Navigator stores lamella positions (XY/Z) and metadata that can be used in FIB‑SEM to refine milling locations, with compatibility noted for downstream software options. This enables consistent 3D ROI targeting and coordinated hand‑off to milling.

Lamella preparation steps include designing trench geometry, performing undercuts with appropriate stage tilts, attaching ice blocks via copper redeposition (“welding”) to the micromanipulator and to the receiving grid, and orienting slices so the lamella face the ion beam. Shallow‑angle polishing with step‑down currents is then applied to reach around 200 nm thickness. These procedures support preparation of HPF tissues and organoids for cryo‑ET, with attention to geometry, orientation, and controlled thinning.

Cryo‑volume EM datasets in organoids exhibit lower contrast and significant charging; nonetheless, contextual structures (nuclei, lipid droplets, mitochondria, microvilli) remain discernible for better targeting. Combining cryo light data with EM helps place milling patterns and assess devitrification before final acquisition.

Future work includes improved vitrification for high‑water‑content organoids (e.g., grown in Matrigel) and further automation of milling and data processing, including machine‑learning‑based, on‑the‑fly segmentation and correlation within the workflow.