Cryoprotectants-assisted plunge freezing of thick brain tissue specimens for targeted physiologically relevant cryo-imaging in situ.

In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. Here, we benchmarked plunge freezing with cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 μm thick. A knock-in mouse model with fluorescent astrocytes allowed for targeted cryo-FIB/SEM volume-EM and cryoET imaging. Prior to cryoET, we generated lamellae in a semi-automated fashion on both liquid metal ion source (LMIS)- and plasma-based cryo-FIB/SEM (pFIB) instrumentation, thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and validated the physiological relevance of our outputs. The proposed pipeline utilizes common vitrification setups, and we expect our approach to become an important step toward democratization of physiologically relevant in situ cryo-imaging studies.