小鼠海马体中厚组织的冷冻电子断层扫描工作流程演示

Overview

This article presents a comprehensive, standardized workflow for cryo-electron tomography (cryo-ET) of thick tissue samples, specifically demonstrated on mouse hippocampus. The protocol integrates rapid high-pressure freezing, precise cryo-focused ion beam (cryo-FIB) milling, and advanced imaging and data processing steps to enable high-resolution, in situ structural analysis of native cellular environments within complex tissues.

Key Study Components

Area of Science

• Structural biology
• Cryo-electron microscopy
• Tissue ultrastructure analysis

Background

• Cryo-ET allows 3D visualization of cellular ultrastructure in a near-native state.
• Application to thick tissues is limited by challenges in vitrification and lamella preparation.
• Existing protocols often lack reproducibility and throughput for complex tissue samples.
• Reliable workflows are needed for in situ analysis of macromolecular complexes within tissues.

Purpose of Study

• To establish a reproducible, end-to-end cryo-ET workflow for thick tissue samples.
• To optimize vitrification and lamella preparation for high-quality imaging.
• To enable high-throughput, artifact-free structural analysis of native tissue environments.

Methods Used

• Rapid dissection and high-pressure freezing (HPF) of tissue using a "waffle" assembly with copper foil.
• Transfer and mounting of tissue onto TEM grids, followed by vitrification within 90 seconds of dissection.
• Cryo-FIB milling for lift-out and thinning of tissue blocks to 120–200 nm lamellae, with over-tilt compensation and stress-relief trenching.
• Standardized conductive coating using sputter platinum and gas injection system (GIS) platinum cap.
• Automated, dose-symmetric tilt-series acquisition using PACEtomo scripts.
• Data processing with AreTomo 3 for motion correction, CTF estimation, alignment, and WBP reconstruction; optional IsoNet for missing-wedge correction.
• Subtomogram averaging and resolution assessment using RELION 4.0.

Main Results

• The workflow consistently produces well-vitrified, uniform lamellae (target thickness 150 ± 40 nm).
• High-throughput tilt-series collection is achieved (typically 5–8 minutes per tilt series).
• Reconstructed tomograms resolve key cellular structures, including mitochondria, ribosomes, cytoskeletal elements, nuclear pore complexes, synaptic features, calcium stores, and myelin sheaths.
• The protocol enables direct visualization and analysis of macromolecular complexes in situ within thick tissue samples.

Conclusions

• This standardized protocol overcomes major challenges in cryo-ET of thick tissues, particularly vitrification and lamella preparation.
• It provides a reproducible, high-throughput route for in situ structural biology studies in complex tissue environments.
• The workflow facilitates detailed analysis of native cellular architecture and macromolecular assemblies directly from tissue.

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