小鼠海马体中厚组织的冷冻电子断层扫描工作流程演示
2026年3月27日
该方案通过将高压冷冻（HPF）与华夫法、低温聚焦离子束（cryo-FIB）提取以及剂量对称的平行采集相结合，标准化了厚组织的冷冻电子断层扫描（cryo-ET），从而在小鼠海马体中实现均匀薄片和玻璃化样本，可重复原 位 分析。
我的研究重点是建立标准化的低温外置流程，以实现对小鼠海马体等厚组织的原位结构分析。现有方法对厚组织缺乏可靠性，因此该协议整合了优化步骤以实现标准化分析。首先，将一滴10升的10%蔗糖溶液滴在干净的玻璃表面上。
快速解剖刚被安乐死的小鼠的海马体。使用锋利的剃须刀片切除0.5至1立方毫米的小组织。用牙签或细刷将组织转移到高压冷冻载体内准备好的铜支撑TEM网格上。
使用2-甲基戊烷保持组织湿润，调整其在网格上的位置，同时减少机械应力以避免损坏网格。然后，用直径3毫米的铜箔片覆盖组织和网格。完成组装时，将第二个高压冷冻载体平放放在顶部，形成密封的华夫饼三明治。
检查冷冻参数，确保仪器没有异常。确保从组织剥离到冷冻的总时间不超过90秒。获取一个新的300网格金网格，作为提拔过程的最终接收网格。
在室温下用立体显微镜轻轻夹入兼容的低温FIB自动网格环。然后，将组装好的AutoGrid直接存放在干燥的烤箱中，温度保持在约40摄氏度，以减少霜冻积累。在连续浸泡液氮过程中，小心地将玻璃化的TEM网格与周围的华夫组件分离。
用预冷却的细尖镊子轻轻撬开两个高压冷冻载具。接着，在预冷的低温加载工作站上，将分离的组织网格安装到第二个低温FIB自动网格环中，并以液氮保护。将两个AutoGrid装入冷冻转移穿梭机样本架的指定槽位。
将每个AutoGrid调整为铣削缺口朝外并垂直放置，以降低铣削角度。将满载航天飞机浸泡在液氮中或保持预冷状态，直到转移到FIB-SEM。用采样棒将采样级装入乐器。
冷冻转移到显微镜后，以低放大的扫描电子显微镜（SEM）导航到组织网格。然后，在电极表面喷溅铂金，30毫安，持续15秒，以提供导电保护。使用气体注入系统沉积约60秒，沉积0.5至1.0微米厚的有机金属铂帽。
在网格表面以30毫安培的压迫压，施加最后一层溅射涂层铂层，持续15秒。利用SEM电子束图像，导航到组织网格中选定的关注区域。为了调整样品进行正面铣削，需将试验台调整为约110度旋转和7度倾斜，以呈现适合离子束接触的样品表面角度。
对于沟槽铣削，利用30千伏电量驱动的离子束，定义围绕目标组织块的矩形沟槽。在距离区块较远处开始铣削，使用相对较高的15纳安电流，以实现快速去除材料。当沟壁接近区块时，将离子束电流降低至7纳安，以实现更精确的铣削控制。
将块的一侧留在散装材料上，形成临时的固定片。对于估计厚度超过20微米的组织块，从背面进行下切铣削步骤，以便最终提取。旋转舞台并调整倾斜角度以接触方块底部。
用聚焦离子束在30千伏和3纳安电流下铣削底部连接。铣削金属块底部，使其紧贴样品块。精密铣削样品与金块之间的接触面，使接触更紧。
将样品连接到针头时，确保金块与目标样品直接接触。利用30千伏和0.5纳安培的聚焦离子束，在界面处进行8到12个连续截面铣削图案，将金块焊接到样品上。要提取组织块，使用聚焦离子束切断连接块块与散装样本的剩余部分。
小心地操作微操作手针，将分离的组织块完全抬离沟槽和周围网格。收回针头，将块体运送到接收格栅。通过微型机械臂指针导航到预冷的300网金接收器网格。
将方块放到选定的网格上。磨掉网格上的小冰晶，使组织块和网格更紧密地附着。把组织块放低，让它只接触网格。
利用离子束将块状物切割出3到4微米厚的切片到网格薄膜上。接着通过铣削牢固焊接切片，在接头两侧使用清洁截面图案，并如前所述重新涂抹导电和保护涂层于网格表面。对于层状变薄，设定阶段倾斜后，确定组织块光滑区域内的最终层状结构位置。
利用表中指定的离子束电流和过度倾斜补偿进行系统铣削。当距离层片1到2微米时，可选择在约1.5微米外铣刨应力缓解沟槽，以防止层板弯曲或卷曲。片状结构准备后，使用冷冻电子显微镜获取图像。
通过华夫饼法高压冷冻处理的组织块呈现出均匀且电子透明的外观。重建的断层扫描解析了细胞环境中的线粒体结构。大分子复合物，如核糖体，在细胞质中被清晰分辨。
重建的断层扫描图中还可见细胞骨架和膜相关细胞特征及核孔复合体。突触结构和特征，包括钙储备和髓鞘结构，在断层扫描中清晰可见。该方案使研究人员能够直接研究复杂组织环境中的原生细胞外部结构。
该冷冻外质实验中最关键的挑战是实现厚组织样本的均匀、无伪质玻璃化。在此程序后，研究人员可以通过相关光和电子显微镜或标记技术定位感兴趣区域，然后进行亚断层平均和结构分析，以解析大分子配合物。
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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.
Frequently Asked Questions
Executive Industry Relevance
High-resolution, in situ structural analysis of thick tissue samples is a critical bottleneck in early discovery and translational research. This standardized cryo-electron tomography (cryo-ET) workflow enables reproducible, artifact-minimized visualization of native cellular architecture directly from complex tissues, supporting predictive confidence in target validation and mechanistic de-risking. The protocol's throughput and reliability position it as a foundational capability for enterprise-scale structural biology and preclinical model development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of macromolecular complexes within native tissue environments.
- Supports mechanistic de-risking by resolving cellular ultrastructure relevant to disease pathways.
- Facilitates functional target validation through high-fidelity visualization of subcellular features.
- Improves predictive confidence for portfolio triage by minimizing sample preparation artifacts.
Screening & Assay Development
- Prepares uniform, electron-transparent lamellae suitable for downstream high-throughput imaging workflows.
- Standardizes sample preparation to ensure reproducibility and quantitative comparability across experiments.
- Enables reliable assessment of compound effects on tissue ultrastructure in screening campaigns.
- Supports platform scalability and reuse for diverse tissue types with minimal adaptation.
Translational & Preclinical Research
- Aligns structural readouts with disease-relevant tissue models for translational biomarker discovery.
- Provides continuity from discovery-stage mechanistic insights to preclinical validation in complex tissues.
- Reduces risk of late-stage biological failure by enabling early detection of ultrastructural phenotypes.
- Supports risk-adjusted advancement decisions based on direct visualization of target engagement.
Pipeline & Workflow Integration
This workflow bridges early discovery, lead identification, and preclinical research by enabling robust, high-throughput structural analysis of thick tissues.
- Discovery Biology: Supports hypothesis testing and pathway clarification through direct visualization of native macromolecular assemblies.
- Screening: Delivers reproducible, quantitative lamellae preparation for reliable downstream imaging and analysis.
- Analytics: Provides high-resolution tomograms and subtomogram averages for comparative condition analysis.
- Translational Research: Facilitates biomarker alignment and disease-relevant model validation using in situ tissue architecture.
- Enterprise Reuse: Establishes a scalable, standardized protocol adaptable across tissue types and research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Delivers standardized, reproducible, and scalable sample preparation and imaging workflows.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by reducing late-stage biological risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of structurally validated targets.
Implementation Considerations
- Requires expertise in cryo-EM, cryo-FIB milling, and high-pressure freezing instrumentation.
- Demands access to advanced analytical infrastructure for automated tilt-series acquisition and data processing.
- Necessitates cross-team standardization of sample handling, vitrification, and lamella preparation protocols.
- Adaptation to other tissue types may require optimization of dissection and freezing parameters.
- Uniform vitrification and artifact minimization are critical for reproducible, high-quality outputs.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:27
High-Pressure Freezing of Thick Tissue for Cryo-Electron Tomography
1:44
Preparation of Receiver and Tissue Grids for Cryo-FIB Lift-Out
4:08
Tissue Block Isolation and Lift-Out
6:56
Block Attachment to the Receiver Grid
7:49
Lamella Thinning
8:36
Results
9:23
Conclusion
