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

Summary

This protocol standardizes cryo-electron tomography (cryo‑ET) for thick tissues by integrating high-pressure freezing (HPF) with the waffle method, cryogenic focus ion beam (cryo‑FIB) lift‑out, and dose‑symmetric, parallel acquisition, yielding uniformly thin lamellae and vitrified specimens for reproducible in situ analysis in the mouse hippocampus.

Abstract

Cryo‑electron tomography (cryo‑ET) enables 3D visualization of native cellular ultrastructure, but its application to thick tissues is limited by the lack of reliable vitrification and low‑throughput lamella preparation. Here, an end‑to‑end workflow is presented that integrates high‑pressure freezing (HPF; ~2100 bar; dissection‑to‑freezing time ≤90 s) using a “waffle” assembly with copper foil to pre‑thin tissue, cryo‑FIB lift‑out, and controlled thinning to 120–200 nm lamellae with over‑tilt compensation. Conductive coating is standardized (sputter‑Pt 30 mA/15 s; GIS cap 0.5–1.0 µm) to minimize curtaining. Automated, dose‑symmetric data acquisition (0°, ±3°, ±6°… to ±60–66°; step size of 2–3°; total dose 100–150 e⁻/Ų) is implemented via PACEtomo scripts, with the starting tilt angle derived from the milling angle and sample orientation. Processing with AreTomo 3 provides functions for motion correction, CTF estimation, alignment, and WBP reconstruction in near‑real time; IsoNet may optionally reduce missing‑wedge anisotropy. Subtomogram averaging (RELION 4.0) follows a pseudo‑subtomogram strategy with resolution reporting based on an FSC cutoff of 0.143. Demonstrated on mouse hippocampus, the workflow routinely produces well‑vitrified samples and uniform lamellae (target thickness 150 ± 40 nm) and achieves high‑throughput tilt‑series collection (typically 5–8 min/tilt series). This protocol offers a reproducible route for studying in situ structural biology directly from complex tissue environments.

Introduction

Cryo-electron tomography (cryo-ET) has emerged as a pivotal technique for visualizing macromolecular complexes and cellular architectures in their native, hydrated state^1,2,3,4,5. By reconstructing three-dimensional volumes from tilt-series of projection images, cryo-ET bridges the gap between cellular context and near-atomic resolution, offering unprecedented insights into structural biology in situ⁶. While cryo‑electron tomography workflows for cellular specimens

Protocol

All procedures involving animal tissue complied with SUSTech IACUC (Protocol SUSTech‑JY202501019). The regents and the equipment used are listed in the Table of Materials.

1. Sample support preparation

1. HPF carrier preparation (see Figure 1A)
   1. Obtain a pair of 6 mm copper HPF carriers (type B). Ensure one of the carriers has a flat surface.
   2. Wet the flat surface of the carrier and proceed to grind. First, use 8000-grit sandpaper until the original machining marks are completely removed. Then, switch to 12000-grit sandpaper and continue grin....

Results

Successful application of this protocol enables the production of high-quality, vitrified tissue lamellae and the subsequent reconstruction of tomograms that reveal native cellular ultrastructure. The following outcomes, demonstrated using mouse hippocampal tissue, are representative of a successful experiment.

Tissue blocks processed by high-pressure freezing with the "waffle" method exhibited a uniform and electron-transparent appearance (Figure 4B,C

Discussion

The workflow presented herein establishes a generalized, optimized, and reproducible pipeline for in situ cryo-electron tomography of native, thick tissues. Its primary contribution lies not in the invention of singular techniques, but in the systematic integration, refinement, and detailed standardization of the entire process, from robust vitrification using a modified waffle-HPF method to a precisely calibrated cryo-FIB lift-out and thinning protocol. This synthesis addresses the critical reproducibility and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Hexadecene	Sigma-Aldrich	H2131	Chemicals & Reagents
2-Methylpentane	Sigma-Aldrich	M65807	Chemicals & Reagents
Clip-ring	Thermo Fisher Scientific	1131750;1188953	Consumables
Copper foil	Fengcheng Metal Materials	1 µm thickness	Consumables
Copper support TEM Grids (100 mesh)	Beijing Zhongjingkeyi Technology Co., Ltd	BZ10021b	Consumables
Cryo-EM	Thermo Fisher Scientific	Krios G4	Equipment
Cryo-FIB	Thermo Fisher Scientific	Aquilos 2	Equipment
Cryo-FIB Autogrid	Thermo Fisher Scientific	1205101	Consumables
Grids (UltrAufoil R 1.2/1.3 300 mesh)	Quantifoil	AltrAufoil	Consumables
High-Pressure Freezer	Leica Microsystems	EM ICE	Equipment
HPF carrier, copper	Leica Microsystems	type B (6 mm)	Consumables
Ion Sputter Coater	Quorum	Q150RS plus	Equipment
Metallographic Polishing Paste	Lab-testec	W0.5	Consumables
Polishing cloth	GORAL	B00809	Consumables
Sandpaper	Eagle Brand Sandpaper	8000-grit, 12000-grit	Consumables
Stereomicroscope	Leica Microsystems	S9i	Equipment
Sucrose	Sigma-Aldrich	200-334-9	Chemicals & Reagents
Tilt-Series Acquisition Software	https://bio3d.colorado.edu/SerialEM/	SerialEM 4.0.9	Software
Tilt-Series Alignment & Reconstruction Software	https://github.com/czimaginginstitute/AreTomo3	AreTomo 3	Software
Tilt-Series Acquisition Software	https://github.com/eisfabian/PACEtomo	PaceTomo	Software
Tilt-Series Alignment & Reconstruction Software	https://github.com/IsoNet-cryoET/IsoNet	IsoNet	Software
Tilt-Series Alignment & Reconstruction Software	https://github.com/cryoem/eman2	EMAN2	Software
Tilt-Series Alignment & Reconstruction Software	https://relion.readthedocs.io/en/release-4.0/index.html	Relion 4	Software