Cryo-Electron Tomography

Cryo-electron tomography (CryoET) is used to resolve biomolecules within their cellular environment down to an unprecedented resolution in the subnanometer range, opening a window to understanding molecular sociology. It is a scale where individual proteins can be identified by their shape alone, without any labeling. But imaging at subnanometer resolution comes with a major challenge: you need to locate and precisely target your sites of interest.

Advanced cryoET solutions from Leica Microsystems use cryo light microscopy for fast, high-resolution imaging and precise transfer of image data, all while maintaining optimal cryo conditions throughout the workflow.

Need assistance?

Contact us for expert advice on solving common cryo-electron tomography workflow challenges.

What is cryo-electron tomography?

Cryo-electron tomography (also known as electron cryotomography) allows protein-protein interactions to be analyzed in three-dimensional molecular resolution in their native and functional state. The sample is imaged in a series of two-dimensional images as it is tilted in a controlled series of positions. The resulting image “slices” can then be combined to produce a 3D reconstruction of the sample.

What are the challenges in a typical CryoET workflow?

The biggest challenge associated with a typical CryoET workflow is related to the difficulty of identifying the precise area of interest containing the cell or protein to be imaged. Repeated failures in targeting can result in multiple repetitions of a time-consuming process, which ultimately wastes costly electron microscope (EM) imaging time. Two other challenges in the workflow include ensuring sample quality and ice thickness are consistent throughout, as well as keeping samples adequately vitrified before they are transferred to the cryo TEM.

What are the steps in a CryoET workflow?

The technique involves the preparation of samples, which are on electron microscopy grids and then rapidly plunge frozen into liquid nitrogen to vitrify the sample and prevent ice crystal formation.

To perform high-resolution cryo tomography the imaged slice thickness of the specimen should not be bigger than 300 nm. For observation of “thicker” parts of the specimens, like cell bodies, the sample must be thinned. Beside cryo ultramicrotomy, Focused-Ion-Beam (FIB) Milling using a dedicated or multi-modal Cryo Scanning Electron Microscope is the method of choice. Two ion beam windows are positioned in such a way that a thin ice sheet (lamella) of about 200 nm thickness is created at the area of interest to make it accessible for Cryo ET.

The prepared samples can now be scanned with the cryo transmission electron microscope and afterwards, the data reconstruction process must take place to reconstruct the 2D images into a single 3D model.

How can CryoET challenges be overcome with cryo light microscopy?

Cryo light microscopy can positively contribute to the CryoET workflow in two important ways.

Firstly, light microscopy helps to assess the quality of the sample. The cryo light microscope gives a quick overview of the freezing quality and ice thickness of the sample as well as whether the sample distribution is optimal for further processing. Leica’s cryo solutions ensure that samples are kept safe and viable during these steps.

Secondly, the cryo light microscope’s greatest potential for greater workflow efficiency is the ability to target the structure of interest much more precisely before the time and cost intensive CryoEM preparations begin. Leica’s cryo light microscopy solutions enable image and coordinate export of the targeted structure towards subsequent EM steps, thereby greatly reducing EM imaging time.

Introducing Coral Cryo: the effective CryoET workflow

Leica's dedicated 3D cryo-electron tomography workflow solutions overcome typical challenges by ensuring sample viability, quality checks and most of all, a precise and reliable 3D targeting mechanism. Make use of our optimized hardware, including cryostage and shuttle, in conjunction with state-of-the-art CryoET targeting software, as well as a variety of seamless integration and transfer options to cryo FIB or VCT stages.

Find out more

EM Cryo CLEM THUNDER Imager

The THUNDER Imager EM Cryo CLEM is a cryo light microscope featuring opto-digital THUNDER technology. It provides the imaging data and secure cryo conditions you need for successful experimental investigations concerning structural biology. Precisely identify cellular structures of interest thanks to high resolution, haze-free imaging with THUNDER technology, then transfer the specimen seamlessly to your EM.

Find out more

HeLa with F-actin stain (mcherry), TGN46 (GFP) and DNA (Hoechst 33342). Sample Courtesy of Dr. Marie-Charlotte Domart & Dr. Lucy Collinson, The Francis Crick Institute, London UK

What to choose: Cryo Widefield or Cryo Confocal?

Feature

Confocal

THUNDER (Widefield)

Sensitivity   *
Speed   *
Lateral resolution ** *
Axial resolution ** *
Optimization of excitation and emission of dyes for cryogenic imaging conditions ** *
Suppression of Autofluorescence (Sample or Carbon layer) *  
Targeting and export 3D 2D

seamless Cryo-Electron Tomography workflow

Unlock precise 3D volume targeting

In this article, find out how the seamless Cryo-Electron Tomography workflow Coral Cryo by Leica Microsystems uses confocal super resolution to target your structure of interest more precisely. The workflow reduces and optimizes the number of workflow steps, improves the sample loading and transfer and so enhances the productivity of the CryoET workflow.

Find out more

Overlay of a superresolved 3D confocal image with top SEM view (left panel) and FIB view (right panel). Overlay was performed using the beads as landmarks for the correlation. HeLa cells labeled as follows: nuclei by Hoechst, blue; mitochondria by MitoTracker Green, green; lipid Droplets by Bodipy and Crimson Beads, red). Scale bar: 20 μm. Cells kindly provided by Ievgeniia Zagoriy and SEM/FIB images kindly provided by Herman Fung, Mahamid-Group, EMBL-Heidelberg, Germany.

Related Articles

Read our latest articles about Cryo-Electron Tomography

The knowledge portal of Leica Microsystems offers scientific research and teaching material on the subjects of microscopy. The content is designed to support beginners, experienced practitioners and scientists alike in their everyday work and experiments.

More Articles

Projection of a confocal z-stack before and after LIGHTNING applied. Sum159 cells, human breast cancer cells kindly provided by Ievgeniia Zagoriy, Mahamid Group, EMBL Heidelberg, Germany. Blue – Hoechst - indicates nuclei, Green – MitoTracker Green – mitochondria, and red – Bodipy - lipid droplets.

New Imaging Tools for Cryo-Light Microscopy

LIGHTNING super-resolution detection concept and TauSense technology facilitate better cryogenic fluorescence imaging for advanced cryo-correlative workflows. The quality of fluorescence microscope…
Correlation of markers in the LM and the FIB image.

How to Target Fluorescent Structures in 3D for Cryo-FIB Milling

This article describes the major steps of the cryo-electron tomography workflow including super-resolution cryo-confocal microscopy. We describe how subcellular structures can be precisely located in…
LNG-non-LNGHeLa cells labeled with light blue –Hoechst, Nuclei

Precise 3D Targeting for EM Imaging - Access What Matters

Find out how the seamless cryo-electron tomography workflow Coral Cryo uses confocal super resolution to target your structure of interest more precisely.

How to Successfully Perform Live-cell CLEM

The Leica Nano workflow provides a streamlined live-cell CLEM solution for getting insight bout structural changes of cellular components over time. Besides the technical handling described in the…

How to Successfully Implement Coral Life

The live-cell  CLEM workflow allows you to capture dynamic information related to a relevant biological process as it happens and put these observations into their ultrastructural context. The Leica…

Advancing Cellular Ultrastructure Research

Freeze-fracture and freeze-etching are useful tools for studying flexible membrane-associated structures such as tight junctions or the enteric glycocalyx. Freeze-fracture and etching are two…

The Cryo-CLEM Journey

This article describes the Cryo-CLEM technology and the benefits it can provide for scientists. Additionally, some scientific publications are highlighted. Recent developments in cryo electron…
The EM ICE Nano loading area

How to Keep Your Samples Under Physiological Conditions

The Coral Life workflow combines dynamic data with the best possible sample fixation by high pressure freezing. However, good sample preservation won’t help if your cells are stressed by temperature…

Webinar Coral Life Workflow

With the workflow Coral Life, searching for the needle in the haystack is a thing of the past. Take advantage of correlative light and electron microscopy to identify directly the right cell at the…
3D reconstruction of an intercellular bridge in a C. elegans embryo

Download EM Workflow Solutions Booklet

This publication is a compilation of appropriate workflows for the most frequently used sample preparation methods, like Correlative Methodologies, Optogenetics & Electro-Physiology, Surface Analysis,…

Interested to know more?

Talk to our experts. We are happy to answer all your questions and concerns.

Contact Us

Do you prefer personal consulting? Show local contacts

Scroll to top