Cryo-electron tomography (cryo-ET) is an incredibly powerful technique that allows the study of cellular structures at high resolution in their near-native state [1]. Cryo-ET involves imaging a vitrified cellular sample as it is tilted in a transmission electron microscope, which allows the reconstruction of a 3D volume or tomogram. This technique can resolve the 3D structures of intracellular organelles and protein complexes within their cellular context at sub-nanometer resolution [2,3]. Doing this enables a detailed understanding of the location, interactions, and function of biomolecules.

In this blog, we'll discuss the challenges in the cryo-ET workflow and introduce you to METEOR, an integrated cryo-CLEM system designed to streamline the cryo-ET process. We'll explore how METEOR, with its innovative design and dedicated software, is revolutionizing how we prepare samples for cryo-ET. You'll also learn how researchers are using METEOR to guide lamella milling, ensuring that the ROIs are preserved and accurately targeted for high-resolution imaging.

If you're a researcher grappling with cryo-ET or bioimaging personnel eager to learn about the latest in cellular imaging technology, this blog is for you. Without further ado, let’s begin!

Challenges in the cryo-ET workflow

However, the cryo-ET workflow comes with significant challenges. To acquire high-quality tomograms, the sample needs to be thinned down to a thickness of just 200-300 nm, which is typically achieved through cryo-focused ion beam (cryo-FIB) milling. Identifying the region of interest (ROI) for milling is often facilitated by correlative light and electron microscopy (cryo-CLEM), which uses cryo-fluorescence light microscopy (cryo-FLM) to precisely localize the ROI [2,4,5].

The cryo-CLEM workflow, however, is time-consuming and fraught with technical difficulties. Sample transfer between the fluorescence microscope and the cryo-FIB/SEM increases the risk of ice contamination and sample damage. Correlating the fluorescence and electron microscopy data can also be challenging, and sample movement during FIB milling can cause the initial correlation to be lost, resulting in the production of lamellae that lack the ROI and are therefore useless. 

Say hello to METEOR - An integrated cryo-CLEM system

A study published in Microscopy Today 2021 by Delmic, in collaboration with researchers from the Max Planck Institute of Biochemistry and the Human Technopole, aimed to tackle these challenges. The study utilized METEOR, an integrated cryo-CLEM system, for this purpose. Designed to be directly integrated into the cryo-FIB/SEM chamber, Delmic’s METEOR system reduces the number of handling steps, minimizing contamination and damage, and streamlining the cryo-ET workflow. It also allows for easier sample inspection after milling, improving the success rate of obtaining the desired ROI. 

METEOR is a top-down widefield FLM designed for integration with a cryo-FIB/SEM. Its objective lens is positioned parallel to the FIB column, enabling a seamless transition between FIB milling and FLM imaging.

For in-vacuum fluorescence imaging, the excitation and emission light paths are directed in and out of the FIB/SEM chamber. As shown in Figure 1, the light path includes a dichroic mirror that reflects the LED excitation light through a vacuum window, which interfaces with the vacuum and fits existing FIB/SEM ports. Inside the vacuum, the objective lens, mounted on a stage with a 31 mm travel range for focusing and z-stack acquisition, transmits the excitation light to the sample. Emitted light returns through the vacuum window and dichroic mirror, passes through a single band-pass filter in a customizable filter wheel, and reaches the high-quality camera to form an image. 

ODEMIS, a dedicated software developed for METEOR, is intuitive and open-source, providing control over FLM settings and the FIB/SEM stage. It simplifies navigation, focusing, and switching between imaging modes. Additionally, ODEMIS allows the creation of an overview atlas of the entire grid, facilitating the understanding of the sample's larger context. The software also supports integrated FLM <> SEM correlation, enabling seamless comparison and analysis of fluorescence light microscopy and scanning electron microscopy data.

How researchers used METEOR to streamline the cryo-ET workflow

METEOR's flexibility allows it to be used in various workflows, such as guiding lamella milling of plunge-frozen samples (refer to Figure 2). In this process, a stitched overview map is created to identify regions of interest (ROIs), and high-quality z-stacks are taken of the ROIs. The overview map and z-stacks are subsequently correlated with the SEM map to identify the exact milling position. After milling, METEOR can be used to confirm the presence of the ROIs. 

FLM-guided lamella targeting and milling of yeast cells

To demonstrate METEOR's potential, researchers used the integrated cryo-CLEM system for guided lamella milling in a yeast strain with eGFP-Ede1 overexpression, as detailed by Wilfling et al [14]. The integrated FLM allowed monitoring of the ROI's fluorescence during milling. Figure 3 illustrates the process, from ROI identification to milling control and the final polished image, with pink arrows highlighting the ROI's presence throughout. This ensured the END condensates were intact for cryo-TEM transfer.

FLM-guided tomography improved the ease of END condensate visualization

Guided tomography, facilitated by the integration of fluorescence light microscopy (FLM) with cryo-electron tomography (cryo-ET), significantly enhances the visualization and analysis of cellular structures like END condensates. The process begins with locating the grid under low-magnification electron microscopy (EM), where an FLM/TEM overlay (Figure 4A) is used to precisely select the region for tomogram acquisition. This overlay ensures that the region of interest (ROI) is accurately identified and targeted, which is crucial for obtaining high-resolution images that reveal detailed structural information.

The importance of guided tomography lies in its ability to maintain the vitreous state of the ROIs, confirming that in situ FLM imaging does not compromise the integrity of the lamella. This is essential because any devitrification or damage to the sample could lead to loss of structural details and inaccurate data. By ensuring the ROIs remain vitreous, guided tomography allows for the faithful reconstruction of the cellular structures in their near-native state.

The tomograms generated through this process revealed several key structural properties of the END condensates. For instance, the condensates exhibited a distinct teardrop-like shape near the plasma membrane, with a noticeable exclusion of ribosomes and the surrounding endoplasmic reticulum (ER) (Figure 4B). These structural insights are invaluable for understanding the organization and function of these cellular compartments.

For detailed insights into the END condensates and Ede1's role, see Wilfling et al.'s study [6].

Why METEOR is the way forward

The study demonstrated how an integrated fluorescence light microscope (FLM) can be used to guide the milling of lamellae for cryo-electron microscopy (cryo-EM) on the same sample stage. This integrated cryo-correlative microscopy approach using the METEOR, offers several advantages.

1. Integrated workflow - METEOR combines FLM and cryo-FIB/SEM in one system, reducing the need for sample transfers and minimizing contamination risks.
2. Increases efficiency - METEOR allows in situ imaging within the cryo-FIB/SEM chamber, streamlining the workflow and saving time.
3. Data correlation and accurate ROI targeting - METEOR provides a direct correlation of fluorescence and electron microscopy images during milling, ensuring accurate ROI targeting.
4. Improved sample yield - METEOR enables fluorescence-guided milling, confirming the presence of target proteins in lamellae and increasing the success rate of preparing high-quality samples for TEM imaging.
5. Superior imaging quality - METEOR is equipped with high-efficiency cameras (up to 95% quantum efficiency) and high NA objectives (up to 0.9), providing superior resolution and targeting efficiency.
6. User-friendly advanced software - Odemis is an open-source software that offers unique features like stitched overview images, autofocus functionality, easy sample navigation, and integrated SEM and FLM correlation, unmatched by competitors.
7. Customizable configuration - Allows selection of objectives, cameras, and filters tailored to specific research needs, unlike the fixed configurations of alternative suppliers.
8. Future-proof system - Modular design supports hardware upgrades, our new designs are always backward compatible and our software gets updated quarterly. This ensures access to the latest technological advancements, maintaining long-term value.

This article is based on the study here - Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ

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