ZEISS Correlative Cryo Workflow

Your Solution for TEM Lamella Preparation and Volume Imaging under Cryogenic Conditions

Cryogenic microscopy allows the examination of cellular structures in their near-to-native state, as the ultrastructure of cells and tissues can be preserved free of artifacts and cellular processes are stopped instantaneously. However, cryogenic microscopy presents users with complex challenges, such as time-consuming preparation and imaging procedures, devitrification, ice contamination or loss of samples – even more if you want to correlate the obtained data across imaging modalities.

ZEISS Correlative Cryo Workflow connects widefield, laser scanning, and focused ion beam scanning electron microscopy in a seamless and easy-to-use procedure. The solution provides hardware and software optimized for the needs of correlative cryogenic workflows, from localization of fluorescent macromolecules to high-contrast volume imaging and on-grid lamella thinning for cryo electron tomography.

Correlative Cryo Workflow accessories

Imaging the near-to-native state

  • Seamless cryogenic workflow across multiple modalities
  • Sample protection against devitrification and ice contamination
  • High-resolution fluorescence imaging
  • High-contrast volume imaging and 3D reconstruction
  • Targeted on-grid lamella thinning for cryo TEM applications
  • Multipurpose use for cryogenic and room temperature applications

A simplified workflow to help you focus on your research

With ZEISS Correlative Cryo Workflow, you master the challenging combination of different imaging modalities under cryo conditions. The workflow solution connects light and electron microscopy, enabling volume imaging and efficient production of TEM lamellae. Dedicated accessories simplify the workflow and facilitate a safe transfer of cryo samples between the microscopes. Data management is assured by ZEN Connect, which keeps your data in context throughout the workflow. A series of processing tools help you enhance the imaging results.

Correlative Cryo Workflow in the Lab
Double-labelled yeast cells (CNM67-tdTomato and NUP-GFP).
Double-labelled yeast cells (CNM67-tdTomato and NUP-GFP). LSM image (left) and Crossbeam image (right). Sample courtesy M. Pilhofer, ETH Zürich, Switzerland

Superior components to give you best-in-class data quality

Thanks to cryo-compatible objectives and the high sensitivity of the Airyscan detector, ZEISS LSM systems enable you to detect proteins and cellular structures at high resolution while gentle illumination and constant low temperatures prevent your samples from devitrification. The ZEISS Crossbeam FIB-SEM lets you enjoy high-contrast volumetric imaging – even without heavy metal staining applied to your samples. Both modalities provide valuable functional and structural information that can give you a thorough understanding of ultrastructure, whether or not you follow up with TEM studies.

Multipurpose solutions to maintain your imaging facility’s productivity

Unlike other solutions, the ZEISS microscopes involved in the workflow can be used not only for cryogenic microscopy, but also for room temperature applications, which is particularly advantageous when the microscopes are not being fully utilized for cryogenic experiments. Converting the instruments from cryogenic to room temperature usage is done quickly and doesn’t require technical expertise. This flexibility gives users more time for their experiments. Imaging facilities benefit from better utilization and a faster return on investment.

Core Imaging Facility with Cryo equipment

ZEISS Correlative Cryo Workflow at a glance

Correlative cryo-workflow

Advantages

Evaluation of sample quality and prevention of sample damage

Loss of samples, ice contamination and de-vitrification are well-known problems in cryo microscopy. ZEISS Correlative Cryo Workflow is designed to protect your precious vitrified samples against many conceivable pitfalls which can occur during this ambitious workflow.

ZEISS Cryo Accessory Kit together with the imaging capabilities of ZEISS LSM/Airyscan and ZEISS Crossbeam mitigate the risk of losing or destroying your sample while working under cryo conditions.

Prior to managing your sample in the workflow, vitrification is a challenge in itself. Despite recent development of vitrification technologies, samples are often still covered under a thick ice layer or only partially vitrified and show areas of non-amorphous ice. Poor vitrification destroys the ultrastructure of cells and tissues. These areas can be identified only in a TEM unless your light microscope or the FIB-SEM provide methods for sample evaluation early in the workflow.

ZEISS LSMs provide such evaluation capability by enabling different contrast methods. The outstanding contrast performance of ZEISS Crossbeam also allows reliable assessment of the sample quality. This will save you time and improve experiment efficiency.

HPF carriers with vitrified samples
HPF carriers with vitrified samples (click for more details)
TEM grids with vitrified samples.
TEM grids with vitrified samples. (click for more details)

Ice thickness measurement and efficient ROI targeting

Ice thickness measurement is crucial for judging the quality of samples and to locate the cells of interest within the vitrified specimen. By means of the light microscope, your sample can be validated easily. Reflected light and confocal fluorescence imaging give the first hints about the quality and let you clearly localize promising cells.

Spiderweb-fuzzy patterns of the fluorescent signal often indicate bad freezing. Furthermore, plunge-frozen samples will show different freezing quality and preservation within one sample. The information about ice thickness and ice quality enables the time-saving pre-selection of cells before moving on to the next step within the correlative cryo workflow.

Plunge-frozen Hela cells (Histone 2-GFP labeled) illustrating ideal conditions for further imaging. The ice layer is around 6.8 μm thick and covers the vitrified cells. These cells are perfectly suited for further FIB-SEM analysis.

Solution Overview

ZEISS Cryo Accessory Kit

ZEISS Correlative Cryo Workflow allows the use of various sample carriers. Whether you use TEM grids, AutoGrid, sapphire discs or HPF planchets, you can count on the Cryo Accessory Kit to enable easy loading, transfer and storage of your sample. A collection of items and tools supports safe sample handling throughout the entire workflow. The components are compatible with:

  • Linkam CMS196V³ cryo-correlative microscopy stage
  • Quorum PP3010Z cryo system
ZEISS Cryo Accessory Kit
ZEISS Cryo Accessory Kit

Easy sample transfer and safe sample handling inside ZEISS Crossbeam

ZEISS Correlative Cryo Workflow comes with Quorum PP3010Z, a highly automated, easy to use, gas-cooled cryo preparation system.

  • The turbo-molecular pumped cryo preparation chamber includes tools for controlled, automatic sublimation and sputter coating.
  • From the cryo preparation chamber connected directly to the ZEISS Crossbeam chamber, the vitrified sample is transferred onto a highly stable cold stage for imaging and milling.
  • Cold trapping in the cryo preparation chamber and Crossbeam chamber protects the sample from ice contamination.
  • Continuous cooling for at least 24 hours is ensured by the CHE3010 off-column cooling system.
  • All Quorum cryo components are controlled by the Prepdek® workstation, including the vacuum storage tube for the cryo transfer device and the TEM prep slusher for the ZEISS loading station.
ZEISS Crossbeam: Rotatable cryo substage
ZEISS Crossbeam: Rotatable cryo substage

The most reliable imaging modalities combined

Cryogenic widefield and confocal microscopy

Cryogenic widefield and confocal microscopy:
ZEISS Axio Imager / ZEISS LSM 900/980 with Airyscan 2
ZEISS Axio Imager, the light microscope of choice for the ZEISS Correlative Cryo Workflow, can be equipped with the cryo stage CMS196V3 from Linkam. Depending on your requirements, you can configure the Axio Imager as:

  • a widefield system (with Apotome 3 to acquire 3D datasets)
  • an LSM 900/980 with Airyscan 2 for high-resolution confocal imaging

The hardware is designed to prevent devitrification and ice contamination during imaging. Objectives are available with a range from 5x to 100x to support imaging from low magnification overview to high-resolution. Different illumination methods, like reflected or transmitted light mode, enable investigation of your sample from different perspectives to provide extra information about ice thickness and sample quality. Thanks to the sensitive Airyscan detector, which allows very gentle illumination, high resolution cryogenic imaging becomes possible.

Both the LSM and the widefield microscope are multipurpose tools that can be converted quickly from cryo to room temperature experiments and vice versa without compromising image quality.

Cryogenic SEM volume imaging and TEM lamella preparation

Cryogenic SEM volume imaging and TEM lamella preparation:
ZEISS Crossbeam 350/550/550L
FIB-SEM technology is well-established in various disciplines, but also found its way into life sciences both as a tool for cutting biological samples with the highest precision and because its imaging capabilities are ideal for unveiling the ultrastructure.

ZEISS Crossbeam was designed to give you highest usability and best image contrast. Even with unstained vitrified samples, this FIB-SEM generates high-contrast images at cryogenic temperatures, allowing the investigation of the ultrastructure of cells and tissues, and making cellular compartments clearly visible.

Typically, the low acceleration voltages required for cryogenic SEM imaging come with the trade-off of low contrast. ZEISS Crossbeam generates high contrast even at low acceleration voltages, a result of the unique interaction between the Gemini electron optics and the detectors. This configuration also opens the possibility to observe the imaging and milling procedure in real time – you can precisely control the milling process and ensure targeted on-grid thinning of ultrathin TEM lamellae.

ZEISS Crossbeam can be used as a multipurpose tool without any compromise in performance.

Keeping everything together: A well-aligned software package

To ensure a streamlined correlative cryo workflow and that the various components work together seamlessly, the software platforms involved were extended to include cryo-specific functions. Additional software modules have been developed to address the challenges arising from correlative cryogenic microscopy.

  • ZEISS ZEN
  • ZEISS ZEN Connect
  • ZEN EM Processing Toolbox
  • SmartSEM and SmartFIB
  • Cryo Drift Reduction
Correlative cryo dataset in ZEISS ZEN Connect
Correlative cryo dataset in ZEISS ZEN Connect

Applications

ZEISS Correlative Cryo Workflow at Work

Cell Biology: Identification of rare events

Spindle pole bodies are difficult to localize within yeast cells. They are small and rarely occurring structures. ZEISS Correlative Cryo Workflow lets you precisely identify and image such cellular structures in the near-to-native state. The LSM with the Airyscan detector makes the identification of these structures even easier so further details can be imaged. All images – from a large overview of the entire cell to high-resolution images of these tiny structures – are organized in a ZEN Connect project, providing all data needed to re-locate these cellular structures in the FIB-SEM.

Using the Crossbeam, TEM lamella of the identified regions can be prepared for cryo electron tomography. Volume imaging is possible as well. Furthermore, the workflow solution allows you to reconnect all data after image acquisition. Images from the Crossbeam or tomograms from the TEM can be combined with the LSM data and can be rendered in three-dimensional context.

Yeast cells labeled with NUP (nuclear pore complex)-GFP and CNM67-tdTomato.
Sample and tomogram courtesy of M. Pilhofer, ETH Zürich, Switzerland

LM and EM dataset – from the grid overview to the region of interest identified for further TEM tomography
FIB image of the prepared lamella; lamella thickness: 230 nm
FIB image of the prepared lamella; lamella thickness: 230 nm
Segmented and reconstructed tomogram

Cell Biology: Correlative 3D volume imaging

Once cellular structures such as spindle pole bodies are identified in the LSM system, the superior imaging quality of ZEISS Crossbeam allows targeting and analyzing the ultrastructure using cryogenic volume imaging. Even with low acceleration voltages, Crossbeam enables high-contrast imaging of non-stained, vitrified samples while protecting the sample from damage. High-resolution images acquired with the LSM and high-contrast images from the Crossbeam facilitate a precise image overlay. Once the region of interest is relocated in the Crossbeam using ZEN Connect, 3D datasets of the identified cells were acquired. Two spindle pole bodies were targeted within the correlative volume. The orientation of individual microtubules becomes clearly visible in the high-contrast images according to the cutting direction of the FIB. Further cell compartments could be identified in the 3D volume.

Sample courtesy of M. Pilhofer, ETH Zürich, Switzerland

Overlay of a high-resolution LSM/Airyscan image with a high-contrast Crossbeam image acquired under cryogenic conditions. The overlay was done with ZEN Connect.
3D volume reconstruction of yeast cells and segmentation of the nucleus (dark blue) as well as several mitochondria.
Longitudinally sectioned spindle pole body
Longitudinally sectioned spindle pole body within the nuclear membrane (top) and cross-sectioned microtubules outside the nuclear membrane (bottom). Image step size of the acquired stack: 50 nm

Cancer Research

Cancer cells exhibit a strong phenotype towards mitochondrial fission which potentially explains their resistance to drugs. Chemical fixation methods often create artifacts such as the accumulation of mitochondria which could be misinterpreted as fission events. Cryo fixation avoids these artifacts and preserves samples in the near-to-native state.

The example shows adenocarcinoma cells plunge-frozen on sapphire disks. LSM data already emphasize a dense mitochondrial network with increased fission subsequently confirmed by the Crossbeam data. After imaging with LSM and Airyscan, the vitrified sample was transferred to the Crossbeam. ZEN Connect was used to relocate the regions of interest, to overlay the respective dataset after acquisition, and to organize all images acquired.

Plunge-frozen adenocarcinoma cells grown on sapphire discs.
Plunge-frozen adenocarcinoma cells grown on sapphire discs. All regions of interest are shown in context in ZEN Connect.
3D dataset of one adenocarcinoma cell showing the strong mitochondrial fission pattern.
3D dataset of one adenocarcinoma cell showing the strong mitochondrial fission pattern.
Auto-segmented network of mitochondria in a subvolume of a Crossbeam dataset.

Plant Science

The response of plants to changing environmental conditions, such as increasing salinity, is an important research topic in plant science. Plants commonly show stress reactions as they cope with these changing conditions. One effect that can be observed on the ultrastructural level is the formation of so-called stromules, long tubular extensions in plastids.

The ZEN Connect project shows images of different imaging modalities: LSM was employed to localize stomata and internalized plastids using the autofluorescence of the sample. After successful re-localization of the region of interest, the LSM image was overlaid with an SEM overview image of the selected stoma. A FIB image stack of the stoma was acquired. The EM dataset revealed increased stromule formation in the plastids.

Sample courtesy: B. Franzisky, University of Hohenheim, Germany

Stomata and internalized plastids were identified with an LSM using the autofluorescence of the sample.
Stomata and internalized plastids were identified with an LSM using the autofluorescence of the sample. The selected stoma was re-located and imaged with the Crossbeam.
Stromules are clearly visible in the section planes acquired with the Crossbeam.
Stromules are clearly visible in the section planes acquired with the Crossbeam.
3D reconstruction and segmentation of the FIB image stack reveals the morphology of the plastids. The reconstruction shows stromules closely interacting with mitochondria.

Developmental Biology: Investigation of mitotic cells in C. elegans

Whole C. elegans worms were fixed by HPF and embryonic cells in metaphase were imaged in situ by cryo-fluorescence microscopy. The screened worms were then heavy-metal stained by freeze substitution, resin-embedded and sectioned so that the same volume could be located and imaged at high resolution, with high contrast, by the Crossbeam. Using this workflow, the targeted metaphase could be successfully reconstructed. Additionally, this approach allowed serendipitous discoveries: an adjacent intriguing punctate fluorescence signal was able to be correlated to a putative autophagosome.

Thus, cryo-fluorescence microscopy of high-pressure frozen thick samples can be used to trap and image transient cellular structures in a near-to-native state; appropriate processing and subsequent correlative volume EM imaging then allows the reconstruction of these targeted architectures at high resolution and in 3D.

Courtesy of Kedar Narayan, National Cancer Institute / NIH and Frederick National Laboratory for Cancer Research, USA

Top: The worm was imaged under cryogenic temperature with an LSM / Airyscan system before freeze substitution. Bottom: The embedded and stained worm was then imaged with a Crossbeam.
Top: The worm was imaged under cryogenic temperature with an LSM / Airyscan system before freeze substitution. Bottom: The embedded and stained worm was then imaged with a Crossbeam.
Reconstruction of cellular structures such as an autophagosome (AP) or the genome in different mitotic phases (*cell in metaphase, # cell in telophase).
Reconstruction of cellular structures such as an autophagosome (AP) or the genome in different mitotic phases (*cell in metaphase, # cell in telophase).
3D reconstruction of cellular structures

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ZEISS Correlative Cryo Workflow

Your Solution for TEM Lamella Preparation and Volume Imaging under Cryogenic Conditions

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