ZEISS Correlative Cryo Workflow

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

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

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.

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: C. Burkhardt, NMI Reutlingen, Germany

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