ZEISS Correlative Cryo Workflow

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.

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.

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}

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}