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Microsystems for Cryomicroscopy

Periodic Reporting for period 2 - MICROCRYO (Microsystems for Cryomicroscopy)

Reporting period: 2020-08-01 to 2022-01-31

The ERC project MICROCRYO aims to vastly improve our ability to study dynamic processes in cells and organelles by developing new methods for correlating live-cell imaging, cryogenic light microscopy, and electron cryomicroscopy with millisecond temporal and nanometer spatial resolution.
Despite rapid progress in the field, much of the potential of microscopy at cryogenic temperature today is still untapped due to limitations in sample preparation. First, vitrification technologies for cryomicroscopy have evolved only incrementally since the 1960s and cannot be combined with many of the sophisticated live imaging methods that have emerged over the past decade. Second, while the synergy of light and electron cryomicroscopy is extremely powerful, cryomicroscopy with light is still in its infancy. Finally, new technologies for ultra-rapid heating and cooling of single cells are needed to systematically advance our understanding of reversibility in the cryopreservation of e.g. stem cells, oocytes, or sperm cells.
This project aims to create a microfluidic technology platform for the direct vitrification of cells in the light microscope by ultra-rapid cooling with millisecond time resolution. The cells will then be imaged at high resolution using electron microscopy and advanced modes of light microscopy combined with new optics adapted to cryogenic conditions. Ultimately, we aim to elucidate if and under which conditions cryofixation can be reversed by ultra-rapid warming such that dynamic cellular processes resume unperturbed.
We expect that our research program will help to gain new insights into the structural and molecular basis of dynamic processes in cells. Understanding these connections is an important step toward identifying the causes of many diseases and devising effective therapies.
The project is designed along three main aims. Aim 1 is to advance a new paradigm for cryofixation that is based on microfluidics. Efficient fabrication technologies for the microfluidic ultra-rapid freezing devices have been established. Furthermore, we identified the technological constraints and the key parameters that determine the maximum size of biological systems for the approach. Heat transfer calculations predict that cooling rates at least two to three orders of magnitude greater than with conventional cryomicroscopy stages are attainable. Aim 2 addresses the lack of cryo-compatible immersion objectives and immersion media that match the optical performance of oil immersion at room temperature. Based on our previous work, we have been working to increase the numerical aperture of cryogenic immersion objectives and to enhance the stability during operation. Aim 3 investigates conditions under which cryofixation by ultra-rapid cooling is reversible. Work on this aim is ongoing.
In this reporting period, important conceptual insights have been gained. These now enable the parametric optimization of microfluidic ultra-rapid cooling devices for specific applications. In addition, newly created transfer technologies now provide an interface between microfluidic ultra-rapid cooling and established cryo-EM workflows. Similarly, a correlative light and electron microscopy (CLEM) workflow using immersion objectives has been established. Hardware and software components that are necessary to investigate perturbations of biological samples by ultra-rapid cooling and ultra-rapid warming have been implemented. Ultimately, we expect to create a platform technology that can be used to correlate live-cell imaging, cryogenic light microscopy, and electron cryomicroscopy with millisecond temporal and nanometer spatial resolution.
A microtechnology platform for correlating live-cell imaging, cryo-light microscopy, and cryo-EM