Periodic Reporting for period 4 - MICROCRYO (Microsystems for Cryomicroscopy)
Periodo di rendicontazione: 2023-08-01 al 2025-01-31
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 temperatures today is still untapped due to limitations in sample preparation. First, vitrification technologies for cryomicroscopy have evolved only incrementally since the 1960s. They cannot be combined with many of the sophisticated live imaging methods that have emerged over the past decade. Second, while the symbiosis 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 are then 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.
Despite rapid progress in the field, much of the potential of microscopy at cryogenic temperatures today is still untapped due to limitations in sample preparation. First, vitrification technologies for cryomicroscopy have evolved only incrementally since the 1960s. They cannot be combined with many of the sophisticated live imaging methods that have emerged over the past decade. Second, while the symbiosis 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 are then 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, which we were able to confirm. Aim 2 addresses the lack of cryo-compatible immersion objectives and immersion media that match the optical performance of oil immersion at room temperature. To this end, a new platform for cryogenic light microscopy based on immersion objectives with high numerical aperture and aberration correction was established. The new technology supports widefield, confocal, and modern super-resolution methods with cryogenic optical performance on par with room-temperature systems. Using this technology, interesting perspectives for cryogenic light microscopy exploiting the unique low-temperature photophysics of fluorescent dyes and proteins were explored. Aim 3 investigates conditions under which cryofixation by ultra-rapid cooling is reversible. Excellent preservation of the structure of a variety of native suspension cells, including human erythrocytes, could be demonstrated.
The results of the project have been disseminated through publication in scientific articles and conferences.
The results of the project have been disseminated through publication in scientific articles and conferences.
Several conceptual and technological advances were made in this project. Microsystems technology was employed for the first time to create microenvironments far from thermal equilibrium. These open new avenues for preparing and imaging vitrified biological objects with light and electron microscopy. In particular, our approach enabled the optimization of microfluidic ultra-rapid cooling devices, which allow cryofixation by ultra-rapid cooling during live imaging. 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 necessary to investigate perturbations of biological samples by ultra-rapid cooling and ultra-rapid warming have been implemented. These new methods were used successfully to cryopreserve human erythrocytes at low cryoprotectant concentrations and investigate their state by super-resolution cryomicroscopy. We expect the platform technology established in this project, connecting live-cell imaging, cryogenic light microscopy, and electron cryomicroscopy, will enable new investigations into the connections between structure and function in cell biology, biomechanics, and structural biology.