Ultrarapid cryo-fixation during live observation on a fluorescence microscope
Bildgebung und Mikroskopie
Fluorescence micro- and nanoscopy has the potential to resolve dynamically established patterns of molecular reactivity inside living cells down to the nm scale. This potential is however mitigated by photon collection that is fundamentally limited by photochemical reactivity and motional blur. This limit set by the photophysical properties of fluorophores cannot be surpassed by better detectors or stronger illumination. A solution to reach practically unlimited photon collection times is halting photoreactivity and bypassing motional blur by virtually instant fixation of cells at a particular instant in time by extreme rapid cooling to a temperature below -136 °C. This ultra-high cooling speed is necessary to maintain water out-of-equilibrium to prevent mechanical damage by ice crystal formation and to avoid decay of the energized microscopic biomolecular patterns. Performing ultra-rapid cryo-arrest directly on a microscope enables virtually instant fixation of native molecular patterns at any timepoint during the observation of their reaction dynamics at physiological temperatures.
Our scientists have developed an ultrarapid cryo-arrest directly on a multimodal fluorescence microscope that preserves molecular activity patterns during observation of their dynamics in living cells at any timepoint. By mounting of the sample directly on a diamond with superior thermal conductivity, cooling speeds of up to 200,000 °C/s to -196 °C can be generated, which prevents ice crystal formation and preserves cells even without application of cryoprotectants. This allows imaging of dynamic processes before cryo-arrest in combination with precise molecular pattern determination at multiple resolutions and modalities within the same cells under cryo-arrest. Ultrarapid cryo-arrest overcomes the fundamental resolution barrier imposed by motional blur and photochemical reactivity and enables observation of native molecular distributions and reaction patterns that are not resolvable at physiological temperatures.
We are now looking for either a licensing partner, or a collaboration partner to further develop this project.
The PCT application was filed ind 2022.
Huebinger et al. (2021). Science Advances. DOI: 10.1126/sciadv.abk0882
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Dr. Ingrid Kapser-Fischer
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