Huebinger Jan, Grecco Hernan, Masip Martín E, Christmann Jens, Fuhr Günter R, Bastiaens Philippe I H
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str.11, 44227 Dortmund, Germany.
Department of Physics, FCEN, University of Buenos Aires and IFIBA, CONICET, Buenos Aires, Argentina.
Sci Adv. 2021 Dec 10;7(50):eabk0882. doi: 10.1126/sciadv.abk0882.
Imaging molecular patterns in cells by fluorescence micro- or nanoscopy has the potential to relate collective molecular behavior to cellular function. However, spatial and spectroscopic resolution is fundamentally limited by motional blur caused by finite photon fluxes and photobleaching. At physiological temperatures, photochemical reactivity does not only limit imaging at multiple scales but is also toxic to biochemical reactions that maintain cellular organization. Here, we present cryoprotectant-free ultrarapid cryo-arrest directly on a multimodal fluorescence microscope that preserves the out-of-equilibrium molecular organization of living cells. This allows the imaging of dynamic processes before cryo-arrest in combination with precise molecular pattern determination at multiple scales within the same cells under cryo-arrest. We both experimentally and theoretically show that ultrarapid cryo-arrest overcomes the fundamental resolution barrier imposed by motional blur and photochemical reactivity, enabling observation of native molecular distributions and reaction patterns that are not resolvable at physiological temperatures.
通过荧光显微镜或纳米显微镜对细胞中的分子模式进行成像，有可能将集体分子行为与细胞功能联系起来。然而，空间和光谱分辨率从根本上受到有限光子通量和光漂白引起的运动模糊的限制。在生理温度下，光化学反应性不仅限制了多尺度成像，而且对维持细胞组织的生化反应有毒害作用。在这里，我们展示了直接在多模态荧光显微镜上进行无冷冻保护剂的超快速冷冻捕获，它保留了活细胞的非平衡分子组织。这使得在冷冻捕获之前对动态过程进行成像，并结合在冷冻捕获状态下对同一细胞内多个尺度的精确分子模式进行测定成为可能。我们通过实验和理论证明，超快速冷冻捕获克服了运动模糊和光化学反应性所带来的基本分辨率障碍，能够观察到在生理温度下无法分辨的天然分子分布和反应模式。
