Cryogenic single-molecule fluorescence imaging

Abstract

Cryo-fixation techniques, including cryo-electron and cryofluorescencemicroscopy, enable the preservation of biologicalsamples in a near-native state by rapidly freezing theminto an amorphous ice phase. These methods prevent thestructural distortions often caused by chemical fixation, allowingfor high-resolution imaging. At low temperatures, fluorophoresexhibit improved properties, such as extended fluorescencelifetimes, reduced photobleaching, and enhanced signal-tonoiseratios, making single-molecule imaging more accurateand insightful. Despite these advantages, challenges remain,including limitations in numerical aperture of objectives andcryo-stage for single-molecule imaging, which can affect photondetection and spatial resolution. Recent advancements atlow temperatures have mitigated these issues, achieving resolutionsat the nanometer scale. Looking forward, innovationsin super-resolution techniques, optimized fluorophores, andArtificial Intelligence (AI)-based data analysis promise to furtheradvance the field, providing deeper insights into biomoleculardynamics and interactions. In this mini-review, we will introducelow-temperature single-molecule fluorescence imaging techniquesand discuss future perspectives in this field.