Cryo-fixation techniques, including cryo-electron and cryofluorescence microscopy, enable the preservation of biological samples in a near-native state by rapidly freezing them into an amorphous ice phase. These methods prevent the structural distortions often caused by chemical fixation, allowing for high-resolution imaging. At low temperatures, fluorophores exhibit improved properties, such as extended fluorescence lifetimes, reduced photobleaching, and enhanced signal-tonoise ratios, making single-molecule imaging more accurate and insightful. Despite these advantages, challenges remain, including limitations in numerical aperture of objectives and cryo-stage for single-molecule imaging, which can affect photon detection and spatial resolution. Recent advancements at low temperatures have mitigated these issues, achieving resolutions at the nanometer scale. Looking forward, innovations in super-resolution techniques, optimized fluorophores, and Artificial Intelligence (AI)-based data analysis promise to further advance the field, providing deeper insights into biomolecular dynamics and interactions. In this mini-review, we will introduce low-temperature single-molecule fluorescence imaging techniques and discuss future perspectives in this field. [BMB Reports 2025
58(1): 2-7].