ArticleMethods and applications in fluorescence2026
Challenges and limitations for live cell imaging in extreme cold.
Article in Methods and applications in fluorescence, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Many ecosystems thrive in near-0 °C conditions, and the mechanisms supporting life in these conditions remain understudied due to the challenges in reproducing such environments in laboratory conditions. One such example is polar organisms, that have adapted their entire lifecycle to operate below freezing temperatures through largely unknown cellular adaptations. As rapid polar warming threatens these species, elucidating their survival strategies is increasingly urgent. Fluorescence-based optical microscopy has been central to the understanding of the dynamic processes sustaining life at the cellular level, yet most imaging approaches have been developed and validated for conditions near mammalian physiological temperatures. Imaging at low temperature introduces a distinct physical regime in which molecular motion, membrane organisation, protein conformational dynamics, and fluorophore photophysics are fundamentally altered. As a result, imaging tools, fluorescent probes, and super-resolution methods optimised at 37 °C often fail when applied near 0 °C, or they report biased information. Here, we examine the conceptual, technical, and practical challenges associated with live-cell fluorescence microscopy at cold temperatures. We discuss when and why common imaging modalities and labelling strategies break down, and how probe behaviour becomes tightly coupled to local changes in physicochemical environment. We offer a perspective on new biological questions that become accessible for study with a microscopy platform optimised for imaging in cold conditions. We highlight trade-offs in current temperature-control strategies and identify unmet needs in fluorophore design, instrument engineering, and quantitative standards. By framing cold microscopy as a distinct operational regime rather than an extension of conventional live-cell imaging, this perspective aims to guide the development of robust tools for studying biological systems near-0 °C conditions.
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