A High-Resolution Microscopy System for Biological Studies of Cold-Adapted Species Under Physiological Conditions

The Antarctic seabed harbors significant biodiversity, and almost 90% of oceanic environments are permanently below 5 °C (i.e., deep sea and polar regions). However, organisms whose entire lifecycle occurs around 0 °C are understudied, leaving this large and diverse proportion of the global biome poorly understood. To address this question at the cellular level, tools are required for high-resolution imaging of biological systems under physiological conditions. This poses severe technical challenges. High-resolution imaging objectives require short working distances and immersion media, causing rapid heat transfer from the microscope to the sample. This affects the viability of live specimens and the interpretability of results. Here, we present a method for high-fidelity imaging of live biological samples at temperatures of around, or below, 0 °C. It relies on hardware additions to traditional microscopy, namely as a cooling collar, 10% ethanol as an immersion medium, and nitrogen flow to reduce condensation It can be straightforwardly implemented on different microscopy modalities, including super-resolution imaging. The method is demonstrated in live cell cultures derived from Antarctic fish and highlights the need to maintain physiological conditions for these fragile samples. Future applications include evolutionary biology, biophysics and biotechnology.