Quantifying UV-induced photodamage for longitudinal live-cell imaging applications of deep-UV microscopy.

Abstract

Deep-UV microscopy enables high-resolution, label-free molecular imaging by leveraging biomolecular absorption properties in the UV spectrum. Recent advances in UV-imaging hardware have renewed interest in this technique for quantitative live cell imaging applications. However, UV-induced photodamage remains a concern for longitudinal dynamic imaging studies. Here, we quantify UV phototoxicity with several cell types at notable UV wavelengths. We find that the fluence required for cell death via UV phototoxicity with continuous UV exposure varies with cell type and wavelength from ∼0.5µJ/µm² to 2µJ/µm², but is independent of typical illumination power/radiant flux of UV microscopy (e.g., 0.1-20 nW/µm²). We also show results from fractionation studies that reveal cell repair following UV exposure, which increases the tolerance to UV radiation by a factor of 2 or more, depending on the fractionation paradigm. Results further show that UV tolerance exceeds ANSI guidelines for maximum permissible exposure. Finally, we calculate imaging limits for a typical application of UV microscopy, such as hematology analysis. Together, this work provides UV fluence thresholds that can serve as guidelines for nondestructive, longitudinal, and dynamic deep-UV microscopy experiments.