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bioRxiv · 10.1101/2024.02.08.579549

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

Nelson, T.·Vargas-Hernandez, S.·Freire, M.·Cheng, S.·Gustavsson, A.-K.
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Abstract

Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise, 3D nanoscale studies of a wide range of cellular structures. Here we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi-and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D singlemolecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

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Nelson, T., Vargas-Hernandez, S., Freire, M., Cheng, S., Gustavsson, A.-K.. 2024-02-12. Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells. https://doi.org/10.1101/2024.02.08.579549

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