Multiplexed and Millimeter-Scale Fluorescence Nanoscopy of Cells and Tissue Sections via Prism-Illumination and...

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) of around 50 μm × 50 μm at once and more recently up to ∼200 μm × 200 μm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints to the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ∼520 μm × 520 μm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm² sample areas can be completed in as little as 40 min per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality, particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ∼10⁶ caveolae structures in ∼1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.