Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm....Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm.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 singlemolecule localization with up to∼520μm×520μm single FOVs and 25−40 nm lateral resolutions.Through stitching,nanoscopic imaging over mm^(2)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,PRIMEPAINT will be useful for building multiscale,Google-Earth-like views of biological systems.展开更多
基金supported by the Cancer Early Detection Advanced Research(CEDAR)Center of the OHSU Knight Cancer Institutesupported in part by the OHSU Knight Cancer Institute,the Damon Runyon Cancer Research Foundation,the Cancer Systems Biology Consortium from the National Cancer Institute(CSBC,grant number U54 CA209988,PI:Joe W.Gray)the National Institute of General Medical Sciences(grant number R01 GM132322,PI:X.N.).
文摘Fluorescence nanoscopy has become increasingly powerful for biomedical research,but it has historically afforded a small field-ofview(FOV)of around 50μm×50μm at once and more recently up to∼200μm×200μm.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 singlemolecule localization with up to∼520μm×520μm single FOVs and 25−40 nm lateral resolutions.Through stitching,nanoscopic imaging over mm^(2)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,PRIMEPAINT will be useful for building multiscale,Google-Earth-like views of biological systems.
