Multiplexed and millimeter-scale fluorescence nanoscopy of cells and tissue sections via prism-illumination and microfluidics-enhanced DNA-PAINT

Abstract

Superresolution microscopy (SRM) has become an enabling tool for biomedical research. A major limitation of SRM, however, is the small field-of-view (FOV), typically ~50μm x 50μm and up to ~200μm x 200μm in recent attempts, hampering its use in imaging large cell populations or clinical tissues. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for efficient, multiplexed SRM across millimeter-scale FOVs. Unlike existing SRM, PRIME-PAINT uses prism-type illumination for robust DNA-PAINT with single FOVs up to ~0.5mm x 0.5mm. Through stitching, imaging >1 mm2 FOVs can be completed in as little as an hour per target. The on-stage microfluidics not only facilitates multiplexing but enhances image quality, particularly for 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. Thus, we expect PRIME-PAINT to be useful toward building multiscale, Google-Earth-like views of biological systems.

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

“…Our microfluidic chips consist of a polydimethylsiloxane (PDMS) channel [36][37][38][39][40][41] with a 3D nanoprinted metalized insert which reflects the LS into the sample and enables convenient perfusion of solutions during multi-target Exchange-PAINT imaging. Previously developed single-objective LS systems have utilized various reflection mechanisms such as a micromirror 42 , an AFM cantilever mirror 43 , pyramidal microcavities 44 , a glass prism 45 , and a microfluidic chamber with metalized side walls 46 to redirect the LS into the sample. However, these systems use imaging chambers that do not offer design flexibility 18,[42][43][44]46 , active solution exchange 18,[42][43][44] , or compatibility with epi-illumination and transmission microscopy 42,46 .…”

Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA‐PAINT‐ERS

“…Additionally, we will discuss several critical steps in immunostaining to ensure that the sample is prepared with high quality for best DNA‐PAINT‐ERS imaging results. These steps should be readily adapted to other types of affinity agents such as nanobodies (Koester, Tao, Szczepaniak, Rames, & Nan, 2022) and sample formats such as clinical tissue sections (Rames et al., 2022).…”

DNA-FRET Constructs Enable Multiplexed Fluorescence Detection at the Single-Molecule Level

“…This approach is compatible with other multiplexing strategies such as additional excitation lasers, 8 novel fluorophore types, orientational control to influence dye polarization, and DNA-PAINT. 9,10 Alternative molecular scaffolds could also be used to create FRETfluors with appealing properties. Particularly, xeno-nucleic acids (XNAs) with synthetic backbones are more resistant to nuclease digestion and thus suitable for use in complex biological mixtures.…”