Microfluidics-Enhanced 3D DNA-Based Points Accumulation for Imaging in Nanoscale Topography

Single-molecule localization microscopy (SMLM) enables the examination of biological samples at the nanoscale. DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a flexible SMLM-based approach with a broad capability for multiplexing. However, low signal-to-background ratio and the solution exchange process present challenges for three-dimensional (3D) multitarget DNA-PAINT imaging. In this work, we present DNA-PAINT in microfluidics for enhanced 3D imaging under simple wide-field epi-illumination. We show that imaging in a microfluidic chip reduces background fluorescence and demonstrate flow-associated improvements in number of localizations and signal photon counts. To provide a perspective on the mechanisms underlying the flow-associated enhancements, we perform numerical simulations to model mass transport, binding kinetics, and photobleaching in DNA-PAINT imaging with microfluidics. Finally, we demonstrate microfluidics-enhanced three-target multiplexed DNA-PAINT imaging, revealing 3D architecture and spatial relationships between cellular targets. Microfluidics-enhanced DNA-PAINT is built on commercially available components with minimal instrumentation, is compatible with standard wide-field illumination strategies, and can be implemented in a broadly accessible manner, facilitating improved nanoscale studies across a wide range of biological and biomedical applications.