Bibliographic Data

Abstract

DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) is a potent variant of single-molecule localization microscopy (SMLM) which is highly effective for multiplexed super-resolution imaging. It achieves localization precision down to nanometers in the lateral direction. However, its routine axial localization precision is approximately three-fold lower as compared to the lateral localization precision. Recently, a technique known as Metal-Induced Energy Transfer (MIET) has been introduced, offering excellent axial resolution at the nanometer scale up to 200 nm above a surface. MIET is characterized by a low entry barrier, as its sole technical requirement is the availability of a fluorescence lifetime imaging modality. In this study, we harness the synergy between the exceptional axial resolution provided by MIET and the lateral resolution achieved with DNA-PAINT (MIET-PAINT) to accomplish multitarget 3D super-resolution imaging. We implemented MIET-PAINT using a wide-field fluorescence lifetime imaging microscope. We validated our technique by measuring the height of emitters placed on top of spacers of known thicknesses. We then demonstrated multiplexed MIET-PAINT imaging of fixed cells to visualize mechanotransduction proteins in the focal adhesion complex (FAC) and the cytoskeleton. We explored the structural arrangement of paxillin, zyxin, and actin stress fibers in U2OS cells and discovered that MIET-PAINT can reliably address multiple targets, providing lateral and axial nanometerscale resolution.