Super resolution imaging by programmable autonomous blinking.

摘要

Current far-field super-resolution techniques offer unprecedented spatial resolution, however, the identification and quantification of multiple molecules that cannot be spatially resolved remains challenging, mainly hampered by the lack of a comprehensive kinetic model for stochastic dye blinking, undercounting due to imperfect dye labeling of molecules, photobleaching, and limited multiplexing capabilities. Here I have developed and validated a quantitative multiplexing super-resolution approach, based on programmable autonomous blinking of a nucleic acid probe.;I use the transient binding of short fluorescently labeled oligonucleotides (technique named DNA-PAINT) for simple and easy-to-implement multiplexed 2D and 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. To achieve multiplexing we developed Exchange-PAINT that allows sequential imaging of multiple target molecules using only a single dye and a single laser source. For first time we experimentally have demonstrated super-resolution imaging of 10 synthetic DNA structures and 4 organelles imaging in cells by targeting 4 specific proteins. For single molecular counting we developed a method called quantitative PAINT or qPAINT, that enables counting by analyzing the predictable binding kinetics of the DNA imager strands with their targets molecules. We precisely benchmarked qPAINT using synthetic DNA nanostructures with a defined number of binding sites, and showed that qPAINT can count integer numbers of molecules with a precision of ∼90 % over a large dynamic range (10 to 150 molecules). High counting precision and accuracy was maintained when imaging cell surface receptor clusters, mRNA molecules in fixed cells and protein clusters from nucleoporines that form the nuclear pore complex (NCP). We also applied this new approach for analyzing the complex interactions of 5 receptor tyrosine kinases (RTKs) simultaneously within their native cellular context in a breast cancer cell line and finally for in situ visualization of single-copy regions of the genome in mouse fibroblasts.