Single-molecule high-resolution imaging with photobleaching

TLDR

Conventional light microscopy is limited to ~200 nm resolution, while spectroscopic methods such as FRET cannot resolve distances beyond 10 nm, leaving a 10–100 nm gap in optical measurement capability. The study aims to locate two fluorescent dyes and determine their separation with 5‑nm precision by exploiting the quantal photobleaching of single molecules. By fitting images taken before and after photobleaching of one dye, the authors simultaneously localize both dyes and compute their separation. They achieve nanometer‑scale resolution, measuring 10–20 nm separations between fluorophores on DNA, thereby bypassing the Rayleigh limit and offering potential for biophysical and genomic applications such as high‑density SNP mapping.

Abstract

Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a “gap” in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10–20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.