Single-image measurements of monochromatic subdiffraction dimolecular separations

TL;DR

This paper introduces SMID, a method for measuring subdiffraction separations between identical fluorescent molecules using a single millisecond exposure image, achieving nanometer precision and high temporal resolution.

Contribution

The study presents a novel single-image approach for measuring subdiffraction separations between identical fluorophores with nanometer accuracy and millisecond temporal resolution.

Findings

Measuring separations down to 40 nm with nanometer precision.

Achieved millisecond temporal resolution in separation measurements.

Enabled single-image analysis of molecular dimers.

Abstract

Measuring subdiffraction separations between single fluorescent particles is important for biological, nano-, and medical-technology studies. Major challenges include (i) measuring changing molecular separations with high temporal resolution while (ii) using identical fluorescent labels. Here we report a method that measures subdiffraction separations between two identical fluorophores by using a single image of milliseconds exposure time and a standard single-molecule fluorescent imaging setup. The fluorophores do not need to be bleached and the separations can be measured down to 40 nm with nanometer precision. The method is called single-molecule image deconvolution -- SMID, and in this article it measures the standard deviation (SD) of Gaussian-approximated combined fluorescent intensity profiles of the two subdiffraction-separated fluorophores. This study enables measurements of (i) subdiffraction dimolecular separations using a single image, lifting the temporal resolution of seconds to milliseconds, while (ii) using identical fluorophores. The single-image nature of this dimer separation study makes it a single-image molecular analysis (SIMA) study.