Single-image separation measurements of two unresolved fluorophores

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.