Send to

Choose Destination
Opt Express. 2010 Aug 2;18(16):16628-39. doi: 10.1364/OE.18.016628.

Single-image separation measurements of two unresolved fluorophores.

Author information

Department of Physics, Washington University, St Louis, MO 63130, USA.


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.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Optical Society of America Icon for PubMed Central
Loading ...
Support Center