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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editor: Gregory W. Faris
  • Vol. 5, Iss. 12 — Sep. 30, 2010

Single-image separation measurements of two unresolved fluorophores

Shawn H. DeCenzo, Michael C. DeSantis, and Y. M. Wang  »View Author Affiliations

Optics Express, Vol. 18, Issue 16, pp. 16628-16639 (2010)

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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.

© 2010 Optical Society of America

OCIS Codes
(100.6640) Image processing : Superresolution
(110.2960) Imaging systems : Image analysis
(180.2520) Microscopy : Fluorescence microscopy

ToC Category:
Imaging Systems

Original Manuscript: March 17, 2010
Revised Manuscript: July 2, 2010
Manuscript Accepted: July 15, 2010
Published: July 23, 2010

Virtual Issues
Vol. 5, Iss. 12 Virtual Journal for Biomedical Optics

Shawn H. DeCenzo, Michael C. DeSantis, and Y. M. Wang, "Single-image separation measurements of two unresolved fluorophores," Opt. Express 18, 16628-16639 (2010)

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