Open AccessAutofocusing system based on optical astigmatism analysis of single-molecule images
Author(s) -
Wonseok Hwang,
Sangsu Bae,
Sungchul Hohng
Publication year - 2012
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.20.029353
Subject(s) - optics , astigmatism , numerical aperture , detector , optical tweezers , laser , aperture (computer memory) , physics , wavelength , acoustics
Single-molecule fluorescence imaging has greatly contributed to our understanding of many bio-molecular systems. While reactions occurring in the range of several minutes can be readily studied using conventional single-molecule fluorescence microscopes, data acquisition for longer time scales is hindered by the focal drift of high numerical aperture objectives, which should be corrected in real time. Here, we developed a robust autofocusing system based on optical astigmatism analysis of single-molecule images. Compared to the previously developed methods, our approach has a merit of simplicity in that neither fiducial makers nor an additional laser-detector system is required. As a demonstration, we observed B-Z transition dynamics occurring for several hours.