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Lee, Ja Yil
School of Life Sciences
Research Interests
  • Investigated eukaryotic homologous recombination.

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Characterization of the evanescent field in objective-based total-internal-reflection fluorescence (TIRF) microscopy

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Title
Characterization of the evanescent field in objective-based total-internal-reflection fluorescence (TIRF) microscopy
Author
Lee, Ja YilKim, Shul-KeeHong, Seok-Cheol
Keywords
Evanescent field;  Z distance measurement;  Total-internal-reflection fluorescence microscopy
Issue Date
2007-05
Publisher
KOREAN PHYSICAL SOC
Citation
JOURNAL OF THE KOREAN PHYSICAL SOCIETY, v.50, no.5, pp.1340 - 1345
Abstract
Owing to its single-molecule sensitivity, objective-based total-internal-reflection fluorescence microscopy has been used to study various biological phenomena, such as conformation changes of a single biomolecule and localization and movement of single molecules inside a cell. In the technique, fluorophores associated with biomolecules are excited by an evanescent field formed near the water/coverglass interface (typically, within 100 to 200 nm). Here, we characterized the axial profile of the evanescent field. By using a prism-based scheme and applying Snell's law, we experimentally measured the incident angle of the excitation light and calculated the penetration depth of the evanescent field. The penetration depth was also acquired experimentally by measuring the fluorescence from a fluorescent bead for the excitation light at various incident angles. These independent approaches permitted highly accurate characterization of the evanescent field profile. This field profile provided the information on the distribution of the evanescent field along the axial direction, which will be useful in studying with this technique various biological phenomena as it provides a means to probe the axial dimension precisely.
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DOI
10.3938/jkps.50.1340
ISSN
0374-4884
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SLS_Journal Papers
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