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  Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy

Niederauer, C., Blumhardt, P., Mücksch, J., Heymann, M., Lambacher, A., & Schwille, P. (2018). Direct characterization of the evanescent field in objective-type total internal reflection fluorescence microscopy. Optics Express, 26(16), 20492-20506. doi:10.1364/OE.26.020492.

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© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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 Creators:
Niederauer, Christian1, Author              
Blumhardt, Philipp1, Author              
Mücksch, Jonas1, Author              
Heymann, Michael1, Author              
Lambacher, Armin2, Author              
Schwille, Petra1, Author              
Affiliations:
1Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565169              
2Fässler, Reinhard / Molecular Medicine, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565147              

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Free keywords: SINGLE SECRETORY GRANULES; CELL-SURFACE TOPOGRAPHY; LIVE CHROMAFFIN CELLS; CORRELATION SPECTROSCOPY; 3-DIMENSIONAL LOCALIZATION; PARTICLE TRACKING; TIRF MICROSCOPY; SUPERRESOLUTION; CALIBRATION; DYNAMICSOptics;
 Abstract: Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution. (c) 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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Language(s): eng - English
 Dates: 2018
 Publication Status: Published in print
 Pages: 15
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: ISI: 000440803600066
DOI: 10.1364/OE.26.020492
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Title: Optics Express
  Abbreviation : Opt. Express
Source Genre: Journal
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Publ. Info: Washington, DC : Optical Society of America
Pages: - Volume / Issue: 26 (16) Sequence Number: - Start / End Page: 20492 - 20506 Identifier: ISSN: 1094-4087
CoNE: https://pure.mpg.de/cone/journals/resource/954925609918