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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 21 — Oct. 21, 2013
  • pp: 25291–25306

Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution

Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar  »View Author Affiliations


Optics Express, Vol. 21, Issue 21, pp. 25291-25306 (2013)
http://dx.doi.org/10.1364/OE.21.025291


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Abstract

Fluorescence microspectroscopy (FMS) with environmentally sensitive dyes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. For filter-based experimental systems, where signal at different wavelengths is acquired sequentially, photostability has been required in addition. Herein, we systematically analyzed our spectral fitting models and bleaching correction algorithms which mitigate both limitations. We showed that careful analysis of data acquired by stochastic wavelength sampling enables nanometer spectral peak position resolution even for highly photosensitive fluorophores. To demonstrate how small spectral shifts and changes in bleaching rates can be exploited, we analyzed vesicles in different lipid phases. Our findings suggest that a wide range of dyes, commonly used in bulk spectrofluorimetry but largely avoided in microspectroscopy due to the above-mentioned restrictions, can be efficiently applied also in FMS.

© 2013 Optical Society of America

OCIS Codes
(000.2170) General : Equipment and techniques
(000.4430) General : Numerical approximation and analysis
(070.4790) Fourier optics and signal processing : Spectrum analysis
(180.2520) Microscopy : Fluorescence microscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence
(110.4234) Imaging systems : Multispectral and hyperspectral imaging

ToC Category:
Microscopy

History
Original Manuscript: August 12, 2013
Revised Manuscript: October 7, 2013
Manuscript Accepted: October 8, 2013
Published: October 16, 2013

Citation
Iztok Urbančič, Zoran Arsov, Ajasja Ljubetič, Daniele Biglino, and Janez Štrancar, "Bleaching-corrected fluorescence microspectroscopy with nanometer peak position resolution," Opt. Express 21, 25291-25306 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-21-25291


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