Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

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Abstract:

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti: sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

Keywords: FLIM; FLUORESCENCE LIFETIME IMAGING MICROSCOPY; INSTRUMENT RESPONSE FUNCTION; IRF; LIFETIME STANDARDS; TWO-PHOTON EXCITATION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370210792081000

Affiliations: 1: Center for Commercialization of Fluorescence Technologies (CCFT), Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107; Dept. of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, 20-031 Lublin, Poland 2: Dept. of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland 3: Center for Commercialization of Fluorescence Technologies (CCFT), Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107 4: Center for Commercialization of Fluorescence Technologies (CCFT), Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107; Dept. of Cell Biology and Anatomy, UNTHSC, Fort Worth, Texas 76107

Publication date: August 1, 2010

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