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Fluorescence instrument response standards in two-photon time-resolved spectroscopy.

Rafal Luchowski1, Mariusz Szabelski, Pabak Sarkar

  • 1Center for Commercialization of Fluorescence Technologies, Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107, USA. rafal.luchowski@unthsc.edu

Applied Spectroscopy
|August 20, 2010
PubMed
Summary
This summary is machine-generated.

This study introduces picosecond lifetime standards for two-photon excitation, using rose bengal, pyridine 1, and LDS 798. These fluorophores enable accurate fluorescence lifetime measurements in multiphoton spectroscopy and imaging microscopy.

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Area of Science:

  • Spectroscopy and Photochemistry
  • Biophysics and Imaging

Background:

  • Accurate fluorescence lifetime measurements are crucial for advanced imaging techniques like fluorescence lifetime imaging microscopy (FLIM).
  • Traditional methods using scatterers as references for multiphoton excitation can be unreliable due to detector sensitivity variations, especially in the near-infrared spectrum.

Purpose of the Study:

  • To identify and validate suitable picosecond fluorescence lifetime standards for two-photon excitation.
  • To provide reliable references for accurate deconvolution of fluorescence lifetime data in multiphoton spectroscopy and FLIM.

Main Methods:

  • Investigated the fluorescence properties of rose bengal, pyridine 1, and LDS 798 under Ti:sapphire femtosecond laser excitation.
  • Measured fluorescence emission spectra covering the visible to near-infrared range (550–850 nm).
  • Demonstrated the use of these fluorophores for measuring instrument response functions and reconvolving fluorescence lifetime data.

Main Results:

  • Rose bengal, pyridine 1, and LDS 798 exhibit suitable fluorescence properties for picosecond lifetime standards in two-photon excitation.
  • The selected fluorophores provide reliable references, overcoming limitations of scatterers related to detector spectral sensitivity.
  • Accurate reconvolution of measured lifetimes was achieved using the proposed standards.

Conclusions:

  • Picosecond lifetime standards are essential for precise fluorescence lifetime measurements in multiphoton applications.
  • Rose bengal, pyridine 1, and LDS 798 are proposed as effective standards for two-photon excitation.
  • These standards will enhance the accuracy and applicability of multiphoton spectroscopy and FLIM.