Fluorescence instrument response standards in two-photon time-resolved spectroscopy.
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).
Luchowski
Rafal
R
Center for Commercialization of Fluorescence Technologies, Dept. of Molecular Biology & Immunology, UNTHSC, Fort Worth, Texas 76107, USA. rafal.luchowski@unthsc.edu
Szabelski
Mariusz
M
Sarkar
Pabak
P
Apicella
Elisa
E
Midde
Krishna
K
Raut
Sangram
S
Borejdo
Julian
J
Gryczynski
Zygmunt
Z
Gryczynski
Ignacy
I
D005456:Fluorescent Dyes / Q000737:chemistry*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes