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)
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