Time-resolved two-photon excitation fluorescence spectroscopy and microscopy using a high repetition rate streak camera

Abstract

We present a time-resolved two-photon excitation fluorescence spectroscopy and a simultaneous time-and spectrumresolved multifocal multiphoton microscopy system that is based on a high repetition rate picosecond streak camera for providing time-and spectrum-resolved measurement and imaging in biomedicine. The performance of the system is tested and characterized by the fluorescence spectrum and lifetime analysis of several standard fluorescent dyes and their mixtures. Spectrum-resolved fluorescence lifetime images of fluorescence beads are obtained. Potential applications of the system include clinical diagnostics and cell biology etc. DOIFluorescence spectroscopy and microscopy have been applied widely in biomedicine and some other fields to study the interactions between electromagnetic radiation and matters and the properties of excited states of atoms and molecules. There are many fluorescence parameters, including fluorescence intensity, spectral profile, peak wavelength, quantum yield, polarization and fluorescence lifetime etc. whose combined measurement can provide complementary information about the object under study. For example, fluorescence spectrum measurement can distinguish different molecular species and contrast different fluorophores in samples. Because fluorescence lifetime is sensitive to the local environment of the fluorophores, many physiological parameters including pH, Ca 2+ , Na + and pO 2 can be quantified via fluorescence lifetime measurement. Fluorescence lifetime contrast is particularly useful in identifying fluorophores with significantly overlapping spectra. Multiphoton excitation fluorescence spectroscopy and microscopy have particularly gained popularity in biomedicine because of many advantages associated with the nonlinear multiphoton excitation [1] , e.g., reduced photobleaching and photodamage, enhanced penetration depth and high signal-to-noise ratio imaging of biological specimens in three dimensions at submicron resolution. Simultaneous measurement of two-photon excitation fluorescence spectra and lifetimes has been applied widely in biology and clinics, e.g., protein-protein interaction study [2] , early cancer diagnosis [3] and tissue discrimination [4] . In order to make full use of the power from a femtosecond Ti: Sapphire laser and reduce the acquisition time in multiphoton fluorescence microscopy, a novel method that employs parallel excitation and detection has been implemented in multifocal multiphoton microscopy (MMM), which uses a microlens array [5,6] or a diffraction optical element [7] to produce an array of high numerical aperture foci on the sample for simultaneous multiphoton excitation and delivers video-rate imaging at high spatial resolution. Recently, fluorescence lifetime imaging (FLIM) has been demonstrated in MMM configuration by several groups [8,9] .Time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy are usually implemented in three configurations, i.e. time-correlated single-photon counting (TCSPC) [10] , time-ga...