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J. Qu, L. Liu, B. Guo, H. Niu; Time-Resolved Two-Photon Excitation Fluorescence Spectroscopy for Biomedicine. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4246.
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Two-photon excitation fluorescence and time-resolved fluorescence spectroscopy are powerful tools in biomedicine. The purpose of this work is to develop a novel time-resolved two-photon excitation fluorescence spectroscopy system that has the advantages of reduced photobleaching and photodamage, enhanced penetration depth and high signal-to-noise ratio measurement of biological specimens at submicron resolution.
The time-resolved two-photon excitation fluorescence spectroscopy system mainly consists of a titanium: sapphire laser, a pulse picker, a polychromator and a high repetition rate streak camera. The titanium: sapphire laser provides mode-locked, ultrafast femtosecond pulses whose repetition rate is reduced to 1MHz with the pulse picker. The output from the pulse picker is focused onto the sample for excitation by two-photon absorption. The emitted fluorescence is dispersed by a polychromator and imaged onto the slit of the streak camera. The spectrally resolved fluorescence lifetime image of the excited spot on the sample is obtained with a fiberoptically taper coupled and cooled CCD camera.
Calibration of the system shows that it has temporal and spectral resolutions of 6ps and 2nm respectively. A few fluorescent dyes, such as Rhodamine B, Rhodamine 6G, Rose Bengal and Coumarin 314, and their mixtures are used to test the system. The results show that the combination of lifetime and spectrum measurement using two photon excitation provides a unique differentiation of fluorescence dyes with overlapping spectra but different lifetimes or vice versa. The potential applications of the system in biomedicine are demonstrated with a few biological samples, such as the FAD solution, gastric smooth muscle tissue and highly differentiated adenocarcinoma of stomach etc.
We report the development of a novel time-resolved two-photon excitation fluorescence spectroscopy system. Detailed performance of the system has been validated using several standard fluorescent dyes and some bio-samples. Fluorescence spectra and lifetimes of the tested compounds are found to be in good agreement with the values reported in literatures.
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