Abstract
We have developed a multiphoton microscopy (MPM) system using a 12-fs Ti:sapphire laser with adjustable dispersion precompensation in order to examine the impact of pulse duration on nonlinear optical signals. The efficiencies of two-photon-excited fluorescence (TPEF) and second harmonic generation (SHG) were studied for various pulse durations, measured at the sample, ranging from approximately 400 fs to sub-20 fs. Both TPEF and SHG increased proportionally to the inverse of the pulse duration for the entire tested range. Because of improved signal-to-noise ratio, sub-20-fs pulses were used to enhance MPM imaging depth by approximately 160%, compared to 120-fs pulses, in human skin.
Highlights
Multiphoton microscopy, combining two-photon excited fluorescenceTPEFand second harmonic generationSHG, has become an important tool for high-resolution, noninvasive imaging of thick biological tissues.[1,2,3] the efficien
Xu and Webb examined the impact of pulse duration on TPEF and observed an inverse relationship for pulses longer than 90 fs.[5]
In Müller et al.,[6] optical pulses of 15-fs duration were achieved at the focal plane of high numerical apertureNAobjectives and a significant increase in TPEF was observed
Summary
Multiphoton microscopy, combining two-photon excited fluorescenceTPEFand second harmonic generationSHG, has become an important tool for high-resolution, noninvasive imaging of thick biological tissues.[1,2,3] the efficien-. Femtosecond pulsed lasers are required in order to increase the peak intensity of excitation. The excitation efficiencies of TPEF and SHG both depend quadratically on the peak intensity of the excitation laser beam.
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