Research Highlights

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 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.

Figure 2 shows the intensities of the TPEF and SHG signals excited from samples with laser pulses of different durations from 400 fs to sub-20 fs measured by intensity autocorrelations. When the pulse duration was varied, the average laser power was maintained at a constant level. The SHG intensities from the rat-tail tendon were measured in both forward and backward propagation directions for the interest of studying the directionality of SHG generation. The SHG signals reported are generally detected in backward direction if not otherwise specified. Figure 2(a) shows the TPEF and SHG signals obtained with the 10X objective. Figure 2(b) shows that multiphoton signals obtained with the 40X objective are similar to those obtained with the 10X objective. These results demonstrate that significant improvement in excitation efficiency can be achieved by shortening the laser pulses. Such improvement continues as the pulse duration is shortened to the sub-20-fs regime. This result holds true for both high and low NA objectives. Our experimental result agrees well with the theoretical prediction. The significant increase in TPEF and SHG signal intensities using ultrashort laser pulse excitation has important implications for nonlinear microscopy. As the excitation efficiency is improved, we can potentially image deeper into thick tissues and increase frame rates.

In conclusion, we have studied the effects of pulse duration on the generation of TPEF and SHG signals at the tight focusing of objectives in nonlinear optical microscopy. Both the TPEF and SHG efficiencies are found to increase proportionally to the inverse of the pulse duration even in the sub-20-fs regime. Practical advantages of using sub-20-fs pulses for multiphoton microscopy are demonstrated by increased tissue penetration and reduced integration times.

Fig. 1 Schematics of the experimental setup. The block in the dashed lines is inserted when interferometric autocorrelation is measured. BS: beam splitter; DM: dichroic mirror; F: filter; L: lens; O: objective; P: prism; PMT: photomultiplier tube.

Fig. 2 Intensities of TPEF and SHG versus pulse duration obtained with (a) 10× and (b) 40× objectives. The squares are for TPEF signals from dilute flourescein solution. The circles and triangles are for SHG signals from rat-tail tendon in forward and backward detections, respectively.

Fig. 3 TPEF and SHG spectra from human skin obtained at various depths excited with (a) sub-20-fs and (b) 120-fs pulses, respectively. Peak values of the TPEF and SHG spectra versus penetration depth obtained with corresponding (c) sub-20-fs and (d) 120-fs pulses. The insertions show the (f) TPEF and (e) SHG images of cellular and fibrous collagen structures at ~40-µm and ~80-µm depths, respectively. The scale bars are 10 µm.

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