Abstract
.Two-photon microscopy (2PM) has revolutionized biomedical imaging by allowing thin optical sectioning in relatively thick biological specimens. Because dispersive microscope components in 2PM, such as objective lens, can alter temporal laser pulse width (typically being broader at the sample plane), for accurate measurements of two-photon absorption properties, it is important to characterize pulse duration at the sample plane. We present a simple modification to a two-photon microscope light path that allows for second-harmonic-generation-based interferometric autocorrelation measurements to characterize ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting (TCSPC). We show that TCSPC can be used as a simple and versatile method to estimate the zero time delay step value between two adjacent ultrafast laser pulses for these measurements. To demonstrate the utility of this modification, we measured the Coherent Chameleon-Ultra II Ti:sapphire laser pulse width at the sample plane using a air, air, or water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was , , and (, ), respectively.
Highlights
We present a second-harmonic generation (SHG)-based interferometric autocorrelation technique to characterize ultrafast laser pulse duration at the sample plane using a simple modification to a two-photon microscope light path
The “decay-like” feature observed in time-resolved SHG traces, shown in Fig. 2(a), is caused by a convolution with the instrument response function of our time-correlated single-photon counting (TCSPC) system, and the full width at half maximum (FWHM) of an SHG signal from a SING ultrafast laser pulse can be used to estimate the instrument response function of the system, in this case, ∼95 ps
The mean and standard deviation (SD) values from six Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics on 08 Nov 2021 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use measurements for the 10×, 40×, and 63× objective lenses were 154 Æ 32, 165 Æ 13, and 218 Æ 27 fs, respectively. These results indicate that the pulse duration values for the 10× and 40× objective lenses were significantly shorter than the 63× objective lens (p < 0.01, by analysis of variance), indicating that the 63× objective lens induces more pulse broadening than the 10× or 40× objective lenses
Summary
The efficacy of 2P excitation is dependent on the laser wavelength, its average power, repetition rate, and pulse width. Power, and repetition rate are readily measured using standard laboratory equipment, ultrafast laser pulse width, usually ranging between 70 and a few hundred femtoseconds in duration, can be measured using highly specialized equipment. Commercial autocorrelators used to measure pulse width are expensive and typically measure the pulse width as the laser beam leaves the laser, not at the sample plane of a two-photon microscope. Group velocity dispersion (due to microscope dispersive optical components) can alter pulse width, which is, characteristically, broader at the sample plane.[8,9,10,11]
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