<p>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 10 × air, 40 × air, or 63 × water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was 154 ± 32, 165 ± 13, and 218 ± 27 fs (<italic>n</italic> = 6, mean ± standard deviation), respectively.</p>.
Keywords: interferometric autocorrelation; multiphoton microscopy; pulse duration; second-harmonic generation; time-correlated single-photon counting.