Measuring two-photon microscopy ultrafast laser pulse duration at the sample plane using time-correlated...

Area of Science:

• Biomedical Imaging
• Ultrafast Optics
• Microscopy

Background:

• Two-photon microscopy (2PM) enables deep tissue imaging but is affected by pulse broadening from optical components.
• Accurate measurement of laser pulse duration at the sample plane is essential for quantitative two-photon absorption studies.

Purpose of the Study:

• To present a simple modification for characterizing ultrafast laser pulse duration at the sample plane in 2PM.
• To validate the use of time-correlated single-photon counting (TCSPC) for pulse duration measurements in 2PM.

Main Methods:

• A modified two-photon microscope light path enabling second-harmonic-generation-based interferometric autocorrelation.
• Utilized TCSPC to estimate the zero time delay step between laser pulses.
• Measured pulse widths using 10× air, 40× air, and 63× water-immersion objectives.

Main Results:

• Successfully characterized ultrafast laser pulse width at the sample plane.
• Demonstrated TCSPC as a versatile method for pulse duration estimation.
• Measured pulse widths of 154±32 fs (10× air), 165±13 fs (40× air), and 218±27 fs (63× water-immersion) at 950 nm.

Conclusions:

• The presented modification offers a straightforward approach to measure pulse duration in 2PM.
• Accurate pulse width characterization is vital for reliable two-photon absorption measurements.
• This method enhances the precision of quantitative analysis in 2PM-based research.