Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

Area of Science:

• Biomedical optics
• Microscopy
• Developmental biology

Background:

• Improving imaging speed in multiphoton microscopy is crucial for live biological studies.
• Light-sheet illumination offers advantages for fast *in vivo* imaging.
• Photoperturbation effects in multiphoton light-sheet microscopy require further investigation.

Purpose of the Study:

• To investigate and quantify photoperturbation in multiphoton light-sheet microscopy.
• To establish guidelines for balancing signal enhancement and photoperturbation.
• To optimize laser parameters for faster and clearer live tissue imaging.

Main Methods:

• Utilizing zebrafish embryo heart rate as a sensitive indicator of photoperturbation.
• Analyzing heart rate variations in response to laser power, pulse frequency, and wavelength.
• Optimizing laser pulse frequency to maximize signal-to-noise ratio and minimize phototoxicity.

Main Results:

• Zebrafish embryo heart rate effectively probes linear and nonlinear photoperturbations.
• Guidelines derived for optimizing laser parameters to balance signal and photoperturbation.
• Achieved a one-order-of-magnitude signal enhancement by optimizing laser pulse frequency.

Conclusions:

• Multiphoton light-sheet microscopy can achieve significantly faster live tissue imaging.
• Understanding and mitigating photoperturbation is key to high-speed microscopy.
• This work provides a framework for optimizing imaging parameters in sensitive biological samples.