26th Australian Institute of Physics Congress and the Australian and New Zealand Conference on Optics and Photonics

Optimising Repetition Rate in Two-Photon Light-Sheet Fluorescence Microscopy for Enhanced Signal Efficiency and Reduced Photodamage

Not scheduled

20m

Belinda Hutchinson Building (The University of Sydney )

Contributed Oral ANZOS | Photonics and Optics (ANZCOP)

Speaker

Bailey Hoare (Adelaide University)

Description

Two-photon light-sheet fluorescence microscopy (2P-LSFM) enables rapid volumetric imaging with excellent optical sectioning and deep tissue penetration. Most 2P-LSFM systems use excitation sources at ~80 MHz; however, given the finite fluorescence lifetime of common fluorophores, lower repetition rates may improve signal generation by concentrating the same average power into fewer, higher-energy pulses. This strategy can increase signal-to-noise ratio (SNR) while reducing average power delivered to the sample [1], although gains may be limited by saturation and nonlinear photodamage [2].

To investigate this trade-off, we developed a 2P-LSFM platform incorporating two co-aligned femtosecond laser sources at 920 nm: a conventional 80 MHz oscillator (Chameleon Vision-S) and a tunable low-repetition-rate laser operating from 100 kHz to 2 MHz (Cronus-3P, Light Conversion), enabling direct comparison of excitation efficiency across a largely unexplored low-repetition-rate regime. Fluorescent microspheres served as a controlled imaging target to quantify SNR as a function of excitation power and repetition rate.

We found that repetition rates between 500 kHz and 2 MHz achieved equivalent SNR to conventional 80 MHz excitation while requiring up to four-fold lower average power at the sample. This reflects enhanced signal efficiency per unit delivered energy and a corresponding reduction in thermal loading. Complementary photobleaching measurements further characterised the relationship between excitation conditions and photostability, guiding optimisation of long-term imaging experiments. Ongoing studies extend this analysis to live biological specimens to assess photodamage and viability under low-repetition-rate excitation.

These findings demonstrate that laser repetition rate is a critical, underutilised parameter for optimising 2P-LSFM performance. By balancing signal generation against photobleaching and photodamage, low-repetition-rate excitation offers a practical route to improving imaging efficiency while preserving sample integrity. Implications for next-generation multiphoton light-sheet systems, including large field-of-view Bessel-beam implementations, will be discussed.

References
Gasparoli FM, et al. OSA Continuum. 2020;3:2935.
Maioli V, et al. Biomedical Optics Express. 2020;11:6012.