Advancing Whole-Drosophila-Brain Functional Imaging with Precise Pulse Control in Three-Photon Fluorescence Microscopy
Shih-Hsuan Chia1*, Je-Chi Jang1, Chen Chi-Wen1, Hen Chang2, Lu-Ting Chou1, Ting-Chen Chang3, Chung-Ming Chen3
1Institute of Biophotonics, National Yang Ming Chiao Tung University, Taipei, Taiwan
2Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan
3Department of Physics, National Taiwan University, Taipei, Taiwan
* Presenter:Shih-Hsuan Chia, email:shchia@nycu.edu.tw
Three-photon fluorescence microscopy is a potent technique for deep-tissue imaging in challenging, highly scattering live specimens. Nevertheless, slow imaging rates and the risk of sample damage have limited its utility. In this study, we present an innovative solution to overcome these challenges, achieving efficient excitation and enabling high-temporal-precision functional imaging of the complete drosophila brain.
Our approach involves the use of a 24-MHz mode-locked Cr:forsterite oscillator, allowing fine-tuning of laser pulse duration while maintaining high sub-MW peak power. This precise pulse duration control enables efficient monitoring of deep-tissue calcium responses following electric shock stimulations, even at a high video rate (512x512 pixels) with an impressive temporal precision of 0.03 seconds.
Furthermore, through the optimization of pulse durations, we explore signal-to-background ratios at various tissue depths. This investigation sheds light on previously elusive scattering parameters, specifically the variation in scattering paths at different depths. This advancement enriches our understanding of biotic tissue properties in in vivo settings, opening up new avenues for deep-tissue imaging research.
In summary, our innovative approach significantly enhances the capabilities of three-photon fluorescence microscopy, offering profound insights into deep-tissue imaging and advancing its applications in the field of biomedical research. This work has the potential to shape the future of deep-tissue imaging and foster further discoveries in the domain.
Keywords: Three-Photon Fluorescence Microscopy, Whole-Drosophila-Brain Imaging , Ultrafast imaging, Ultrafast nonlinear optics