Rapid volumetric imaging with Bessel-Beam three-photon microscopy.

Bingying Chen,Zhigang Zhang,Yanhui Hu,Heping Cheng,Chris Xu,Haitao Wu,Guoqing Chen,Yulong Li,Meijun Pang,Aimin Wang,Yunfeng Zhang,Liangyi Chen,Jianzhi Zeng,Xiaoshuai Huang,Zhe Zhao,Dongzhou Gou,Zhuan Zhou

doi:10.1364/boe.9.001992

Abstract

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

Highlights

Multi-photon microscopy (MPM) holds the promise of unique and impactful applications in cell biology and neuroscience research, where it can enable the high-resolution imaging of the neuronal structures buried deep inside the intact brain [1,2,3]
Given that a single neuron often has dendrites that are sparsely distributed in a 3D volume, Bessel beam scanning is an excellent fast imaging method for obtaining volumetric information about the neuronal structures and activities
The femtosecond laser at 1300 nm could be switched to a home-built femtosecond Nd:fiber laser, which delivered up to 800 mW, 150-fs pulses at 930 nm with a repetition rate of 50 MHz [15, 16]

Summary

Introduction

Multi-photon microscopy (MPM) holds the promise of unique and impactful applications in cell biology and neuroscience research, where it can enable the high-resolution imaging of the neuronal structures buried deep inside the intact brain [1,2,3]. “In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain,” Nat. Methods 14(4), 388–390 (2017). “910nm femtosecond Nd-doped fiber laser for in vivo two-photon microscopic imaging,” Opt. Express 24(15), 16544–16549 (2016).

Results

Conclusion