Millisecond scale two-photon volumetric scanning microscopy
Po-Yuan Wang1*, Yu-Hsuan Tsai1, Chien-Sheng Wang1, Hung-Yu Chen1, Chih-Wei Liu2, Ruei-Huang Chen3,4, Vijay Raj Singh5, Peter T. C. So5, Chia-Fu Chou2, Li-An Chu4,6, Ann-Shyn Chiang4,6,7, Shi-Wei Chu1,4,8
1Department of Physics, National Taiwan University, Taipei, Taiwan
2Institute of Physics, Academia Sinica, Taipei, Taiwan
3Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu, Taiwan
4Brain Research Center, National Tsing Hua University, Hsinchu, Taiwan
5Laser Biomedical Research Center, G. R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA
6Department of Biomedical Engineering & Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan
7Kavli Institute for Brain and Mind, University of California, San Diego, CA, USA
8Molecular Imaging Center, National Taiwan University, Taipei, Taiwan
* Presenter:Po-Yuan Wang,
     It is crucial to obtain rapid dynamic processes in 3D organism, such as millisecond-scale action potentials of individual neurons in the whole brain, leading to the request of high-speed volumetric imaging with subcellular spatial resolution. To allow study of brain emergent property, we combined millimeter penetration two-photon microscopy with sub-millimeter brain of Drosophila. However, even with the state-of-the-art volumetric two-photon microscopy, the temporal resolution to resolve every single neuron in a whole brain is far from millisecond.
     Here, we demonstrate a multifocal volumetric multiphoton microscopy that achieves more than 500 Hz volume rate, to our knowledge the fastest two-photon volumetric imaging. The axial and lateral imaging speeds are enhanced via a tunable acoustic gradient index (TAG) lens that offers 70-kHz high- speed axial scanning, and a diffractive optical element (DOE) to generate 32-foci, respectively. This high-speed volumetric imaging system paves the way toward inter-neuronal action potential observation inside a whole Drosophila brain.

Keywords: high speed, 3D image, two-photon, Drosophila