Millisecond-scale volumetric imaging microscopy

Yu-Hsuan Tsai^1*, Chien-Sheng Wang¹, Yu-Feng Chien¹, Jyun-Yi Lin¹, Shi-Wei Chu^1,2

¹Department of Physics, Natioanl Taiwan University, Taipei, Taiwan
²Molecular Imaging Center, National Taiwan University, Taipei, Taiwan

* Presenter:Yu-Hsuan Tsai, email:r06222013@ntu.edu.tw

Capturing highly dynamic processes in three-dimension is crucial for biological study, especially for in vivo brain imaging. For example, action potential, which is the main signal transmission mechanism among neurons, typically has temporal width within few milliseconds. In order to catch this rapid 3D dynamics, millisecond scale volumetric imaging is indispensable. Among current imaging techniques, two-photon microscopy appears to be a suitable candidate because it not only provides subcellular spatial resolution but also optical sectioning ability to monitor three-dimensional structure. However, the state-of-the-art volumetric imaging speed of two-photon microscopy is far below millisecond scale. Although light-sheet and light-field microscopies may boost imaging speed, they are based on wide-field detection, and thus are limited to transparent sample, not feasible for dense neural structures such as Drosophila brain. Other scanning-based techniques, such as liquid lens and acousto-optic deflector, cannot reach millisecond-scale volume rate due to their limitation on axial or lateral scanning speed.
In this study, we proposed a 32-channel scanning system which significantly enhances lateral scanning speed by 32 times using diffractive optical element (DOE). For axial speed, the 32-channel system is combined with a tunable acoustic gradient index lens (TAG) that could offer 100 kHz ultrafast axial scanning, so we can now achieve 548 Hz volume imaging rate. To our knowledge, this is the record-high two-photon volumetric imaging speed, which should be fast enough to capture action potential propagations inside a neural network. This innovative high-speed volumetric imaging system paves the way for millisecond scale neural dynamics study in living brains.

Keywords: multi-photon microscopy, multi-focal microscopy, fast focus-tunable lens, three-dimensional microscopy