Iyer Rishyashring R, Sorrells Janet E, Yang Lingxiao, Renteria Carlos A, Chaney Eric J, Tehrani Kayvan F, Spillman Darold R, Boppart Stephen A
Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, USA.
Department of Electrical and Computer Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, USA.
Optica. 2024 Sep 20;11(9):1352-1367. doi: 10.1364/optica.532367. Epub 2024 Sep 19.
The technologies to examine the neuronal microenvironment label free remain critically underexplored. There is a gap in our knowledge of underlying metabolic, biochemical, and electrophysiological mechanisms behind several neurological processes at a cellular level, which can be traced to the lack of versatile and high-throughput tools to investigate neural networks. In this paper, four label-free contrasts were explored as mechanisms to study neuronal activity, namely, scattering, birefringence, autofluorescence from metabolic cofactors and molecules, and local biochemistry. To overcome challenges of observing neuronal activity spanning three orders of magnitude in space and time, microscopes had to be developed to simultaneously capture these contrasts quickly, with high resolution, and over a large FOV. We developed versatile autofluorescence lifetime, multiharmonic generation, polarization-sensitive interferometry, and Raman imaging in epi-detection (VAMPIRE) microscopy to simultaneously observe multiple facets of neuronal structure and dynamics. The accelerated computational-imaging-driven acquisition speeds, the utilization of a single light source to evoke all contrasts, the simultaneous acquisition that provides an otherwise impossible multimodal dynamic imaging capability, and the real-time processing of the data enable VAMPIRE microscopy as a powerful imaging platform for neurophotonics and beyond.
用于无标记检测神经元微环境的技术仍未得到充分探索。在细胞水平上,我们对几种神经过程背后潜在的代谢、生化和电生理机制的认识存在差距,这可归因于缺乏用于研究神经网络的通用且高通量工具。在本文中,探索了四种无标记对比机制来研究神经元活动,即散射、双折射、代谢辅因子和分子的自发荧光以及局部生物化学。为了克服在空间和时间上跨度达三个数量级观察神经元活动的挑战,必须开发能够快速、高分辨率且在大视场范围内同时捕捉这些对比的显微镜。我们开发了用于落射检测的通用自发荧光寿命、多谐波产生、偏振敏感干涉测量和拉曼成像(VAMPIRE)显微镜,以同时观察神经元结构和动力学的多个方面。加速的计算成像驱动采集速度、利用单个光源激发所有对比、同时采集提供了原本不可能的多模态动态成像能力以及数据的实时处理,使VAMPIRE显微镜成为神经光子学及其他领域的强大成像平台。