Murakoshi Hideji, Ueda Hiromi H, Goto Ryuichiro, Hamada Kosuke, Nagasawa Yutaro, Fuji Takao
Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan.
Department of Physiological Sciences, The Graduate University for Advanced Studies, Hayama, Kanagawa, 240-0193, Japan.
Biomed Opt Express. 2022 Dec 19;14(1):326-334. doi: 10.1364/BOE.477322. eCollection 2023 Jan 1.
Multiphoton microscopy has enabled us to image cellular dynamics . However, the excitation wavelength for imaging with commercially available lasers is mostly limited between 0.65-1.04 µm. Here we develop a femtosecond fiber laser system that produces ∼150 fs pulses at 1.8 µm. Our system starts from an erbium-doped silica fiber laser, and its wavelength is converted to 1.8 µm using a Raman shift fiber. The 1.8 µm pulses are amplified with a two-stage Tm:ZBLAN fiber amplifier. The final pulse energy is ∼1 µJ, sufficient for imaging. We successfully observe TurboFP635-expressing cortical neurons at a depth of 0.7 mm from the brain surface by three-photon excitation and Clover-expressing astrocytes at a depth of 0.15 mm by four-photon excitation.
多光子显微镜使我们能够对细胞动力学进行成像。然而，市售激光器用于成像的激发波长大多限制在0.65 - 1.04 µm之间。在此，我们开发了一种飞秒光纤激光系统，该系统能产生波长为1.8 µm、脉宽约150 fs的脉冲。我们的系统以掺铒石英光纤激光器为起点，利用拉曼位移光纤将其波长转换为1.8 µm。1.8 µm的脉冲通过两级掺铥氟锆酸盐（Tm:ZBLAN）光纤放大器进行放大。最终脉冲能量约为1 µJ，足以用于成像。我们通过三光子激发成功观察到距脑表面0.7 mm深处表达TurboFP635的皮质神经元，并通过四光子激发成功观察到距脑表面0.15 mm深处表达Clover的星形胶质细胞。
