Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China.
College of Physics and Energy, Shenzhen University, Shenzhen, China.
J Biophotonics. 2019 Jun;12(6):e201800423. doi: 10.1002/jbio.201800423. Epub 2019 Mar 13.
Three-photon microscopy excited at the 1700-nm window (roughly covering 1600-1840 nm) is especially suitable for deep-brain imaging in living animals. To match the brain refractive index, D O has been exclusively used as the immersion medium. However, the hygroscopic property of D O leads to a decrease of transmittance of the excitation light and as a result a decrease in three-photon signals over time. Solutions such as replacing D O from time to time, wrapping both the objective lens and the immersion D O, and sealing D O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D O and silicone oil immersion show that silicone oil immersion yields 17% higher three-photon signal in third-harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three-photon fluorescence imaging of vasculature up to 1460 μm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long-span three-photon brain imaging excited at the 1700-nm window.
三光子显微镜在 1700nm 窗口激发（大致覆盖 1600-1840nm）特别适用于活体动物的大脑深层成像。为了匹配大脑折射率，D O 被专门用作浸液介质。然而，D O 的吸湿性会导致激发光的透过率降低，从而导致三光子信号随时间衰减。已经证明了一些解决方案，例如定期更换 D O、包裹物镜和浸液 D O 以及用石蜡液密封 D O，这些都会增加系统的复杂性。基于我们最近对浸液油的特性分析，我们提出使用硅油作为 D O 的潜在替代品，用于大脑深层成像。在 1600nm 激发下，我们使用 D O 和硅油浸液进行的对比深层大脑成像显示，在白质中的三次谐波成像中，硅油浸液产生的三光子信号高 17%。此外，硅油浸液还能够在活体小鼠大脑中进行三光子荧光血管成像，深度可达 1460μm（机械深度），帧率为 2 帧/秒。结合非吸湿性的物理特性，硅油有望用于在 1700nm 窗口激发的长距离三光子大脑成像。
