Shanghai Jiaotong University, China.
Exp Eye Res. 2010 Aug;91(2):308-14. doi: 10.1016/j.exer.2010.06.007. Epub 2010 Jun 15.
Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW fs laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 mum diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 h after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas.
使用超快脉冲激光器从眼睛产生的非线性光学 (NLO) 信号的成像由于使用具有较慢扫描速度的传统显微镜而仅限于离体组织的研究。本研究的目的是评估一种新型高扫描率检眼镜在使用附加的飞秒激光时生成 NLO 信号的能力。使用新设计的海德堡双光子激光检眼镜和附加的 25mWfs 激光（中心波长为 780nm、脉冲宽度为 75fs 和重复率为 50MHz）在活体麻醉白化兔中产生和成像 NLO 信号。为了评估双光子激发荧光 (TPEF) 信号的产生，首先用蓝绿色荧光 FluoSpheres（1 微米直径）标记培养的兔角膜成纤维细胞 (RCF)，然后将细胞微注射到中央角膜中。在注射后 6 小时，通过反射和 TPEF 成像可以检测到 RCF 细胞的团块。到第 6 天，通过 TPEF 证实含有荧光微球的 RCF 显示出更分散的形态，并且已经从原始注射部位迁移。总体而言，这项研究表明使用 NLO 显微镜连续检测活体完整角膜中的 TPEF 信号的潜力。我们得出的结论是，进一步改进双光子激光检眼镜应该会开发出一种重要的新临床仪器，能够检测来自患者角膜的 NLO 信号。
