High-Resolution, Noninvasive, Two-Photon Fluorescence Measurement of Molecular Concentrations in Corneal Tissue
High-Resolution, Noninvasive, Two-Photon Fluorescence Measurement of Molecular Concentrations in Corneal Tissue
复制标题
DOI：
10.1167/iovs.10-6620
复制
发表时间：
2011-04-01
影响因子：
4.4
通讯作者：
Knox, Wayne H.
中科院分区：
文献类型：
--
作者：
Cui, Liping;Huxlin, Krystel R.;Knox, Wayne H.
目的.对局部应用于眼表后的活体角膜中荧光分子的浓度和扩散分布进行高分辨率、非侵入性、校准测量。使用800 nm飞秒激光进行双光子荧光（TPF）轴向扫描测量。在孔载玻片中测试由荧光素钠（Na-FI;浓度范围，0.01%-2.5%）和核黄素（浓度范围，0.0125%-0.1%）组成的校准溶液，并评估TPF信号。然后用不同分子量（MW）的Na-Fl、核黄素或荧光素葡聚糖（Fl-d）处理保存在角膜储存介质中并具有完整或去除的角膜上皮的切除的猫眼球30分钟。然后立即使用校准的TPF测量这些分子在中央角膜深度的浓度。我们的TPF系统的轴向分辨率为6 μ m，TPF信号和低浓度的大多数荧光团之间观察到线性关系。用Na-Fl或核黄素处理的完整角膜表现出仅约20 μ m的可检测的穿透深度，而当在荧光团应用之前去除上皮时，穿透深度约为400至600 μ m。完整角膜的峰值浓度是去除上皮后的一半。用2，000，000 MW Fl-d处理的清创角膜显示出156.7 μ m的半最大穿透深度，而3，000 MW葡聚糖处理的半最大穿透深度为384 μ m。高分子右旋糖酐的峰浓度是低分子右旋糖酐的四分之一。TPF是一种有效的、高分辨率的、非侵入性的方法，用于量化荧光分子在角膜中的扩散和浓度。（Invest Ophthalmol维斯科学。2011;52：2556-2564）DOI：10.1167/iovs.10-6620
PURPOSE. To perform high-resolution, noninvasive, calibrated measurements of the concentrations and diffusion profiles of fluorescent molecules in the live cornea after topical application to the ocular surface.METHODS. An 800-nm femtosecond laser was used to perform two-photon fluorescence (TPF) axial scanning measurements. Calibration solutions consisting of sodium fluorescein (Na-Fl; concentration range, 0.01%-2.5%) and riboflavin (concentration range, 0.0125%-0.1%) were tested in well slides, and TPF signals were assessed. Excised feline eyeballs preserved in corneal storage medium and with either intact or removed corneal epithelia were then treated with Na-Fl, riboflavin, or fluorescein dextran (Fl-d) of different molecular weight (MW) for 30 minutes. Calibrated TPF was then used immediately to measure the concentration of these molecules across the central corneal depth.RESULTS. The axial resolution of our TPF system was 6 mu m, and a linear relationship was observed between TPF signal and low concentrations of most fluorophores. Intact corneas treated with Na-Fl or riboflavin exhibited a detectable penetration depth of only approximately 20 mu m, compared with approximately 400 to 600 mu m when the epithelium was removed before fluorophore application. Peak concentrations for intact corneas were half those attained with epithelial removal. Debrided corneas treated with 2,000,000 MW Fl-d showed a half-maximum penetration depth of 156.7 mu m compared with 384 mu m for the 3,000 MW dextran. The peak concentration of the high MW dextran was one quarter that of the lower MW dextran.CONCLUSIONS. TPF is an effective, high-resolution, noninvasive method of quantifying the diffusion and concentration of fluorescent molecules across the cornea. (Invest Ophthalmol Vis Sci. 2011;52:2556-2564) DOI:10.1167/iovs.10-6620