Department of Ophthalmology, University of Colorado Denver, Aurora, Colorado, USA.
Invest Ophthalmol Vis Sci. 2013 May 1;54(5):3094-101. doi: 10.1167/iovs.13-11642.
To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques.
Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm.
The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes.
CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.
在不应用外源性荧光标记的情况下，使用非侵入性、非破坏性技术，对完整小鼠眼球的细胞和非细胞结构进行成像。
使用两种非线性光学技术：相干反斯托克斯拉曼散射（CARS）和双光子自发荧光（TPAF）对新鲜眼球进行成像。从巩膜约位于角膜缘和视神经之间的中间区域采集横切横向切片和连续的平面（矢状面）矢状切片。成像从巩膜表面进行到深度约 60μm。
TPAF 通道中明显存在巩膜内胶原纤维的荧光信号；巩膜胶原纤维没有组织，呈随机堆积。巩膜包含约 3 至 15μm 直径的无 TPAF 和 CARS 荧光区域，可能代表小血管或巩膜成纤维细胞。视网膜色素上皮层的强烈点状 CARS 信号大小和形状与视黄醇储存酯一致。通过富含脂质的质膜的 CARS 信号可以识别杆状外节。
CARS 显微镜可用于在不使用荧光染料或外源性表达重组蛋白的情况下对哺乳动物视网膜的外部区域进行成像。随着技术的进步，CARS/TPAF 可能代表一种非侵入性成像视网膜的新途径，并可能补充目前在临床实践中使用的方式。
