Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.
Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.
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相干反斯托克斯拉曼散射（CARS）显微镜：一种视网膜成像新技术。
DOI：
10.1167/iovs.13-11642
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发表时间：
2013
影响因子：
4.4
通讯作者：
Lei,TimC
中科院分区：
文献类型：
--
作者：
Masihzadeh,Omid;Ammar,DavidA;Kahook,MalikY;Lei,TimC
目的：使用非侵入性、非破坏性技术，在不应用外源荧光标记的情况下，对完整小鼠眼睛中视网膜的细胞和非细胞结构进行成像。方法：使用两种非线性光学技术对新鲜去核的小鼠眼睛进行成像：相干反斯托克斯拉曼散射（CARS）和双光子自发荧光（TPAF）。从角膜缘和视神经之间大约中间的巩膜区域收集横断面横截面和连续的平面（正面）矢状切片。成像从巩膜表面进行至约60μm的深度。结果：巩膜内胶原纤维的荧光信号在TPAF通道中明显；巩膜胶原纤维没有组织，呈随机排列。巩膜包含缺乏 TPAF 和 CARS 荧光的区域，直径约为 3 至 15 μm，可以代表小血管或巩膜成纤维细胞。来自视网膜色素上皮层的强烈点状 CARS 信号具有视黄基存储酯的大小和形状。杆外节可以通过来自富含脂质的质膜的 CARS 信号来识别。结论：CARS 显微镜可用于对哺乳动物视网膜的外部区域进行成像，而无需使用荧光染料或外源表达的重组蛋白。随着技术的进步，CARS/TPAF 可能代表一种无创视网膜成像的新途径，并可能补充目前临床实践中使用的模式。
Purpose.: 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.Methods.: 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.Results.: 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.Conclusions.: 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.
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