Doheny Eye Institute and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Invest Ophthalmol Vis Sci. 2013 Jul 18;54(7):4813-20. doi: 10.1167/iovs.12-11235.
In situ 2-photon excitation fluorescence (TPEF) of the human trabecular meshwork (TM) reveals beams of heterogeneous autofluorescence (AF) comprising high intensity fluorescent fibers (AF-high) on a background of lower intensity fluorescence (AF-low). To determine the sources of this AF heterogeneity, we imaged human TM to characterize AF, second harmonic generation (SHG) for collagen, and eosin-labeled fluorescence identifying elastin.
Corneoscleral rims retained after corneal transplantation were incubated with and without eosin, and imaged by TPEF. TPEF was collected through multiphoton bandpass filters to obtain AF, SHG (collagen bandwidth), and eosin-labeled fluorescence images. For qualitative comparisons, near-simultaneous image acquisition pairs of AF-SHG (+/-eosin coincubation), AF-eosin, and SHG-eosin were captured. For quantitative comparisons, multiple regions of interest (ROI) were defined in separate TM beam regions within the uveal and corneoscleral meshwork for image acquisition pairs of AF-SHG (without eosin coincubation) and SHG-eosin. We defined 18 ROI within each acquisition pair as the basis for Manders colocalization analysis. Perfect colocalization was defined as a Manders coefficient (Mcoeff) of 1.
Qualitatively and quantitatively, AF-low colocalized with SHG (Mcoeff=1), but not SHG signal-voids. AF-high colocalized with SHG signal-voids (Mcoeff=1), but not the SHG signal. Like AF-high, eosin-labeled fluorescence qualitatively and quantitatively colocalized (Mcoeff=1) with SHG signal-voids, but not the SHG signal.
Heterogeneous AF in human TM is comprised of high intensity signal originating from elastin fibers in beam cores and lower intensity signal originating from collagen. These findings are relevant to interpreting structural extracellular matrix signals in AF images of the TM.
人眼小梁网（TM）的共聚焦 2 光子激发荧光（TPEF）显示出不均匀的自发荧光（AF）束，这些束由高强度荧光纤维（AF-high）和背景下的低强度荧光（AF-low）组成。为了确定这种 AF 异质性的来源，我们对人 TM 进行了成像，以表征 AF、用于胶原蛋白的二次谐波产生（SHG）和识别弹性蛋白的曙红标记荧光。
角膜移植后保留的角巩膜缘在孵育时有和没有曙红的情况下进行成像，并通过 TPEF 进行成像。TPEF 通过多光子带通滤波器收集，以获得 AF、SHG（胶原蛋白带宽）和曙红标记荧光图像。为了进行定性比较，同时采集了 AF-SHG（+/-曙红共孵育）、AF-曙红和 SHG-曙红的近同时图像采集对。为了进行定量比较，在虹膜和角巩膜小梁网的不同 TM 光束区域中定义了多个感兴趣区域（ROI），用于 AF-SHG（无曙红共孵育）和 SHG-曙红的图像采集对。我们将每个采集对中的 18 个 ROI 定义为 Manders 共定位分析的基础。完美共定位定义为 Manders 系数（Mcoeff）为 1。
定性和定量地，AF-low 与 SHG 共定位（Mcoeff=1），但与 SHG 信号缺失不共定位。AF-high 与 SHG 信号缺失共定位（Mcoeff=1），但与 SHG 信号不共定位。与 AF-high 一样，曙红标记的荧光定性和定量地与 SHG 信号缺失共定位（Mcoeff=1），但与 SHG 信号不共定位。
人 TM 中的不均匀 AF 由高强度信号组成，源自束核中的弹性纤维，以及源自胶原的低强度信号。这些发现与解释 TM 的 AF 图像中的结构细胞外基质信号有关。
