Novel Immunofluorescence Methods for Retinal Research

描述（由申请人提供）：由于人类视网膜富含自体荧光化合物，如脂褐素，因此在基础眼科研究或眼科病理学临床评价期间，使用免疫荧光方法定位组织切片中的分子是不切实际的;对于视网膜色素上皮细胞尤其如此。人类视网膜内的免疫定位仅限于老式的组织化学方案，其特征在于低分辨率、低灵敏度和差的对比度。我们假设，内源性自体荧光在人类视网膜切片可以大大减少或消除利用某些物理性质的无机纳米粒子含有稀土镧系元素。我们测试这一假设的目的是建立新的技术来研究人类视网膜的基础上使用镧系纳米粒子。在第一个目标中，人视网膜组织切片将用针对视网膜抗原的第一步抗体标记，然后用与含有镧系元素（Eu 3+或Tb 3+）的纳米颗粒共价连接的第二步试剂标记，其表现出非常大的斯托克斯位移（~300 nm）和600微秒的荧光寿命。使用闪光灯和时间门控触发器结合时间调制相机，我们将只收集寿命较长的镧系元素发射。为了测试该方法的效用，将在来自对照和糖尿病视网膜病变患者的病理学样品中检查与视网膜的不同部分反应的一组抗体。在第二个目标中，我们将利用第一步抗体标记的样品，然后用含有上转换磷光体（UCP）的纳米颗粒标记抗体。反斯托克斯显微镜将用于在视网膜上对这些纳米颗粒进行成像，而不会混淆自体荧光。将在980 nm处提供激发以促进含Er 3+的UCP的反斯托克斯或“上转换的”绿色荧光发射。通过使用红外激发，视网膜中不会发生内源性自发荧光物质的激发，因为：视网膜不吸收这种红外光，并且视网膜不显示Er 3+的独特物理性质（长荧光寿命和反斯托克斯发射）。我们预计，这些创新的免疫荧光显微镜方法将被广泛用于眼科和眼科病理学的临床实践。此外，这些研究将为新一代人类视网膜生物学研究奠定基础，这些研究以前被自体荧光所掩盖，例如受体和通道定位，酶易位，氧化剂损伤和蛋白质-蛋白质相互作用等。 公共卫生相关性：这项研究计划将导致新方法的发展，进行眼科研究和眼科病理学。因此，它可能有助于眼科研究的新发现以及改善患者护理。

DESCRIPTION (provided by applicant): As human retinas are rich in auto fluorescent compounds, such as lipofuscin, it is not practical to use immunofluorescence methods to localize molecules in tissue sections during basic eye research or in clinical evaluations of eye pathology; this is especially true for the retinal pigment epithelium. Immunolocalization within human retinas is limited to old-fashioned histochemical protocols, which are characterized by low resolution, low sensitivity, and poor contrast. We hypothesize that endogenous auto fluorescence in human retinal sections can be greatly reduced or eliminated by taking advantage of certain physical properties of inorganic nanoparticles containing rare earth lanthanides. Our objective in testing this hypothesis is to establish novel techniques to study the human retina based upon the use of lanthanide nanoparticles. In the first aim, human retinal tissue sections will be labeled with first step antibodies directed against retinal antigens, followed by labeling with a second-step reagent covalently linked with nanoparticles containing lanthanides (Eu3+ or Tb3+), which exhibit a very large Stokes shift (~300 nm) and fluorescent lifetimes of 600 microseconds. Using a flash-lamp and a time-gated trigger in conjunction with a temporally modulated camera, we will collect only the longer-lived lanthanide emission. To test this method's utility, a panel of antibodies reactive with different portions of the retina will be examined in pathology samples from control and diabetic retinopathy patients. In the second aim, we will utilize samples labeled with first step antibodies followed by antibodies tagged with nanoparticles containing up-converting phosphors (UCPs). Anti-Stokes microscopy will be used to image these nanoparticles on retina without confounding auto fluorescence. Excitation will be provided at 980 nm to promote anti-Stokes or "up-converted" green fluorescence emission of Er3+-containing UCPs. By using infrared excitation, no excitation of endogenous auto fluorescent species in the retina will take place because: the retina does not absorb this infrared light and the retina does not display the unique physical properties of Er3+ (long fluorescence lifetime and anti-Stokes emission). We anticipate that these innovative immunofluorescence microscopy methods will be broadly used in ophthalmology and in the clinical practice of ocular pathology. Moreover, these studies will lay the groundwork for a new generation of human retinal biology studies that were previously obscured by auto fluorescence, such as receptor and channel localization, enzyme translocation, oxidant damage, and protein-protein interactions, among others. PUBLIC HEALTH RELEVANCE: This research program will lead to the development of new methods to conduct eye research and ocular pathology. Consequently, it will likely contribute to new discoveries in eye research as well as improved patient care.