Novel Immunofluorescence Methods for Retinal Research

描述（由申请人提供）：由于人类视网膜中含有丰富的自荧光化合物，如脂褐素，在眼科基础研究或眼科病理临床评估中，使用免疫荧光方法定位组织切片中的分子是不切实际的；视网膜色素上皮尤其如此。人类视网膜内的免疫定位仅限于老式的组织化学方案，其特点是低分辨率，低灵敏度和差对比度。我们假设，利用含有稀土镧系元素的无机纳米颗粒的某些物理性质，可以大大减少或消除人视网膜切片中的内源性自荧光。我们测试这一假设的目的是建立基于镧系纳米粒子使用的研究人类视网膜的新技术。在第一个目标中，人类视网膜组织切片将用第一步针对视网膜抗原的抗体进行标记，然后用第二步试剂与含有镧系元素（Eu3+或Tb3+）的纳米颗粒共价连接进行标记，其表现出非常大的斯托克斯位移（~300 nm）和荧光寿命为600微秒。使用闪光灯和时间门控触发器与临时调制相机相结合，我们将只收集较长寿命的镧系元素发射。为了测试这种方法的实用性，将在对照和糖尿病视网膜病变患者的病理样本中检测一组与视网膜不同部分反应的抗体。在第二个目标中，我们将利用第一步抗体标记的样品，然后使用含有上转换荧光粉（UCPs）的纳米颗粒标记的抗体。将使用抗斯托克斯显微镜对这些纳米颗粒在视网膜上成像，而不会混淆自身荧光。在980 nm处提供激发，以促进含Er3+的UCPs的反stokes或“上转换”绿色荧光发射。通过红外激发，不会激发视网膜内的内源性自荧光物质，因为：视网膜不吸收这种红外光，视网膜没有显示Er3+独特的物理性质（长荧光寿命和抗stokes发射）。我们期待这些创新的免疫荧光显微镜方法将广泛应用于眼科和眼科病理的临床实践。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，这些研究以前被自身荧光所掩盖，如受体和通道定位，酶易位，氧化损伤和蛋白质-蛋白质相互作用等。

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.