Wallrabe Horst, Stanley Michael, Periasamy Ammasi, Barroso Margarida
University of Virginia, Department of Biology, Gilmer Hall, 057, Charlottesville, Virginia 22904, USA.
J Biomed Opt. 2003 Jul;8(3):339-46. doi: 10.1117/1.1584444.
One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.
单光子和双光子荧光共振能量转移（FRET）显微镜技术，结合不同带宽的发射滤光片和一种新型光谱溢出校正算法（PFRET算法），为定量测量内吞膜中受体聚集提供了基础。带宽更宽的发射滤光片可增加FRET信号及相应的溢出。采用PFRET校正算法进行处理会使校正水平提高，能量转移效率（E%）值具有可比性，从而验证了我们基于算法的方法。E%与受体、供体水平以及供体：受体（D:A）比率之间的关系用于表征内吞膜中受体-配体复合物的分布。除了用于检测聚集的标准测试（E%与受体水平无关）外，我们还描述了第二个参数：E%随供体水平和D:A比率增加而呈负相关。我们提出了供体几何排斥假说以解释这一现象。单光子和双光子FRET显微镜检测表明，聚合免疫球蛋白A受体-配体复合物在极化的Madin-Darby犬肾细胞顶端内吞膜内聚集成簇。
