SPCE Imaging Microscopy

描述（由申请人提供）：我们提出利用表面等离子体耦合发射（SPCE）的现象来显著改善细胞和细胞膜的图像。SPCE技术是基于激发的荧光团偶极耦合到金属层中的表面等离子体，随后是定向偏振辐射进入玻璃相。SPCE辐射保留了荧光团的光谱特性。我们的初步数据表明，激发的荧光团附近的连续半透明银膜可以有效地耦合到表面等离子体和“发射”在玻璃基板后面的金属在明确定义的角度。这项新技术将有助于研究许多膜包埋蛋白，如通道，载体，泵和受体在细胞表面的功能，包括胞吐和胞吞的过程中参与质膜和细胞内室之间的膜蛋白的再循环。选择性地可视化活细胞中的这些过程和亚膜结构的能力将极大地有助于提高对细胞生理学和病理生理学基本原理的理解。在第一阶段（R21），我们将通过设计实验SPCE系统（载玻片、耦合光学器件、样品、沉积、检测）来优化条件，以产生最大的SPCE信号和最小的不需要的原液背景。在第二阶段（R33），我们将开发一个功能SPCE显微镜成像系统。该原型将用于研究在玻璃/金或玻璃/银/二氧化硅基底上生长的荧光团标记的肺上皮A549细胞。

DESCRIPTION (provided by applicant): We propose to utilize the phenomenon of surface plasmon-coupled emission (SPCE) to significantly improve images of cells and cell membranes. The SPCE technology is based on a coupling of excited fluorophore dipoles to the surface plasmons in metallic layer followed by a directional polarized radiation into a glass phase. The SPCE radiation preserves the spectral properties of fluorophores. Our preliminary data demonstrate that excited fluorophores near continous semi-transparent silver films can efficiently couple to surface plasmon and "emit" in the glass substrate behind the metal at sharply defined angles. This new technology will be useful for studying multitude of membrane-embedded proteins, such as channels, carriers, pumps and receptors function at the cell surface, including processes of exocytosis and endocytosis which are involved in the recycling of membrane proteins between the plasma membrane and intracellular compartments. Ability to selectively visualize such processes and sub-membrane structures in living cells would be tremendously helpful in improving the understanding of the fundamental principles of cell physiology and pathophysiology. In Phase I (R21) we will optimize conditions by engineering the experimental SPCE system (slides, coupling optics, sample, depositions, detection) to yield the maximum SPCE signal and minimum of unwanted bulk solution background. In Phase II (R33) we will develop a functional SPCE microscope imaging system. This prototype will be used to study fluorophore-labeled lung epithelial A549 cells grown on a glass/gold or glass/silver/silica substrates.