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. ? ? ?
| Matveeva, Evgenia G; Gryczynski, Ignacy; Barnett, Anne et al. (2007) Metal particle-enhanced fluorescent immunoassays on metal mirrors. Anal Biochem 363:239-45 |
