Plasmonic microscopy for biomedical imaging

This chapter describes plasmonic microscopy, which, through surface plasmons (SPs), offers high sensitivity and local electromagnetic (EM) field enhancement to satisfy the requirements of biomedical imaging in real time. For a better understanding, the surface plasmon resonance (SPR) phase and fluorescence-enhanced microscopes based on a prism or objective-coupled attenuated total reflection (ATR) configuration are demonstrated. First of all, an SPR phase imaging system based on modified Mach-Zehnder phase-shifting interferometry (PSI) is adopted to detect DNA hybridization. In addition, a common-path PSI technique via an electro-optic modulator (liquid crystal phase retarder) provides long-term phase imaging stability for real-time kinetic studies in biomolecular interaction analysis (BIA). Furthermore, surface plasmon-enhanced-total internal reflection fluorescence (SPE-TIRF) microscopy provides bright live-cell images via surface plasmon-enhanced fluorescence (SPEF), and has been successfully 412used for the real-time observation of the thrombomodulin protein of a living cell membrane. In order to exploit SPE-TIRF microscopy, an ultrafast laser is also adopted. With the combination of SP enhancement and two-photon excitation, TIRF microscopy features brighter and more contrasted fluorescence membrane images. Furthermore, a theoretical model based on the Fresnel equation and classical dipole radiation modeling is employed to investigate the SP enhancement and quenching of two-photon excited fluorescence. The local electric field enhancement, fluorescence quantum yield, and fluorescence emission coupling yield via SPs are theoretically analyzed at different dielectric spacer thicknesses between the fluorophore and the metal film. Ultimately, with the combination of the SPR phase and SPE-TIRF techniques, plasmonic microscopy simultaneously enables the sensitive phase and fluorescence-enhanced images of living-cell contacts on the surface of a biosubstrate.