Surface Plasmon-coupled Fluorescence Microscope to Study Ion Channel Dynamics
The long term objective of this project is the development of an optical detection system based on surface plasmon resonance to study the dynamics of membrane proteins with special emphasis in voltage gated ion channels such as the Na and K channels that are responsible for the generation and propagation of the nerve impulse. Channel proteins are labeled in specific sites with fluorescent probes using cysteine chemistry and the fluorescence is detected by the proposed optical setup. Fluorescence changes, produced by quenching or energy transfer are indicators of local environmental changes and thus they follow conformational changes within the protein as the channel undergoes transitions from the closed to the open state. The optical apparatus uses a hemispherical lens that couples an incoming laser beam on a glass chip that has a thin (50 nm) siver layer where the biological preparation lies separated by a thin (10 nm) layer of silicon oxide. The correct angle of excitation induces plasmon resonance in the metal and enhances the fluorescence of fluorophores labeling the channel. The detection is done from the biological preparation side or from the excitation side. In the second case the signal to noise ratio is expected to be much larger because the coupled emission comes from a region limited to 20 nm and, as it is directional, a specially designed optics collects most of the light on a photodetector. The testing of the optical sytem is done on labeled ion channels expressed in mammalian cells or in supported bilayers. In the second case, the supported bilayer is made with liposomes containing purified labeled channels. The voltage across the bilayer is changed taking advantage of the silver layer of the plasmon chip. A modification of the optical system is also proposed to image the biological preparation to follow the time course of the fluorescence of individual molecules in response to voltage pulses that change the conformation of the channel. The understanding of conformational dynamics of channel proteins is a crucial step in the design of drugs or therapies needed to ameliorate or cure several neurological deseases produced by abnormal function of ion channels. The optical system developed in this application is aimed at developing a new microscope that is especially designed to detect conformational changes of ion channels with improved resolution, higher sensitivity and improved rejection of spurious fluorescence than presently available devices.