We have constructed an instrument that utilizes a recently developed system for interfacial microscopy. It is known that a light wave that is totally internally reflected at an interface of two transparent media with different refractive indices (e.g. glass and water or medium) forms a film of light (called evanescent field) at the surface of the low refractive index material. We have applied this principle, using an optical waveguide to generate an evanescent field on top of the waveguide surface. The depth of this evanescent field is ~100 nm. This film of light can be used as a surface-bound illumination source. The light is able to excite a fluorescent dye, if the appropriate excitation wavelength is chosen (similar to total internal reflection fluorescence [TIRF] microscopy). Because the illumination takes place within the first 100 nm above the waveguide surface, only this spatial region is visualized. No fluorescence signals from further away interfere with the surface features. Therefore, the method is ideally suited for thin film quality control and for imaging cell-substrate interactions. We call this method “waveguide evanescent field fluorescence” (WEFF) microscopy. Using the instrument, we have obtained images of Langmuir-Blodgett deposited lipid films and of osteoblast-like cells (labeled with DiI plasma membrane specific dye) grown directly on the glass surface of the waveguide. By exchanging the camera for a spectrometer, the set-up can be converted quickly to an interfacial spectroscopic tool. The samples are freely accessible from the top, so that additional tools like micropipettes and patch-clamp microelectrodes can be used. We plan to use this system to study the kinetics of cell-substrate interactions and their regulation by physiological and pathological stimuli. The effects of surface functionalization and pharmaceutical agents on cell adhesion can also be readily assessed.
