Waveguide evanescent field fluorescence (WEFF) microscopy is an alternative to total internal reflection fluorescence (TIRF) microscopy. WEFF microscopy allows imaging of contact regions between biological cells and their substratum and ultra-thin films on substrates by illuminating with an evanescent field. Typically a monolayer of a mixture of lipids and proteins called “lung surfactant” covers the lung air-water interfaces. The lung surfactant provides low surface tension which is necessary for proper lung function and to minimize the required work for breathing. Lung surfactant in vivo is a complex mixture of lipids and proteins which is difficult to prepare as an artificial biological sample. Various mixtures of lipids have been used to simulate the lipid components in artificial lung surfactant. Langmuir monolayers at the air–water interface are an accessible model for studies of lung surfactants at the air-water interface. We have chosen this artificial lung surfactant system, which is known to be demixed on a Longmuir-Blodgett-trough, as a model system for studying the advanced imaging possibilities of WEFF microscopy. Longmuir-Blodgett technique was implemented to fabricate monolayers of the model system of lung surfactant on waveguides at various surface pressures. These monolayers were imaged by WEFF microscopy. The WEFF images were compared with images taken by a commercially available standard epi-fluorescence microscope. Comparison included contrast, photo bleaching and sensitivity. Compared to the standard fluorescence microscope, WEFF microscope images show much higher contrast, sensitivity and lower photobleaching. A fine structure in the condensed domains and the liquid phase were observed for the first time.
