Fluorescence interference-contrast microscopy of cell adhesion on oxidized silicon

Standing modes of light in front of the reflecting surface of silicon modulate the excitation and emission of fluorescent dyes. This effect was used to determine the distance of a biomembrane from an oxidized silicon chip. The membrane of a red blood cell (ghost) was stained with a cyanine dye and attached with poly-lysine to a surface structured with microscopic steps of silicon dioxide on silicon. The system was illuminated in a microscope. The fluorescence intensity of the membrane depended on the height of the steps. The data were fitted by an optical theory which accounts both for the interference of the exciting light and for the interference of the emitted light at a finite aperture. The distance between the membrane and the silicon dioxide was determined to be 12 nm. PACS: 33.50.D; 68.35.G; 78.66; 87.22 Nerve cells and silicon devices can be joined by electrical induction when the cell membrane is attached closely to an oxidized chip [1]. Recording as well as stimulation of neuronal activity from oxidized silicon have been reported [2, 3]. The strength of coupling depends on the width of the electrolyte which separates the insulating layers of the cell membrane and the silicon dioxide. This width was estimated to be in the range 10–100 nm on the basis of the coupling experiments themselves [4]. No direct measurements are available. Usually the attachment of cells to surfaces is studied by reflection interference contrast microscopy (IRM/RICM) [5– 9] or by total internal reflection fluorescence microscopy (TIRFM) [10–15]. In both methods the cell is illuminated by visible light through a transparent support. In the RICM method the light is reflected from the substrate/electrolyte and electrolyte/cell interfaces and gives rise to an interference pattern. In the TIRFM method the cell is illuminated under the condition of total reflection; the membrane or electrolyte are labelled with a fluorescent dye which is excited by the evanescent wave. ∗Correspondence author RIC-microscopy and TIRF-microscopy cannot be used for cells on silicon, which is not transparent in the visible range. For that reason a new method of fluorescence interference contrast (FLIC) microscopy was proposed to map the distance between a membrane and oxidized silicon [16]. FLIC-microscopy takes advantage of the Wiener effect [17– 19], the interference of incident and reflected light above a mirror. The standing modes of the electromagnetic field above the surface of silicon modulate the excitation and the emission of a fluorescent dye which is dispersed in an adjacent solvent or bound to a macromolecule or membrane. Some features of FLIC-microscopy were tested with a dry monomolecular film [16]. In this paper we consider FLIC-microscopy as a tool to study cell adhesion. It is applied to determine the distance between a biomembrane and a silicon chip. As a test system we used ghosts of human erythrocytes because of their homogeneous membrane. In the first part we describe the concept of the method and explain the experimental technique. In the second part we present the data and discuss the results. 1 Materials and methods First we consider the principles of FLIC-microscopy as applied to cell adhesion. Then we describe the fabrication of the chips, the preparation of stained erythrocyte membranes, the attachment of the cells to the chip, the photometric set-up, the optical theory and the evaluation of the data. 1.1 Principles of FLIC-microscopy Silicon reflects visible light. The interference of the incident light with the reflected light gives rise to standing modes of the electromagnetic field. As a result the electronic excitation of a dye molecule depends on its position in front of the mirror. By analogy, the fluorescence emission of a dye molecule is affected due to the interference of light which is emitted with and without reflection, or in other words due to emission into an unoccupied standing mode of the electromagnetic field.