Dye distance mapping using waveguide evanescent field fluorescence microscopy and its application to cell biology

Previous studies have measured the distance between cells and the substratum at sites of adhesion via the emission of a fluorescent dye and waveguide methods. Here, we demonstrate a novel approach to measure the position of fluorescent dyes above a waveguide surface in the 10-200 nm distance range throughout an entire area, yielding a 2D dye distance map or a 3D contour plot. The dye is located in a multilayered Langmuir Blodgett (LB) film or in the plasma membrane of a cell. Waveguide evanescent field fluorescence (WEFF) images obtained using two different waveguide modes are employed allowing, with a simple mathematical approach, the calculation of dye distance maps. Ultra-thin steps made using LB technology, adhesion distances and the bending of the plasma membrane between focal adhesions of osteoblastic cells are shown as examples. The errors are discussed. False color representation of a dye distance map with four osteoblasts. The inset represents an overexposed WEFF image of the same field of view.