Journal of Biophotonics
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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.