![]() The fluorophore position cannot be simply read off of a single measured FLIC curve. As mentioned before, the theoretical model describes the relative fluorescence intensity measured versus the fluorophore height. The silicon dioxide thickness is very important in making accurate FLIC measurements. This is a diagram of an example FLIC experimental setup with silicon, three oxide layers and a fluorescently labeled lipid bilayer (the yellow stars represent fluorophores. With the sample system built, all that is needed is an epifluorescence microscope and a CCD camera to make quantitative intensity measurements. ![]() On top of the oxide is placed the fluorescently labeled specimen, such as a lipid membrane, a cell or membrane bound proteins. An oxide layer is then thermally grown on top of the silicon wafer to act as a spacer. The fact that it is not an equality relation will have a significant effect on the experimental procedure.Ī silicon wafer is typically used as the reflective surface in a FLIC experiment. The observed fluorescence intensity, I F L I C and the distance of the fluorophore above the reflective surface. They derived a relationship between the observed fluorescence intensity and the distance of the fluorophore from a reflective silicon surface. The optical theory underlying FLIC was developed by Armin Lambacher and Peter Fromherz. an artificial lipid bilayer, or a living cell membrane or the structure of fluorescently labeled proteins on a surface.įLIC optical theory General two layer system This allows for the nanometer height measurements.įLIC microscope is well suited to measuring the topography of a membrane that contains fluorescent The resulting interference between the direct and the reflected light leads to a double sin 2 modulation of the intensity, I, of a fluorescent object as a function of distance, h, above the reflecting surface. Fluorescence interference contrast (FLIC) microscopy is a microscopic technique developed to achieve z-resolution on the nanometer scale.įLIC occurs whenever fluorescent objects are in the vicinity of a reflecting surface (e.g.
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