Axial Nanometer Distances Measured by Fluorescence Lifetime Imaging Microscopy

We present a novel fluorescence lifetime imaging microscopy technique to measure absolute positions of fluorescent molecules within 100 nm above a metalized surface based on distance-dependent fluorescence lifetime modulations. We apply this technique to fluorescently labeled microtubules as optical probes with various unlabeled proteins attached. By measuring the fluorescence lifetimes, we obtain the position of the microtubules and therefore determine the geometrical size of the attached proteins with nanometer precision.