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The process of spontaneous emission can be dramatically modified by optical microstructures and nanostructures. We have studied the modification of fluorescence dynamics using a variable thickness polymer spacer layer fabricated using layer-by-layer self-assembly with nanometer accuracy. The change in fluorescence lifetime with spacer layer thickness agrees well with theoretical predictions based on the modified photonic density of states (PDOS), and yields consistent values for the fluorophores’ intrinsic fluorescence lifetime and quantum yield near a dielectric as well as a plasmonic interface. Based on this observation, we further demonstrate that self-assembled fluorophores can be used to probe the modified PDOS near optical microstructures and nanostructures.