Photonic crystal enhanced fluorescence (PCEF) has been demonstrated as an effective means for amplifying the excitation provided to surface-bound fluorescent molecules while simultaneously enhancing fluorescence emission collection efficiency. Optimal coupling of a fluorophore-exciting light source to the PC occurs with the use of collimated plane waves, as utilized in a special-purpose fluorescence microscope specifically designed for coupling with PCEF surfaces. However, PCEF surfaces are also capable of coupling light from focused sources, such as those used in commercially available confocal laser scanners, but with a reduction in the obtainable enhancement factor. Using computer simulations and experimental measurements, we describe the interaction between the resonant bandwidth of a PCEF device surface and the optical design of the detection instrumentation that is used to provide fluorescence excitation. We show that highly collimated illumination is required for achieving the greatest PCEF enhancement factors, but at the expense of poor tolerance to nonuniformities in resonant wavelength across the PCEF surface. To overcome this limitation, we demonstrate a fixed wavelength/multiple incident angle scanning detection system that is capable of measuring every pixel in a PCEF fluorescence image under conditions that optimize resonant excitation efficiency. Finally we discuss the enhanced excitation mechanism for photonic crystal enhanced fluorescence in the context of photobleaching. We show that the photobleaching rate of dye molecules on the photonic crystal surface is accelerated by 30x compared to an ordinary glass surface, but substantial signal gain is still evident, even after extended periods of continuous illumination at the resonant condition.
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