Surface-Enhanced Two-Photon Excitation Fluorescence of Various Fluorophores Evaluated Using a Multiphoton Microscope

We present a rapid, low-power testbed for the detection and imaging of fluorescent probes utilizing two-photon excitation fluorescence (2PEF). The 2PEF signal from fluorophores commonly used in biological imaging have been enhanced using plasmonic substrates that have been designed to have a high plasmonic resonance over a narrow band that matches the source. The samples were illuminated and imaged using a low-power, in-house multiphoton microscope. A green fluorescent protein (bfloGFPa1), chlorophyll, or rhodamine 6G were deposited onto the plasmonic substrates and their 2PEF signals were measured relative to planar samples. The fluorescence intensities from the green fluorescent protein, chlorophyll, and rhodamine were enhanced by factors of 50, 8, and 4, respectively, with the structured substrates (relative to the planar substrates).