The resolution of single-molecule super-resolution imaging is limited by the brightness of the probes. Fluorescent proteins (FPs), widely used labels in biological imaging, have particularly lower brightness and photon yield compared to dyes. Fortunately, in the enhanced electromagnetic field about a resonantly excited plasmonic nanoparticle, the excitation efficiency, radiative rate and non-radiative rate of nearby fluorescent molecules—including FPs—can be enhanced. However, the net fluorescence improvement is a complicated tradeoff between radiative and non-radiative enhancements. Single-molecule studies are necessary to fully understand this relationship by unraveling the heterogeneities hidden in ensemble experiments. Plasmon-enhanced FPs will be especially useful for biological imaging. However, strong coupling from nano-antenna has also been shown to shift the fluorescence emission up to tens of nanometers; as a result, super-resolved localization accuracy is compromised by this plasmonic effect. To better understand and overcome this problem, dSTORM (direct Stochastic Optical Reconstruction Microscopy) is used here to quantitatively measure the separation distance-dependent mislocalizations in the nanoparticle-fluorophore coupled system.<br/>We have measured the interactions of gold nanorods with single molecules of the organic Cy3.5 dye and the red FP mCherry using PAINT (Point Accumulation for Imaging in Nanoscale Topography). For both fluorophores, the highest fluorescence enhancement (1.5-fold and 3.4-fold, respectively) was achieved at ~10 nm spacer thickness. We have further studied as a function of distance the interactions of photoactivatable FPs with an isotropic gold nanosphere. Silica shells of precisely controlled thicknesses were used as nanometer-scale spacers between the nano-antenna and covalently conjugated FPs. Finally, we have investigated the emission mislocalization effect by linking dye molecules to the surface of gold nanospheres through rigid, tunable double-stranded DNA. With dSTORM, the apparent emission positions of single molecules were mapped in 2D and 3D, and compared with the actual positions to reveal the coupling effect at different separation distances. We have found that even up to 70 nm separation distance, the localization positions are shifted 45 nm towards the nano-antenna.

Single-Molecule super-resolution study of distance-dependent plasmonic fluorescence enhancement and emission mislocalization

253rd National Meeting (2017)

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