Schematic representation of selection of emission from a specific fluorophore in the proximity of the AuNP.

<p>The AuNP is shown schematically in yellow with regions of plasmonic enhancement in green. (A)In part A, the direction of linear polarization of the laser light is horizontal: particle 1 is not in the region of enhancement but particle 2 is. Therefore most of the fluorescence will arise from particle 2. (B)If the polarization of the laser beam is vertical, only the fluorescence from particle 1 will be enhanced.</p

Emission spectrum of the same AuNP of Fig 2 with a polarization angle of 45° excitation wavelength of (A) 740 nm, (B) 840nm and (C) 890nm.

<p>In C and B the SHG signal is very strong. The broad emission spectrum maximum depends in the wavelength of excitation.</p

Solution fluctuation spectroscopy of 1 nM AuNP nanoparticles and 100nM EGFP.

<p>(A) Autocorrelation function of 1nM suspension of gold nanoparticles using a laser at 488nm and detecting the particle in confocal reflection (blue line) and of 100 nM EFGP excited at 488nm and viewed with an emission bandpass at 525/20 (red curve). When the AuNPs and the EGFP where together in the same sample the two autocorrelation curves remain almost identical. (B) Cross-correlation between the two channels (green curve) and the fit (black) using the same parameters for the fit of the autocorrelation curve of the sample with the AuNPs alone. (C) Intensity fluctuations in the reflection channel showing spikes due to the AuNPs passing in the volume of excitation.</p

Spectrum of a AuNP excited at 4 different wavelengths and with an orientation of 45 degrees for the linear polarizer.

<p><b>This sample was added with a solution of 100nM EGFP</b>. (A) EGFP is minimally excited at 740 nm and we see the typical spectrum of the metallic nanoparticle. (B) at 790nm we broad emission of the nanoparticle shifts toward the red and a band starts to appear in the 520nm region typical of EGFP. (C) at 840nm the SHG signal starts to be observed. (D) at 890 nm the fluorescence from the EGFP is strongly enhanced and this fluorescence is clearly distinguishable in the 529 nm region. At this excitation wavelength there is also a very strong SHG signal.</p

DIC image of AUNPs.

<p>(A) DIC image. The large structure on the bottom is an impurity over the glass surface that was used to guide the recognition of the same area for the fluorescence image. (B) Fluorescence image of a small region of the slide with fixed AuNPs excited at 890 nm with emission band pass filter 560–650 nm. The image in B is strongly contrasted to make the AuNP visible. A threshold of 20 levels above the background was applied to the fluorescence image to select only the fluorescent AuNPs. (C) Overlay of the DIC and fluorescence image. All particles in B have a corresponding DIC signal in A, but not all dots in A have a corresponding fluorescence dot in B.</p

1 nM AuNP suspension in the presence of 100nM mCherry.

<p>The excitation wavelength was 740 nm. In all graph the vertical axis is in intensity units from the Andor camera. (A) Intensity trace of a small region of the overall data collection. The intensity is obtained by the integrating the spectrum from 420nm to 680nm. A total of 500,000 spectra were collected every 200 microseconds. Occasional burst are observed along the intensity trace above the average spectral intensity due to mCherry in solution. (B) The average spectrum of 80 intensity bursts. (C) Average spectrum of the regions of the trace without bursts. The spectra in B and C are very similar.</p

1 nM AuNP suspension in the presence of 100nM EGFP.

<p>The excitation wavelength was 890 nm. In all graph the vertical axis is in intensity units from the Andor camera. (A) Intensity trace of a small region of the overall data collection. The intensity is obtained by the integrating the spectrum from 420nm to 680nm. A total of 500,000 spectra were collected every 200 microseconds. Occasional burst are observed along the intensity trace above the average spectral intensity due to EGFP in solution. (B) The average spectrum of 144 intensity bursts. (C) Average spectrum of the regions of the trace without bursts. The spectra in B and C are very similar.</p

Emission spectrum of EGFP and mCherry in a region of the sample with no AuNP.

<p>The excitation wavelengths were chosen to maximally excite each of the fluorophores, 890nm and 740nm, respectively.</p