Confocal imaging of single metallic nanoparticles
Recently, the imaging of single metallic nanoparticles using light microscopy has attracted increasing interest. Individual gold particles, for example, have been used to excite or probe single dye molecules.1 These particles have also seen wide use in biomedical research,2 and are promising candidates for labelling biological subnuclei organelles.
Up to now, several methods have been reported for imaging single metallic nanoparticles.3,4 However, none of these allow individual particle detection, or provide direct information about shape, size, or orientation without the help of sophisticated image analysis tools. Here, we describe a novel method that uses confocal microscopy to distinguish particle shape and orientation from a single confocal image that relies on interpreting single-particle scattering patterns.5
These patterns are obtained from raster-scans of inverted confocal microscope images (see Figure 1). We mostly used azimuthally and radially polarized doughnut laser beams (produced per the procedure describe in Dorn et al.6) for particle polarizability excitation, which influences the diffraction patterns. Although these patterns are diffraction-limited—the focal-spot diameter is about 500 nm—they contain information about the geometry, size and orientation of particles an order of magnitude smaller. This information can be probed through particle polarizability, which was found to change strongly with excitation wavelength.
The metallic nanoparticles were attached to a glass coverslip below a fixed immersion-oil objective with a 1.35 numerical aperture. Using an avalanche photodiode as a detector, we collected both the light reflected by the coverslip-sample interface, and the light scattered by the metallic particles (see Figure 2, left side), which gives rise to the interference patterns seen in Figures 1and2(a)and(c).
We imaged spherical, rod-shaped and triangular gold nanoparticles, as well as rod-shaped silver nanoparticles, homogeneously dispersed on the glass in some cases, and embedded in polyvinyl alcohol polymer in others. We also made in situ topology measurements to ensure that the detected optical signal came from a single particle.5 Simulations run to quantify the individual particle scattering patterns show that metallic nanoparticles can be clearly detected, and that gold nanospheres and nanorods can be differentiated—compare Figure 2(a) and (b) with Figure 2(c) and (d)—as can gold nanotriangles (not shown).
A single confocal image was found to be sufficient for determining the 2D orientation of a metallic nanorod, as Figure 2(a) and (b) show. Further, comparisons of simulations with observations indicate that it is possible to determine the orientation of metallic nanorods in 3D.
In summary, we have developed an original method to determine the shape, the size and orientation of individual metallic nanoparticles using confocal microscopy. We have been able to characterize metallic nanospheres with diameters of about 10 nm, and can determine the orientation of nanorods with principal axes measuring about 15 nm × 45 nm. We are presently trying to estimate the 3D orientations of metallic nanoparticles, and to optimize the method in order to detect particles diameters smaller than 5nm. This method is potentially useful for determining the relative orientation of a molecular structure placed in the vicinity of a metallic nanorod. Another promising application is the use of silver and gold nanorods as non-bleaching sensors for topological measurements in a biological environment.
We wish to acknowledge all the colleagues who contributed to this work (see reference5) and Tina Züchner for providing the experimental results presented in this article. We are also grateful to DFG (ME1600 6–1, 2) for financial support.
