Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy
2015-06-23T00:00:00Z (GMT) by
Optical microscopy modalities that achieve spatial resolution beyond the resolution limit have opened up new opportunities in the biomedical sciences to reveal the structure and kinetics of biological processes on the nanoscale. These methods are, however, mostly restricted to fluorescence as contrast mechanism, which limits the ultimate spatial resolution and observation time that can be achieved by photobleaching of the fluorescent probes. Here, we demonstrate that Raman scattering provides a valuable contrast mechanism for optical nanoscopy in the form of super-resolution structured illumination microscopy. We find that nanotags, <i>i.e.</i>, gold and silver nanoparticles that are capable of surface-enhanced Raman scattering (SERS), can be imaged with a spatial resolution beyond the diffraction limit in four dimensions alongside and with similar excitation power as fluorescent probes. The highly polarized nature of super-resolution structured illumination microscopy renders these nanotags elliptical in the reconstructed super-resolved images, which enables us to determine their orientation within the sample. The robustness of nanotags against photobleaching allows us to image these particles for unlimited periods of time. We demonstrate this by imaging isolated nanotags in a dense layer of fluorophores, as well as on the surface of and after internalization by osteosarcoma cells, always in the presence of fluorescent probes. Our results show that SERS nanotags have the potential to become highly multiplexed and chemically sensitive optical probes for optical nanoscopy that can replace fluorophores in applications where fluorescence photobleaching is prohibitive for following the evolution of biological processes for extended times.
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