Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy

Abstract

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.e., 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.

Super-Resolution Structured Illumination Microscopy

“…Recently, even other sources of incoherent photons, such as gold and silver nanoparticles capable of surface-enhanced Raman scattering, have been demonstrated to provide excellent contrast for SR-SIM imaging. 46 The main applications are, however, found in the biological sciences, where initially fixed cellular structures and, more recently, even living cells have been imaged by SR-SIM. For fixed cell imaging, one can choose from the widest selection of fluorophores that are either initially expressed by the cells themselves, or added after fixation to specifically stain cellular structures of interest by immunofluorescence or other mechanisms of specific molecular binding.…”

Sensing Glucose in Urine and Serum and Hydrogen Peroxide in Living Cells by Use of a Novel Boronate Nanoprobe Based on Surface-Enhanced Raman Spectroscopy

“…24–27 Moreover, SERS probes are commercially available or obtained from simple synthesis, thus avoiding the complicated and expensive design and synthesis of traditional fluorescence probes. Unfortunately, direct detection of H 2 O 2 remains a challenge to SERS-based schemes due to the intrinsically small Raman cross section of the molecule.…”

“…Instead, surface-enhanced Raman spectroscopy (SERS) , is an extremely sensitive technique − and shows excellent photostability and biocompatibility when gold nanoparticles (AuNPs) are used as the plasmonic substrate. − Moreover, SERS probes are commercially available or obtained from simple synthesis, thus avoiding the complicated and expensive design and synthesis of traditional fluorescence probes. Unfortunately, direct detection of H 2 O 2 remains a challenge to SERS-based schemes due to the intrinsically small Raman cross section of the molecule.…”

Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

“…FairSIM was primarily designed to provide single-slice reconstructions of SR-SIM systems working with both two-beam interference (utilized by many home-built and total internal reflection-excited fluorescence (TIRF)-based systems) and three-beam interference (utilized in all commercially available systems) for pattern generation. This development was motivated by the need for both a rapid and flexible single-slice reconstruction mode for our own commercial SR-SIM system 30 , and a stand-alone reconstruction tool for our home-built, two-beam illumination SIM set-ups.…”