Multiplexed two-photon excitation spectroscopy of single gold nanorods.

Plasmonic metallic nanoparticles are commonly used in (bio-)sensing applications because their localized surface plasmon resonance is highly sensitive to changes in the environment. Although optical detection of scattered light from single particles provides a straightforward means of detection, the two-photon luminescence (TPL) of single gold nanorods (GNRs) has the potential to increase the sensitivity due to the large anti-Stokes shift and the non-linear excitation mechanism. However, two-photon microscopy and spectroscopy are restricted in bandwidth and have been limited by the thermal stability of GNRs. Here, we used a scanning multi-focal microscope to simultaneously measure the two-photon excitation spectra of hundreds of individual GNRs with sub-nanometer accuracy. By keeping the excitation power under the melting threshold, we show that GNRs were stable in intensity and spectrum for more than 30 min, demonstrating the absence of thermal reshaping. Spectra featured a signal-to-noise ratio of >10 and a plasmon peak width of typically 30 nm. Changes in the refractive index of the medium of less than 0.04, corresponding to a change in surface plasmon resonance of 8 nm, could be readily measured and over longer periods. We used this enhanced spectral sensitivity to measure the presence of neutravidin, exploring the potential of TPL spectroscopy of single GNRs for enhanced plasmonic sensing.

Coating gold nanorods with self-assembling peptide amphiphiles promotes stability and facilitates in vivo two-photon imaging.

Gold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing, and in vivo imaging. However, as-synthesised GNRs need to be modified with a biocompatible stabilising coating in order to be employed in these fields as the ligands used to stabilise GNRs during synthesis are toxic. An issue is that GNR performance in the aforementioned techniques can be affected by these modified coatings. For example if coatings are too thick then GNR entry into cells, or their sensitivity in sensing applications, can be compromised. Here we show that thiolated peptide amphiphiles (PAs) can act as GNR stabilisers and provide a thin and highly-stable coating under physiologically relevant conditions. Additionally, all tested PAs formed highly ordered (51.8-58.8% ß-content), and dense (2.62-3.87 peptides per nm2) monolayers on the GNR surface. Moreover, the PA-coated GNRs demonstrated no cytotoxicity in vitro and, via injection in zebrafish embryos, the behavior and cellular interactions of such PA-coated GNRs were visualised in vivo, in real time, with two-photon (2P) microscopy.

Adsorption of titanium dioxide nanoparticles onto zebrafish eggs affects colonizing microbiota.

Teleost fish embryos are protected by two acellular membranes against particulate pollutants that are present in the water column. These membranes provide an effective barrier preventing particle uptake. In this study, we tested the hypothesis that the adsorption of antimicrobial titanium dioxide nanoparticles onto zebrafish eggs nevertheless harms the developing embryo by disturbing early microbial colonization. Zebrafish eggs were exposed during their first day of development to 2, 5 and 10 mg TiO2 L-1 (NM-105). Additionally, eggs were exposed to gold nanorods to assess the effectiveness of the eggs' membranes in preventing particle uptake, localizing these particles by way of two-photon microscopy. This confirmed that particles accumulate onto zebrafish eggs, without any detectable amounts of particles crossing the protective membranes. By way of particle-induced X-ray emission analysis, we inferred that the titanium dioxide particles could cover 25-45 % of the zebrafish egg surface, where the concentrations of sorbed titanium correlated positively with concentrations of potassium and correlated negatively with concentrations of silicon. A combination of imaging and culture-based microbial identification techniques revealed that the adsorbed particles exerted antimicrobial effects, but resulted in an overall increase of microbial abundance, without any change in heterotrophic microbial activity, as inferred based on carbon substrate utilization. This effect persisted upon hatching, since larvae from particle-exposed eggs still comprised higher microbial abundance than larvae that hatched from control eggs. Notably, pathogenic aeromonads tolerated the antimicrobial properties of the nanoparticles. Overall, our results show that the adsorption of suspended antimicrobial nanoparticles on aquatic eggs can have cascading effects across different life stages of oviparous animals. Our study furthermore suggests that aggregation dynamics may occur that could facilitate the dispersal of pathogenic bacteria through aquatic ecosystems.

Light-triggered switching of liposome surface charge directs delivery of membrane impermeable payloads in vivo.

Surface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching of liposome surface charge, from neutral to cationic, in situ and in vivo (embryonic zebrafish). Prior to light activation, intravenously administered liposomes, composed of just two lipid reagents, freely circulate and successfully evade innate immune cells present in the fish. Upon in situ irradiation and surface charge switching, however, liposomes rapidly adsorb to, and are taken up by, endothelial cells and/or are phagocytosed by blood resident macrophages. Coupling complete external control of nanoparticle targeting together with the intracellular delivery of encapsulated (and membrane impermeable) cargos, these compositionally simple liposomes are proof that advanced nanoparticle function in vivo does not require increased design complexity but rather a thorough understanding of the fundamental nano-bio interactions involved.