Spectroscopic Properties of Gold Nanoparticles at the Single‐Particle Level in Biological Environments

Labeling cells and tissues with fluorescent probes, such as organic dyes and quantum dots (Qdots) is a widespread and successful technique for studying molecular dynamics both in vitro and in vivo. However, those probes usually suffer from undesirable photophysical/photochemical processes, such as blinking and photobleaching, limiting their utilization. The main challenges in fluorescent probe design are to improve their absorption/emission properties, and to provide higher stability against photobleaching. In the last few years, metallic nanoparticles (NPs) of various sizes, shapes, and compositions have been used as a new alternative for cellular microscopy. This is in part because—unlike common organic dyes and Qdots—metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright, and their luminescence spans over the visible spectrum. These characteristics make them attractive contrast agents for cell imaging both in vitro and in vivo. For these reasons, the emission of metallic NPs in bulk solutions has already been extensively characterized. In contrast with bulk experiments, where billions of molecules are measured simultaneously, single‐particle techniques allow the observation of characteristics and dynamical processes otherwise hidden in the measured average. A full understanding of the photophysical properties of the NPs is critical when they are used for single‐molecule applications. Photophysical processes can be a source of artifacts if they are not interpreted accordingly, and thus a careful characterization of these labels at the single‐particle level became crucial for the correct interpretation of the experimental results. Herein, we study some of their unique optical properties at the single‐particle level and show examples that illustrate their intrinsic heterogeneity when used in biological environments.