Nanotechnology and biophotonics provide the life sciences with unique methods for investigating a wide of range of biological systems at unprecedented levels of precision and control. The behaviour of cells is determined by their nanoscale constituents, including organelles and other nanoscale molecular complexes such as ribosomes, receptors and macromolecules. The interaction of these constituents with engineered nanostructures allows their visualization and control.

Optical imaging assisted by molecular-specific labelling with photoluminescent nanoparticles (NPs) is one of the most significant applications of these nanomaterials. This direct, minimally invasive approach makes it possible to investigate the cellular morphology and various processes in living cells and/or tissues in their full biological context. Their detection sensitivity is high, reaching the regime of single molecules. Single-molecule imaging unobscured by ensemble averaging is a significant tool for understanding complex biological systems. Single-molecule imaging is required, for example, when studying molecular trafficking events associated with cell signalling via the activation of membrane receptors, and in studies of individual receptors and ligands. These studies place stringent demands on the fluorophore performance: fluorescence intermittency (blinking) and irreversible light-induced transitions to dark states (photobleaching) pose significant limitations. Dark-state transitions are particularly undesired in single-molecule studies and also in super-resolution microscopy [for example, stimulated emission depletion microscopy (STED)], which require high illumination intensities. Typical organic dye fluorophores survive only about one million excitation–emission cycles before undergoing an irreversible transition to the dark state (photobleaching), and this means that the continuously emitting fluorophore persists for only ∼3 ms (assuming the fluorescence lifetime τ = 3 ns and saturation excitation conditions). Longer wavelength fluorophores such as Cy5 and Cy7, Alexa Fluor 750 and CF dyes, whose excitation–emission wavelengths fall into the biological tissue transparency window (wavelength range 700–1300 nm) (Figure 10.1(a)) are especially prone to photobleaching.