Biofunctionalization of Fluorescent Nanoparticles

The current revolution in life sciences is strongly linked to the availability of sophisticated new experimental tools that enable the manipulation of biomolecules and the study of biological processes at the molecular level using state-of-the-art imaging techniques, such as single molecule imaging. Optical microscopy is fundamental to furthering our understanding of the structural, organizational, and dynamic properties of biological systems because a wide variety of complementary, non-invasive optical techniques exemplified by wide-field microscopy techniques, such as brightfield, darkfield, phase contrast, and DIC exist. Among these optical detection techniques, fluorescence microscopy is particularly important because it facilitates highly sensitive and specific imaging experiments. In addition, more sophisticated imaging approaches such as confocal and near-field imaging provide the opportunity for 3D and sub-diffraction limit imaging, respectively. Optical microscopy is now sensitive enough to track individual molecules if they are conjugated to appropriate imaging probes. Traditionally, such single molecule probes were mm-size colloidal particles and single fluorophores [1]. Colloidal probes such as gold or fluorescently labeled polystyrene beads are typically much larger (0.1–1 mm) than the biomolecule to be studied. However, fluorescent dyes, though smaller, show pronounced photo-instabilities, including blinking (due to fluorescence intensity fluctuations) and photobleaching, thus complicating single molecule tracking experiments and other fluorescence-based long-term studies. From the above description, further progress in the field of optical single molecule imaging obviously depends on the availability of appropriate labels that combine small size and high photostability with the ability to be used in multicolor studies. Fluorescent nanoparticles fulfill these important criteria. To be used in a biological environment, these nanoprobes need to be biofunctionalized appropriately, which remains a significant challenge. The main focus of this chapter is to provide an overview of recent developments addressing the bioconjugation of fluorescent nanoparticles and their surface modification using biocompatible coatings. Section 1.2 summarizes the different types