ABSTRACT
The recently emerged field of single-molecule detection (SMD) has experienced rapid growth over recent years. This explosion has been fueled by the promise of the development of practical new technologies for chemical analysis that achieve the ultimate level of sensitivity, particularly for biological molecules. The field of SMD has also allowed the implementation of experiments designed to study the basic molecular properties of isolated molecules, as opposed to the properties provided by the ensemble average of bulk solutions [1]. It was Hirschfield in 1976 [2] who first reported on the microscopic observation of single molecules of γ-globulin adsorbed on a glass slide and labeled with 80-100 fluorescein molecules. In the case of solutions, Peck et al. [3] detected fluorescence from single phycoerythrin molecules, a multi-fluorophore phycobiliprotein having a fluorescence yield equivalent to about 100 rhodamine molecules [4]. Strictly speaking, the first detection of fluorescence from a single fluorophore in liquid solution was not achieved until 1990. At this time, Shera et al. [5] reported on the observation of fluorescence bursts from single fluorophores in a flow cell. Since then, many other approaches for the detection of single molecules in solution have appeared, including the use of levitated microdroplets [6], drops attached to a microscope objective [7], and, more recently, the use of infrared excitation [8], which is the subject of this chapter.
