Colorful Molecular Diagnostics

Most diagnostic assays used daily in clinical laboratories are based on identifying and quantifying the presence of specific molecules that serve as markers of pathologic conditions and physiological status; however, these molecules are neither visualized nor counted directly. Instead, amplified signals generated through various strategies [e.g., real-time PCR, enzyme-linked assays (ELISAs), or simply blood culture] quantify the molecules of interest in an indirect fashion. These bioassays provide easy and robust detection on affordable instrumentation, but their amplification procedures also are usually associated with additional costs, additional time to results, increased labor, and a certain error rate. Furthermore, most of these methods monitor a single marker in a single sample. Consequently, methods that detect molecular markers directly are highly desirable. In most cases, this quest involves fluorescence spectroscopy and imaging, owing to its flexibility, low cost, and sensitivity in visualizing and characterizing single molecules in very small sample volumes (1). Until recently, single-molecule fluorescence detection was not a trivial task, because the signals generated from an individual molecule (for example, a fluorophore attached to a protein or DNA) were extremely weak. Only custom-built microscopes featuring the best combination of illumination sources, fluorescent probes, and ultrasensitive detectors could provide the sensitivity required to “see” and count single fluorescent molecules. Improvements introduced to facilitate studies of biological mechanisms in vitro and in vivo have made single-molecule microscopy instruments more sensitive, streamlined, and affordable. In fact, such instruments are leading the race for third-generation DNA sequencing (2, 3). In this issue of the Journal, Yim et al. (4) introduce a different application of single-molecule fluorophore …