However, it wasn't until the early 1940s that Albert Coons developed a technique for labeling antibodies with fluorescent dyes, thus giving birth to the field of immunofluorescence. Most notable among these early fluorescent dyes were the substituted xanthenes, fluorescein and rhodamine B, and the biaminated acridine derivative, acridine orange.įluorochromes were introduced to fluorescence microscopy in the early twentieth century as vital stains for bacteria, protozoa, and trypanosomes, but did not see widespread use until the 1920s when fluorescence microscopy was first used to study dye binding in fixed tissues and living cells. However, several synthetic dye classes synthesized during this period, based on the xanthene and acridine heterocyclic ring systems, proved to be highly fluorescent and provided a foundation for the development of modern synthetic fluorescent probes. Although these dyes were highly colored and capable of absorbing selected bands of visible light, most were only weakly fluorescent and would not be useful for the fluorescence microscopes that would be developed several decades later. Among these were pararosaniline, methyl violet, malachite green, safranin O, methylene blue, and numerous azo (nitrogen) dyes, such as Bismarck brown. The history of synthetic fluorescent probes dates back over a century to the late 1800s when many of the cornerstone dyes for modern histology were developed. In addition, fluorescent probes have been widely applied to genetic mapping and chromosome analysis in the field of molecular genetics. Fluorescent dyes are also useful in monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. Other probes are employed to monitor dynamic processes and localized environmental variables, including concentrations of inorganic metallic ions, pH, reactive oxygen species, and membrane potential. Many fluorescent probes are constructed around synthetic aromatic organic chemicals designed to bind with a biological macromolecule (for example, a protein or nucleic acid) or to localize within a specific structural region, such as the cytoskeleton, mitochondria, Golgi apparatus, endoplasmic reticulum, and nucleus. Biological laser scanning confocal microscopy relies heavily on fluorescence as an imaging mode, primarily due to the high degree of sensitivity afforded by the technique coupled with the ability to specifically target structural components and dynamic processes in chemically fixed as well as living cells and tissues.
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