The Development of Photostabile Fluorescent Reporters
Many seminal advances in modern molecular biology and medicine are possible because of the availability of sophisticated fluorescent reporter compounds that, with exquisite selectivity and sensitivity, provide the means to observe structure-function relationships in biological systems. Although this use of organic fluorophore reporters is not new, it remains a dynamic and growing technology that continues to be used to solve important new problems. Currently there is considerable interest in employing this methodology in single molecule detection to study individual events in biochemicals. As is the case with any technology, the use of fluorescent probes has limitations that may frustrate the experimentalist from taking full advantage of the promise of the method. One of the most severe is the constraint related to fluorophore photodecomposition (bleaching) which sets strict limits both on the time that chromophores can be monitored and on the illumination intensity that can be used to interrogate the sample. As a consequence, researchers are often precluded from exploiting one of the most powerful tenets of fluorescence spectroscopy which teaches that the signal strength observed for a fluorophore (within limits) increases linearly with the intensity of the excitation.
Renewed demands for dyes better suited for fluorescence reporting has stimulated us to initiate a research program in collaboration with our Rowland colleague Dr. Amit Meller, a biophysicist with expertise in single molecule detection, with the goal of developing a series of improved fluorophores having much better light- and dark-stabilities, more ideal photophysical characteristics (i.e. high quantum yields that are invariant with temperature or environment) and which collectively have absorption and emission bands that span the visible and near-infrared spectral regions.