The use of fluorescent dyes has effectively replaced radioactivity as the tagging method of choice for biological assays. The ability to make full use of the fluo rescent molecule and all its properties should be the benchmark of any fluorescence-based assay technology. While most assay strategies make use of only a single fluorescent property, the broad applicability of a screening system requires that flexibility of assay design be extended to the readout technology by including a variety of detection modes. While most scientists may associate the use of fluorescence in biological assay systems solely with the measurement of fluorescence intensity, the measurement of additional fluorescent properties such as lifetime, polarization, and quenching can yield a wealth of information from a single measurement. This ability to collect multiple data points per measurement not only provides an internal control but also contributes to screening efficiency by enabling rapid multiparameter evaluation of compound-target interactions.
Confocal microscopic optical systems form the foundation of the readout technologies employed in the EVOscreen system. Using confocal optics, submi-croliter miniaturization can be accomplished without any loss of signal quality (Fig. 3). The highly focused confocal optics reduce the detection volume to one femtoliter. This means that the detection volume, roughly the size of a bacterial cell, is much smaller than a eukaryotic cell or the standard miniaturized assay volumes of one microliter. Whereas other methodologies often suffer from the increased contribution of surface effects and from drastically reduced signal-to-noise ratios when assay volumes are reduced, the confocal optics employed for all EVOscreen readout technologies eliminates such concerns.
Furthermore, confocal detection technology enables the use of homogeneous assay methods, i.e., those that eliminate washing or so-called ''sandwich'' strategies. This is a great advantage in screening in that it allows biological systems to be evaluated in close to their natural in vivo state. Building on this confocal fluorescence detection platform, the EVOscreen ® system described fulfills all the demands made of a HTS system in the combinatorial age.
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