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We report on the development of a flow system integrated with a microscope that facilitates the simultaneous optical trapping and fluorescence excitation/detection of micron-sized samples that have been tagged with fluorescent probes. This system, when used in conjunction with nucleotide fluorescence labeling and DNA fragment cleavage procedures, offers the potential for the rapid sequencing of DNA fragments attached to microsphere 'handles.' Using a Nd:YAG laser (1064 nm) as the trapping beam, latex microspheres were stably trapped in a flow stream, where flow velocities in the range of 1 - 10 mm/s were achieved. Such flow velocities are commensurate with those required to stretch, and measure fluorescence from, DNA strands in high speed sequencing applications. High spatial resolution (approximately 1 micrometer) and high signal-to-noise ratios (greater than 103) were achieved using a high N.A. objective lens for trapping and fluorescence detection within a confocal microscope geometry. In this system, samples could be displaced with the scanning laser trap by up to plus or minus 25 micrometers off the trapping beam axis in the sample plane while maintaining, at the same time, a large fluorescence detection efficiency. At a trapping depth of 20 micrometer below the chamber surface, a laser power of 100 mW was sufficient to hold a 2 micrometer diameter microsphere in a flow stream having a velocity of 1 mm/s while its fluorescence was measured. The results of a systematic study which investigates the effects of trapping efficiency, trapping depth, flow velocity, and tweezers holding time in a 500 micrometer by 500 micrometer flow microchamber system are reported. The application of this technique to the confinement and detection of fluorescence-labeled DNA nucleotides is also described.
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