High-throughput scanning confocal microscope for single molecule analysis

An automated and programmable confocal microscope that can acquire upwards of 103 single-molecule fluorescence resonance energy transfer (FRET) time traces is presented. The microscope augments the capabilities of current instruments by employing a flow cell that allows full control of the sample’s chemical environment, automatic locating and probing of single molecules, and an automatic focusing feature that enables the unattended scanning of large areas of the sample. This microscope performs routine acquisition of thousands of single-molecule events, in a highly reproducible fashion. These capabilities were utilized in recording the distributions of FRET efficiencies arising from control DNA molecules specifically labeled with internal donor-acceptor reporters.