Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)

A PC plug-in card for on-line time resolved fluorescence detection of singl e dye molecules based on a new time-correlated single photon counting (TCSP C) module is described. The module contains all electronic components const ant fraction discriminators (CFDs), time-to-amplitude converter (TAC), anal og-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate o f 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 mu s without t ime gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a c onfocal microscope, we studied bursts of fluorescence photons from individu al dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microca pillary with an inner diameter of 0.5 mm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented tech nique permits the counting and identification of all labeled analyte molecu les present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times. (C) 1999 American Institute of Physic s. [S0034-6748(99)03503-0].