Real-Time Detection of Single Molecules in Solution by Confocal Fluorescence Microscopy

We report real-time detection of single fluorescent molecules in solution with a simple technique that combines confocal microscopy, diffraction-limited laser excitation, and a high-efficiency photon detector. The probe volume, ~5.0 x 10^-16 L, is defined latitudinally by optical diffraction and longitudinally by spherical aberration. With an unlimited excitation throughput and a low background level, this technique allows fluorescence detection of single rhodamine molecules with a signal-to-noise ratio of ~10 in 1 ms, which approaches the theoretical limit set by fluorescence saturation. Real-time measurements at a speed of 500 000 data points/s yield single-molecule fluorescence records that not only show the actual transit time of a particular molecule across the probe volume but also contain characteristically long (~50 μs) and short (~4 μs) dark gaps. Random-walk simulations of single fluorescent molecules provide evidence that these long and short dark periods are caused mainly by boundary recrossing motions of a single molecule at the probe volume periphery and by intersystem crossing into and out of the dark triplet state. We have also extended the use of confocal fluorescence microscopy to study individual, fluorescentiy tagged biomolecules, including deoxynucleotides, single-stranded primers, and double-stranded DNA. The achieved sensitivity permits dynamic structural studies of individual λ-phage DNA molecules labeled with intercalating fluorescent dyes; the results reveal largeamplitude DNA structural fluctuations that occur on the millisecond time scale.