REAL-TIME DETECTION OF SINGLE-MOLECULES IN SOLUTION BY CONFOCAL FLUORESCENCE MICROSCOPY

We report real-time detection of single fluorescent molecules in solut ion with a simple technique that combines confocal microscopy, diffrac tion-limited laser excitation, and a high-efficiency photon detector. The probe volume, similar to 5.0 x 10(-16) L, is defined latitudinally by optical diffraction and longitudinally by spherical aberration. Wi th 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 similar to 10 in 1 ms, which approach es the theoretical limit set by fluorescence saturation. Real-time mea surements at a speed of 500 000 data points/s yield single-molecule fl uorescence records that not only show the actual transit time of a par ticular molecule across the probe volume but also contain characterist ically long (similar to 50 mu s) and short (similar to 4 mu s) dark ga ps. Random-walk simulations of single fluorescent molecules provide ev idence that these long and short dark periods are caused mainly by bou ndary recrossing motions of a single molecule at the probe volume peri phery and by intersystem crossing into and out of the dark triplet sta te. We have also extended the use of confocal fluorescence microscopy to study individual, fluorescently tagged biomolecules, including deox ynucleotides, single-stranded primers, and double-stranded DNA. The ac hieved sensitivity permits dynamic structural studies of individual la mbda-phage DNA molecules labeled with intercalating fluorescent dyes; the results reveal large-amplitude DNA structural fluctuations that oc cur on the millisecond time scale.