Detection and identification of single molecules in solution

Soper, Steven A.; Davis, Lloyd M.; Shera, E.B.

doi:10.1364/josab.9.001761

Cited by 135 publications

(81 citation statements)

References 21 publications

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“…Previously, measurements of fluorescence lifetimes for single molecules in solution have been achieved by the use of a flowing sample stream, for which one fluorescence lifetime was apparent (19,20). We present the results from experiments in which the fluorescence lifetimes of individual molecules undergoing Brownian motion have been measured by confocal illumination and detection (3,5).…”

mentioning

confidence: 99%

Conformational transitions monitored for single molecules in solution.

Edman

Mets

Rigler

1996

Proc. Natl. Acad. Sci. U.S.A.

311

265

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Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, singlephoton counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.A new era of molecular analysis was begun by the introduction of extremely sensitive methods to detect and characterize species at the single-molecule level by spectroscopic means. Several techniques to trace individual fluorescent particles in solution have been reported over the last 12 years (1-9). Single-molecule detection (SMD) in solids and on surfaces has also been accomplished (10-14); however, many features important for chemical and biological systems can only be realized in the liquid phase, as is pointed out by reports regarding applications in DNA sequencing (15, 16) or detection and selection of rare events in diagnostics and biotechnology (17). Another important application for the SMD technique is the study of reaction dynamics at the singlemolecule level.Wang and Wolynes (18) have recently reported how measurements of single molecules can make it possible to gain information about phenomena that cannot be easily understood when a large number of molecules are observed simultaneously. A complex behavior (e.g., a distribution of a physical entity) will not provide information on whether all molecules share a common distribution or whether each molecule gives its own specific contribution to the distribution seen for many molecules. Measurements of single molecules can be decidedly important in obtaining such information.Previously (22), but we used it as a sensitive instrument -to provide precise statistical characteristics of the system. Single-Molecule Measurements. To achieve SMD, we measured repeatedly for short periods (t). The ratio of the measurement time to the characteristic passage time of a molecule through the VE is defined as T, the relative measuring time. By selecting only those measurements that experienced a large time integral of the detected fluorescence, we could effectively...

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mentioning

confidence: 99%

Conformational transitions monitored for single molecules in solution.

Edman

Mets

Rigler

1996

Proc. Natl. Acad. Sci. U.S.A.

311

265

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“…The higher accuracy provided by the SPTM in the measurement of the short lifetime components makes it possible to determine more accurately the single molecule lifetime probability distribution. Furthermore, the faster time response of the SPTM makes it possible to reduce more effectively the background by means of the lifetime gating [7], [25]. In fact, in cases where the Raman signal is the major background source in the single molecule experiment, the background level can be reduced to 1/20 without losing any fluorescence signal.…”

Section: Resultsmentioning

confidence: 99%

Photon-Timing Detector Module for Single-Molecule Spectroscopy With 60-ps Resolution

Rech

Luo

Ghioni

et al. 2004

IEEE J. Select. Topics Quantum Electron.

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“…Because the data are stored in the time-tagged time-resolved mode that allows for recording every detected photon with its individual timing and detection channel information, it is possible to detect photons that arrive at the detector some ps after the excitation pulse. In this way, Rayleigh scattered light can be removed by early time-gating [27].…”

Section: Comparison Of Correlated Fluorescence Fluctuation Traces Witmentioning

confidence: 99%

“…In fluorescence fluctuation methods, the fluctuations are measured against a background noise of Raman and Rayleigh scattered light from the medium or high dark current of the detector. The first approach that enabled measuring single fluorescent molecules in solution was called time-gated discrimination [26,27]. The detector was on only for a controlled time interval during a possible fluorescent burst from a single molecule.…”

Section: Introductionmentioning

confidence: 99%

Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

Földes-Papp¹,

Liao²,

You³

et al. 2009

CPB

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Abstract:We first report on the development of new microscope means that reduce background contributions in fluorescence fluctuation methods: i) excitation shutter, ii) electronic switches, and iii) early and late time-gating. The elements allow for measuring molecules at low analyte concentrations. We first found conditions of early and late time-gating with time-correlated single-photon counting that made the fluorescence signal as bright as possible compared with the fluctuations in the background count rate in a diffraction-limited optical set-up. We measured about a 140-fold increase in the amplitude of autocorrelated fluorescence fluctuations at the lowest analyte concentration of about 15 pM, which gave a signal-to-background advantage of more than two-orders of magnitude. The results of this original article pave the way for single-molecule detection in solution and in live cells without immobilization or hydrodynamic/electrokinetic focusing at longer observation times than are currently available.

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Detection and identification of single molecules in solution

Cited by 135 publications

References 21 publications

Conformational transitions monitored for single molecules in solution.

Conformational transitions monitored for single molecules in solution.

Photon-Timing Detector Module for Single-Molecule Spectroscopy With 60-ps Resolution

Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

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