Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique

Sauer, Markus; Angerer, Bernhard; Han, K.-T.; Zander, C.

doi:10.1039/a901411j

Cited by 44 publications

(47 citation statements)

References 24 publications

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“…Recently (20,21), we demonstrated that differences of about 1 ns in fluorescence lifetime are sufficient to identify various single dye molecules in aqueous solution with high classification probabilities using less than 100 collected photon counts. Scanning confocal fluorescence microscopy images obtained from single Cy5 (a) and JA242 (b) molecules adsorbed on silanized cover slides are shown on the left hand side of Fig.…”

Section: Resultsmentioning

confidence: 99%

“…phenomena that are hidden in ensemble experiments due to averaging. In addition, single-molecule methods are ideally suited to study subpopulations (17)(18)(19)(20)(21) and to monitor reaction pathways of individual members (22)(23)(24) in a heterogeneous mixture. Fluorescence lifetimes of individual immobilized molecules have been studied by time-correlated single-photon counting (TCSPC) using near-field (25) and far-field optical microscopy (5,6).…”

Section: Introductionmentioning

confidence: 99%

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Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

et al. 2000

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We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06± 0.37 ns (Cy5) and 3.89± 0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short offstates t off of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield Y ISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

et al. 2000

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“…With respect to analytical purposes as well as for single molecule DNA-sequencing it is of great interest to achieve a most efficient detection of all molecules within a sample volume [34][35][36][37][38]. In this case a sample survey is not sufficient, but a complete counting of all molecules within a sample volume is the ultimate goal.…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Dörre¹,

Stephan²,

Eigen

2001

Single Mol.

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In this article we describe a detailed comparison between single molecule detection in three different types of microchannels. The overall detection efficiencies and the signal-to-noise ratios are compared. A new data evaluation technique that is used is explained in detail. The average number of photons per molecule is calculated from the measurements and the detection properties of each channel with respect to the diffusional characteristics of the dye molecules are investigated. From the detection efficiencies advantages and drawbacks of the individual methods are elucidated as well as possible applications of the techniques are illustrated.

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“…SMD has been applied for monitoring molecular diffusion in free solutions, 9 studying absorption and desorption of protein molecules at a solid/liquid interface, 10,11 finding anomalous radial migration of DNA molecules in capillary electrophoresis. 12,13 In addition to fundamental investigations, SMD has been extensively applied for biological or biochemical areas, such as high throughput DNA screening in electrophoresis, 14,15 single molecule immunoassay, 16 DNA sizing and sorting, [17][18][19][20] DNA mapping 21,22 and in situ hybridization. 23 The single molecule detection technique was also used to probe the flow profile.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous measurements of the flow velocities in a microchannel by wide/evanescent field illuminations with particle/single molecules

et al. 2005

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A laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. Fluorescent microbeads with a diameter of 175 nm were used to calibrate the system, and the collisions of microbeads with channel walls were directly observed. The 175 nm microbeads velocities in the main flow and at 275 nm from the bottom of the channel were measured. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory when the diameter of a microbead was considered. The errors caused by Brownian diffusion in our measurement were negligible compared to the flow velocity. Single lDNA molecules were then used as a flowing tracer to measure the velocities. The velocity can be obtained at a distance of 309.0 ¡ 82.6 nm away from bottom surface of the channel. The technique may be potentially useful for studying molecular transportation both in the center and at the bottom of the channel, and interactions between molecules and microchannel surfaces. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.

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Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique

Cited by 44 publications

References 24 publications

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Simultaneous measurements of the flow velocities in a microchannel by wide/evanescent field illuminations with particle/single molecules

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