Single Molecule Rotational and Translational Diffusion Observed by Near-Field Scanning Optical Microscopy

Ruiter, A.G.T.; Veerman, J.A.; García-Parajo, María F.; Hulst, Niek F. van

doi:10.1021/jp971066s

Cited by 110 publications

(157 citation statements)

References 32 publications

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“…The similarity is an elegant manifestation of the ergodic principle of statistical physics. 07.79.Fc, Single molecule microscopy and spectroscopy give new and unique insight in the complex behavior of individual emitters on the nanometer scale [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Measurements on individual molecules have two distinct advantages.…”

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confidence: 99%

Time-Varying Triplet State Lifetimes of Single Molecules

et al. 1999

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It is found that triplet state lifetimes and intersystem crossing yields of individual molecules embedded in a polymer host at room temperature are not constant in time. The range over which the triplet lifetime of a single molecule varies during long observation times shows a strong similarity with the distribution of lifetime values obtained during short observation times of many individual molecules dispersed in space. The similarity is an elegant manifestation of the ergodic principle of statistical physics. 07.79.Fc, Single molecule microscopy and spectroscopy give new and unique insight in the complex behavior of individual emitters on the nanometer scale [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Measurements on individual molecules have two distinct advantages. First, phenomena that are usually hidden due to ensemble averaging can be directly observed, such as spectral and rotational jumps [1][2][3][4][5][6][7][8], photon (anti-)bunching [7,[9][10][11][12][13], and discrete photobleaching. Second, the monitoring of single molecule fluorescence forms a powerful way to probe the dynamics of the local environment on a nanometer scale. Therefore, single molecule detection allows the inhomogeneity of the ensemble to be directly related to the real time dynamics of the heterogeneity of the environment. In contrast, alternative techniques to study the composition of an inhomogeneously broadened ensemble like spectral hole-burning or pump-probe spectroscopies still average over a subset of the ensemble.For the purpose of this Letter, fluorescent molecules are considered as a three-level system. Besides the repetitive transitions between the singlet ground state ͑S 0 ͒ and the lowest singlet excited state ͑S 1 ͒ giving rise to fluorescence, the molecule has a small chance to undergo intersystem crossing (ISC) from S 1 to the lowest excited triplet state ͑T 1 ͒. As long as the T 1 state remains occupied, the S 0 2 S 1 transition does not occur and the fluorescence is interrupted temporarily. After decaying to S 0 the molecule starts fluorescing again. As a result, the fluorescence photons are emitted in bunches separated by dark periods when the molecule is in T 1 . This so-called photon bunching can be investigated in two ways. First, autocorrelation of the time intervals between detected photon counts yields the typical duration of the dark periods and thus the T 1 lifetime [7]. The disadvantage of this indirect method is that it yields only a mean value for the T 1 lifetime. Second, integration of the detected fluorescence photons over time intervals shorter than the duration of the dark periods can identify the time length of each excursion to T 1 in a direct way [11][12][13]. We have used the second method to obtain time-resolved T 1 state dynamics.The first measurements on the T 1 state of individual molecules were performed at cryogenic temperatures [1,[7][8][9]11]. It was found that T 1 lifetimes and intersystem crossing rates could vary among different molecules, which was attributed to loc...

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Time-Varying Triplet State Lifetimes of Single Molecules

et al. 1999

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“…The total detection efficiency of the microscope is estimated to be 1-2%. 17 To determine the fluorescence polarization, we used a scheme with two avalanche photodiode detectors (Figure 1), as was also used in by Ruiter et al 5 A polarization beam-splitting cube splits the two linear polarization components of the fluorescence onto the photodiodes. The photon signals of both detectors are counted simultaneously by two independent counters.…”

Section: Methodsmentioning

confidence: 99%

“…Two images of both linear polarization components in the microscope were simultaneously acquired, as reported elsewhere. 5 By calculating the inverse tangents from the relation of the intensities in the two detection channels for each pixel, we deduced an image of the polarization distribution. Figure 6 shows an image of the x-and y-polarized light and the corresponding polarization image, where in the polarization images the low intensities of the fluorescence are blanked out.…”

Section: Number Of Fluorescing Molecules: Simulation Vs Experimentmentioning

confidence: 99%

“…The microscopic observation of fluorescence from single chromophores adsorbed on surfaces or in thin films or membranes [1][2][3][4][5][6][7] and in fluids 8,9 at room temperature and low temperatures 10,11 gives direct insight into several processes that are not accessible in ensemble measurements, where fluorescence is detected from many molecules simultaneously. For example, bleaching of a single molecule can appear in one step 2,4 after several fluorescence cycles, the number of which may vary statistically between different molecules, whereas a fluorescence bleaching curve of a molecule ensemble decays continuously and nearly exponentially in time.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescence Blinking and Photobleaching of Single Terrylenediimide Molecules Studied with a Confocal Microscope

Göhde¹,

Fischer²,

Fuchs³

et al. 1998

J. Phys. Chem. A

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University of Groningen A. Herrmann and K. Mu 1llenMax-Planck-Institut für Polymerforschung, D-55128 Mainz, Germany ReceiVed: April 16, 1998; In Final Form: June 9, 1998 Single terrylenediimide molecules diluted in a 20-nm-thick polyvinylbutyral polymer film were localized and observed by scanning confocal fluorescence microscopy. A modular and compact confocal microscope and the high optical stability of the molecules allowed a repeated imaging and observation over >5 h at room temperature. Most of the molecules showed several "on-off-on" transitions (blinking) on a time scale from seconds to hours, before permanent bleaching occurred. We determined that >1.5 × 10 7 fluorescence photons are emitted from the most-stable molecules before the final bleaching step occurs. Despite the "onoff-on" transitions, however, the overall change in fluorescence intensity, either integrated over each image of a time series or summed for several individual molecules, resembled an exponential-like decay, familiar from measurements of many-molecule ensembles. We also observed the polarization of the fluorescence from single molecules during excitation with circular polarized light. From these measurements, possible rotations of the molecular dipoles were studied. Over a span of 5 h, the polarization angle in most cases did not change by >15-20°. This may explain the slow and small intensity changes but excludes molecular rotation as a reason for the blinking behavior.

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“…SNOM looks particularly suitable for this purpose due to the strong localization of the field emitted by the probe; moreover, due to the presence of transverse and longitudinal field components close to the probe apex, it has been used for the determination of the three-dimensional (3D) orientation of single fluorophores by controlling the polarization of the excitation light [29]. Aperture SNOM probes have been employed for fluorescence studies on biological molecules and polymers [24,[28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

Lotito

Sennhauser²,

Hafner³

et al. 2011

PIER

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Abstract-We present a numerical analysis of the interaction between novel scanning near field optical microscopy probes based on an asymmetric structure and a single fluorescent molecule. Our finite element analysis shows how such near field probes can be effectively used for high resolution detection of single molecules, in particular those with a longitudinal dipole moment. At the same time, fluorescent molecules can be exploited as point-like probes of the single vectorial components of the near field distribution at the probe apex, providing a powerful tool for near field probe characterization.

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Single Molecule Rotational and Translational Diffusion Observed by Near-Field Scanning Optical Microscopy

Cited by 110 publications

References 32 publications

Time-Varying Triplet State Lifetimes of Single Molecules

Time-Varying Triplet State Lifetimes of Single Molecules

Fluorescence Blinking and Photobleaching of Single Terrylenediimide Molecules Studied with a Confocal Microscope

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

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