Light quenching and fluorescence depolarization of rhodamine B and applications of this phenomenon to biophysics

Lakowicz, Joseph R.; Gryczyński, Ignacy; Богданов, В. Л.; Kuśba, Józef

doi:10.1021/j100052a055

Cited by 52 publications

(45 citation statements)

References 27 publications

(23 reference statements)

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“…In order to get full information about the ultrafast dynamics, we here develop the isotropic (54.7°) and anisotropic (parallel and vertical) femtosecond fluorescence depletion methods, where the polarizations of dumping beams with respect to that of pumping beam are selected by rotating a zero-level half-wave plate to obtain isotropic (54.7°) and anisotropic (parallel and vertical) results, respectively. The time-resolved anisotropic decay r(t) was calculated from the decay curves under the condition of parallel polarizations (I || ) and perpendicular polarizations (I ⊥ ) of dumping beam relative to the polarization of pumping beam according to eq 3: 33,35,36 The factor G accounts for the difference in sensitivities for the detection of signal in the perpendicular-and parallelpolarized configurations, where G (G ) I ⊥ /I || , when the excitation is vertically polarized) is estimated about 1.02. The instrument response function is determined according to the 1 + 1′ two photon excited fluorescence method as described before, 10,11,34 in which, the IRF (instrumental response function) up to 90 fs is achieved.…”

Section: Methodsmentioning

confidence: 99%

Determination of the Formation of Dark State via Depleted Spontaneous Emission in a Complex Solvated Molecule

et al. 2007

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We present a general two-color two-pulse femtosecond pump-dump approach to study the specific population transfer along the reaction coordinate through the higher vibrational energy levels of excited states of a complex solvated molecule via the depleted spontaneous emission. The time-dependent fluorescence depletion provides the correlated dynamical information between the monitored fluorescence state and the SEP "dumped" dark states, and therefore allow us to obtain the dynamics of the formation of the dark states corresponding to the ultrafast photoisomerization processes. The excited-state dynamics of LDS 751 have been investigated as a function of solvent viscosity and solvent polarity, where a cooperative two-step isomerization process is clearly identified within LDS 751 upon excitation.

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Section: Methodsmentioning

confidence: 99%

Determination of the Formation of Dark State via Depleted Spontaneous Emission in a Complex Solvated Molecule

et al. 2007

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“…Stimulated emission forces excited molecules to an upper vibrational level of the ground state, whose ultrafast vibrational decay (2) prevents re-excitation by the same beam. As a result, fluorescence can be entirely stopped (3)(4)(5)(6). ''Engineering'' of the fluorescence spot, or point-spread-function (PSF) by stimulated emission depletion, was predicted to improve the resolution in the transverse direction (3,5), and initial experiments with nanocrystals indeed confirmed an improvement by a factor of 1.3 (6).…”

mentioning

confidence: 99%

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

Klar

Jakobs

Dyba

et al. 2000

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

1,641

1,263

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The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90 -110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details. It is generally assumed that the extent of the focal spot of a microscope is not reducible beyond the wavelength of light , because diffraction demands an extent of at least ͞3 in the lateral and in the axial direction. In consequence, for more than a century, focused light was abandoned whenever higher spatial definitions were required. The diffraction limit also stimulated the invention of electron, scanning probe, and nearfield optical microscopy, but these techniques are largely surface-bound and not able to image the intact cellular interior. Therefore, far-field light microscopy, encompassing the conventional and confocal fluorescence microscope, remained the most widely applied microscopy in biological research (1); however, the limited spot size hinders the visualization of fine subcellular structures.We now report the generation of fluorescence focal spots of substantially reduced extent and their application to threedimensional imaging. For the first time, the axial and lateral resolution of a lens is found to be in the same range (90-110 nm). Confocal sectioning is improved up to 5-fold. The improvement is attained by quenching through stimulated emission the excited fluorophores at the rim of the excitation focal spot with a beam whose wavelength is in the red edge of the fluorophore emission spectrum. Stimulated emission forces excited molecules to an upper vibrational level of the ground state, whose ultrafast vibrational decay (2) prevents re-excitation by the same beam. As a result, fluorescence can be entirely stopped (3-6). ''Engineering'' of the fluorescence spot, or point-spread-function (PSF) by stimulated emission depletion, was predicted to improve the resolution in the transverse direction (3, 5), and initial experiments with nanocrystals indeed confirmed an improvement by a factor of 1.3 (6). However, this moderate improvement was achieved along a single direction only, and it remained unclear whether it would be effective in biological imaging. Physical Principles and SetupOur setup used two synchronized trains of laser pulses: a train of visible pulses was used for excitation and a near-infrared counterpart for stimulated emission depletion (STED). Each excitation pulse was immediately followed by a STED pulse. The pulses originally stemmed f...

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“…After vibrational relaxation, which occurs on a (sub)picosecond time scale, organic fluorophores emit within a few nanoseconds with a probability given by the fluorescence quantum yield. Alternatively, the S 1 !S 0 transition can be enforced by light, that is by stimulated emission.[10] Stimulated emission has been employed to probe the temporal evolution of the excited state in a pump-probe fashion, as well as to alter the fluorescence lifetime and anisotropy [11,12] of organic fluorophores in solution. Furthermore, stimulated emission depletion (STED) of the excited state has been introduced in fluorescence microscopy [13,14] to break the diffraction resolution barrier, [15,16] but has recently also been used to measure cross sections in the bulk.…”

mentioning

confidence: 99%

Absolute Optical Cross Section of Individual Fluorescent Molecules

Kastrup

Hell

2004

Angew Chem Int Ed

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Besides having revealed transitions masked in the bulk, [1,2] optical spectroscopy of single molecules has enabled the observation of elementary molecular quantum events, such as the absorption and emission of a photon. [3][4][5][6] Single photon transitions are quantified by a cross section that can be regarded as the photon capture area of a molecule. Therefore, absolute optical cross sections can readily be measured in the bulk from the decrease (by absorption) or the gain (by stimulated emission) of the number of photons in a beam. [7] Unfortunately, this method is not applicable to individual molecules, because the resulting change in intensity is negligible. If the fluorescence quantum efficiency Q em is known, the molecular absorption cross section can be calculated from the fluorescence emission.[8] However, this method re-introduces the hallmark of ensemble averaging, because Q em is known only from the bulk. As a result, the absolute photon capture area of a specific molecule has remained elusive.Here we report on the measurement of absolute cross sections of stimulated emission at room temperature. No a priori information about the individual molecule is needed. In addition, we demonstrate the instant control of a molecules excited state by light and thus, as a matter of fact, also the viability of pump-probe experiments [9] with individual molecules at room temperature.Molecular optical absorption usually takes place from the vibrationally relaxed ground state S 0 to a hot Franck-Condon state S 1 * of the first excited singlet state (Figure 1 a). After vibrational relaxation, which occurs on a (sub)picosecond time scale, organic fluorophores emit within a few nanoseconds with a probability given by the fluorescence quantum yield. Alternatively, the S 1 !S 0 transition can be enforced by light, that is by stimulated emission.[10] Stimulated emission has been employed to probe the temporal evolution of the excited state in a pump-probe fashion, as well as to alter the fluorescence lifetime and anisotropy [11,12] of organic fluorophores in solution. Furthermore, stimulated emission depletion (STED) of the excited state has been introduced in fluorescence microscopy [13,14] to break the diffraction resolution barrier, [15,16] but has recently also been used to measure cross sections in the bulk. [17] We attained single molecule sensitivity with a scanning confocal setup fed by two synchronized pulse trains: a first pulse at a wavelength of l = 565 nm for excitation was followed by a red-shifted pulse at l = 778 nm for STED, both adapted to the spectrum (Figure 1 b) of the xanthene dye JA 26 (1). Efficient STED demands a STED pulse duration t of about 15 ps, [13,15] because this is significantly shorter than the nanosecond lifetime of S 1 , but much longer than the subpicosecond vibrational decay of S 0 * . Thus the S 1 $S 0 * system is effectively depleted. Figure 2 illustrates the ability of the STED pulse to "switch off" single fluorescent molecules embedded in a poly(vinyl alcohol) film. The image was ...

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Light quenching and fluorescence depolarization of rhodamine B and applications of this phenomenon to biophysics

Cited by 52 publications

References 27 publications

Determination of the Formation of Dark State via Depleted Spontaneous Emission in a Complex Solvated Molecule

Determination of the Formation of Dark State via Depleted Spontaneous Emission in a Complex Solvated Molecule

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

Absolute Optical Cross Section of Individual Fluorescent Molecules

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