Fluorescence of coumarins and xanthenes after two-photon absorption with a pulsed titanium–sapphire laser

Fischer, A.; Cremer, Christoph; Stelzer, Ernst H. K.

doi:10.1364/ao.34.001989

Cited by 190 publications

(132 citation statements)

References 21 publications

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“…Experimentally, intersystem crossing and photobleaching cause severe deviation from the expected power law dependence of the excited fluorescence. Photobleaching may account for the nonquadratic dependence of TPE fluorescence in some earlier reports (44). Using a high-n.a.…”

Section: Consequences Of the Photophysics Of Multiphoton Excitationmentioning

confidence: 94%

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Zipfel

Shear

et al. 1996

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

1,114

801

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Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.Molecular two-photon excitation (TPE) was predicted by Goppert-Mayer in 1931 (1). Experimental observations of multiphoton processes awaited the invention of pulsed ruby lasers in 1960. Closely following the demonstration of secondharmonic generation (SHG), the first demonstration of nonlinear optics, two-photon absorption was utilized by Kaiser and Garrett to excite fluorescence emission in CaF2:Eu3+ (2).Three-photon excited fluorescence was observed and the three-photon absorption cross section for naphthalene crystals was estimated by Singh and Bradley in 1964 (3). Subsequently, multiphoton excitation and fluorescence has been used in molecular spectroscopy of various materials (4-8).A significant biological application of multiphoton excitation began with the invention of two-photon laser scanning microscopy (TPLSM) by Denk, Strickler, and Webb in 1990 (9). Originally devised for localized photochemical activation of caged biomolecules, TPE of photochemical polymer crosslinking also has provided a means for high-density threedimensional optical data storage at 1012 bits/cm3 (10).This article on multiphoton excitation is motivated by the emergence of TPLSM as a powerful new microscopy for three-dimensionally resolved fluorescence imaging of biological samples (11,12). The development of TPLSM has been propelled by rapid technological advances in laser scanning microscopy (LSM) (13), fluorescence probe synthesis, modelocked femtosecond lasers (14, 15), and computational threedimensional image reconstruction (16). Recently, threephoton excited fluorescence and its potential applications in imaging have also been reported for several fluorescent dyes (17)(18)(19)(20). Effective implementation of nonlinear laser microscopy, however, requires k...

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Section: Consequences Of the Photophysics Of Multiphoton Excitationmentioning

confidence: 94%

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Zipfel

Shear

et al. 1996

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

1,114

801

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“…[272,273] For example, artificially enhanced effective TPA cross sections were reported with nanoor picosecond excitations. [274] Consequently, a pulse duration in the nanosecond range (which is comparable to typical excited-state lifetimes) is too long to provide reliable measurements of TPA properties. It has been demonstrated and commonly accepted that only pulses in the femtosecond regime assure accurate σ 2 estimates.…”

Section: Techniques: Concepts Assets and Drawbacksmentioning

confidence: 99%

Enhanced Two‐Photon Absorption of Organic Chromophores: Theoretical and Experimental Assessments

et al. 2008

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“…[35,36,74,75] Depopulation processes of each electronic excited state are characterized by the rate constants k f , k IC , and k ISC for de-excitation of S 1 by fluorescence, internal conversion, and intersystem crossing to T 1 , respectively, leading to an overall fluorescence lifetime of t 0 = 1/k 0 = 1/(k f +k IC +k ISC ). k T , k Sn1 , and k Tn1 denote the de-excitation rate constants of T 1 , S n , and T n , respectively.…”

Section: Molecular Photokinetic Model Of Photobleachingmentioning

confidence: 99%

Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

2005

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Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

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Fluorescence of coumarins and xanthenes after two-photon absorption with a pulsed titanium–sapphire laser

Cited by 190 publications

References 21 publications

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

Enhanced Two‐Photon Absorption of Organic Chromophores: Theoretical and Experimental Assessments

Molecular Photobleaching Kinetics of Rhodamine 6G by One‐ and Two‐Photon Induced Confocal Fluorescence Microscopy

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