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

Xu, Chris; Zipfel, Warren R.; Shear, Jason B.; Williams, Rebecca M.; Webb, Watt W.

doi:10.1073/pnas.93.20.10763

Cited by 1,114 publications

(835 citation statements)

References 38 publications

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“…For in vivo imaging of fluoresceinated molecules, the multiphoton illumination wavelength ( ex) was tuned to 900 nm (Xu et al, 1996;Zipfel et al, 2003a). Nonlinear emissions were collected in epifluorescence mode and separated from the excitation beam directly after the objective with a 670DCXXRU long-pass dichroic (Chroma Technology, Rockingham, VT).…”

Section: Materials and Methods In Vivo Multiphoton Microscopymentioning

confidence: 99%

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

et al. 2005

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Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. Key words: growth plate; vasculature; multiphoton microscopy; endochondral ossificationBone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. Chondrocytic differentiation in the growth plate begins with clonal expansion of stem cells, continues during cellular proliferation, and ends with cellular enlargement, followed by death and replacement by bone. The kinetics of chondrocytic activity in each stage and of the regulatory transitions between them determine the rate of bone elongation achieved. As in any cellular growth system, nutrients are required to sustain the process, and multiple endocrine and paracrine inputs regulate at each stage. For growth plate cartilage, the role of the extracellular matrix also must be considered, not only as a substantive contributor to elongation per se, but through its potential role as a communication link among chondrocytes of the growth plate, and between growth plate chondrocytes and cells of surrounding tissues such as the peri-

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Section: Materials and Methods In Vivo Multiphoton Microscopymentioning

confidence: 99%

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

et al. 2005

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“…Depending on the nature of this coupling, the ground state or the excited state may be either localized or delocalized, and specific optical properties may be either enhanced or suppressed. 44 Among the many NLO effects, two-photon absorption (TPA) has become very popular during the past decade owing to its wide-ranging applications such as two-photon laser scanning microscopy, [45][46][47] photodynamic therapy, 48 optical power limitation, 4,5 microfabrication, [49][50][51][52] or 3D optical data storage. [53][54][55][56] Depending on the applications, two-photon chromophores have to satisfy different kinds of requirements.…”

Section: Introductionmentioning

confidence: 99%

Effects of (Multi)branching of Dipolar Chromophores on Photophysical Properties and Two-Photon Absorption

et al. 2005

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To investigate the effect of branching on linear and nonlinear optical properties, a specific series of chromophores, epitome of (multi)branched dipoles, has been thoroughly explored by a combined theoretical and experimental approach. Excited-state structure calculations based on quantum-chemical techniques (timedependent density functional theory) as well as a Frenkel exciton model nicely complement experimental photoluminescence and one-and two-photon absorption findings and contribute to their interpretation. This allowed us to get a deep insight into the nature of fundamental excited-state dynamics and the nonlinear optical (NLO) response involved. Both experiment and theory reveal that a multidimensional intramolecular charge transfer takes place from the donating moiety to the periphery of the branched molecules upon excitation, while fluorescence stems from an excited state localized on one of the dipolar branches. Branching is also observed to lead to cooperative enhancement of two-photon absorption (TPA) while maintaining high fluorescence quantum yield, thanks to localization of the emitting state. The comparison between results obtained in the Frenkel exciton scheme and ab initio results suggests the coherent coupling between branches as one of the possible mechanisms for the observed enhancement. New strategies for the rational design of NLO molecular assemblies are thus inferred on the basis of the acquired insights.

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“…Multiphoton excitation of chromophores in biological imaging 1,2 and quantitative fluorescence measurements 3 reduces background luminescence and has the potential to increase sensitivity and selectivity. Multiphoton excitation is usually used to excite the same fluorescent species as normally obtained under conventional one photon ultraviolet or visible illumination.…”

Section: Introductionmentioning

confidence: 99%

Investigation of multiphoton-induced fluorescence from solutions of 5-hydroxytryptophan

Bisby

Arvanitidis

Botchway

et al. 2003

Photochem Photobiol Sci

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It is reported (Shear et al, Photochem.Photobiol. 65, 931 (1997) ) that multiphoton near infrared excitation of 5-hydroxytryptophan results in a transient product with green fluorescence. Visible fluorescence from multiphoton excitation enables detection of 5-hydroxytryptophan with extremely high sensitivity and also has potential applications in imaging of biological systems and investigation of protein dynamics. The characteristic fluorescence at 500 nm has now also been observed on one-photon photolysis of solutions at 308 nm, followed by excitation of fluorescence at 430 nm. Fluorescence was observed in aerated and deaerated solutions and in the presence of ascorbate. Enhancement of fluorescence was observed on addition of ethanol. Transient absorption experiments with 308 nm photolysis showed the formation of three transient species. In the presence of ascorbate the radical formed by photoionisation was removed, revealing a long-lived species (  > 1 ms) with a similar absorption spectrum, which is ascribed to the fluorescing species. Fluorescence induced by multiphoton excitation had a lifetime of 910 ± 10 ps and was also unaffected by ascorbate. In the presence of organic solvents there was an increase in fluorescence lifetime, but a decrease in overall fluorescence intensity. The fluorescence intensity and fluorescence lifetime both decreased in acidic solution (pH < 3). It is suggested that the fluorescence originates not from the 5-indoxyl radical, but from a transient product formed by intramolecular rearrangement.2

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

Cited by 1,114 publications

References 38 publications

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

Effects of (Multi)branching of Dipolar Chromophores on Photophysical Properties and Two-Photon Absorption

Investigation of multiphoton-induced fluorescence from solutions of 5-hydroxytryptophan

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