Multiphoton-induced Fluorescence Research Articles

Ultrasensitive detection of a responsive fluorescent thymidine analogue in DNA via pulse-shaped two-photon excitation.

Fluorescent base analogues (FBAs) are versatile nucleic acid labels that can replace a native nucleobase, while maintaining base pairing and secondary structure. Following the recent demonstration that free FBAs can be detected at the single-molecule level, the next goal is to achieve this level of detection sensitivity in oligonucleotides. Due to the short-wavelength absorption of most FBAs, multiphoton microscopy has emerged as a promising approach to single-molecule detection. We report the multiphoton-induced fluorescence of 5-(5-(4-methoxyphenyl)thiophen-2-yl)-6-aza-uridine (MeOthaU), a polarity-sensitive fluorescent thymidine analogue, as a nucleoside, and in two single-stranded deoxyribo-oligonucleotides, with and without their complementary strands. Ensemble steady-state and time-resolved measurements in dioxane, following one-photon and two-photon excitation, reveals both strongly and weakly emissive species, assigned as rotamers, while in Tris buffer there are additional non-emissive states, which are attributed to tautomeric forms populated in aqueous environments. The two-photon (2P) brightness for MeOthaU is highest as the free nucleoside in dioxane (10 GM) and lowest as the free nucleoside in Tris buffer (0.05 GM). The species-averaged 2P brightness values in DNA are higher for the single strands (0.66 and 0.82 GM for sequence context AXA and AXT, respectively, where X is MeOthaU) than in the duplex (0.31 and 0.25 GM for AXA and AXT, respectively). Using 2P microscopy with pulse-shaped broadband excitation, we were able to detect single- and double-stranded oligos with a molecular brightness of 0.8-0.9 kHz per molecule. This allowed the detection of as few as 7 DNA molecules in the focus, making it the brightest responsive FBA in an oligonucleotide reported to date.

Influence of the Irregularity of the Molecular Structure on the Chiral Expression of Poly(fluorene)s

In this report, the influence of the irregularity of the molecular structure on the chiral expression of 9,9-dialkylpoly(fluorene)s is investigated. For this purpose, different 9,9-dialkylpoly(fluorene)s─one homo-9,9-dialkylpoly(fluorene) and five alternating 9,9-dialkylpoly(fluorene)s─were synthesized according to an A2+B2 polymerization. The side chains of the 9,9-dialkylpoly(fluorene)s were chosen in such a way that a systematical increase in irregularity in the molecular structure of the 9,9-dialkylpoly(fluorene)s was obtained. As the organization of 9,9-dialkylpoly(fluorene)s is governed by an interplay between inter- and intramolecular interactions, we hypothesize that with the increase in molecular irregularity, the organization is more governed by intramolecular interactions and that the intermolecular interactions are suppressed. UV–vis, circular dichroism, and fluorescence measurements confirm this hypothesis. Furthermore, the actual process toward the chiral expression was monitored using a unique setup comprising in situ real-time transmission, optical rotation, static light scattering, third-harmonic scattering, and multiphoton-induced fluorescence experiments, which reveals that different pathways are followed in very regular and very irregular samples.

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAthaU). We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAthaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.

Multiphoton-induced fluorescence spectrum of sulfur dioxide

The laser-induced fluorescence spectrum of sulfur dioxide (SO2) in the range of 200–460 nm has been obtained with the dyer laser (579 nm) pulsed by an Nd:YAG laser as the excitation source. The transition process, both X1A1→C1B2, excited by three photons and X1A1→A1A2, excited by two photons, has been evaluated. The relaxation kinetics of the excited molecules in the states of C1B2 and A1A2 have been analyzed on the basis of the excitation spectrum in the range of 560–580 nm. The spectral sequence rules are assigned, and it can be calculated that the symmetric stretching vibration and the bending vibration frequencies of the C1B2 state are ω1=942.9 cm−1, ω2=369.8 cm−1, and the bending vibration frequencies of the A1A2 state are ω2=493.4 cm−1. The analysis of the time-resolved spectrum proved that the relaxation process, C1B2→A1A2→X1A1, and the phosphorescent process, a3B1→X1A1, exist simultaneously.

Observation of Multiphoton‐Induced Fluorescence from Graphene Oxide Nanoparticles and Applications in In Vivo Functional Bioimaging

Lightening organelles: A femtosecond laser can excite multiphoton-induced luminescence of graphene oxide nanoparticles. The flow, distributions, and clearance of intravenously injected GO-PEG nanoparticles in the blood vessel of mice could be observed clearly by two-photon imaging. The 3D distribution of microinjected GO-PEG nanoparticles in a mice brain could also be reconstructed with two-photon microscopy.

Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

Three-photon absorption enhancement in symmetrical charge transfer pull–pull fluorene derivatives

We report the multiphoton-induced upconversion fluorescence of 2,7-bisbenzothiazole-9,9-didecylfluorene (Acceptor-π-Acceptor) in hexane solution, pumping at wavelengths from 300 to 1200 nm, and its three-photon absorption cross-section at 1095 nm ( σ 3 ′ = 355 × 10 - 78 cm 6 s 2 photon - 2 for the transition S 0 → S 1). The multiphoton-induced fluorescence emission and the absorption measurements of 2,7-bisbenzothiazole-9,9-didecylfluorene in hexane were performed with a tunable OPG pumped by a Nd-YAG picosecond laser. It is demonstrated that the strong symmetrical electron-transfer from the electron rich fluorene core to the two electron-withdrawing benzothiazole end groups enhances σ 3 ′ . An increase of approximately four and nine fold, with respect to its Donor-π-Donor and Acceptor-π-Donor counterparts, respectively, was observed.

High Throughput Screening with Multiphoton Excitation.

Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two-photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and of calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.

Synthesis and Nonlinear Optical, Photophysical, and Electrochemical Properties of Subphthalocyanines

Novel boron(III) subphthalocyanines (SubPcs) soluble in organic solvents containing a variety of donor and acceptor substituent groups have been synthesized by boron trihalide-induced cyclotrimerization of adequately substituted derivatives of phthalonitrile in 1-chloronaphthalene. The choice of the substituents on the 1,2-dicyanobenzene derivatives has been made taking into account the high reactivity of the Lewis acid BCl3 toward many functional groups. Considering this limitation, we set out to synthesize phthalodinitriles equipped with iodo, nitro, alkyl- or arylthio, alkyl- or arylsulfonyl groups that are sufficiently stable under the required reaction conditions and also provide an easily accessible set of acceptor/donor substituents. The quadratic and cubic hyperpolarizabilities of these compounds as well as their linear optical and electrochemical properties have been measured by several techniques, including EFISH (at two wavelengths), HRS, and THG, steady-state and time-resolved absorption and fluorescence, laser-induced optoacoustic calorimetry, time-resolved near-infrared emission spectroscopy, and cyclic voltammetry. βHRS has been measured at 1.46 μm, where the contamination from the multiphoton-induced fluorescence can be ruled out. βHRS reachs high values that markedly depend on substitution. It shows a clear enhancement with the acceptor character of the substituents, the highest values being obtained for the compounds bearing the strongest acceptor groups. They are comparable or even superior to many efficient second-order compounds. A main outcome of these results is that an adequate choice of the substituents offers a promising route for optimization of the quadratic response of the SubPcs. This kind of compounds is less prone to aggregation than their expanded analogues, the phthalocyanines, fluoresces with quantum yields ca. 0.25, lower than those typical for phthalocyanines, and has larger triplet quantum yields. The triplet-state lifetime is in the 100-μs time range, long enough for efficient oxygen quenching. Indeed, subphthalocyanines sensitize singlet molecular oxygen, O2(1Δg), with quantum yields ranging from 0.23 to 0.75. The ground-state oxidation potentials are similar to those of phthalocyanines, while the reduction potentials are clearly more negative; i.e., they are more difficult to reduce. In contrast, electronically excited subphthalocyanines are more easily oxidized than the corresponding phthalocyanines by ca. 500 mV which results in lower photostability, especially in polar solvents.

Time-resolved nonlinear fluorescence spectroscopy using femtosecond multiphoton excitation and single-photon timing detection

We have developed a time-correlated single-photon timing nonlinear fluorometer for recording the fluorescence decay times and rotational correlation times of molecular probes using 120 fs regeneratively amplified Ti:Sapphire laser excitation via simultaneous non-resonant absorption of two or more near infrared photons. A microchannel plate photomultiplier giving 70 ps impulse response is used for detection. Studies on 1,6-diphenylhexatriene, rhodamine 6G and p-terphenyl in propylene glycol demonstrate two- and three-photon induced fluorescence characteristics. The radiative properties for one- and multiphoton excitation were found to be identical. However, the time-zero anisotropy observed for multiphoton excitation was larger than for one-photon excitation, indicating an increased degree of orientation of excited molecules after multiphoton absorption. The results reveal the potential of multiphoton-induced fluorescence anisotropy in the study of the structure and dynamics of microheterogeneous systems (i.e. biomembranes, porous matrices etc) by selecting the excitation wavelength and class of probe molecule.

Single-shot UV autocorrelator that uses a two-photon-induced photoacoustic signal in water

A single-shot autocorrelation of an ultrashort UV pulse is measured by detecting a two-photon-induced photoacoustic signal in water. A supersonic wave representing the autocorrelation function is detected by a polyvinylidene fluoride piezoelectric film with a spatial resolution of less than 60 microm. The detection sensitivity is higher than that of a multiphoton-induced fluorescence method by approximately 2 orders of magnitude.

Exact results for the emission from one and two atoms in an ideal cavity at multiphoton resonance

The authors examine the emission from the systems of one or two two-level atoms in an ideal cavity with one mode at multiphoton resonance. Exact results for the two-time dipole correlation function and the time-dependent spectra of multiphoton-induced fluorescence are presented.