One-photon Excited Fluorescence Research Articles

Small molecule probes with strong one and two-photon excited fluorescence for bioimaging

Two-photon excited fluorescence (TPEF) is widely sought after in bioimaging applications due to its exceptional deep tissue penetration and excellent high spatiotemporal resolution. In this paper, benzothiazole and benzimidazole are linked to N, N-diphenylaniline, respectively, through pyridine vinyl. Two new D-π-A (electron donor-π conjugated bridges-electron acceptor) probes (P1, P2) were designed and synthesized. The molecular structures were completely characterized through FT-IR, 1H NMR, 13C NMR, ESI-MS, and X-ray single crystal diffraction. The probes have good one-photon excited fluorescence and two-photon excited fluorescence. Experimental test results indicate that they can be fluorescence probes for biological imaging of HePG2 and juvenile zebrafish. In addition, the fluorescent probes also have the ability to target and locate cancer cell mitochondria.

Variational calculus approach to Zernike polynomials with application to FCS

Zernike polynomials are a sequence of orthogonal polynomials that play a crucial role in optics and, in particular, modeling microscopy systems. Introduced by Frits Zernike in 1934, they are particularly useful in expressing wavefront aberrations and, thus, imperfections of imaging systems. However, their origin and properties are rarely discussed and proven. Here, we present a novel approach to Zernike polynomials using variational calculus and apply them to describe aberrations in fluorescence microscopy. In particular, we model the impact of various optical aberrations on the performance of one-photon and two-photon excitation fluorescence microscopy.

Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

Development of hydrogel-based standards and phantoms for non-linear imaging at depth.

Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques. We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth. Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers. These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and , respectively. Once fabricated, the phantoms were found to be stable for many months. The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.

Optical properties, bioimaging and theoretical calculation of a Zn(II) complex based on triphenylamine derivative

A luminescent material with various optical properties based on a triphenylamine Zn(II) complex is described. The ultraviolet–visible absorption, one-photon excited fluorescence (OPEF) and two-photon excited fluorescence (TPEF) of the complex indicate that the material has good OPEF and TPEF properties. And the results of one- and two-photon HepG2 cells imaging experiments show the potential of the complex in fluorescence microscopy bioimaging. The experimental Stokes shift and the FWHM (full-width at half-maximum) in different solvents were correlated with the rMPI polarity of the solvent, and the perfect Boltzmann curves were obtained, where the Boltzmann correlation between Stokes shift and solvent polarity is reported for the second time. But the Boltzmann correlation between FWHM and solvent polarity is reported for the first time. In addition, the computational results indicate that, the covalent bond within the salt ZnBr2 is strengthened by the coordination, and the newly formed coordination bond Zn-N is stronger than the original covalent bond Zn-Br.

In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors.

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is to extend the excitation and emission wavelengths to the second near-infrared window (NIR-II) at >1,000 nm, also called the short-wavelength infrared window. Here we show biocompatible core-shell lead sulfide/cadmium sulfide quantum dots emitting at ~1,880 nm and superconducting nanowire single-photon detectors for single-photon detection up to 2,000 nm, enabling a one-photon excitation fluorescence imaging window in the 1,700-2,000 nm (NIR-IIc) range with 1,650 nm excitation-the longest one-photon excitation and emission for in vivo mouse imaging so far. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~1,100 μm through an intact mouse head, and enabled non-invasive cellular-resolution imaging in the inguinal lymph nodes of mice without any surgery. We achieve in vivo molecular imaging of high endothelial venules with diameters as small as ~6.6 μm, as well as CD169 + macrophages and CD3 + T cells in the lymph nodes, opening the possibility of non-invasive intravital imaging of immune trafficking in lymph nodes at the single-cell/vessel-level longitudinally.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.

Synthesis and optical properties of linear and branched styrylpyridinium dyes in different environments

A common motif in the design of organic dye compounds is that of an electron donor and an acceptor moiety linked through a conjugated bridge, but more complicated structures can also be built using such motifs as building blocks. Following these principles, we synthesized two styrylpyridinium dyes, a linear one and a branched molecule composed of three units of the mono moiety connected by benzyl group and investigated their photophysical properties in different environments: their linear absorption, one-photon excited fluorescence and two-photon absorption properties. The compound with the symmetrically substituted phenyl core shows a redshift of the absorption and fluorescence bands. Spectrally resolved two-photon absorption cross section measurements carried out by femtosecond Z-scan technique show that the value for the trimer is over twelve times larger than that for the monomer, and not just three times as expected from the molar mass increase. Large two-photon absorption (2PA) values were measured in the near infrared (NIR) region. The prototropic study indicates that the protonation of the amino group blocks a lone pair on the nitrogen atom, making the photoinduced electron transfer process from the amine group to the pyridinium ring ineffective. This results in the reduction of the long-wavelength absorption with a simultaneous increase of the band in the short-wavelength region of the spectrum and a decrease in the fluorescence intensity. The behavior of dyes in the presence of β-cyclodextrin was also studied based on NMR, UV–Vis, and fluorescence spectroscopy. The performed experiments indicated the formation of 1:1 inclusion complex in aqueous solution.

Two-photon flow cytometry with laser scanning two-dimensional airy beams

Flow cytometry is an essential technique in biomedical discovery for cell counting, cell sorting, and biomarker detection. In vivo flow cytometers based on one-photon or two-photon excited fluorescence have been developed for over a decade. One drawback of the laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is limited to the focal spot due to the short Rayleigh range of focused Gaussian beams. Hence, the sampling volume is much smaller than in one-photon flow cytometers, making it challenging to count or detect rare circulating cells in vivo. Non-diffracting light waves like Bessel beams and Airy beams have narrow intensity profiles with an effective spot size as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making them an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Airy beams rather than Gaussian beams is side lobes Airy beams have, which contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using 2D Airy beams to form a light-sheet that intersects the blood vessel a microfluidic channel, which was used to model a blood vessel. The setup can successfully detect and count flowing fluorescent microspheres in a microchannel.

Zn(II) complexes based on functional organic ligands: Two-photon activity, theoretical calculation and bioimaging

An organic ligand (3-[4-(diphenylamino)styryl] pyridine, L) was synthesized. By complexation of L with ZnBr2 and Zn(NCS)2 respectively in methanol and dichloromethane, two complexes ZnBr2L2 (dye1) and Zn(NCS)2L2 (dye2) were prepared. The two dyes exhibit good one- and two-photon fluorescence performance with high fluorescence quantum yields and large two-photon absorption cross section values. The mechanism of one-photon absorption and one-photon excited fluorescence based on computational results was obtained by TDDFT/CAM-B3LYP method. The results of living cell imaging experiments exhibit their compatibility in cytoplasm of HepG2 cells and show their potential in fluorescence microscopy bioimaging

Rational design, facile synthesis, and linear/nonlinear optical properties of novel two-photon absorption stilbene derivatives with different configurations

Four novel two-photon absorption compounds (1MOBI, 2MOHA, 3BrBI, and 4BrHA), which have assembled stilbene π-bridge, different electron acceptors and donors, were synthesized conveniently via the one-step Horner-Wadsworth-Emmons reaction. Their configurations are A-π-D-π-A, D′-π-D-π-D′, A′-π-A-π-A′, and D-π-A-π-D, respectively. Their linear and nonlinear optical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. The results demonstrate that the compounds with dihexylamino donors exhibit good optical properties. 2MOHA and 4BrHA exhibit large two-photon absorption cross-sections (2183 and 1737 GM) in DMF, which are larger than those of many known stilbene derivatives. The relationships between the structures and the optical properties were studied aided with the time-dependent density functional theory calculations.

Crystal structure and optical property of a Cadmium(II) complex based on triphenylamine derivative—Theoretical and experimental investigation

A triphenylamine derivative (E)-N,N-diphenyl-4-(2-(pyridin-2-yl)vinyl)benzenamine (L) was synthesized, and the corresponding cadmium complex CdI2L2 crystal was obtained and solved by single crystal X-ray diffraction analysis. The experimental resutls of one photon absorption (OPA) and one photon excited fluorescence (OPEF) indicate that coordination of CdI2 only has slight effect on the spectrum peak position relative to the free ligand. Besides the Stokes shift, the full width half maximum of OPEF and the variation of the “peak span between solvents” from OPA to OPEF can also indicate the electron transition direction of the excitation process. The laser experiments show that both the ligand and complex have considerable two photon excited fluorescence (TPEF) emission. The experimental Stokes shift is correlated with the computational solvent polarity rMPI, and the results illustrate that the curve of “experimental Stokes shift vs. solvent rMPI” is exactly the Boltzmann S-curve. The computational results also indicate that the static interaction between the CdI2L2 molecules in crystal has strong effect on OPA properties. Considering the weak coordination bond strength of the complex CdI2L2, the S1→S0′ fluorescence emission may occur on both ligands, although the S0→S1′ excitation only occurs on the right ligand.

Novel yellow- to red-emitting fluorophores: Facile synthesis, aggregation-induced emission, two-photon absorption properties, and application in living cell imaging

Four novel yellow-to red-emitting fluorophores with a 1-(2-hydroxyethyl) pyridinium core and different electron-donating terminal moieties (N-methylcarbazole, N,N-dibutylbenzenamine, N-methylpyrrole, and 2-methylfuran) were designed and facilely synthesized via the one-step Knoevenagel condensation. All these compounds (HPs) were characterized by 1H NMR, 13C NMR, and HRMS. Their photophysical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. And the density functional theory calculations were conducted to analyze the electronic structures of HPs. The two-photon absorption cross-sections (δ) values of HPs measured by the Z-scan technique were determined to be as large as 1354 (1HP-CZ), 4462 (2HP-BA), 836 (3HP-PR), and 2944 GM (4HP-FU) in DMSO. The two-photon action cross-sections (Φ × δ) values of 1HP-CZ and 2HP-BA in H2O measured by the two-photon induced fluorescence method were about 50 GM. 1HP-CZ, 3HP-PR, and 4HP-FU also exhibited good water solubility. Meanwhile, it was found that 2HP-BA exhibited notable aggregation-induced emission characteristic in DMSO/H2O mixture. The aggregate particle size distribution of 2HP-BA was measured by the dynamic light scattering method, and the aggregation characteristic of 2HP-BA was observed by the transmission electron microscopy. Besides, 2HP-BA also exhibited red emission. Then, HPs with relatively low cytotoxicity were used for one- and two-photon excited fluorescence imaging in living HepG2 cells. The results indicate that HPs are potential candidates in the bioimaging field due to their photophysical properties and biocompatibility.

Two photon excited fluorescence and hyper Rayleigh scattering of Protoporphyrin IX

The two-photon excited fluorescence (TPEF) cross-section of Protoporphyrin IX (PpIX) in solution is determined at different excitation wavelengths. Maxima observed at 620 and 634 nm are associated with two different forms of PpIX already observed in one-photon excited fluorescence studies. In order to further provide insights into these two forms, first hyperpolarizabilities are also reported for these two forms with corresponding depolarization ratio further demonstrating the existence of specific symmetry differences. The static and the frequency dependent first hyperpolarizability were also calculated using Gaussian 09 and compared to the experimental data. This work shows that PpIX can exhibit two forms that might be targeted in applications like oncology clinical agents.

Aggregation-Induced Plasmon Coupling-Enhanced One- and Two-Photon Excitation Fluorescence by Silver Nanoparticles.

Plasmon coupling-induced intense local electrical field in the gap of closely packed metal nanoparticles (NPs) has been known capable of significantly enhancing optical properties of chromophores. Here, we have investigated aggregation-induced plasmon coupling-enhanced one-photon excitation (1PE) and two-photon excitation (2PE) fluorescence of dyes using Ag NPs of three different sizes (20, 36, and 48 nm). The fluorescence of a model dye, Rhodamine B isothiocyanate (RiTC), was prequenched by attaching to Ag NPs and subsequently enhanced upon forming aggregates of Ag NPs. It was found that aggregates of larger sized Ag NPs gave larger 1PE and 2PE fluorescence enhancement on the basis of free dyes, while aggregates of smaller counterparts displayed larger enhancement on the basis of the corresponding prequenched ones. 1PE and 2PE fluorescence were enhanced by 2.5- and 10.2-fold by aggregated 48 nm Ag NPs compared to free dyes and by 8.0- and 22.5-fold by aggregated 20 nm Ag NPs compared to the quenched ones, respectively. This scheme achieved fluorescence enhancement significantly beyond the level of fluorescence recovery, much larger than conventional turn-on fluorescence probes, which is attractive for developing sensitive fluorescence turn-on-based detection with reduced background.

Lysosome-targetable carbon dots for highly efficient photothermal/photodynamic synergistic cancer therapy and photoacoustic/two-photon excited fluorescence imaging

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT) holds great potential for efficient cancer therapy by inducing reactive oxygen species (ROS) or heat into tumor. Nevertheless, PDT or PTT suffers from some limitations, e.g., PTT requires long-time and high-power laser irradiation to generate enough heat, while the hypoxia microenvironment of tumor and the limit diffuse distance of ROS hamper the efficacy of oxygen-dependent PDT. Here we reported the carbon dots (CDs) which could simultaneously generate singlet oxygen (1O2), hydroxyl radical (OH), and heat under a 635 nm laser irradiation, with a 1O2 generation quantum yield of 5.7% and photothermal conversion efficiency of 73.5% (the highest thus far for CDs). Significantly, the CDs can selectively accumulate in lysosome, which is an ideal organelle for phototherapy because of its key role in sustaining cellular activity and stability. Moreover, the CDs present one-photon excited (OPE) and two-photon excited (TPE) fluorescence, and excellent photoacoustic (PA) imaging capability. Combining the good biocompatibility, the as-prepared CDs was served as multi-functional phototheranostic agent for synergistic PA/fluorescence imaging, and PDT/PTT.

Synthesis, electronic structure, linear and nonlinear photophysical properties of novel asymmetric branched compounds

A series of novel asymmetric branched compounds that utilize a 1,3,5-triazine core and feature D-π-A-(π-D′-π-A′)0-2 configurations (D = donor, A = acceptor, π = conjugated bridge) were designed, successfully synthesized, and fully characterized by 1H NMR, 13C NMR, FT-IR, and HRMS. Their photophysical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and frequency up-converted fluorescence, were systematically investigated in different solvents. With a rise in the polarity of solvents, the peak positions of the one-photon excited fluorescence are red-shifted and the Stokes shifts increase, while the linear absorption wavelengths change slightly. In addition, the target compounds except CZ show the positive solvatokinetic effect. With a rise in the number of branches, the red shifts of the absorption and emission maxima, the hyperchromicity of the molar absorption coefficients, and the decrease of the Stokes shifts are observed. The peripheral electron donors (carbazole, phenothiazine) and acceptors (pyridine, benzimidazole) also exert an important influence on the photophysical properties. Under excitation of 690–930 nm fs laser pulses, all the target compounds emit frequency up-converted fluorescence with the maximal peaks at 471–575 nm, and the two-photon absorption cross-sections in THF are 132 (PTZ), 182 (CZ), 453 (CZ-Py1), 844 (CZ-Py2), 1244 (CZ-BI1), and 2072 (CZ-BI2) GM, respectively. Their two-photon response is found to be nearly additive with respect to the number of branches. The time-dependent density functional theory calculations were conducted to gain an insight into their electronic structures and to better understand the structure-photophysical property relationships. The results clearly indicate the importance of appropriate structural units on the enhancement of two-photon absorption properties.

Isomeric Effect of Mercaptobenzoic Acids on the Synthesis, Stability, and Optical Properties of Au25(MBA)18 Nanoclusters.

We report a simple size focusing, two-step “bottom-up” protocol to prepare water-soluble Au25(MBA)18 nanoclusters, using the three isomers of mercaptobenzoic acids (p/m/o-MBA) as capping ligands and Me3NBH3 as a mild reducing agent. The relative stability of the gas-phase multiply deprotonated Au25(MBA)18 ions was investigated by collision-induced dissociation. This permitted us to evaluate the possible isomeric effect on the Au–S interfacial bond stress. We also investigated their optical properties. The absorption spectra of Au25(MBA)18 isomers were very similar and showed bands at 690, 470, and 430 nm. For all Au25(MBA)18 isomeric clusters, no measurable one-photon excited fluorescence under UV–vis light was found, in neither solid- nor solution-state. The two-photon excited emission spectra and first hyperpolarizabilities of the clusters were also determined. The results are discussed in terms of the possible isomeric effect on excitations within the metal core and the possibility of charge transfer excitations from the ligands to the metal nanocluster.

Two‐Photon‐Absorption Properties of Pyrene‐Based Dipolar D‐π‐A Fluorophores

AbstractTwo‐photon absorption (TPA) properties of pyrene‐based derivatives are rare as a result of the limited number of controllable synthetic methods to prepare them. However, these materials are in great demand given their potential practical application in photics and biological imaging. Herein, we present a set of pyrene‐based dipolar donor‐π‐acceptor (D‐π‐A) fluorophores with a wide‐range of color tuning and large TPA cross‐sections (up to 2200 GM at 780 nm) by regioselective substitution at the 1,3‐ and 6,8‐positions under the perspective of theoretical analysis. The linear and nonlinear optical properties of these compounds have been studied. The near‐identical emission wavelengths between the two‐photon‐excited fluorescence (TPEF) and one‐photon excited fluorescence (OPEF) indicated that they are generated from the same fluorescent excited state by either one‐ or two‐photon excitation. With the exception of the strong donor [N(CH3)2] appended fluorophore, the TPEF exhibited a large red‐shift compared with the OPEF spectra as a result of the complicated working mechanisms in operation, including intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT). All fluorophores exhibit high two‐photon cross‐sections (or two‐photon brightness, δΦ), especially for 1348 GM, which indicated that these materials can be used as colorants for probe and bioimaging applications.

Super-resolution fluorescence microscopy by stepwise optical saturation.

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.