Two-photon Fluorescence Spectra Research Articles

Carbonized Polymer Dot Probe for Two-Photon Fluorescence Imaging of Lipid Droplets in Living Cells and Tissues.

As a dynamic and multifunctional organelle, lipid droplets (LDs) are essential in maintaining lipid balance and transducing biological signals. LD accumulation and catabolism are closely associated with energy metabolism and cell signaling. In order to easily trace LDs in living cells, a novel carbonized polymer dot (CPD)-based fluorescent nanoprobe is reported to serve the needs of LD-targeting imaging. This probe exhibits the advantages of excellent biocompatibility, simple preparation, good lipophilicity, and high compatibility with commercial dyes. Transient absorption spectroscopy was employed to discuss the luminescence mechanism of CPDs, and the results indicate that the excellent fluorescence property and the environment-responsive feature of our CPDs are derived from the intramolecular charge transfer (ICT) characteristics and the D-π-A structure that possibly formed in CPD. This nanoprobe is available for one-photon fluorescence (OPF) and two-photon fluorescence (TPF) imaging and is also practicable for staining LDs in living/fixed cells and lipids in tissue sections. The staining process is completed within several seconds, with no washing step. The intracellular LDs involving the intranuclear LDs (nLDs) can be selectively lit up. This probe is feasible for visualizing dynamic interactions among LDs, which suggests its great potential in revealing the secret of LD metabolism. The in situ TPF spectra were analyzed to determine surrounding microenvironment according to the polarity-responsive feature of our CPDs. This work expands the applications of CPDs in biological imaging, helps design new LD-selective fluorescent probes, and has implications for studying LD-related metabolism and diseases.

Unveiling the Molecular Dynamics in a Living Cell to the Subcellular Organelle Level Using Second-Harmonic Generation Spectroscopy and Microscopy.

Second-harmonic generation (SHG) microscopy has been proved to be a powerful method for investigating the structures of biomaterials. SHG spectra were also generally used to probe the adsorption and cross-membrane transport of molecules on lipid bilayers in situ and in real time. In this work, we applied SHG and two-photon fluorescence (TPF) spectra to investigate the dynamics of an amphiphilic ion with an SHG and TPF chromophore, D289 (4-(4-diethylaminostyry)-1-methyl-pyridinium iodide), on the surface of human chronic myelogenous leukemia (K562) cells and the subcellular structures inside the cells. The adsorption and cross-membrane transport of D289 into the cells and then into the organelles such as mitochondria were revealed. SHG images were also recorded and used to demonstrate their capability of probing molecular dynamics in organelles in K562 cells. This work demonstrated the first SHG investigation of the cross-membrane transport dynamics on the surface of subcellular organelles. It may also shed light on the differentiation of different types of subcellular structures in cells.

Modeling of Carbon Monoxide Two-Photon Laser-Induced Fluorescence (LIF) Spectra at High Temperature and Pressure.

In this study, quantitative model of two-photon excitation and fluorescence spectra of carbon monoxide based on up-to-date spectroscopic constants collected during an extensive literature survey was developed. This semi-classical model takes into account Hönl-London factors, quenching effects (collisional broadening and shift), ionization and stark effect (broadening and shift), whereas predissociation is neglected. It was specifically developed to first reproduce with a high confidence level the behavior of our experimental spectra obtained from laser-induced fluorescence (LIF) measurements, and then to allow us to extrapolate the fluorescence signal amplitude in other conditions than those used in these experiments. Synthetic two-photon excitation and fluorescence spectra of CO were calculated to predict the fluorescence signal at high pressures and temperatures, which are representative of gas turbine operating conditions. Comparison between experimental and calculated spectra is presented. Influence of temperature on both excitation and fluorescence spectra shapes and amplitudes is well reproduced by the simulated ones. It is then possible to estimate flame temperature from the comparison between experimental and calculated shapes of numerical excitation spectra. Influence of pressure on both excitation and fluorescence spectra was also investigated. Results show that for temperature below 600 K and pressure above 0.1 MPa, the usual Voigt profile is not suitable to reproduce the shape of the excitation spectrum. We found that the Lindholm profile is well suited to reproduce the pressure-dependence of the spectrum in the range 0.1 to 0.5 MPa at 300 K, and 0.1 to 0.7 MPa at 860 K. Beyond 0.7 MPa, in this temperature range, it is shown that the Lindholm profile does no longer match the spectral profiles, in particularly the red wing. Further analyses taking into account the line mixing phenomenon at higher pressure are thus discussed.

Combination of [12]aneN3 and Triphenylamine-Benzylideneimidazolone as Nonviral Gene Vectors with Two-Photon and AIE Properties.

Three nonviral gene vectors, TPA-BI-A/B/C, have been designed and synthesized by the combination of one or two hydrophilic [12]aneN3 moieties and two-photon fluorescent triphenylamine-benzylideneimidazolone (TPA-BI) units through different ester linkage. Spectroscopic characterization demonstrated that TPA-BI-A/B/C had strong aggregation-induced emissions (AIE), large Stokes shifts (230, 284, and 263 nm), and large two-photon absorption cross sections (δ2PA) (67, 592, and 80 GM). Gel electrophoresis indicated that the three compounds completely condensed DNA at 15 μM in the presence of DOPE, and showed the lipase- and pH-triggered reversible release of DNA and the fluorescent recognition of the different lengths of ssDNA and dsDNA. The optimal TPA-BI-C/DOPE-mediated luciferase and GFP activity was 146% and 290% higher than those of Lipo2000. The transfection process of DNA could be traced clearly through one- and two-photon fluorescence spectra, and displayed in a 3D-video. TPA-BI-C/DOPE successfully transfected the GFP gene into zebrafish, which was superior to Lipo2000 (192%). In conclusion, TPA-BI-C/DOPE is the first nonviral gene vector with the abilities of pH/lipase enzyme responsiveness, one/two-photon fluorescent tracking of intracellular delivery of DNA, and successful transfection in vivo and in vitro, even better than Lipo2000.

Synthesis, photophysical properties, and DNA-binding of novel A-π-D-π-A' two-photon absorption chromophores

Three novel two-photon absorption chromophores (1BI, 2TAZ, and 3PyB), which have trans-1,4-divinylbenzene π-bridge, methoxy donors, and different acceptors, were synthesized. Their structures were elucidated by means of hydrogen-1 nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, Fourier transform infrared spectroscopy, and high-resolution mass spectrometry. Their photophysical properties, including linear absorption, single-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. The structure-property relationships were studied aided with the density functional theory calculations. In addition, the absorption, single- and two-photon fluorescence spectra of 3PyB-ctDNA were examined to evaluate whether 3PyB could be used as a DNA stain. The possible interaction mechanism of 3PyB-ctDNA was also studied by the NaCl titration and the viscosity experiment. The results indicate that 3PyB may serve as a potential DNA fluorescent probe.

A highly selective and sensitive fluorescence probe with A-π-D-π-A structure for detection of Ag+

A novel triphenylamine derivative, 4,4′-bis-([1,10]phenanthrolin-2-yl) imidazo -triphenylamine (IPTPA) with an A-π-D-π-A structure, has been synthesized and characterized. The UV absorption, single and two-photon fluorescence spectra showed that IPTPA was highly sensitive to Ag+ with a lower detection limit of 3.97 nmoL/L and a moderate binding constant of 2.16 × 105/M at room temperature. The two-photon active cross-section of IPTPA was calculated with a value of 94.68 GM at 800 nm in the tris-HCl buffer solution. The binding mechanism between IPTPA and Ag+ were explored by the Job plot, ESI-MS and 1H NMR. The A-π-D-π-A structure of IPTPA was substituted by the A-π-D-π-D structure of IPTPA-Ag to improve the fluorescence. Furthermore, the fluorescence microscopy images indicated that IPTPA could detect Ag+ in living cells.

Fano interference in two-photon transport

We present a general input-output formalism for the few-photon transport in multiple waveguide channels coupled to a local cavity. Using this formalism, we study the effect of Fano interference in two-photon quantum transport. We show that the physics of Fano interference can manifest as an asymmetric spectral line shape in the frequency dependence of the two-photon correlation function. The two-photon fluorescence spectrum, on the other hand, does not exhibit the physics of Fano interference.

New ESIPT-Inspired Photostabilizers of Two-Photon Absorption Coumarin–Benzotriazole Dyads: From Experiments to Molecular Modeling

In this work, a variety of new coumarin–benzotriazole dyads are synthesized to study the photostabilities under the irradiation of broad UV light as well as near-infrared (near-IR) laser respectively inspired by efficient excited state intramolecular proton transfer (ESIPT). The corresponding esterified dyads and the coumarin dyes are prepared as the references. The intramolecular hydrogen bond in the target dyads is confirmed by 1H NMR spectra and UV/visible absorption spectra. The internal proton transfer in the excited states of the target dyads under single photon excitation and near-IR laser irradiation is demonstrated by the measurements of one-photon fluorescence spectra and two-photon upconverted fluorescence spectra, respectively. The photostabilities of the target dyads under broad UV light irradiation and near-IR laser irradiation in various organic solvents with saturated air, oxygen, or argon are studied. The theoretical evidence of ESIPT of the target dyads is provided by the molecular geome...

The fluorescence and dynamics properties in phenoxy-phthalocyanines liquid

We investigated the one/two-photon fluorescence and excited state dynamics properties of two synthesized phenoxy-phthalocyanines (Pc1 and Pc2) using mild reaction coordination method. The results show that the fast decay component in the time-resolved fluorescence technique dynamics comes from the intramolecular vibrational relaxation, the slower ones from the internal conversion. Furthermore, in comparison with one-photon fluorescence spectra, the red shift of two-photon fluorescence spectra can be explained by the reabsorption effect of molecules. The samples are expected to be a potential candidate for optical applications and photodynamic therapy.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

BackgroundDelignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison.ResultsFluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed.ConclusionsOur results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.

Synthesis, crystal structure, optical properties, DNA-binding and cell imaging of an organic chromophore

An organic chromophore (4-[ N, N- bis(2′-chloroethyl)anilino] ethenyl N-methyl pyridinium- N- methyl iodide) L was synthesized and fully characterized by elemental analysis, IR, 1HNMR spectra and single crystal X-ray diffraction analysis. One and two-photon fluorescence spectra for it were studied. Interestingly, L exhibited obvious two-photon absorption in the range from 700–850 nm in DMSO solution. The interactions between the chromophore and calf thymus DNA were investigated by absorption, fluorescence spectra and viscosity experiments. The results showed that the chromophore could interact with DNA in the mode of intercalation and be effectively used as fluorescent staining dye for cell imaging applications. Furthermore, the cell viability data for MCF-7 (Human breast carcinoma) cells indicated that the low-micromolar concentrations of the L are essentially non-toxic and can be applied in brighter two-photon fluorescent (TPF) bioimaging.

Combined influences of chromatic aberration and scattering in depth-resolved two-photon fluorescence endospectroscopy

The influence of chromatic aberration of an objective lens in two-photon fluorescence (TPF) endospectroscopy of scattering media has been systematically investigated through both experiments and numerical simulations. Experiments were carried out on a uniform 3D scattering gelatin phantom embedded with TiO2 granules (to mimic tissue scattering) and fluorescein-tagged polystyrene beads. It was found that fluorescence spectral intensity and lineshape varied as a function of depth when measured with a gradient-index (GRIN) lens which has severe chromatic aberration. The spectral distortion caused by the chromatic aberration became diminishing as the imaging depth increased. Ray tracing analysis and Monte Carlo simulations were carried out to study the interplay of chromatic aberration and scattering in the depth-resolved TPF spectra. The simulation results suggest that the collected fluorescence signals from deeper layers included more out-of-focus photons that experienced a few or multiple scatterings, which diminish the influence of chromatic aberration on the measured TPF spectra. The simulated collection efficiencies of TPF at different wavelengths and depths can be used to properly recover the true depth-resolved TPF spectra of a relatively uniform scattering medium.

Two-photon fluorescence imaging of DNA in living plant turbid tissue with carbazole dicationic salt

Three carbazole dicationic salts, namely 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), 9-ethyl-3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (9E-BMVC) and 9-ethyl-3,6-bis(1-hydroxyethyl-4-vinylpyridinium)carbazole diiodide (9E-BHVC), were synthesized successfully. Their photophysical properties were evaluated by absorption, one- and two-photon fluorescence spectra, and their higher fluorescence intensity and larger two-photon excited fluorescence action cross-sections (Φ×δ) in the presence of DNA than those in the absence of DNA give them good DNA two-photon light-switch properties. Furthermore, their ability to image nuclei in living plant cells and turbid tissues by using two-photon excited fluorescence was carefully studied, and the experimental results indicate that these dicationic salts can exclusively label nuclei in intact living plant cells and tissues. In particular, 9E-BHVC exhibits optimized DNA labeling performance. Very importantly, compared to DAPI, 9E-BHVC can be used to carry out deeper observation using the same incident power, or can be used to obtain usable fluorescent images by using a lower incident power.

Ultrafast nonlinear optical properties of dye-doped PMMA discs irradiated by 40 fs laser pulses

The two-photon absorption (TPA) characteristics of PMMA discs doped with three different dyes were studied using an fs-pulsed Ti-Sapphire laser as the pump source, and employing the open-aperture Z-scan technique. TPA cross-sections obtained for PMMA doped with the dyes PM597, DCM and rhodamine 6G–rhodamine B (co-doped) were found to be equal to 24.7, 33.3 and 32.3 GM, respectively (1 GM=10 −50 cm 4 s phot −1 mol −1). Furthermore, two-photon fluorescence was measured for the samples containing DCM and rhodamine 6G–rhodamine B (co-doped). Compared to the one-photon fluorescence spectrum, the peaks in the two-photon fluorescence spectrum were red shifted and the extent of red shift increased with increasing doping concentration. We have also observed that the red shift in the two-photon fluorescence peak of the samples in the solid form is much larger than that in the solution state. This phenomenon could be explained by a twisted intra-molecular charge transfer model.

RESULTS OF ONE THOUSAND CONSECUTIVE BIPOLAR TRANSURETHRAL RESECTION IN SALINE SYSTEM(TURIS)

The two-photon absorption (TPA) characteristics of PMMA discs doped with three different dyes were studied using an fs-pulsed Ti-Sapphire laser as the pump source, and employing the open-aperture Z-scan technique. TPA cross-sections obtained for PMMA doped with the dyes PM597, DCM and rhodamine 6G–rhodamine B (co-doped) were found to be equal to 24.7, 33.3 and 32.3 GM, respectively (1 GM=10−50 cm4 s phot−1 mol−1). Furthermore, two-photon fluorescence was measured for the samples containing DCM and rhodamine 6G–rhodamine B (co-doped). Compared to the one-photon fluorescence spectrum, the peaks in the two-photon fluorescence spectrum were red shifted and the extent of red shift increased with increasing doping concentration. We have also observed that the red shift in the two-photon fluorescence peak of the samples in the solid form is much larger than that in the solution state. This phenomenon could be explained by a twisted intra-molecular charge transfer model.

Multiphoton Upconversion Fluorescence Properties of Heteroaromatic Push-Pull Polymer

Two novel heteroaromatic push-pull fluorescence polymers poly [(2,5-diphenylene-1,3,4-oxadiazole) -4,4′-vinylene-alt-N,N′-bis(4-phenylenevinylene) -phenylamine](P1) and poly [(2,5-diphenylene-1,3,4-oxadiazole) -4,4′-vinylene-alt-N-ethyl-3,6-carbazolevinylene](P2) were synthesized by the Wittig reaction and the Heck reaction. Polymers P1 and P2 have decomposition temperatures of 373 and 412 ℃,respectively,and exhibit good thermal stabilities. Their electrochemical properties were explored by cyclic voltammetry. The highest occupied molecular orbital(HOMO) energy levels of P1 and P2 were-5.39 and-5.81 eV,respectively,and the lowest unoccupied molecular orbital(LUMO ) energy levels of P1and P2were-2.81 and-3.09 eV,respectively. Three-photon and two-photon upconversion fluorescence spectra of both polymers were measured using a femtosecond Ti:sapphire that was pumped by a femtosecond laser at 1250 and 800 nm. Strong three-photon upconversion fluorescence with maximum emission wavelengths of 510 nm for P1 and 491 nm for P2 in THF solution were observed. By pumping at 800 nm,strong two-photon upconversion fluorescences at 511 nm for P1 and 495 nm for P2 in THF were measured. Maximum peaks of single-photon fluorescence in THF were at 503 nm for P1 and at 475 nm for P2 with fluorescence quantum yields of 0.80 for P1 and 0.31 for P2. The effect of solvent polarity on multi-photon fluorescence was studied. Increased solvent polarity were found to induce a marked red-shift of the emission band in polymers P1 and P2.

Autofluorescence of epithelial tissue: single-photon versus two-photon excitation

We instrumented a combined fluorescence spectroscopy and imaging system to characterize the single- and two-photon excited autofluorescence in epithelial tissue. Single-photon fluorescence (SPF) are compared with two-photon fluorescence (TPF) measured at the same location in epithelial tissue. It was found that the SPF and TPF signals excited at corresponding wavelengths are similar in nonkeratinized epithelium, but the SPF and TPF spectra in the keratinized epithelium and the stromal layer are significant different. Specifically, the comparison of SPF signals with TPF signals in keratinized epithelial and stromal layers shows that TPF spectral peaks always have about 15-nm redshift with respect to SPF signals, and the TPF spectra are broader than SPF spectra. The results were generally consistent with the SPF and TPF measurements of pure nicotinamide adenine dinucleotide, flavin adenine dinucleotide, keratin and collagen, the major fluorophores in epithelium and stroma, respectively. The double peak structure of TPF spectra measured from keratinized layer suggests that there may be an unknown fluorophore responsible for the spectral peak in the long wavelength region. Furthermore, the TPF signals excited in a wide range of wavelengths provide accurate information on epithelial structure, which is an important advantage of TPF over SPF spectroscopy in the application for the diagnosis of tissue pathology.

The synthesis and optical properties of three stilbene-type dyes

Three “D–π–A” stilbene-type dyes, namely trans-4-[ p-( N, N-di (2-hydroxyethyl))-amino-styryl]- N-methyl pyridinium iodide (DHEASPI-C 1), trans-4-[ p-( N, N-di (2-hydroxyethyl))-amino-styryl]- N-octyl pyridinium bromide (DHEASPBr-C 8) and trans-4-[ p-( N, N-di (2-hydroxyethyl))-amino-styryl]- N-dodecyl pyridinium bromide (DHEASPBr-C 12) were synthesized and their optical properties in different environments were investigated. Red shifts occurred in both the one-photon and two-photon fluorescence spectra from DHEASPI-C 1 to DHEASPBr-C 8 and to DHEASPBr-C 12 as the alkyl group in the terminal acceptor was changed from methyl through octyl to dodecyl. When a 1064 nm, <130 fs mode-locked Nd: YAG laser pulse was pumped, their lasing locations occurred at 613–624 nm. When DHEASPI-C 1 was used as reference, the two-photon absorption cross-sections ( σ s) of DHEASPBr-C 8 and DHEASPBr-C 12 were 8.02 and 13.5, respectively.

Molecular rovibrational dynamics investigated by two-photon wavepacket interferometry with phase-locked pulse pairs

Time-resolved two-photon fluorescence spectra have been investigated based on wavepacket interferometry (WPI), and the wavepacket dynamics of the excited states for 4-dicyanomethylene-2-methyl-6- p-dimethyl-aminostryryl-4H-pyran (DCM) is determined by phase-locked femtosecond pulse pairs. A relative phase between the femtosecond pulse pairs can be maintained as the delay line scanning, and so the two-photon fluorescence signals will be observed to appear periodically recurring features. It indicates the constructive or destructive interference between two-photon wavepackets on the molecular excited states. The experimental results show that the phase-locked WPI has the potential applications in the wavepacket dynamics of the complicated molecular systems.

Carbazole-based 1D and 2D hemicyanines: synthesis, two-photon absorption properties and application for two-photon photopolymerization 3D lithography

One and two dimensional (1D and 2D) carbazole based hemicyanines, where methyl pyridinium, methyl indolium and methyl benzothiazolium were used as acceptor group, were synthesized by Knoevenagel condensation. One-photon absorption, fluorescence and two-photon fluorescence spectra were investigated. The experimental results indicated that the different ionic acceptors affect their one-photon and two-photon properties. Among them, 2D methyl pyridinium carbazole derivatives exhibited low quantum yields and large two-photon absorption cross sections more than 1600 GM. The synthesized compounds were used as photoinitiator of two-photon photopolymerization (TPP), and three-dimensional (3D) microstructure was successfully fabricated by TPP 3D lithography. They could be utilized as effective two-photon polymerization photoinitiators.