Two-photon Fluorescence Spectroscopy Research Articles

Revealing natural fluorescence in transparent insect wings by linear and nonlinear optical techniques

For most natural organisms, the physical, chemical and biological aspects of fluorescence emission are poorly understood. For example, to the best of our knowledge, fluorescence from the transparent wings of any of the 3000 known species of cicadas has never been reported in the literature. These wings are known to exhibit anti-reflective properties arising from quasi-periodic arrays of nipples. Our study, using linear and nonlinear optical techniques, including spectrofluorimetry, two-photon fluorescence spectroscopy and Second Harmonic Generation (SHG), reveals the fluorescence properties in the wings the grey and the common cicadas (Cicada orni and Lyristes (Tibicen) plebejus, respectively), as well as the broad-bordered bee hawk-moth (Hemaris fuciformis). The study suggests that fluorescence would be more widespread in transparent insect wings than what was previously believed. Comparing this result to the fluorescence emission from the wings of the Bornean damselfly (Vestalis amabilis), we inferred that this emission probably arises from resilin, a protein reported to enhance wing flexibility. Moreover, the nonlinear optical investigation of the insects’ wings provided further insight into wing structure, indicating that multiphoton techniques add valuable information for the analysis of insect integuments. The strong SHG signal detected from the wing veins implies that these veins are materially organised in a non-centrosymmetric and hence non-random fashion.

Dimensions of fluorescence kinetic concentration of doped morphology homologs synthesized by TCPP and UiO-66 MOF

Metal-organic frameworks (MOFs) have attracted significant attention thanks to their excellent physical and chemical properties. Metal-organic frames are widely used in optics. For example, metal-organic frameworks can be used in fluorescent probes to detect and treat diseases, and can also be used to make solid-state light-emitting devices. Considering the high stability and easy synthesis of Zr-based UiO-66 MOF as well as the strong fluorescent emission of porphyrin molecules, the TCPP-incorporated UiO frameworks would inherit these advantages and exhibit intriguing optical performance. Therefore, in this paper, TCPP was doped into UiO-66, and the optical properties of the resulting MOF structure were observed. To obtain the optimal doping condition, three doping concentrations of TCPP were used. Besides the structural and morphological characterization by X-ray diffraction and scanning electron microscopy, more attentions have paid on the optical testing based on absorption and fluorescence spectroscopy, two-photon fluorescence spectroscopy and time-resolved spectroscopy. As a result, the TCPP-doped MOFs exhibited good fluorescence properties and satisfied our previous expectations of nonlinear optical properties and two-photon absorption. This work would pave a new way in constructing various MOFs-based solid-state devices with widespread optical applications.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) and super-resolution fluorescence imaging with a supramolecular biopolymer for the controlled tagging of polysaccharides.

A new supramolecular polysaccharide complex, comprising a functionalised coumarin tag featuring a boronic acid and β-d-glucan (a natural product extract from barley, Hordeum Vulgare) was assembled based on the ability of the boronate motif to specifically recognise and bind to 1,2- or 1,3-diols in water. The complexation ratio of the fluorophore : biopolymer strand was determined from fluorescence titration experiments in aqueous environments and binding isotherms best described this interaction using a 2 : 1 model with estimated association constants of K2:1a1 = 5.0 × 104 M-1 and K2:1a2 = 3.3 × 1011 M-1. The resulting hybrid (denoted 5@β-d-glucan) was evaluated for its cellular uptake as an intact functional biopolymer and its distribution compared to that of the pinacol-protected coumarin boronic acid derivative using two-photon fluorescence lifetime imaging microscopy (FLIM) in living cells. The new fluorescent β-d-glucan conjugate has a high kinetic stability in aqueous environments with respect to the formation of the free boronic acid derivative compound 5 and retains fluorescence emissive properties both in solution and in living cells, as shown by two-photon fluorescence spectroscopy coupled with time-correlated single photon counting (TCSPC). Super-resolution fluorescence imaging using Airyscan detection as well as TM AFM and Raman spectroscopy investigations confirmed the formation of fluorescent and nano-dimensional aggregates of up to 20 nm dimensions which self-assemble on several different inert surfaces, such as borosilicate glass and mica surfaces, and these aggregates can also be observed within living cells with optical imaging techniques. The cytoplasmic distribution of the 5@β-d-glucan complex was demonstrated in several different cancer cell lines (HeLa and PC-3) as well as in healthy cells (J774.2 macrophages and FEK-4). Both new compounds (pinacol protected boronated coumarin) 5-P and its complex hybrid 5@β-d-glucan successfully penetrate cellular membranes with the minimum morphological alterations to cells and distribute evenly in the cytoplasm. The glucan biopolymer retains its activity towards macrophages in the presence of the coumarin tag functionality, demonstrating the potential of this natural β-d-glucan to act as a functional self-assembled theranostic scaffold capable of mediating the delivery of anchored small organic molecules with imaging and drug delivery applications.

Atomic frequency reference at 1033 nm for ytterbium (Yb)-doped fiber lasers and applications exploiting a rubidium (Rb) 5S1/2 to 4D5/2 one-colour two-photon transition.

We demonstrate a two-photon transition of rubidium (Rb) atoms from the ground state (5S1/2) to the excited state (4D5/2), using a home-built ytterbium (Yb)-doped fiber amplifier at 1033 nm. This is the first demonstration of an atomic frequency reference at 1033 nm as well as of a one-colour two-photon transition for the above energy levels. A simple optical setup is presented for the two-photon transition fluorescence spectroscopy, which is useful for frequency stabilization for a broad class of lasers. This spectroscopy has potential applications in the fiber laser industry as a frequency reference, particularly for the Yb-doped fiber lasers. This two-photon transition also has applications in atomic physics as a background- free high- resolution atom detection and for quantum communication, which is outlined in this article.

Two-Photon and Time-Resolved Fluorescence Spectroscopy as Probes for Structural Determination in Amyloid-β Peptides and Aggregates

The development of new sensitive methods for the detailed collection of conformational and morphological information about amyloids is crucial for the elucidation of critical questions regarding aggregation processes in neurodegenerative diseases. The combined approach of two-photon and time-resolved fluorescence spectroscopy described in this report interrogates the early conformational dynamics seen in soluble oligomers of amyloid-β(1-42). Concentration-dependent aggregation studies using two-photon absorption show enhanced sensitivity toward conformational changes taking place in the secondary structure of the amyloid peptide as aggregation proceeds. Fluorescence lifetimes and changes in anisotropy values indicate Förster-type energy transfer occurring as a function of aggregation state. The sensitivity of our two-photon methodology is compared to that of circular dichroism (CD) spectroscopy, and the results indicate that the two-photon absorption cross-section method exhibits superior sensitivity. A theoretical model is developed that, together with electronic structure calculations, explains the change in cross section as a function of aggregation in terms of interacting transition dipoles for aggregates showing stacked or parallel structures. This suggests that the two-photon method provides a sensitive alternative to CD spectroscopy while avoiding many of the inherent challenges particular to CD data collection. The implication of this finding is significant, as it indicates that a two-photon-based technique used in conjunction with time-resolved fluorescence might be able to reveal answers to conformational questions about amyloid-β(1-42) that are presently inaccessible with other techniques.

Precision measurement of the hyperfine structure of 8d2D3/2 state of 133Cs by the radio-frequency phase modulation technique

We report the measurement of the hyperfine structure in the 8d2D3/2 state of 133Cs isotope by Doppler free two-photon fluorescence spectroscopy in a gas cell. The hyperfine level separations were measured by the radio-frequency phase modulation technique using a frequency stabilized Ti:sapphire laser. The frequency separation between various hyperfine levels of the excited state has been measured with a precision of ~60kHz. From the measured hyperfine separations of the excited state, we have determined the magnetic dipole coupling constant (A) and the electric quadrupole coupling constant (B) for the 8d2D3/2 state. The determined hyperfine structure constants are A=3.98 (0.01) MHz and B=−0.03 (0.09) MHz. Our deduced magnetic dipole coupling constant (A) value is thus by far the most precise and was found to be in good agreement with the 3σ level with the earlier reported values. This work reports the electric quadrupole coupling constant (B) value for the 8d2D3/2 state for the first time.

Two-Photon Fluorescence Spectroscopy and Imaging of 4-Dimethylaminonaphthalimide Peptide and Protein Conjugates

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.

Hyperfine structure of the7d2D3/2level in cesium measured by Doppler-free two-photon spectroscopy

We report the measurement of the hyperfine structure in the 7$d$ ${}^{2}{D}_{3/2}$ state of ${}^{133}$Cs isotope by Doppler-free two-photon fluorescence spectroscopy in a gas cell. The hyperfine level separations were measured using a frequency-stabilized Ti:sapphire laser locked to one of the hyperfine transitions and an acousto-optic modulator locked to another hyperfine transition. The frequency separation between various hyperfine levels has been measured with a precision of \ensuremath{\sim}100 kHz. From the measured hyperfine separations of the excited state, we have determined the magnetic dipole coupling constant ($A$) and electric quadrupole coupling constant ($B$) for the 7$d$ ${}^{2}{D}_{3/2}$ state. The determined hyperfine structure constants are $A=7.38$ (0.01) MHz and $B=\ensuremath{-}0.18$ (0.1) MHz. The values measured are found to be in good agreement with the earlier reported results using stepwise excitation process.

Two-photon fluorescent immunosensor for androgenic hormones using resonant grating waveguide structures

Abstract An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methylboldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 μg l −1 , two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conjugates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB antibody. The immunoreagents are immobilized onto a resonant Ta 2 O 5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The developed immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.

Measurement of the hyperfine splitting of the 9 S1/2 level in Cesium by Doppler-free two-photon spectroscopy

The hyperfine splitting for the 133Cs isotope for the 6s 2S1/2→9s 2S1/2 transition has been measured using Doppler-free two-photon fluorescence spectroscopy in a gas cell for the first time to our knowledge. The hyperfine splitting between the two components was measured to be 8754 (1)MHz and the magnetic dipole coupling constant (A) for the excited 9s 2S1/2 level was determined to be 109.7 (3)MHz for the 133Cs isotope. The measured values are in good agreement with the earlier reported results.

Phosphorus Ligand Imaging with Two-Photon Fluorescence Spectroscopy: Towards Rational Catalyst Immobilization

Spotless catalysts: Ligand immobilization was studied by two-photon fluorescence microscopy with a fluorescent nixantphos ligand as probe (see picture). In the immobilization process ligand aggregates form in solution and are deposited on the support, where they appear as bright spots in fluorescence images. Preventing their formation leads to spotless catalysts with superior catalytic properties.

Publisher's Note: Measurement of the cesium7dD3∕22hyperfine coupling constants in a thermal beam using two-photon fluorescence spectroscopy [Phys. Rev. A77, 062505 (2008)

Publisher's Note: Measurement of the cesium7dD3∕22hyperfine coupling constants in a thermal beam using two-photon fluorescence spectroscopy [Phys. Rev. A77, 062505 (2008)

Comparison of nonlinear absorption in three similar dyes: Polymethine, squaraine and tetraone

We performed a detailed experimental investigation and quantum-chemical analysis of two-photon absorption (2PA) spectra of symmetrical cationic polymethine and neutral squaraine and tetraone dyes having nearly identical terminal groups and similar conjugation length. Squaraine and tetraone structures differ from polymethines in that their conjugated backbone includes an electron accepting bridge. Frequency degenerate 2PA spectra of these molecules are measured by two-photon fluorescence spectroscopy and the Z-scan technique. Comparing 2PA spectra of polymethine with squaraine and tetraone structures, we find that we can access considerably larger 2PA cross-sections (⩾8000 × 10 −50 cm 4 s/photon) in the squaraine and tetraone molecules due to an increase in the density of final states introduced by the electron accepting bridge in the conjugated chain. We discuss the difference in electronic structure for these molecules and introduce a link between 2PA and excited-state absorption spectra. The results of these experiments combined with agreement of quantum-chemical calculations moves us closer to a predictive capability for the relation between molecular structure and nonlinear optical properties.

Two-photon Excited Fluorescence of Nitrogen-Vacancy Centers in Proton-Irradiated Type Ib Diamond

Two-photon fluorescence spectroscopy of negatively charged nitrogen-vacancy [(N-V)-] centers in type Ib diamond single crystals have been studied with a picosecond (7.5 ps) mode-locked Nd:YVO(4) laser operating at 1064 nm. The (N-V)- centers were produced by radiation damage of diamond using a 3 MeV proton beam, followed by thermal annealing at 800 degrees C. Prior to the irradiation treatment, infrared spectroscopy of the C-N vibrational modes at 1344 cm(-1) suggested a nitrogen content of 109 +/- 10 ppm. Irradiation and annealing of the specimen led to the emergence of a new absorption band peaking at approximately 560 nm. From a measurement of the integrated absorption intensity of the sharp zero-phonon line (637 nm) at liquid nitrogen temperature, we determined a (N-V)- density of (4.5 +/- 1.1) x 10(18) centers/cm3 (or 25 +/- 6 ppm) for the substrate irradiated at a dose of 1 x 1016) H(+)/cm(2). Such a high defect density allowed us to observe two-photon excited fluorescence and measure the corresponding fluorescence decay time. No significant difference in the spectral feature and fluorescence lifetime was observed between one-photon and two-photon excitations. Assuming that the fluorescence quantum yields are the same for both processes, a two-photon absorption cross section of sigma(TPA) = (0.45 +/- 0.23) x 10(-50) cm(4).s/photon at 1064 nm was determined for the (N-V)- center based on its one-photon absorption cross section of sigma(OPA) = (3.1 +/- 0.8) x 10(-17) cm2 at 532 nm. The material is highly photostable and shows no sign of photobleaching even under continuous two-photon excitation at a peak power density of 3 GW/cm(2) for 5 min.

Time-resolved two-photon excitation fluorescence spectroscopy and microscopy using a high repetition rate streak camera

We present a time-resolved two-photon excitation fluorescence spectroscopy and a simultaneous time-and spectrum-resolved multifocal multiphoton microscopy system that is based on a high repetition rate picosecond streak camera for providing time-and spectrum-resolved measurement and imaging in biomedicine. The performance of the system is tested and characterized by the fluorescence spectrum and lifetime analysis of several standard fluorescent dyes and their mixtures. Spectrum-resolved fluorescence lifetime images of fluorescence beads are obtained. Potential applications of the system include clinical diagnostics and cell biology etc.

Experimental and theoretical approaches to understanding two-photon absorption spectra in polymethine and squaraine molecules

We performed a detailed experimental investigation and quantum-chemical analysis of two-photon absorption (2PA) spectra of a series of symmetrical cationic polymethines and neutral squaraines having similar structures. Degenerate 2PA spectra of these molecules are taken by using two-photon fluorescence spectroscopy and the Z-scan technique. All measurements are made with 150 fs laser pulses of 1 kHz repetition rate in the tuning range of 520-2100 nm(0.6-2.4 eV). Comparing 2PA spectra of polymethines and squaraines, we find that we can access considerably larger 2PA cross sections (≥8600×10−50 cm4s/photon) in the squaraines owing to narrower linear absorption spectra and an increase in the density of unoccupied molecular orbitals introduced by the squaraine acceptor group in the conjugated chain.

Two-photon evanescent-volume wave monitoring of desorbing Cs atoms

The two-photon fluorescence spectroscopy in the vicinity of the surface has been performed for Cesium atoms. One of the laser beams was directed perpendicularly to the surface, whereas the second one excited an evanescent wave propagating along the surface. To demonstrate its potential, this technique has been applied for a qualitative comparative study of Cs atom desorption from a glass surface versus the same surface coated with a PDMS film. That way the fluorescence line originating primarily from desorbing atoms could be spectrally isolated. The desorption flux from polymer coated surfaces is found to be significantly smaller as compared to the flux from a plain glass surface.

Two-Photon Fluorescence Spectroscopy of Individually Trapped Pseudoisocyanine J-Aggregates in Aqueous Solution

We have investigated a pseudoisocyanine dye aqueous solution including nanometer-sized J-aggregates by combining optical trapping and two-photon fluorescence spectroscopy. By focusing an intense near-infrared laser into an 8 x 10(-3) M solution, the intense fluorescence from J-aggregates for a few to tens of seconds is observed intermittently, indicating that individual J-aggregates are trapped in and diffuse out from a focal spot. The peak position and full width at half-maximum of the J-band are different from each other. By measuring 171 J-aggregates, it was found that J-aggregates can be classified largely into two groups. The existence of two kinds of groups of J-aggregates could be attributed to the difference in the nucleation process, which is affected by the substrate. J-aggregates possessing a J-band of a narrower bandwidth in a shorter wavelength region are trapped for a longer period of time, indicating that highly ordered J-aggregates are trapped for a longer period of time because of their high polarizability.

Time-resolved two-photon excitation fluorescence spectroscopy

A novel time-resolved two-photon excitation fluorescence spectroscopic system based on a high repetition rate streak camera and capable of simultaneous measurement of fluorescence spectrum and lifetime of the samples is presented. The system has high temporal (6.5—200 ps) and spectral (1—3 nm) resolutions and permits rapid data acquisition and reliable and reproducible lifetime and spectrum determinations. The system is tested with standard fluorescent dyes (rhodamine 6G, coumarin 314 and their mixtures) and the lifetimes and spectra obtained agree well with those reported in the literatures. Experimental results indicate that our system provides a unique contrast mechanism for discriminating multi-fluorophore or multi-component of biological tissues through spectrally and temporally resolved information and may find extensive applications in multispectral fluorescence lifetime imaging and fluorescence resonance energy transfer imaging.

Two-Photon Fluorescence Spectroscopy and Microscopy of NAD(P)H and Flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections ( σ 2P) increase rapidly at wavelengths below 800 nm, and the maximum σ 2P of LipDH is ∼5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., ∼410–490 nm and ∼510–650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the σ 2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.