Two-photon Excitation Spectra Research Articles

Ultrafast supercontinuum sculpting for two-photon spectroscopy and microscopy of ratiometric fluorescent indicators

We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy.

Push-pull conjugated chromene-derivatives for potential bio-imaging applications. Synthesis, X-Ray and DFT studies, one- and two-photon photophysical properties

Dicyanomethylene-4H-benzopyran derivatives dyes containing D-π-A-type structure have been prepared and fully characterized. The Z configuration of all three structures has been confirmed by SCXRD studies. Dyes 1–6 present a strong absorption in the 450–550 nm region with ε values over 43 000 M−1 cm−11–4 showed fluorescence properties while 5 and 6 are very weakly fluorescent. The electronic structure of the first singlet excited state S1 was investigated to further explain the evolution of fluorescence quantum yields. The two-photon excitation spectra were recorded with a similar behaviour upon two-photon excitation, with two absorption bands, one in the 800–850 nm region with σ2 of 200–300 GM and another one at about 1000 nm with σ2 of 300–400 GM. Dyes 3 and 4 have shown interesting properties (both in intensity and FLIM modes) in two-photon excited microscopy.

Measurement of three-photon excitation cross-sections of fluorescein from 1154 nm to 1500 nm.

Measurements of three-photon action cross-sections for fluorescein (dissolved in water, pH ∼11.5) are presented in the excitation wavelength range from 1154 to 1500 nm in ∼50 nm steps. The excitation source is a femtosecond wavelength tunable non-collinear optical parametric amplifier, which has been spectrally filtered with 50 nm full width at half maximum band pass filters. Cube-law power dependance is confirmed at the measurement wavelengths. The three-photon excitation spectrum is found to differ from both the one- and two-photon excitation spectra. The three-photon action cross-section at 1154 nm is more than an order of magnitude larger than those at 1450 and 1500 nm (approximately three times the wavelength of the one-photon excitation peak), which possibly indicates the presence of resonance enhancement.

A computational two-photon fluorescence approach for revealing label-free the 3D image of viruses and bacteria.

Current solutions for bacteria and viruses identification are based on time-consuming technics with complex preparation procedures. In the present work, we revealed label-free the presence of free viral particles and bacteria with a computational two-photon fluorescence (C-TPF) strategy. Six bacteria were tested: Escherichia coli, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas fluorescens, Bacillus subtilis, and Clostridium perfringens. The two families of viral particles were the herpes virus with the cytomegalovirus (CMV, 300 nm of diameter) and the coronavirus with the SARS-CoV-2 (100 nm of diameter). The instrumental and computational pipeline FAMOUS optimized the produced 3D images. The origin of the fluorescence emission was discussed for bacteria regarding to their two-photon excitation spectra and attributed to the metabolic indicators (FAD and NADH). The optical and computational strategy constitute a new approach for imaging label-free viral particles and bacteria and paves the way to a new understanding of viral or bacterial ways of infection.

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range.

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.

Combined contributions of carotenoids and chlorophylls in two-photon spectra of photosynthetic pigment-protein complexes-A new way to quantify carotenoid dark state to chlorophyll energy transfer?

Transitions into the first excited state of carotenoids, Car S1, are optically forbidden in conventional one-photon excitation (OPE) but are possible via two-photon excitation (TPE). This can be used to quantify the amount of Car S1 to Chlorophyll (Chl) energy transfer in pigment-protein complexes and plants by observing the chlorophyll fluorescence intensity after TPE in comparison to the intensity observed after direct chlorophyll OPE. A parameter, ΦCoupling Car S1-Chl, can be derived that directly reflects relative differences or changes in the Car S1 → Chl energy transfer of different pigment-protein complexes and even living plants. However, very careful calibrations are necessary to ensure similar OPE and TPE excitation probabilities and transition energies. In plants, the exact same sample spot must be observed at the same time. All this is experimentally quite demanding. ΦCoupling Car S1-Chl also corrects intrinsically for direct chlorophyll TPE caused by larger chlorophyll excesses in the complexes, but recently it turned out that in certain TPE wavelengths ranges, its contribution can be quite large. Fortunately, this finding opens also the possibility of determining ΦCoupling Car S1-Chl in a much easier way by directly comparing values in TPE spectra observed at wavelengths that are either more dominated by Cars or Chls. This avoids tedious comparisons of OPE and TPE experiments and potentially allows measurement at even only two TPE wavelengths. Here, we explored this new approach to determine ΦCoupling Car S1-Chl directly from single TPE spectra and present first examples using known experimental spectra from Cars, Chl a, Chl b, LHC II, and PS 1.

Multiplexed two-photon excitation spectroscopy of single gold nanorods.

Plasmonic metallic nanoparticles are commonly used in (bio-)sensing applications because their localized surface plasmon resonance is highly sensitive to changes in the environment. Although optical detection of scattered light from single particles provides a straightforward means of detection, the two-photon luminescence (TPL) of single gold nanorods (GNRs) has the potential to increase the sensitivity due to the large anti-Stokes shift and the non-linear excitation mechanism. However, two-photon microscopy and spectroscopy are restricted in bandwidth and have been limited by the thermal stability of GNRs. Here, we used a scanning multi-focal microscope to simultaneously measure the two-photon excitation spectra of hundreds of individual GNRs with sub-nanometer accuracy. By keeping the excitation power under the melting threshold, we show that GNRs were stable in intensity and spectrum for more than 30min, demonstrating the absence of thermal reshaping. Spectra featured a signal-to-noise ratio of >10 and a plasmon peak width of typically 30nm. Changes in the refractive index of the medium of less than 0.04, corresponding to a change in surface plasmon resonance of 8nm, could be readily measured and over longer periods. We used this enhanced spectral sensitivity to measure the presence of neutravidin, exploring the potential of TPL spectroscopy of single GNRs for enhanced plasmonic sensing.

Characterizing photostability of red fluorescent proteins under multiphoton excitation

Red fluorescent proteins (RFPs) and biosensors built upon them provide an attractive advantage for two-photon laser microscopy (2PLM) because they can report from deeper layers of tissue compared to green fluorescent proteins. The two-photon excitation spectra of RFPs consist of two different transitions, a weak one, at 1000-1200 nm (S0->S1), and a strong one, at 740-800 nm (S0->Sn). Therefore, using 740-800 nm excitation looks more efficient. Here we study the multiphoton bleaching rate of RFP mCherry in these two spectral regions.

Rashba splitting in organic\u2013inorganic lead\u2013halide perovskites revealed through two-photon absorption spectroscopy

The Rashba splitting in hybrid organic–inorganic lead–halide perovskites (HOIP) is particularly promising and yet controversial, due to questions surrounding the presence or absence of inversion symmetry. Here we utilize two-photon absorption spectroscopy to study inversion symmetry breaking in different phases of these materials. This is an all-optical technique to observe and quantify the Rashba effect as it probes the bulk of the materials. In particular, we measure two-photon excitation spectra of the photoluminescence in 2D, 3D, and anionic mixed HOIP crystals, and show that an additional band above, but close to the optical gap is the signature of new two-photon transition channels that originate from the Rashba splitting. The inversion symmetry breaking is believed to arise from ionic impurities that induce local electric fields. The observation of the Rashba splitting in the bulk of HOIP has significant implications for the understanding of their spintronic and optoelectronic device properties.

Trivial Excitation Energy Transfer to Carotenoids Is an Unlikely Mechanism for Non-photochemical Quenching in LHCII

Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favors excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD) trajectories about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely its linear absorption (LA), transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII free energy surface. We show that trivial, Coulomb-mediated energy transfer to S1 is an unlikely quenching mechanism, with pigment movements insufficiently pronounced to switch the system between quenched and unquenched states. Modulation of S1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is possibly an artifact of quantum chemical over-estimation of S1 oscillator strength and the real mechanism likely involves short-range interaction and/or non-trivial inter-molecular states.

Two-photon excitation fluorescent spectral and decay properties of retrograde neuronal tracer Fluoro-Gold

Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of Fluoro-Gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of Fluoro-Gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of Fluoro-Gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the two-photon excitation spectrum of Fluoro-Gold between 720 and 990 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of Fluoro-Gold labeled facial motor nuclei. Optimal two-photon excitation of Fluoro-Gold within microscope tuning range occurred at 720 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution.

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity – due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra –, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.

Preface to the special issue dedicated to Professor Richard P. Van Duyne (1945–2019)

Preface to the special issue dedicated to Professor Richard P. Van Duyne (1945–2019)

Two-photon absorption and excitation spectroscopy of carotenoids, chlorophylls and pigment-protein complexes.

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than ∼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

Isoenergetic two-photon excitation enhances solvent-to-solute excited-state proton transfer.

Two-photon excitation (TPE) is an attractive means for controlling chemistry in both space and time. Since isoenergetic one- and two-photon excitations (OPE and TPE) in non-centrosymmetric molecules are allowed to reach the same excited state, it is usually assumed that they produce similar excited-state reactivity. We compare the solvent-to-solute excited-state proton transfer of the super photobase FR0-SB following isoenergetic OPE and TPE. We find up to 62% increased reactivity following TPE compared to OPE. From steady-state spectroscopy, we rule out the involvement of different excited states and find that OPE and TPE spectra are identical in non-polar solvents but not in polar ones. We propose that differences in the matrix elements that contribute to the two-photon absorption cross sections lead to the observed enhanced isoenergetic reactivity, consistent with the predictions of our high-level coupled-cluster-based computational protocol. We find that polar solvent configurations favor greater dipole moment change between ground and excited states, which enters the probability for TPE as the absolute value squared. This, in turn, causes a difference in the Franck-Condon region reached via TPE compared to OPE. We conclude that a new method has been found for controlling chemical reactivity via the matrix elements that affect two-photon cross sections, which may be of great utility for spatial and temporal precision chemistry.

Synthesis of Two‐Dimensional Perovskite by Inverse Temperature Crystallization and Studies of Exciton States by Two‐Photon Excitation Spectroscopy

AbstractTwo‐dimensional (2D) organic–inorganic hybrid perovskites (OIHPs), a natural multiple‐quantum‐well structure with quasi‐2D electronic properties, have recently emerged as a promising class of semiconducting materials for photovoltaic and optoelectronic applications. However, facile synthesis of high‐quality 2D OIHPs single crystals is still lacking. The layer dependence of the exciton binding energy of (C4H9NH3)2PbI4 (C4PI), a widely studied 2D OIHP, is still debated. Herein, a novel synthesis technique based on inverse temperature crystallization in a binary‐solvent system is used to prepare 2D OIHPs and a systematic study of excitonic states of the synthesized 2D OIHPs by two‐photon excitation (TPE) spectroscopy is conducted. The obtained TPE spectra indicate that the exciton binding energies are similar for C4PI nanosheets and bulk crystals with different number of layers, most likely due to the intrinsically weak interlayer coupling. Further, the dark excitonic 2p states of (C6H5(CH2)2NH3)2PbI4 (PEPI) and C4PI are also observed by TPE spectroscopy. The results provide a novel synthesis protocol and insight into exciton properties of 2D OIHPs.

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.

An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3–5.0 μm for excitation wavelengths of 730–1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)–poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.

Absorption, Fluorescence, and Two-Photon Excitation Ability of 5-Phenylisolidolo[2,1-a]quinolines.

We report on the absorption, fluorescence, and two-photon excitation spectra of a series of 5-phenylisoindolo[2,1-a]quinoline dyes. Depending on the substituents, we observed increasing two-photon absorption cross sections, with values up to 56 GM@973 nm, which are similar to those of the enhanced green fluorescent protein and fluorescein, common fluorescent chromophores.

Absolute CO number densities measured using TALIF in a non-thermal plasma environment

We report first measurements of time-resolved absolute CO number densities and rotational temperatures in a non-thermal CO2 plasma environment using TALIF with a nanosecond pulsed laser. Two-photon excitation spectra from the (v′ = 0) ← (v″ = 0) Q-branch are recorded and fitted to extract rotational temperatures. Absolute number densities are determined from the frequency-integrated excitation spectrum. The plasma under investigation is a pulsed glow discharge operated at a frequency of 60 Hz with an plasma-on time of 5 ms per plasma cycle, 50 mA plasma current and a pressure of 6.67 mbar. CO number densities range from m−3 to m−3, while rotational temperatures range from 370 ± 40 K to 700 ± 70 K at the beginning and end of the plasma-on phase, respectively. Our results show fair agreement with literature data.