Efficient Two-photon Excitation Research Articles

Femtosecond two-photon LIF imaging of atomic hydrogen in high-pressure methane-air flames

Atomic hydrogen (H) imaging studies in combustion systems have been limited thus far to atmospheric- or low-pressure flames. The commonly used excitation scheme for two-photon laser-induced fluorescence (TPLIF) of H involves 205-nm deep-ultra-violet (DUV) laser pulses generated using nanosecond (ns), picosecond (ps), or femtosecond (fs) laser systems. Ultrashort pulse, fs excitation schemes are more attractive because of the efficient two-photon excitation and interference-free kHz-rate imaging capability. In this work, we implemented a home-built, direct fourth harmonic generation (FHG) laser system to generate high-energy fs pulses for H imaging in CH4/air flames at pressures up to 10 bar. The high-energy FHG output was capable of overcoming transmission losses of DUV laser pulses through thick optical windows of the pressure vessel. Signal interferences such as photoionization, photolytic production, amplified spontaneous emission, and background chemiluminescence were avoided or minimized during data acquisition and subsequent data processing steps. A quadratic dependence of the H TPLIF signal on the laser energy was observed. The increase in the pressure from 1 to 10 bar showed a rapid decay of the fs H TPLIF signal although it should scale linearly with increasing H number density with pressure ideally at equilibrium conditions. Besides a detailed quenching correction, laser absorption and fluorescence signal trapping remain major issues at elevated pressures. The measured fluorescence signals are compared with the equilibrium H number densities and local H concentrations calculated using the Cantera (0D) and UNICORN (2D) flame codes, respectively, for a range of equivalence ratios, and possible reasons for discrepancies are discussed. Experimentally obtained 1D and 2D spatial distributions of H are well predicted by the UNICORN flame model. Steps towards quantitative single-shot H TPLIF imaging measurements in high-pressure flames are also discussed. Overall, the fs TPLIF imaging technique coupled with a high-efficiency home-built FHG system is a promising diagnostic approach for H imaging in high-pressure flames.

Compact multicolor two-photon fluorescence microscopy enabled by tailorable continuum generation from self-phase modulation and dispersive wave generation.

By precisely managing fiber-optic nonlinearity with anomalous dispersion, we have demonstrated the control of generating plural few-optical-cycle pulses based on a 24-MHz Chromium:forsterite laser, allowing multicolor two-photon tissue imaging by wavelength mixing. The formation of high-order soliton and its efficient coupling to dispersive wave generation leads to phase-matched spectral broadening, and we have obtained a broadband continuum ranging from 830 nm to 1200 nm, delivering 5-nJ pulses with a pulse width of 10.5 fs using a piece of large-mode-area fiber. We locate the spectral enhancement at around 920 nm for the two-photon excitation of green fluorophores, and we can easily compress the resulting pulse close to its limited duration without the need for active pulse shaping. To optimize the wavelength mixing for sum-frequency excitation, we have realized the management of the power ratio and group delay between the soliton and dispersive wave by varying the initial pulse energy without additional delay control. We have thus demonstrated simultaneous three-color two-photon tissue imaging with contrast management between different signals. Our source optimization leads to efficient two-photon excitation reaching a 500-µm imaging depth under a low 14-mW illumination power. We believe our source development leads to an efficient and compact approach for driving multicolor two-photon fluorescence microscopy and other ultrafast investigations, such as strong-field-driven applications.

High-repetition-rate krypton tagging velocimetry in Mach-6 hypersonic flows.

A 100 kHz krypton (Kr) tagging velocimetry (KTV) technique was demonstrated in a Mach-6 Ludwieg tube using a burst-mode laser-pumped optical parametric oscillator system. The single-beam KTV scheme at 212 nm produced an insufficient signal in this large hypersonic wind tunnel because of its low Kr seeding (≤5%), low static pressure (∼2.5torr), and long working distance (∼1m). To overcome these issues, a new scheme using two excitation beams was developed to enhance KTV performance. A 355 nm laser beam was combined with the 212 nm beam to promote efficient two-photon Kr excitation at 212 nm, and increase the probability of 2 + 1 resonant-enhanced multiphoton ionization by adding a 355 nm beam. A signal enhancement of approximately six times was obtained. Using this two-excitation beam approach, strong long-lasting KTV was successfully demonstrated at a 100 kHz repetition rate in a Mach-6 flow.

Efficient two-photon excitation stimulated emission depletion nanoscope exploiting spatiotemporal information.

.Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.

Two-photon excited photoluminescence of single perovskite nanocrystals.

Lead-halide perovskite nanocrystals (NCs) have emerged as a novel type of semiconductor nanostructure, attracting great research interests in both fundamental science and practical applications. Here, we compare the optical properties of single CsPbI3 NCs under both one-photon and two-photon excitations, mainly including the photoluminescence (PL) blinking and PL decay dynamics. By means of the PL saturation effect caused by multi-exciton Auger recombination, we have also estimated a two-photon absorption cross section of ∼6.8 × 106 GM for single CsPbI3 NCs. The ability to realize efficient two-photon excitation of single perovskite NCs with strongly suppressed background fluorescence will help not only to promote their bio-imaging and biolabeling applications but also to reveal and manipulate their delicate electronic structures for potential usage in quantum information processing.

Efficient non-degenerate two-photon excitation for fluorescence microscopy.

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

Background-free two-photon fluorescence readout via a three-photon charge-state modulation of nitrogen-vacancy centers in diamond.

We demonstrate that a background-free readout of two-photon fluorescence from nitrogen-vacancy (NV) centers in a strongly fluorescing environment can be accomplished by all-optical means via a multiphoton charge-state modulation of NV centers in a mixture of negatively charged and neutral NV centers. A 100fs, 1060nm output of an ytterbium fiber laser is ideally suited for this modality of multiphoton microscopy, providing, as our experiments show, an efficient two-photon excitation of both NV- and NV0 charge states, but keeping the nonlinearity of n-photon ionization needed for NV-/NV0 charge-state modulation to a minimum, n=3.

Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions.

Efficient two-photon excitation by photonic dimers.

In this Letter, we show that photonic dimers, the quantum mechanical bound states of two photons, enable efficient nonlinear two-photon excitation, primarily due to the Lorentzian energy anti-correlation and to the temporal proximity between the constituent photons. We analytically and numerically demonstrate the order-of-magnitude improvement of the excitation efficiency per photon by the photonic dimers over the ultrashort pulses from a laser. We further show that, owing to the high excitation efficiency, the two-photon transition rate by photonic dimers deviates from the well-known linear intensity dependence at low intensities. Possible approaches for generating the photonic dimers in semiconductor platforms are also investigated.

The Impact of Compressed Femtosecond Laser Pulse Durations on Neuronal Tissue Used for Two-Photon Excitation Through an Endoscope

Accurate intraoperative tumour margin assessment is a major challenge in neurooncology, where sparse tumours beyond the bulk tumour are left undetected under conventional resection. Non-linear optical imaging can diagnose tissue at the sub-micron level and provide functional label-free histopathology in vivo. For this reason, a non-linear endomicroscope is being developed to characterize brain tissue intraoperatively based on multiple endogenous optical contrasts such as spectrally- and temporally-resolved fluorescence. To produce highly sensitive optical signatures that are specific to a given tissue type, short femtosecond pulsed lasers are required for efficient two-photon excitation. Yet, the potential of causing bio-damage has not been studied on neuronal tissue. Therefore, as a prerequisite to clinically testing the non-linear endomicroscope in vivo, the effect of short laser pulse durations (40–340 fs) on ex vivo brain tissue was investigated by monitoring the intensity, the spectral, and the lifetime properties of endogenous fluorophores under 800 and 890 nm two-photon excitation using a bi-modal non-linear endoscope. These properties were also validated by imaging samples on a benchtop multiphoton microscope. Our results show that under a constant mean laser power, excitation pulses as short as 40 fs do not negatively alter the biochemical/ biophysical properties of tissue even for prolonged irradiation.

Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.

An Efficient Multicolor Two-Photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

Two-photon imaging of endogenous fluorescence can provide important physiological and metabolic information from intact tissues in a label-free and non-invasive way. However, imaging of multiple intrinsic fluorophores, such as NADH, FAD, retinoids and porphyrins in living systems is generally hampered by sequential multi-wavelength excitation resulting in long acquisition times and motion artifacts. We report an efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 700-1040nm range, using wavelength mixing. By using two synchronized pulse trains at two different wavelengths, an additional “virtual” two-photon excitation wavelength is generated, and simultaneous excitation of blue, green and red endogenous fluorophores is achieved. This method permits fast and reliable simultaneous imaging of the metabolic coenzymes NADH and FAD to being implemented, overcoming the difficulties associated with their difference in absorption spectra and disparity in concentration. We achieve efficient ratiometric redox imaging and simultaneous efficient two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. Lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states were measured in both reconstructed human skins and live C. elegans worms. Finally, we perform hyperspectral imaging of endogenous fluorophores during early zebrafish development.

Designed Er(3+)-singly doped NaYF4 with double excitation bands for simultaneous deep macroscopic and microscopic upconverting bioimaging.

Simultaneous deep macroscopic imaging and microscopic imaging is in urgent demand, but is challenging to achieve experimentally due to the lack of proper fluorescent probes. Herein, we have designed and successfully synthesized simplex Er(3+)-doped upconversion nanoparticles (UCNPs) with double excitation bands for simultaneous deep macroscopic and microscopic imaging. The material structure and the excitation wavelength of Er(3+)-singly doped UCNPs were further optimized to enhance the upconversion emission efficiency. After optimization, we found that NaYF4:30%Er(3+)@NaYF4:2%Er(3+) could simultaneously achieve efficient two-photon excitation (2PE) macroscopic tissue imaging and three-photon excitation (3PE) deep microscopic when excited by 808 nm continuous wave (CW) and 1480 nm CW lasers, respectively. In vitro cell imaging and in vivo imaging have also been implemented to demonstrate the feasibility and potential of the proposed simplex Er(3+)-doped UCNPs as bioprobe.

Enhanced upconversion of NaYF_4:Er^3+/Yb^3+ phosphors prepared via the rapid microwave-assisted hydrothermal route at low temperature: phase and morphology control

Monophasic NaYF4:Er(3+)/Yb(3+) crystals were synthesized via the microwave-assisted hydrothermal route at 180°C. Microwave heating during the hydrothermal process substantially reduces the duration of reaction for the formation of cubic-NaYF4:Er(3+)/Yb(3+) nanocrystals from 6 h to 30 min. As the duration of the reaction increases, cubic-NaYF4:Er(3+)/Yb(3+) nanocrystals are transformed to uniform hexagonal-NaYF4:Er(3+)/Yb(3+) microprisms because of the enhanced reaction kinetics. Bright upconverted emission from the NaYF4:Er(3+)/Yb(3+) crystal, obtained by the efficient two-photon excitation, is related to crystal structure and morphology. The hexagonal microprisms exhibit better upconversion and are employed in security applications.

Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. From the Clinical EditorThis study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

Picosecond excitation for reduction of photolytic effects in two-photon laser-induced fluorescence of CO

Two-photon laser-induced fluorescence for detection of carbon monoxide (CO) frequently shows interferences by emission from photolytically produced C2 radicals encountered under fuel-rich combustion conditions. Reduced C2 interference for excitation with laser pulses in the picosecond regime is here demonstrated by comparison with excitation using nanosecond pulses for measurements in laminar premixed ethene–air flames. Compared with nanosecond pulses of 8ns duration and 4mJ pulse energy, picosecond pulses of 80ps duration and around 0.5mJ pulse energy gave ∼10 times higher peak power, which allowed for efficient CO excitation and resulted in stronger signal with lower C2 interference. CO fluorescence with picosecond excitation showed a linear to quadratic power dependence, indicating photoionization, whereas a more quadratic dependence was found for the C2 interference. A sub-nanosecond effective lifetime of CO resulted in a rapid fluorescence decay compared with C2 and allowed for efficient reduction in C2 interference by minimizing the detection gate. In addition, interference compensation using time-resolved detection could be demonstrated. Altogether, picosecond pulses provide efficient two-photon excitation of CO in terms of signal strength as well as reduced C2 interference.

量子相関光子による効率的2光子励起の条件

量子相関光子による効率的2光子励起の条件

Efficient selective two-photon excitation by tailored quantum-correlated photons

We theoretically investigate two-photon excitation by correlated photons with energy anticorrelation. A three-level atomic system is used to evaluate selectivity and efficiency of two-photon excitation. It is shown that tailored squeezing of two-photon probability distribution concurrently enhances excitation efficiency and high-contrast selective excitation, without phase manipulation or pulse-shaping techniques. We also discuss candidates to generate such squeezed photons in real material systems.

Microwave spectroscopy of Ag I atoms in Rydberg states: S, P and D terms

Microwave resonance transitions of Ag I atoms laser excited to Rydberg S, P and D states of the configuration 4d10nℓ, for n = 28–39, were measured with MHz accuracy at frequencies from 76 to 363 GHz. Quantum defects and Rydberg–Ritz coefficients were determined based on 47 new resonances, together with doublet fine-structure splittings of P and D terms. An efficient single-laser two-photon excitation path 52S1/2 → 282D5/2 was discovered based on a coincidence with twice the term difference of the transition 52S1/2 → 52P3/2.

Multi-photon photoelectron spectromicroscopy of supported polystyrene spheres

Multi-photon photoemission excited by 100 fs, 400 nm laser pulses leads to an unexpected high contrast in photoelectron images of polystyrene spheres on a platinum substrate. The total, energy-integrated photoelectron yield shows clear signatures of two-photon photoemission from the substrate while photoemission from polystyrene is dominated by one-photon processes for low laser power and multi-photon processes for higher laser power. For excitation with UV light from a conventional Hg arc lamp, we observe a marked energy shift of the photoelectron spectrum of polystyrene with respect to that of the substrate. This shift is related to the different surface potentials of the conductive substrate and the dielectric spheres in the strong electric field of the objective lens of the microscope. Laser illumination causes photoconductivity in polystyrene by efficient two-photon excitation of long-lived states and induces a shifting of the surface potential of the polystyrene spheres. Pump–probe experiments support our conclusion that photoemission from polystyrene takes place from these long-lived intermediate states via a one-photon process for sufficiently low laser power. We suggest that photoelectron spectromicroscopy might be useful as a non-scanning method for fast height profiling of supported dielectric structures.