Third Harmonic Generation Signal Research Articles

Nonlinear Holographic All-Dielectric Metasurfaces.

Nonlinear holographic metasurfaces have been intensively studied due to their potentials in practical applications. So far, nonlinear holographic metasurfaces have only been realized with plasmonic nanoantennas, suffering from high absorption loss and low damage threshold. Herein we propose and experimentally demonstrate a novel mechanism for nonlinear holographic metasurfaces. In contrast with conventional studies, the all-dielectric metasurface is composed of C-shaped Si nanoantennas. The incident laser is enhanced by their fundamental resonance, whereas the generated third-harmonic generation (THG) signals are redistributed to the air gap region via the higher order resonance, significantly reducing the absorption loss at short wavelength and resulting in an enhancement factor as high as 230. After introducing abrupt phase changes from 0 to 2π to the C elements, high-efficiency cyan and blue THG holograms have been experimentally generated with the Si metasurface for the very first time. This research shall shed light on the advances of nonlinear all-dielectric metasurfaces.

Detection of the T cell activation state using nonlinear optical microscopy.

The ability to monitor the activation state of T-cells during immunotherapy is of great importance. Although specific activation markers do exist, their abundance and complicated regulation cannot definitely define the activation state of the cells. Previous studies have shown that Third Harmonic Generation (THG) imaging could distinguish between activated versus resting microglia and healthy versus cancerous cells, mainly based on their lipid-body profiles. In the present study, mitogen or antigen-stimulated T-cells were subjected to THG imaging microscopy. Qualitative and quantitative analysis showed statistically significant increase of THG mean area and intensity in activated versus resting T-cells. The connection of THG imaging to chemical information was achieved using Raman spectroscopy, which showed significant differences between the activation processes and controls, correlating of THG signal area with cholesterol and lipid compounds, but not with triglycerides. The obtained results suggested a potential employment of nonlinear microscopy in evaluating of T-cell activation, which is expected to be largely appreciated in the clinical practice.

Third harmonic generation on silicon surface induced by femtosecond laser

The emission spectra of silicon (Si) surface have been studied by a Ti: sapphire femtosecond laser amplification system. The third optical harmonic and plasma emission were detected. The third harmonic generation (THG) is sensitive to the laser fluence and negatively correlated with the fluence. The nonlinear loss on the THG conversion efficiency at high laser fluence is mainly due to the two-photon absorption (TPA) effect. The micro-nanoparticles around the ablated area increase with decreasing laser fluence, which may be one of the reasons for the enhancement of the THG signal. Compared with the theoretical calculation of the THG (266.67 nm), the slight spectral blue-shift (<2 nm) can be attributed to the nonlinear optical processes of the self-phase modulation.

Probing the origin of highly-efficient third-harmonic generation in plasmonic nanogaps.

Plasmonic structures can precisely localize electromagnetic energy to deep subwavelength regions resulting in significant field enhancement useful for efficient on-chip nonlinear generation. However, the origin of large nonlinear enhancements observed in plasmonic nanogap structures consisting of both dielectrics and metals is not fully understood. For the first time, here we probe the third harmonic generation (THG) from a variety of dielectric materials embedded in a nanogap plasmonic cavity. From comprehensive spectral analysis of the THG signal, we conclude that the nonlinear response results primarily from the dielectric spacer layer itself as opposed to the surrounding metal. We achieved a maximum enhancement factor of more than six orders of magnitude compared to a bare gold film, which represents a nonlinear conversion efficiency of 8.78 × 10-4%. We expect this new insight into the nonlinear response in ultrathin gaps between metals to be promising for on-chip nonlinear devices such as ultrafast optical switching and entangled photon sources.

Enhancement of the efficiency of the third harmonic generation process in ZnO:F thin films probed by photoluminescence and Raman spectroscopy

ZnO:F nanostructures at varying fluorine doping concentrations were prepared by spray pyrolysis technique. Effect of fluorine on structure, surface morphology, optical absorption and linear and nonlinear optical properties of the nanostructures were investigated by photoluminescence (PL), raman spectroscopy, THG (third harmonic generation) and Z-Scan analysis. The gaussian fitting on the PL spectra shows different luminescent centers and increase in the intensity. An increase in the PL emission intensity was observed due to the incorporation of fluorine in ZnO lattice which correlates to the formation of various defect states. Prominent emission in the blue, green and violet region along with a weak UV-violet near band edge (NBE) emission was noted in the nanostructures resulted by the Zn and O vacancy defect. ZnO related phonon modes were observed in the raman spectroscopy along with E2 high mode around 439 cm-1 as the signature peak of wurtzite ZnO nanostructures. Z-scan measurements were performed to determine the absorptive and refractive nonlinearity of ZnO:F thin films. The nonlinear optical properties of the ZnO:F thin films found to be enhanced on fluorine incorporation into ZnO lattice. The third order optical susceptibility χ(3) increased from 3.5 × 10-4 esu to 6.17 × 10-3 esu due to the enhancement of electronic transition to different defect levels formed in the films. The third harmonic generation studies on ZnO:F thin films were investigated using Nd:YAG laser at a wavelength of 1064 nm and 8 ns pulse width. The THG signal intensity has shown an increment upon fluorine incorporation. The highest value of THG signal was obtained for the 1% ZnO:F thin films. The enhancement of third harmonic response shows that ZnO:F nanostructures finds immense applications in photonic device applications.

Enhanced visible light generation in an active microcavity via third-harmonic conversion beyond the non-depletion approximation

We explore the possibility of using an active doubly resonant microtoroid resonator to produce high-efficiency third-harmonic generation (THG) by exploiting optical third-order nonlinearity. In a microresonator, the active fundamental mode is coherently driven with a continuous-wave input laser at the telecommunication wavelength (1550 nm), and then, the visible THG signal (517 nm) is monitored via an individual bus waveguide. We thoroughly compare our results with those obtained from the conventional passive (i.e., loss) microtoroid resonator by a systematic analysis and detailed numerical simulations based on the Heisenberg-Langevin equations of motion. It is shown that the achievable THG spectrum features an ultralow critical input power. The THG power transmission can be significantly enhanced by about three orders of magnitude at a low input power of 0.1 μW as compared with the obtained results in the passive microtoroid resonator THG system. Moreover, the THG efficiency can reach up to 100% with optical critical input power as low as a few microwatts. In turn, the analytical expressions of the critical intracavity intensity of the light in the microcavity, the critical input pump power, and the maximum THG efficiency are obtained. The enhanced THG power transmission and high conversion efficiency are attributed to a gain-induced loss compensation in the microtoroid resonator, reducing the effective loss felt by the resonator photons. With state-of-the art technologies in the field of solid-state resonators, including but not limited to microtoroids, the proposed THG scheme is experimentally realizable.

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy.

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Sealing of Immersion Deuterium Dioxide and Its Application to Signal Maintenance for Ex-Vivo and In-Vivo Multiphoton Microscopy Excited at the 1700-nm Window

Excitation at the 1700-nm window is an effective means for extending imaging depth and imaging modalities in multiphoton microscopy (MPM). To enhance multiphoton signal levels and enable deep-tissue penetration, water immersion has to be replaced by deuterium dioxide (D2O) immersion to boost transmittance at the 1700-nm window. The key problem facing this D2O immersion technique is the hygroscopic nature of D2O, which leads to decrease of MPM signals as time lapses. Here, we demonstrate a simple, yet very effective technique to isolate D2 O from the ambient environment, by sealing it with the paraffin liquid. We demonstrate the application of this technique to MPM signal maintenance in both three-photon fluorescence generation in a fluorescent dye and third-harmonic generation (THG) imaging of biological tissue, excited at the 1700-nm window. Ex-vivo imaging results show that during an imaging session of 5 h, multiphoton signals of both modalities can be maintained with no deterioration due to absorption of water vapor from the environment. Furthermore, we demonstrate in-vivo deep-tissue mouse brain imaging using this technique, in which THG signals can be maintained for at least 5 h. This justifies the applicability and effectiveness of our D2O sealing technique for long-span in-vivo imaging.

Bright AIE Nanoparticles with F127 Encapsulation for Deep‐Tissue Three‐Photon Intravital Brain Angiography

Deep-tissue imaging is of great significance to biological applications. In this paper, a deep-red emissive luminogen 2,3-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl) fumaronitrile (TPATCN) with aggregation-induced emission (AIE) feature is prepared. TPATCN molecules were then encapsulated within a polymeric matrix of Pluronic F-127 to form nanoparticles (NPs). TPATCN NPs exhibit bright three-photon fluorescence (3PF) in deep-red region, together with high chemical stability, good photostability, and biocompatibility. They are further utilized for in vivo 3PF imaging of the brain vasculature of mice, under the excitation of a 1550 nm femtosecond laser. A vivid 3D reconstruction of the brain vasculature is then built with a penetration depth of 875 µm, which is the largest in ever reported 3PF imaging based on AIE NPs. After that, by collecting both of the 3PF and third-harmonic generation signals, multichannel nonlinear optical imaging of the brain blood vessels is further realized. These results will be helpful to study the structures and functions of the brain in the future.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.

Large optical third-order nonlinearities in a switchable Prussian blue analogue

We report on the observation of an efficient coherent up-conversion via third harmonic generation (THG) in Rb0.94Mn[Fe(CN)6]0.98.0.3H2O material. Our THG measurements at fundamental wavelengths ranging from 1200 to 2400 nm show that, in this spectral range, the THG signal overcomes the signal generated by frequency doubling. This demonstrates that this material possesses significant third-order nonlinear optical (NLO) responses. Its effective χ(3) value is at least two orders of magnitude greater than α-Quartz. We also demonstrate that this material exhibits a broad thermal hysteresis loop around room temperature, which makes it possible to simultaneously photo-commute its linear and third-order nonlinear optical properties.

Nonlinear imaging microscopy for assessing structural and photochemical modifications upon laser removal of dammar varnish on photosensitive substrates.

The nonlinear optical microscopy (NLM) modalities of Multi-Photon Excited Fluorescence (MPEF) and Third Harmonic Generation (THG) have been combined in this work to characterize as a function of depth with micrometric resolution the type and extent of morphological and photochemical modifications that take place upon ultraviolet (UV) pulsed laser removal of a dammar varnish layer applied on a photosensitive substrate. The latter consists on a layer of the synthetic polymer poly-methyl methacrylate doped with a photosensitizer, the aromatic compound 1,4-di[2-(5-phenyloxazolyl)] benzene, that strongly fluoresces upon UV light illumination. A number of laser conditions for partial or total elimination of the varnish coating were explored, namely different wavelengths (266, 248 and 213 nm) and pulse durations, in the nanosecond, picosecond and femtosecond ranges. Changes in the MPEF signals upon laser ablation of the outermost varnish layer successfully signpost photochemical modifications of the varnish or of the photosensitive under-layer, and their dependence with the laser ablation parameters, i.e., wavelength and pulse duration. In turn, THG signals mark the presence of layer boundaries and the reduction by laser ablation of the thickness of the varnish coating. The obtained MPEF and THG data are complemented by morphological observation by optical microscopy and measurements of laser induced fluorescence and micro-Raman spectra of the samples before and after laser ablation at the selected laser irradiation conditions. The results acquired through these non-destructive NLM imaging techniques serve to understand the phenomena that are induced upon laser ablation and to determine the best operating conditions that ensure controlled removal of the varnish with minimal morphological and chemical modifications to the under-layers. This research is of direct application to the UV pulsed laser cleaning of paintings and demonstrates the potential of NLM as a novel assessment tool for non-destructive, on line monitoring of the laser cleaning process.

Controlling third harmonic generation with gammadion-shaped chiral metamaterials

We theoretically investigated third harmonic generation (THG) from planar chiral metamaterials consisting of a square array of gammadion-shaped metal-insulator-metal multilayered nanostructures. We show that there exists strong circular dichroism (CD) for THG on the proposed chiral metamaterials. We also demonstrate that geometrically mirroring the gammadion -shaped meta-atoms can result in reversal of the THG-CD effect. Based on these CD effects in the optical nonlinear regime, we propose a design of a Fresnel zone plate (FZP) for intense focusing of the THG signals, in which adjacent zones of the FZP consist of gammadions with mirror symmetry and generate circularly polarized THG with opposite handedness. Furthermore, we demonstrate that the relative phase of the THG can be continuously changed by rotating the gammadion around its rotational axis, which could be used in the FZP to control the polarization of the output THG signals.

Distinction between breast cancer cell subtypes using third harmonic generation microscopy.

Third Harmonic Generation (THG) microscopy as a non-invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2-positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer-specific profiles, correlating lipid raft-corresponding spectra to THG signal, associating thus THG to chemical information. THG imaging of a cancer cell.

Comparison of Signal Detection of GaAsP and GaAs PMTs for Multiphoton Microscopy at the 1700-nm window

Multiphoton microscopy (MPM) enables noninvasive optical imaging into the deep tissue in animal models in vivo . Recently, 1700 nm has been demonstrated as a promising excitation window for deeper penetration. Signal depletion is currently the limiting factor for imaging depth at the 1700-nm window in MPM. As a result, efficient signal detection is an effective means to further boost imaging depth. GaAsP and GaAs photomultiplier tubes (PMTs) are commonly used for signal detection in MPM. Here, we demonstrate a comparison investigation of signal detection of a GaAsP and a GaAs PMT for several modalities of MPM at 1700 nm. The two PMTs are identical in structure, connected to the same electronics, and mounted at exactly the same place in the microscope, facilitating a fair comparison. Our results show that with a 1667-nm excitation, GaAsP PMT is more efficient for signal detection of a 3-photon fluorescence of quantum dot Qtracker 655, third-harmonic generation signal, and a 4-photon fluorescence of fluorescein, whereas GaAs PMT is far superior in detecting a second-harmonic generation signal. The measured results are in good agreement with theoretical calculations based on wavelength-dependent cathode radiant sensitivities. We expect that our results will offer guidelines for PMT selection for MPM at the 1700-nm window.

Third order nonlinear optical features of Bi2Fe4O9 multiferroic near antiferromagnetic phase transitions

We have found substantial increase of the infrared third harmonic generation (THG) for the fundamental wavelength 9.4 μm of microsecond CO2 laser for Bi2Fe4O9 microcrystalline powders. Additional enhancement of the THG signal was achieved by use of Er:glass 20 ns laser generating at 1540 nm before the IR probing of the THG. At the same time the analogous effect for the photoinduced two-photon absorption (TPA) was substantially less. The observed effect is explained within a framework of phenomenological description. Principal role here is played by magneto-electric interactions due to interactions of ferroelectric polarizations and ferromagnetic states. The observed effect may be used for creation of IR laser magneto-optical materials.

Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo.

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.

Intrinsic Biomarker for Oxidative Stress by FLIM

Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed single point Raman spectroscopy which revealed typical lipid droplet Raman-bands. LLS FLIM signature was also observed in mouse perigonadal white adipose tissue (WAT) lipid droplets, which co-localized with THG signal. This further confirms the association of LLS to lipid droplets. We show application of LLS as OS biomarker in cardiology and cancer. An increase of LLS was observed in induced-pluripotent-stem-cell-derived cardiomyocytes under OS due to hypoxia. This correlates to the increase in ROS production due to hypoxia. LLS FLIM signature was also observed in melanoma, a ROS driven cancer. LLS were mostly located in the dendritic projection of the melanoma cells. Comparatively, only a small amount of LLS was displayed by melanocytes. This label-free, non-invasive FLIM-phasor method is a promising imaging technique for OS detection in biological systems, especially for in vivo measurements.Work supported in part by NIH grants P50GM076516,P41GM103540 and UH2TR000481-0.

Third-harmonic generation at the interfaces of a cuvette filled with selected organic solvents.

We report on the third-harmonic generation (THG) of tightly focused femtosecond laser pulses at the interfaces of a cuvette filled with organic solvents. Such a system presents four interfaces separating two materials of different refractive indices and third-order nonlinear susceptibilities where the THG takes place because the symmetry around the focus is broken. We selected two cuvettes (silica and B270 crown glass) filled with different organic solvents (acetone, chloroform, and dimethyl sulfoxide) in order to have a variety of interfaces with different linear and nonlinear optical properties. For some of the peaks, the self-focusing modifies the expected cubic power law dependence for THG and as a consequence the four peak profiles may be quite uneven. Although the THG is due to the electronic part of the nonlinear susceptibility, it can suffer from the influence of the self-focusing effect, a Kerr nonlinearity that can have both instantaneous electronic and slow nuclear contributions. This mixture of two distinct third-order nonlinear processes was never considered for such interfaces. All the THG signals could be understood by taking into account the self-focusing effect. Furthermore, the nonlinear refractive indices, n(2), and third-order nonlinear susceptibilities of the solvents, χ((3)), could be determined simultaneously by the THG signals using the cuvette walls as a reference.