Use Of Third-harmonic Generation Research Articles

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.

The third-order nonlinear optical susceptibility of gold

We critically analyze reported measured values of the third-order nonlinear optical susceptibility χ(3) of bulk gold. Reported values of this quantity span a range of more than three orders of magnitude. Much of this variation results from the use of different measurement procedures which are sensitive to different contributions to the nonlinear optical response. For example, values measured through use of third-harmonic generation or non-degenerate four-wave mixing tend to be significantly lower than those obtained from measurements of the intensity-dependent refractive index. We ascribe this behavior to the fact that the first two processes respond only to “instantaneous” nonlinearities, whereas the nonlinear refractive index has a contribution from the much stronger but much slower “hot electron,” or “Fermi-smearing” mechanism, which has a response time of the order of picoseconds. The data also reveal that the hot-electron contribution has a strong dependence on laser wavelength, because of the turn-on of the 5d to 6sp transition at about 550nm. It is hoped that the compilation presented here will prove useful in establishing what value of χ(3) is most appropriate for adoption under various laboratory conditions.

Ultrafast Modulation of the Plasma Frequency of Vertically Aligned Indium Tin Oxide Rods

Light-matter interaction at the nanoscale is of particular interest for future photonic integrated circuits and devices with applications ranging from communication to sensing and imaging. In this Letter a combination of transient absorption (TA) and the use of third harmonic generation as a probe (THG-probe) has been adopted to investigate the response of the localized surface plasmon resonances (LSPRs) of vertically aligned indium tin oxide rods (ITORs) upon ultraviolet light (UV) excitation. TA experiments, which are sensitive to the extinction of the LSPR, show a fluence-dependent increase in the frequency and intensity of the LSPR. The THG-probe experiments show a fluence-dependent decrease of the LSPR-enhanced local electric field intensity within the rod, consistent with a shift of the LSPR to higher frequency. The kinetics from both TA and THG-probe experiments are found to be independent of the fluence of the pump. These results indicate that UV excitation modulates the plasma frequency of ITO on the ultrafast time scale by the injection of electrons into, and their subsequent decay from, the conduction band of the rods. Increases to the electron concentration in the conduction band of ∼13% were achieved in these experiments. Computer simulation and modeling have been used throughout the investigation to guide the design of the experiments and to map the electric field distribution around the rods for interpreting far-field measurement results.

Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy

Non-linear optical microscopy methods can characterize over time multiple functional properties of engineered tissues during development. Here, we demonstrate how the combined use of third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) imaging can provide direct quantitative biomarkers of adipogenic stem cell differentiation and metabolic state, respectively. Specifically, we imaged over nine weeks silk scaffolds embedded with human mesenchymal stem cells and exposed to either propagation (PM) or adipogenic differentiation media (AM). THG was employed to visualize the formation of lipid droplets. 2PEF was used to assess the metabolic state of the cells through the redox ratio defined based on the endogenous FAD and NADH fluorescence. The redox ratio of cells in the AM scaffold was significantly lower than that in the PM scaffold during week 5 and 9, and correlated with significant increases in lipid-to-cell volume ratio, and number and size of lipid droplets in the AM scaffold. These findings indicate that the decrease in redox ratio during adipogenic differentiation is associated with fatty acid synthesis and lipid accumulation. Our methods therefore enabled us to identify and measure dynamic correlations between lipid droplet formation and cell metabolic state, while providing insight on the spatial heterogeneity of the observed signals.

Synthesis, characterization and third-order non-linear optical properties of novel fluorene monomers and their cross-conjugated polymers

Abstract We designed and synthesized two novel fluorene monomers of D – A – D (donor–acceptor–donor) type ( M1 and M2 ), and their two corresponding polymers ( PM1 and PM2 ) and a copolymer ( CPM ). These high molecular weight, film-forming polymers were obtained from metal-free, superacid-catalyzed reactions of the monomers with N -phenylisatin. The cubic NLO response ( χ (3) ) for these new compounds, in solid thin films, was measured through the use of third-harmonic generation (THG) Maker-Fringes technique at IR wavelengths given values of the order of 10 −12 esu from which, the corresponding second hyperpolarizabilities ( γ ) were estimated to be of the order of 10 −33 esu for monomers and 10 −31 esu for polymers. Second hyperpolarizabilities have also been estimated theoretically at B3LYP/6-31G(d) level of theory in gas phase and related with the electronic structure of the synthesized molecules.

Toward practical application of cholesteric liquid crystals to tunable lasers

To realize practical application of cholesteric liquid crystals (CLCs) to photonic tunable laser devices, two efforts have been made: (1) photopolymerization and (2) use of third-harmonic generation (THG) for excitation. The proposed system is based on a spatial optical-pitch gradient in CLCs and doping of two organic dyes (Forster couple) with a concentration gradient. We obtained position-sensitive laser emissions covering the whole visible region (from 480 nm to 670 nm) in photopolymerized CLC films under excitation at 410 nm by an optical parametric oscillator. Preliminary data using more conventional pumping source (THG of a Nd:YAG laser) are also reported.

Direct imaging through scattering media by use of efficient third-harmonic generation in organic materials

We report on real-time, time-gated, direct imaging through scattering media with an attenuation of 14 mean-free paths by use of third-harmonic generation in the eye-safe and telecommunication-compatible near-IR spectral region (1550 nm).

Ultrashort pulses at 130 nm by third-harmonic generation in a magnesium fluoride crystal

The use of third-harmonic generation of visible pulses in a magnesium-fluoride wedge has been proposed in order to obtain ultrashort pulses with photon energy in the vacuum ultraviolet range. Starting from a 200 fs blue pulse with 18 \ensuremath{\mu}J of energy (which is the second harmonic of the IR pulses produced by a regenerative Ti-Sapphire laser system), ${10}^{3}$ photons were obtained at 9.4 eV, with ${10}^{\ensuremath{-}9}$ conversion efficiency. From the measurements it was inferred that ${\ensuremath{\chi}}^{(3)}(\ensuremath{-}3\ensuremath{\omega};\ensuremath{\omega},\ensuremath{\omega},\ensuremath{\omega})\ensuremath{\approx}0.45\ifmmode\times\else\texttimes\fi{}{10}^{\ensuremath{-}22}{\mathrm{m}}^{2}/{\mathrm{V}}^{2}$ for the third-order susceptibility of magnesium fluoride. The simplicity of the arrangement makes this approach ideal for the generation of the ultraviolet probe required by time and angle-resolved photoemission spectroscopy.

Third harmonic generation as a structural diagnostic of ion-implanted amorphous and crystalline silicon

We demonstrate the use of third harmonic generation as a probe of the bulk symmetry properties of semiconductors. Using 20 ns, 1.06 μm laser pulses we have applied this probe to structural diagnostics of (100) annealed and unannealed Si wafers implanted with boron and arsenic ions at dosages above and below the threshold for amorphization. The presence of free carriers with densities up to 1021 cm−3 is found to increase the intensity but not alter the symmetry characteristics of the third harmonic light; the enhancement in the light level is attributed to a change in the reflectivity.