Harmonic Generation Technique Research Articles

New Strategies for Cellular‐Scale Imaging of Bone and Cartilage

Confocal microscopy can be used to obtain detailed, 3D‐resolved images of cells inside bone and cartilage. However the high scattering coefficients of dense connective tissues often necessitate tissue sectioning for resolving cellular scale detail inside. Multiphoton microscopy enables clearer imaging and greater depth penetration into these tissues and the related technique of second harmonic generation enables acquisition of collagen images at the single‐fibril level in 3D and in some cases in vivo. New improvements in clearing technologies and optical methods such as light sheet microscopy continue to advance the depth in to which dense tissues can be imaged using highly‐specific fluorescent probes. Concurrently, improvements in X‐ray computed tomography, the standard method for 3D medical assessment of bone morphology and lesions, have led to resolutions rivaling those obtained with optical microscopy, in some cases with specifically targeted electron dense contrast agents. Different techniques excel for different specific questions to be addressed within this diverse and powerful imaging landscape.

Complementing Confocal Detection of Antibody‐labeled Lysophosphatidic Acid Receptors in Human Gingivae with Label‐free Second Harmonic Generation Confocal Microscopy Detection of Collagen

We have found that lysophosphatidic acid (LPA) controls the healing and [Ca2+]i responses of human gingival fibroblasts via the LPA1 and LPA3 receptor subtypes, and that LPA3 may modulate their inflammatory responses. We have used immunofluorescence to detect LPA receptor (LPAR) distribution in human gingivae, but that technique does not afford visualization of the state of collagen fiber organization. We hypothesized that using the second harmonic generation (SHG) technique would enable detection of changes in the appearance, collagen fibril density, and fibril organizational structure of human gingiva, and that this technique would complement visualizing LPAR expression by confocal microscopy. Pieces of intact, paraformaldehyde‐fixed, methanol:acetone (1:1) de‐lipidated attached gingivae [~1.25 mm thick (from IRB‐approved, consented donors) normal or with moderate‐severe periodontal disease] were used. Confocal analysis using anti‐LPA1 and anti–LPA3 antibodies confirmed that LPA3 is primarily found in the epithelium, and that nuclear LPA1 expression in the sub‐epithelial connective tissue is greatly diminished in periodontal disease. SHG analysis clearly showed the normal collagen structure and subsequent reduction in the abundance and organization in the collagen fibrils of periodontal gingivae. This multidisciplinary study shows that SHG effectively complements confocal microscopy as a tool to help understand periodontal changes at the level of receptors and collagen organization.

Characterizing Fibrosis in Mouse Kidney Using Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model

Renal tubulointerstitial fibrosis is considered to be the final common pathway for most forms of chronic kidney disease (CKD). Fibrosis has been traditionally characterized by histological studies such as Picro‐Sirius Red (PSR) staining, Masson Trichrome staining or collagen immunohistochemistry. Such methods are limited due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to unilateral ureteral obstruction (UUO), a well‐characterized model of fibrosis. At 21 days, both kidneys were collected. Serial sections of both kidneys were analyzed by PSR staining or FLIM with SHG. Quantification of a whole kidney section from the PSR staining showed 34.32±0.99% area of fibrosis in the injured kidney compared to 5.55±1.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.44±4.21% compared to 2.59±1.98% control kidney). In conclusion, the combined FLIM and SHG images let us establish a criterion for quantitative determination of fibrosis directly from the microscope images.

Tunable enhancement of ferroelectric properties in BaxSr1−xTiO3/La0.7Sr0.3MgO3 heterostructures observed by means of second harmonic generation technique

This work is devoted to research an influence of Ba cation concentration on polarization properties of ferroelectric layer which is included in bilayer structures as functional stratum. We have created bilayer nanocomposites consisting of a ferroelectric Ba(x)Sr(1−x)TiO3 (BST) layer and a ferromagnetic La0.7Sr0.3MnO3 (LSMO) layer with various concentrations of Ba. The epitaxial heterostructure BST/LSMO was fabricated using metal-organic aerosol deposition technique (MAD) on a single-crystal substrate SrTiO3 (001). The structural parameters of the BST/LSMO were defined by X-ray diffraction analysis. The in-plane ferroelectric polarization, measured by means of optical second harmonic generation (SHG) method, was shown to be strongly dependent on the Ba concentration. We consider this effect as structural distortion at room temperatures which close to the structural phase transition and altering the Ba concentration affects the vicinity to the phase transition point.

Non-linear ultrasonic characterisation of heat-damaged cement paste

This paper focuses on the experimental characterisation of the elastic properties of sound and heat-damaged cement paste for different water contents: w/c = 0.3, 0.4 and 0.5. Damage is first assessed from mass density and wave velocity measurements. Linear acoustic methods were carried out but they did not permit an efficient evaluation of the earliest stages of damage. The goal of this research is to assess damage-induced non-linearities in the behaviour of heat-damaged cement paste. The method is based on the higher harmonic generation (HHG) technique. An empirical law is proposed to relate the non-linear elastic parameter β to the cement water content and to temperature. This is expected to give new insights in the field of non-destructive testing (NDT) methods; in particular, non-linear acoustic methods are expected to enable early failure diagnosis and thus reducing maintenance costs of infrastructure.

Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model

2410-Pos Board B547 Characterizing Fibrosis in Mouse Kidney using Auto Fluorescence Flim and SHG in UUO Model Moshe Levi 1 , Evgenia Dobrinskikh 1 , Suman Ranjit 2 , John Montford 1 , Alexander Dvornikov 3 , Allison Lehman 1 , Seth Furgeson 1 , Raphael Nemenoff 1 , Enrico Gratton 2 . Medicine, U of Colorado, Aurora, CO, USA, 2 Biomedical Engineering, U of California, Irvine, CA, USA, 3 Biomedical Engineering, U of California, Aurora, CA, USA. Chronic kidney disease (CKD) arises from a diverse list of renal injuries. Renal fibrosis is considered to be the final common pathway for most forms of CKD and involves glomerular sclerosis and/or interstitial fibrosis. Because of that there is great interest in identifying renal fibrosis in the early stages of CKD to prevent progression. Unilateral ureteral obstruction (UUO) is a well- described model of CKD and renal fibrosis. Kidney injury and fibrosis usually are assessed by histology. Histological methods include Picro-Sirius Red stain- ing, Masson’s Trichrome staining or immunohistochemistry for collagen iso- forms. Quantitation of fibrosis using histologic techniques may be difficult due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to UUO of right kidney. At 21 days, both kidneys were collected, fixed, and paraffin-embedded. The uninjured left kidney was used as a control. Serial sections of both kidneys were analyzed by Picro-Sirius Red staining or FLIM with SHG. Quantification of a whole kidney section from the Picro- Sirius Red staining showed 34.3250.99% area of fibrosis in the left kidney compare to 5.5551.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.4454.21% compared to 2.5951.98% control kidney). Finally the combined FLIM and SHG images let us create a criterion to separate fibrosis directly from the microscope images.

Functionalized Ruthenium Dialkynyl Complexes with High Second-Order Nonlinear Optical Properties and Good Potential as Dye Sensitizers for Solar Cells

Various dipolar π-delocalized Ru(II) dialkynyl complexes were prepared and characterized. Their second-order nonlinear optical (NLO) properties were investigated by the electric-field-induced second harmonic generation (EFISH) technique working in CH2Cl2 solution with an incident wavelength of 1907 nm, whereas the dipole moments were determined by density functional theory (DFT) calculations. All the investigated complexes are characterized by a negative value of μβ1.907 EFISH, in agreement with a negative value of Δμeg (difference of the dipole moment in the excited and ground state) upon excitation. Their second-order NLO response can be easily modulated by the nature of the alkynyl substituents. Besides, the most promising “push–pull” ruthenium diacetylide complex, adequately functionalized for anchoring to TiO2, was tested as photosensitizer in dye-sensitized solar cells (DSSCs).

The effect of chromophores concentration on the nonlinear optical activity of methacrylic copolymers with azochromophores in the side chain

Quadratic nonlinear-optical characteristics of thin films based on methacrylic copolymers with of chromophore-containing monomers incorporated at various concentrations are measured by Second Harmonic Generation technique. Optimal chromophores content is obtained to be about 17 mol%, rather high values of nonlinear-optical coefficient, d33, up to 60 pm/V, are determined.

Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics.

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.

Synthesis, crystal structure and properties of alluaudite-like triple molybdate Na25Cs8Fe5(MoO4)24

A new triple molybdate Na25Cs8Fe5(MoO4)24 was synthesized using solid state reactions and studied with X-ray powder diffraction, second harmonic generation (SHG) technique, differential scanning calorimetry, Mössbauer and dielectric impedance spectroscopy. Single crystals of Na25Cs8Fe5(MoO4)24 were obtained and its structure was solved (the space group P1¯, a=12.5814(5), b=13.8989(5), c=28.4386(9)Å, α=90.108(2), β=90.064(2), γ=90.020(2)°, V=4973.0(3)Å3, Z=2, R=0.0440). Characteristic features of the structure are polyhedral layers composed of pairs of edge-shared FeO6 and (Fe, Na)O6 octahedra, which are connected by bridging МоО4 tetrahedra. The layers share common vertices with bridging МоО4 tetrahedra to form an open 3D framework with the cavities occupied by the Cs+ and Na+ cations. The compound undergoes first-order phase transformation at 642K and above this phase transition, electrical conductivity reaches 10−3–10−2Scm−1. Thus, Na25Cs8Fe5(MoO4)24 may be considered as a promising compound for developing new materials with high ionic conductivity.

Investigation of carrier transit motion in PCDTBT by optical SHG technique

We analyze the carrier transit behavior in poly[N-9’-heptadecanyl-2,7-carbazole-alt-5,5-(4’,7’-di-2-thienyl-2’,1’,3’-benzothiadiazole)] (PCDTBT), which has been reported as a donor material for efficient bulk heterojunction photovoltaic devices. The transfer and transient carrier mobilities in the PCDTBT thin films have been measured and analyzed. The transfer mobility has been measured by the transfer curve of the OFET, whereas the transient mobility is recorded using a time-resolved electric field-induced optical second harmonic generation (TRM-SHG) technique. Using the TRM-SHG technique, the dynamic motion of the charge carriers in the PCDTBT thin films has been directly visualized. We anticipate that the analysis of the carrier motion by TRM-SHG will be effective for the understanding of carrier behavior in PCDTBT thin film and will help to make further improvements in the efficiency of the PCDTBT-based photovoltaic devices.

Highly coherent vacuum ultraviolet radiation at the 15th harmonic with echo-enabled harmonic generation technique

Measurements of highly coherent vacuum ultraviolet radiation are produced with the echo-enabled harmonic generation technique.

Stable Scanning of Dressing Fields on Multiwave Mixing in Optical Ring Cavity

We investigate a novel detection technique to study the coherent control of dressed multiwave mixing (MWM) process. A stable laser dressing field with a wavelength of 480 nm is realized via the second harmonic generation technique. By scanning such dressing field, stable Rydberg MWM signals with high signal-to-noise ratio are observed. With the MWM signals oscillated in a stabilized three-mirror cavity and cavity dressing field scanned, dressed MWM cavity modes are obtained. Thanks to the stable 480 nm and cavity dressing fields, the long-term drift of dressed MWM in cavity can be dramatically reduced to <4 MHz from 100 MHz.

Excited-state dynamics of rhodamine 6G in aqueous solution and at the dodecane/water interface.

The excited-state dynamics of rhodamine 6G (R6G) has been investigated in aqueous solution using ultrafast transient absorption spectroscopy and at the dodecane/water interface using the femtosecond time-resolved surface second harmonic generation (SSHG) technique. As the R6G concentration exceeds ca. 1 mM in bulk water, both R6G monomers and aggregates are excited to a different extent when using pump pulses at 500 and 530 nm. The excited-state lifetime of the monomers is shortened compared to dilute solutions because of the occurrence of excitation energy transfer to the aggregates, which themselves decay nonradiatively to the ground state with a ca. 70 ps time constant. At the dodecane/water interface, both monomers and aggregates contribute to the SSHG signal to an extent that depends on the bulk concentration, the pump and probe wavelengths, and the polarization of probe and signal beams. The excited-state lifetime of the monomers at the interface is of the order of a few picoseconds even at bulk concentrations where it is as large as several nanoseconds. This is explained by the relatively high interfacial affinity of R6G that leads to a large interfacial concentration, favoring aggregation and thus rapid excitation energy transfer from monomers to aggregates.

Label‐free imaging of the regenerating human periodontal ligament and gingival tissue using second harmonic generation confocal microscopy (LB41)

Surgical treatment interventions for treating periodontal disease all disrupt the collagenous structures (gingiva and periodontal ligament) that form the soft‐tissue supporting structures of teeth. We hypothesized that using the second harmonic generation (SHG) technique would enable detection of changes in the appearance, fibril density, and fibril organizational structure of the human periodontal ligament and gingiva during the regenerative process taking place following periodontal surgery. De‐identified archival unstained,de‐paraffinized histological sections (4μM) of teeth with surrounding hard and soft tissues (from normal or various stages of post‐surgical regenerating periodontal structures) were used. It was found that SHG clearly showed the normal organization and the subsequent changes in the collagen fibrils in normal vs.regenerating periodontal soft tissues. This multidisciplinary study shows that SHG can be effectively used to study the organization of collagen in the normal and regenerating periodontal ligament and surrounding gingival tissue. SHG could constitute a potentially useful technique for determining the efficacy of periodontal regenerative strategies, and for assessing / studying peri‐implantitis using punch biopsies.Grant Funding Source: INBRE/BRIN P20GM103427

Monitoring fibrous scaffold guidance of three-dimensional collagen organisation using minimally-invasive second harmonic generation.

The biological and mechanical function of connective tissues is largely determined by controlled cellular alignment and therefore it seems appropriate that tissue-engineered constructs should be architecturally similar to the in vivo tissue targeted for repair or replacement. Collagen organisation dictates the tensile properties of most tissues and so monitoring the deposition of cell-secreted collagen as the construct develops is essential for understanding tissue formation. In this study, electrospun fibres with a random or high degree of orientation, mimicking two types of tissue architecture found in the body, were used to culture human fibroblasts for controlling cell alignment. The minimally-invasive technique of second harmonic generation was used with the aim of monitoring and profiling the deposition and organisation of collagen at different construct depths over time while construct mechanical properties were also determined over the culture period. It was seen that scaffold fibre organisation affected cell migration and orientation up to 21 days which in turn had an effect on collagen organisation. Collagen in random fibrous constructs was deposited in alternating configurations at different depths however a high degree of organisation was observed throughout aligned fibrous constructs orientated in the scaffold fibre direction. Three-dimensional second harmonic generation images showed that deposited collagen was more uniformly distributed in random constructs but aligned constructs were more organised and had higher intensities. The tensile properties of all constructs increased with increasing collagen deposition and were ultimately dictated by collagen organisation. This study highlights the importance of scaffold architecture for controlling the development of well-organised tissue engineered constructs and the usefulness of second harmonic generation imaging for monitoring collagen maturation in a minimally invasive manner.

Measuring selective estrogen receptor modulator (SERM)-membrane interactions with second harmonic generation.

The interaction of selective estrogen receptor modulators (SERMs) with lipid membranes has been measured at clinically relevant serum concentrations using the label-free technique of second harmonic generation (SHG). The SERMs investigated in this study include raloxifene, tamoxifen, and the tamoxifen metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. Equilibrium association constants (Ka) were measured for SERMs using varying lipid compositions to examine how lipid phase, packing density, and cholesterol content impact SERM-membrane interactions. Membrane-binding properties of tamoxifen and its metabolites were compared on the basis of hydroxyl group substitution and amine ionization to elucidate how the degree of drug ionization impacts membrane partitioning. SERM-membrane interactions were probed under multiple pH conditions, and drug adsorption was observed to vary with the concentration of soluble neutral species. The agreement between Ka values derived from SHG measurements of the interactions between SERMs and artificial cell membranes and independent observations of the SERMs efficacy from clinical studies suggests that quantifying membrane adsorption properties may be important for understanding SERM action in vivo.

Second harmonic generation measurements at high pressures on powder samples

This article reviews the most recent results con- cerning second harmonic generation (SHG) experiments of non-phase matchable and phase matchable powder samples at high pressures and explains the pressure de- pendence of the intensity of the SHG signal by correlat- ing it to the ratio between the average coherence length and the average particle size. The examples discussed here include pressure-induced structural changes in quartz, ZnO, ice VII and KIO3. It is shown that the second harmonic generation technique is a unique tool for the detection of pressure-induced structural phase transi- tions. It is laboratory based and allows fast measure- ments. It is complementary to X-ray diffraction and pro- vides additional information about the presence of an inversion center for unknown or controversially dis- cussed structures at high pressure.

An acido-triggered reversible luminescent and nonlinear optical switch based on a substituted styrylpyridine: EFISH measurements as an unusual method to reveal a protonation–deprotonation NLO contrast

Diphenyl-(4-{2-[4-(2-pyridin-4-yl-vinyl)-phenyl]-vinyl}-phenyl)-amine (DPVPA) constitutes a novel acido-triggered reversible luminescent and nonlinear optical switch. Remarkably, for the first time the Electric-Field Induced Second Harmonic generation (EFISH) technique is used to reveal a protonation-deprotonation NLO contrast.

A study on surface symmetry and interfacial enhancement of SrTiO3 by second harmonic generation

The symmetry of the surfaces of SrTiO3 and slightly Nb-doped SrTiO3 crystals was investigated by the optical reflected second harmonic generation technique. The good agreement between experimental and theoretical results of the second harmonic intensity dependence on the azimuth angle indicates that the SrTiO3 (001) surface is with 4mm symmetry and the Nb-doped SrTiO3 (111) surface with 3m symmetry. The measurements of the polarization dependent second harmonic intensity confirm that conclusion. The enhancement of the surface polarization in the structure of SrTiO3 capped La0.9Sr0.1MnO3 films compared with that in the La0.9Sr0.1MnO3 films has been obtained.