Polarization-resolved Second Harmonic Generation Research Articles

Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

Polarization-resolved super-resolution second-harmonic generation imaging based on multifocal structured illumination microscopy.

Polarization-resolved second-harmonic generation (PSHG) microscopy is widely used in investigating the structural and morphological alterations of collagen. However, the resolution of second-harmonic generation (SHG) imaging remains constrained by optical diffraction, resulting in the polarization extraction of collagen characteristics from the average properties of collagen fibers. In this study, multifocal structured illumination microscopy (MSIM) was combined with PSHG to achieve polarization-resolved super-resolution imaging of second-harmonic generation signals. For the first time to our knowledge, periodic structures with an average pitch of 277 nm were observed in mouse tail tendons using optical microscopy, and the orientation angle of fibrils within each period was found to exhibit an alternating arrangement along the axis in a regular pattern.

Global imaging for polarization resolved second harmonic generation of WS2 monolayers

Second-harmonic generation (SHG) is a nonlinear optical effect enhanced by broken crystallin symmetry and is very sensitive to electronic structures. SHG has recently been applied to two-dimensional transition metals dichalcogenides (2D-TMDs). 2D-TMDs have been the focus of much recent research due to their ultrathin scale, high quantum confinement, and energy gap toning ability that results in unique linear or nonlinear optical and electrical properties. WS2 monolayers are well-known 2D TMDs with strong SHG. They have attracted a great deal of interest due to their potential applications in fundamental material characterization and nanophotonic device development. In this study, we grew WS2 monolayers using the chemical vapor deposition (CVD) technique and characterized them with Raman and photoluminescence (PL) spectroscopy. The intense direct excitonic peak A at 630 nm was identified in the PL spectra, while the Raman spectra exhibited the two distinctive modes A1g (at 418 cm−1) and E2g (at 356 cm−1). The monolayers were pumped by an 830 nm circularly polarized and defused pulsed laser to produce the SHG image. global one-shot SH images for different growth shapes were obtained and crystalline domains were identified using polarization-resolved second-harmonic generation imaging (PRSHGI). The defect level was observed to clearly enhance the SHG signal following the increase in broken crystalline centrosymmetric and relaxing the optical selection roles at the valley degree of freedom.

Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy

Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0–F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.

Polarization-resolved second-harmonic generation: a Jones matrix approach

Polarization-resolved second-harmonic generation: a Jones matrix approach

Effect of out of plane orientation on polarization second harmonic generation of single collagen fibrils.

Second harmonic generation (SHG) microscopy has emerged as a powerful technique for visualizing collagen organization within tissues. Amongst the many advantages of SHG is its sensitivity to collagen nanoscale organization, and its presumed sensitivity to the relative out of plane polarity of fibrils. Recent results have shown that circular dichroism SHG (CD-SHG), a technique that has been commonly assumed to reveal the relative out of plane polarity of collagen fibrils, is actually insensitive to changes in fibril polarity. However, results from another research group seem to contradict this conclusion. Both previous results have been based on SHG imaging of collagen fibrils within tissues, therefore, to gain a definitive understanding of the sensitivity of SHG to relative out of plane polarity, the results from individual fibrils are desirable. Here we present polarization resolved SHG microscopy (PSHG) data from individual collagen fibrils oriented out of the image plane by buckling on an elastic substrate. We show through correlation with atomic force microscopy measurements that SHG intensity can be used to estimate the out of plane angle of individual fibrils. We then compare the sensitivity of two PSHG techniques, CD-SHG and polarization-in, polarization-out SHG (PIPO-SHG), to the relative out of plane polarity of individual fibrils. We find that for single fibrils CD-SHG is insensitive to relative out of polarity and we also demonstrate the first direct experimental confirmation that PIPO-SHG reveals the relative out of plane polarity of individual collagen fibrils.

Calibrated χ(2)-tensor polarization-resolved second harmonic generation microscopy for precise tissue imaging

Polarization-resolved second harmonic generation (P-SHG) microscopy is commonly used to analyze the second-order susceptibility, χ(2), tensor, which enables the calculation of the molecular structure of harmonophores. However, despite extensive research on type I collagen, the measured χ(2) ratios vary considerably among published values, which raises the question of whether P-SHG imaging is universally applicable to all tissues containing harmonophores. In this work, we propose that the deviation of χ(2) ratios is primarily due to ignoring the molecular tilt angle and chirality in image analysis. To confirm our hypothesis, we present an analytical model based on C6 symmetry that takes into account these two factors. We also introduce an imaging scheme that splits SHG into X- and Y-polarized image components for χ(2) tensor analysis. Our approach effectively improves the precision of determining χ(2) ratios, depending on how much the two factors affect the P-SHG signals.

Characterization of pathological stomach tissue using polarization-sensitive second harmonic generation microscopy.

Alterations in collagen ultrastructure between human gastric adenocarcinoma and normal gastric tissue were investigated using polarization-resolved second harmonic generation (PSHG) microscopy. Cylindrical and trigonal symmetries were assumed to extract quantitative PSHG parameters, ρ, κ and S, from each image pixel. Statistically significant variations in these values were observed for gastric adenocarcinoma, indicating a higher disorder of collagen. Numerical focal volume simulations of crossing fibrils indicate increased S parameter is due to more intersecting collagen fibrils of varying diameters. These parameters were also able to distinguish between different grades of gastric adenocarcinoma indicating that PSHG may be useful for automated cancer diagnosis.

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy

A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.

Revealing the Structural Organization of Gamma-irradiated Starch Granules Using Polarization-resolved Second Harmonic Generation Microscopy.

Starch is a semi-crystalline macromolecule with the presence of amorphous and crystalline components. The amorphous amylose and crystalline amylopectin regions in starch granules are susceptible to certain physical modifications, such as gamma irradiation. Polarization-resolved second harmonic generation (P-SHG) microscopy in conjunction with SHG-circular dichroism (CD) was used to assess the three-dimensional molecular order and inherent chirality of starch granules and their reaction to different dosages of gamma irradiation. For the first time, the relationship between starch achirality (χ21/χ16 and χ22/χ16) and chirality (χ14/χ16) determining susceptibility tensor ratios has been elucidated. The results showed that changes in the structure and orientation of long-chain amylopectin were supported by the decrease in the SHG anisotropy factor and the χ22/χ16 ratio. Furthermore, SHG-CD illustrated the molecular tilt angle by revealing the arrangement of amylopectin molecules pointing either upward or downward owing to molecular polarity.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

Emergent second-harmonic generation in van der Waals heterostructure of bilayer MoS2 and monolayer graphene.

Van der Waals (vdW) stacking of two-dimensional (2D) materials to create artificial structures has enabled remarkable discoveries and novel properties in fundamental physics. Here, we report that vdW stacking of centrosymmetric 2D materials, e.g., bilayer MoS2 (2LM) and monolayer graphene (1LG), could support remarkable second-harmonic generation (SHG). The required centrosymmetry breaking for second-order hyperpolarizability arises from the interlayer charge transfer between 2LM and 1LG and the imbalanced charge distribution in 2LM, which are verified by first-principles calculations, Raman spectroscopy, and polarization-resolved SHG. The strength of SHG from 2LM/1LG is of the same order of magnitude as that from the monolayer MoS2, which is well recognized with strong second-order nonlinearity. The emergent SHG reveals that the interlayer charge transfer can effectively modify the symmetry and nonlinear optical properties of 2D heterostructures. It also indicates the great opportunity of SHG spectroscopy for characterizing interlayer coupling in vdW heterostructures.

Fast polarization-sensitive second-harmonic generation microscopy based on off-axis interferometry.

We propose polarization-sensitive second-harmonic generation microscopy based on off-axis interferometry (OI-PSHG) by recording the complex field of a wide-field second-harmonic generation (SHG) image and performing polarization measurements. With the ability to record the SHG signals associated with different positions simultaneously, the proposed method exhibits a higher imaging frame rate than raster scanning-based SHG microscopy. The molecular orientation (in terms of their symmetric axis) of tendon collagen fibrils and myosin in muscle is resolved in three dimensions from a subset of polarization-resolved SHG holograms. With the present configuration, it takes approximately 0.01 s to acquire an image with 128 × 128 pixels, which is mainly limited by the excitation power density for wide-field illumination. For the same data throughput using pixel-by-pixel scanning, 0.16-s-long acquisition is required, with the pixel dwell time of 10 µs. Offering the ability to perform wide-field imaging and polarization measurements, the present work lays the foundation for fast SHG microscopy using complex deconvolution and harmonic tomography.

Wavelength dependence of polarization-resolved second harmonic generation from ferroelectric SnS few layers

Two-dimensional group-IV monochalcogenides such as GeS, GeSe, SnS, and SnSe were theoretically predicted as multiferroic materials with two or more ferroic properties. However, most of their bulk crystals are stacked layer by layer with an antiferroelectric manner, which lose the macroscopic in-plane ferroelectricity. In this work, we studied SnS in which the layers are stacked in a ferroelectric manner both experimentally and theoretically. We utilized polarization-resolved second harmonic generation (SHG) microscopy to investigate numerous flakes of ferroelectric SnS few layers on mica substrates. We found the SHG polar patterns dramatically varied in the range of 800 nm and 1000 nm due to the frequency-dependent SHG susceptibilities. First-principles calculations have been performed to study the frequency-dependent and layer-dependent SHG susceptibilities in the ferroelectric SnS with AA and AC stacking orders. The variation trend of calculated SHG polar patterns as a function of frequency agrees well with that of the experimental results. Since polarization-resolved SHG is a noncontact and nondestructive technique to determine the crystal orientation, understanding of its properties is important, especially for monitoring the transition of different ferroic phases.

Gate-Tunable Helical Currents in Commensurate Topological Insulator/Graphene Heterostructures.

van der Waals heterostructures made from graphene and three-dimensional topological insulators promise very high electron mobilities, a nontrivial spin texture, and a gate-tunability of electronic properties. Such a combination of advantageous electronic characteristics can only be achieved through proximity effects in heterostructures, as graphene lacks a large enough spin-orbit interaction. In turn, the heterostructures are promising candidates for all-electrical control of proximity-induced spin phenomena. Here, we explore epitaxially grown interfaces between graphene and the lattice-matched topological insulator Bi2Te2Se. For this heterostructure, spin-orbit coupling proximity has been predicted to impart an anisotropic and electronically tunable spin texture. Polarization-resolved second-harmonic generation, Raman spectroscopy, and time-resolved magneto-optic Kerr microscopy are combined to demonstrate that the atomic interfaces align in a commensurate symmetry with characteristic interlayer vibrations. By polarization-resolved photocurrent measurements, we find a circular photogalvanic effect which is drastically enhanced at the Dirac point of the proximitized graphene. We attribute the peculiar gate-tunability to the proximity-induced interfacial spin structure, which could be exploited for, e.g., spin filters.

Highly Aligned Bacterial Nanocellulose Films Obtained During Static Biosynthesis in a Reproducible and Straightforward Approach.

Bacterial nanocellulose (BNC) is usually produced as randomly‐organized highly pure cellulose nanofibers films. Its high water‐holding capacity, porosity, mechanical strength, and biocompatibility make it unique. Ordered structures are found in nature and the properties appearing upon aligning polymers fibers inspire everyone to achieve highly aligned BNC (A‐BNC) films. This work takes advantage of natural bacteria biosynthesis in a reproducible and straightforward approach. Bacteria confined and statically incubated biosynthesized BNC nanofibers in a single direction without entanglement. The obtained film is highly oriented within the total volume confirmed by polarization‐resolved second‐harmonic generation signal and Small Angle X‐ray Scattering. The biosynthesis approach is improved by reusing the bacterial substrates to obtain A‐BNC reproducibly and repeatedly. The suitability of A‐BNC as cell carriers is confirmed by adhering to and growing fibroblasts in the substrate. Finally, the thermal conductivity is evaluated by two independent approaches, i.e., using the well‐known 3ω‐method and a recently developed contactless thermoreflectance approach, confirming a thermal conductivity of 1.63 W mK−1 in the direction of the aligned fibers versus 0.3 W mK−1 perpendicularly. The fivefold increase in thermal conductivity of BNC in the alignment direction forecasts the potential of BNC‐based devices outperforming some other natural polymer and synthetic materials.

Second harmonic generation microscopy of otoconia.

The origin of second harmonic generation (SHG) signal in otoconia was investigated. SHG signal intensity from otoconia was compared to pure calcite crystals, given calcite is the primary component of otoconia and is known to emit surface SHG. The SHG intensity from calcite was found to be ∼41× weaker than the SHG intensity from otoconia signifying that the SHG signal from otoconia is likely generated from the organic matrix. Furthermore, the SHG intensity from otoconia increased when treated with a chelating agent known to dissolve calcite which confirms that calcite is not the source of SHG. Additionally, polarization-resolved SHG microscopy imaging revealed that the arrangement of the SHG emitters is radial and can form highly ordered domains.

Nonlinear Optical Imaging of In‐Plane Anisotropy in Two‐Dimensional SnS (Advanced Optical Materials 10/2022)

This cover illustration represents a laser field interacting with an ultrathin SnS crystal and the production of a second harmonic generation (SHG) field. Two representative polar diagrams of the polarization-resolved SHG intensity from different SnS crystals, and the fitting with the nonlinear optics model, are also shown, enabling the extraction of quantitative information on the in-plane anisotropy in SnS. For further details see article number 2102776 by Emmanuel Stratakis and co-workers.

Boosting Second-Harmonic Generation in Monolayer Rhenium Disulfide by Reversible Laser Patterning

Active modification and control of transition metal dichalcogenides (TMDs) properties are highly desirable for next-generation optoelectronic applications. In particular, controlling one of the most important characteristics of TMDs─their crystal structure and symmetry─may open means for manipulating their optical nonlinearities and electrical transport properties. Here, we show that a monolayer ReS2, which does not have a broken inversion symmetry due to its stable 1T′-distorted phase and correspondingly shows only weak second-harmonic generation (SHG), can produce a significantly enhanced (∼2 orders of magnitude) SHG upon reversible laser patterning. This enhancement can be explained by the laser-induced transition from centrosymmetric 1T′ to noncentrosymmetric 2H-phase. This hypothesis is confirmed by polarization-resolved SHG measurements, which reveal a gradual change from the 2-fold to 6-fold symmetry profiles upon laser patterning. Additionally, we found that laser patterning of the bilayer ReS2 samples, contrary to the monolayers, leads to a substantially reduced SHG signal. This result corroborates the 1T′-to-2H laser-induced phase transition. Finally, we show that the laser-induced patterning is reversible by heat. These results open a possibility to actively and reversibly control the crystal structure of mono- and few-layer ReS2 and thus its optical and electronic properties.

Polarization-Resolved Extreme-Ultraviolet Second-Harmonic Generation from LiNbO3

Second harmonic generation (SHG) spectroscopy ubiquitously enables the investigation of surface chemistry, interfacial chemistry, as well as symmetry properties in solids. Polarization-resolved SHG spectroscopy in the visible to infrared regime is regularly used to investigate electronic and magnetic order through their angular anisotropies within the crystal structure. However, the increasing complexity of novel materials and emerging phenomena hampers the interpretation of experiments solely based on the investigation of hybridized valence states. Here, polarization-resolved SHG in the extreme ultraviolet (XUV-SHG) is demonstrated for the first time, enabling element-resolved angular anisotropy investigations. In noncentrosymmetric LiNbO_{3}, elemental contributions by lithium and niobium are clearly distinguished by energy dependent XUV-SHG measurements. This element-resolved and symmetry-sensitive experiment suggests that the displacement of Li ions in LiNbO_{3}, which is known to lead to ferroelectricity, is accompanied by distortions to the Nb ion environment that breaks the inversion symmetry of the NbO_{6} octahedron as well. Our simulations show that the measured second harmonic spectrum is consistent with Li ion displacements from the centrosymmetric position while the Nb─O bonds are elongated and contracted by displacements of the O atoms. In addition, the polarization-resolved measurement of XUV-SHG shows excellent agreement with numerical predictions based on dipole-induced SHG commonly used in the optical wavelengths. Our result constitutes the first verification of the dipole-based SHG model in the XUV regime. The findings of this work pave the way for future angle and time-resolved XUV-SHG studies with elemental specificity in condensed matter systems.