Polarization-resolved Second Harmonic Generation Microscopy Research Articles

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.

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.

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.

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.

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.

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.

Influence of hematoxylin and eosin staining on the quantitative analysis of second harmonic generation imaging of fixed tissue sections.

Second harmonic generation (SHG) microscopy has emerged over the past two decades as a powerful tool for tissue characterization and diagnostics. Its main applications in medicine are related to mapping the collagen architecture of in-vivo, ex-vivo and fixed tissues based on endogenous contrast. In this work we present how H&E staining of excised and fixed tissues influences the extraction and use of image parameters specific to polarization-resolved SHG (PSHG) microscopy, which are known to provide quantitative information on the collagen structure and organization. We employ a theoretical collagen model for fitting the experimental PSHG datasets to obtain the second order susceptibility tensor elements ratios and the fitting efficiency. Furthermore, the second harmonic intensity acquired under circular polarization is investigated. The evolution of these parameters in both forward- and backward-collected SHG are computed for both H&E-stained and unstained tissue sections. Consistent modifications are observed between the two cases in terms of the fitting efficiency and the second harmonic intensity. This suggests that similar quantitative analysis workflows applied to PSHG images collected on stained and unstained tissues could yield different results, and hence affect the diagnostic accuracy.

Dual-LC PSHG microscopy for imaging collagen type I and type II gels with pixel-resolution analysis.

Collagen of type I (Col I) and type II (Col II) are critical for cartilage and connective tissues in the human body, and several diseases may alter their properties. Assessing the identification and quantification of fibrillar collagen without biomarkers is a challenge. Advancements in non-invasive polarization-resolved second-harmonic generation (PSHG) microscopy have provided a method for the non-destructive investigation of collagen molecular level properties. Here we explored an alternative polarization modulated approach, dual-LC PSHG, that is based on two liquid crystal devices (Liquid crystal polarization rotators, LPRs) operating simultaneously with a laser scanning SHG microscope. We demonstrated that this more accessible technology allows the quick and accurate generation of any desired linear and circular polarization state without any mechanical parts. This study demonstrates that this method can aid in improving the ability to quantify the characteristics of both types of collagen, including pitch angle, anisotropy, and circular dichroism analysis. Using this approach, we estimated the effective pitch angle for Col I and Col II to be 49.7° and 51.6°, respectively. The effective peptide pitch angle for Col II gel was first estimated and is similar to the value obtained for Col I gel in the previous studies. Additionally, the difference of the anisotropy parameter of both collagen type gels was assessed to be 0.293, which reflects the different type molecular fibril assembly. Further, our work suggests a potential method for monitoring and differentiating different collagen types in biological tissues, especially cartilage or connective tissue.

Quantitative Polarization-Resolved Second-Harmonic-Generation Microscopy of Glycine Microneedles.

Second-harmonic generation (SHG) is a nonlinear optical process that can provide disease diagnosis through characterization of biological building blocks such as amino acids, peptides, and proteins. The second-order nonlinear susceptibility tensor χ(2) of a material characterizes its tendency to cause SHG. Here, a method for finding the χ(2) elements from polarization-resolved SHG microscopy in transmission mode is presented. The quantitative framework and analytical approach that corrects for micrometer-scale morphology and birefringence enable the determination and comparison of the SHG susceptibility tensors of β- and γ-phase glycine microneedles. The maximum nonlinear susceptibility coefficients are d33 = 15 pm V-1 for the β and d33 = 5.9 pm V-1 for the γ phase. The results demonstrate glycine as a useful biocompatible nonlinear material. This combination of the analytical model and polarization-resolved SHG transmission microscopy is broadly applicable for quantitative SHG material characterization and diagnostic imaging.

Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization-resolved second-harmonic generation microscopy.

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra-molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization-resolved second-harmonic generation (P-SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.

Dual- and single-shot susceptibility ratio measurements with circular polarizations in second-harmonic generation microscopy.

Polarization-resolved second-harmonic generation (P-SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z-axis parallel and x-axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P-SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual-shot configuration where the SHG circular anisotropy generated using incident right- and left-handed circularly-polarized light is measured; and a single-shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes-Mueller polarimetry. The dual- and single-shot circular anisotropy measurements can be used for fast imaging that is independent of the in-plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.

Label-Free Non-linear Multimodal Optical Microscopy—Basics, Development, and Applications

Nonlinear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free nonlinear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The nonlinear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF) and lipid droplets in cells (CARS). Again, the nonlinear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved nonlinear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.

Photonic-Crystal-Fiber-Coupled, Hand-Held, Polarization-Resolved Second-Harmonic-Generation Microscope for <italic>In Vivo</italic> Visualization of Dermal Collagen Fibers in Human Skin

Second-harmonic-generation (SHG) microscopy is a powerful tool for in vivo monitoring of collagen fibers in human skin. Furthermore, polarization-resolved SHG microscopy can provide additional insights regarding the direction of collagen fibers, i.e., collagen fiber orientation. However, their practical use in the dermatological field is still limited due to the bulky and complicated setup. In this paper, we constructed a photonic-crystal-fiber (PCF) coupled, hand-held polarization-resolved SHG microscope for in vivo monitoring of collagen fibers in human skin. Fiber delivery of ultrashort pulse light was achieved without significant change of the linear polarization by a large-mode-area PCF, whereas the SHG microscopy setup was enclosed into a hand-held probe head. The combination of PCF with the hand-held probe head largely enhances the flexibility of measurement sites in the human skin.

Polarization-resolved second harmonic microscopy of skeletal muscle in sepsis.

Polarization-resolved second harmonic generation (P-SHG) microscopy is able to probe the sub-micrometer structural organization of myosin filaments within skeletal muscle. In this study, P-SHG microscopy was used to analyze the structural consequences of sepsis, which is the main cause of the critical illness polyneuromyopathy (CIPNM). Experiments conducted on two populations of rats demonstrated a significant difference of the anisotropy parameter between healthy and septic groups, indicating that P-SHG microscopy is promising for the diagnosis of CIPNM. The difference, which can be attributed to a change of myosin conformation at the sub-sarcomere scale, cannot be evidenced by classical SHG imaging.

Polarization-resolved second-harmonic-generation imaging of dermal collagen fiber in prewrinkled and wrinkled skins of ultraviolet-B-exposed mouse.

.Skin wrinkling is a typical symptom of cutaneous photoaging; however, the skin wrinkling depends on not only the actual age but also exposure history to ultraviolet B (UVB) rays in individuals. Therefore, there is considerable need for its assessment technique in vivo in skin cosmetics and antiaging dermatology. Wrinkles always appear as linear grooves in the skin, and dermal collagen fibers play an important role to determine the morphology and mechanical properties of the skin. Therefore, an optical probe sensitive to dermal collagen fiber and its orientation will be useful. Polarization-resolved second-harmonic-generation (SHG) microscopy is a promising approach for in vivo evaluation of collagen fiber orientation because the efficiency of SHG light is sensitive to collagen fiber orientation when the incident light is linearly polarized. We investigate orientation change of dermal collagen fiber in prewrinkled and wrinkled skins of the UVB-exposed mouse model using polarization-resolved SHG microscopy. A polarization anisotropic image of the SHG light indicates that the change of collagen fiber orientation starts in the prewrinkled skin of UVB-exposed mice, then the wrinkle appears. Furthermore, the dominant direction of collagen fiber orientation in the prewrinkled skin is significantly parallel to the wrinkle direction in the wrinkled skin. This result implies that the change of collagen fiber orientation is a trigger of wrinkling in cutaneous photoaging.

Snapshot second-harmonic generation polarimeter based on spectral analysis.

A new high-speed second-harmonic generation (SHG) polarimetric method is reported. It is based on the spectral analysis of the SHG radiation emitted by a nonlinear medium excited with circularly polarized femtosecond laser pulses. The setup uses only passive components for polarization encoding and a fast spectrometer for spectral analysis. The method is validated on a z-cut quartz plate, then on a collagen-rich biological tissue with a view to single image polarization-resolved SHG microscopy.

A Study on Image Quality in Polarization-Resolved Second Harmonic Generation Microscopy

Second harmonic generation (SHG) microscopy represents a very powerful tool for tissue characterization. Polarization-resolved SHG (PSHG) microscopy extends the potential of SHG, by exploiting the dependence of SHG signals on the polarization state of the excitation beam. Among others, this dependence translates to the fact that SHG images collected under different polarization configurations exhibit distinct characteristics in terms of content and appearance. These characteristics hold deep implications over image quality, as perceived by human observers or by image analysis methods custom designed to automatically extract a quality factor from digital images. Our work addresses this subject, by investigating how basic image properties and the outputs of no-reference image quality assessment methods correlate to human expert opinion in the case of PSHG micrographs. Our evaluation framework is based on SHG imaging of collagen-based ocular tissues under different linear and elliptical polarization states of the incident light.

Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy.

This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules.

Detection of imprecise estimations for polarization-resolved second-harmonic generation microscopy.

Second-harmonic generation microscopy can provide estimation of some local molecule distribution properties. However, in order not to get erroneous conclusions, it is important to detect measurements with insufficient precision. Such a detection technique is developed considering an approximation of the ultimate precision provided by the Cramer-Rao bound. This method is characterized and a simple approximation of its detection and false alarm probabilities is developed.

Polarization-resolved second-harmonic generation imaging for liver fibrosis assessment without labeling

We apply the polarization-resolved second-harmonic generation (PR-SHG) microscopy to investigate the changes of collagen typings (type I vs type III) and collagen fibril orientations of liver tissue in bile-duct-ligation (BDL) rat models. The PR-SHG results show that the second-order susceptibility tensor ratios (χ31/χ15 and χ33/χ15) of collagen fibers increase with liver fibrotic progression after BDL surgery, reflecting an increase of the type III collagen component with the severity of liver fibrosis; and the square root of the collagen type III to type I ratio linearly correlates (R2 = 0.98) with histopathological scores. Furthermore, the collagen fibril orientations become more random with liver fibrosis transformation as compared to normal liver tissue. This work demonstrates that PR-SHG microscopy has the potential for label-free diagnosis and characterization of liver fibrosis based on quantitative analysis of collagen typings and fibril orientations.