Forward Second Harmonic Generation Research Articles

Broadband second-harmonic generation based on orientation-patterned II-VI crystals

Broadband second-harmonic generation (SHG) has important applications in many fields, such as femtosecond laser technology, optical communication, and biomedicine. The broadband SHG has been implemented in some crystals; however, it is not clear for forward and backward broadband quasi-phase-matching (QPM) SHGs in orientation-patterned Ⅱ-Ⅵ crystals. In this paper, the characters of orientation-patterned ZnS (OP-ZnS), orientation-patterned ZnSe (OP-ZnSe), and orientation-patterned CdTe (OP-CdTe) are, respectively, studied, including the forward broadband SHG and backward broadband SHG bandwidths at different temperatures. In addition, the influences of temperature on the forward broadband SHG bandwidth are investigated. The forward broadband QPM SHGs of OP-ZnS, OP-ZnSe, and OP-CdTe are obtained, and the backward broadband QPM SHGs cannot be obtained. The influence of temperature on the forward bandwidth is small.

Enhancement of efficiency of second-harmonic generation from MoS2 monolayers in 1D Fibonacci photonic crystals

In this article, the increase of the efficiency of second-harmonic generation (SHG) from MoS2 monolayers embedded in different one-dimensional Fibonacci photonic crystal structures is investigated. The systems contain ZnS, SiO2, and MoS2 layers with the same thickness of ZnS and SiO2 films, which are arranged on the basis of different Fibonacci sequences. The transfer matrix method is used to calculate the forward and backward SHG efficiencies in the undepleted pump approximation. The thicknesses of the ZnS and SiO2 films are changed to obtain the maximum frequency conversion in different structures. Tuning the thicknesses causes the second-harmonic waves generated in each MoS2 monolayer to interfere constructively. Also, the fundamental and second-harmonic wavelengths are both located at the photonic band gap edges, where the density of electromagnetic modes and the nonlinear interaction time are enhanced. Our results show that the SHG efficiencies were increased in quasiperiodic photonic crystals with respect to the periodic structure with the same number of MoS2 layers. In Fibonacci photonic crystal structures there are more geometrical parameters that can be tuned to obtain the highest efficiencies. Furthermore, we used a Bragg mirror at one side of each structure to control the propagation direction of the generated second-harmonic waves and to decrease the backward waves, which led to enhancement of the forward waves.

Collagen organization of renal cell carcinoma differs between low and high grade tumors

BackgroundThe traditional pathologic grading for human renal cell carcinoma (RCC) has low concordance between biopsy and surgical specimen. There is a need to investigate adjunctive pathology technique that does not rely on the nuclear morphology that defines the traditional grading. Changes in collagen organization in the extracellular matrix have been linked to prognosis or grade in breast, ovarian, and pancreatic cancers, but collagen organization has never been correlated with RCC grade. In this study, we used Second Harmonic Generation (SHG) based imaging to quantify possible differences in collagen organization between high and low grades of human RCC.MethodsA tissue microarray (TMA) was constructed from RCC tumor specimens. Each TMA core represents an individual patient. A 5 μm section from the TMA tissue was stained with standard hematoxylin and eosin (H&E). Bright field images of the H&E stained TMA were used to annotate representative RCC regions. In this study, 70 grade 1 cores and 51 grade 4 cores were imaged on a custom-built forward SHG microscope, and images were analyzed using established software tools to automatically extract and quantify collagen fibers for alignment and density assessment. A linear mixed-effects model with random intercepts to account for the within-patient correlation was created to compare grade 1 vs. grade 4 measurements and the statistical tests were two-sided.ResultsBoth collagen density and alignment differed significantly between RCC grade 1 and RCC grade 4. Specifically, collagen fiber density was greater in grade 4 than in grade 1 RCC (p < 0.001). Collagen fibers were also more aligned in grade 4 compared to grade 1 (p < 0.001).ConclusionsCollagen density and alignment were shown to be significantly higher in RCC grade 4 vs. grade 1. This technique of biopsy sampling by SHG could complement classical tumor grading approaches. Furthermore it might allow biopsies to be more clinically relevant by informing diagnostics. Future studies are required to investigate the functional role of collagen organization in RCC.

First-Order Forward and Backward Quasi-Phase Matched Second Harmonic Generation in Silicon-Organic Hybrid Structures

We propose and design a high-efficiency forward and backward second harmonic generation (SHG) in nonlinear polymer filled silicon slot waveguide known as silicon-organic hybrid (SOH) waveguides. The spatial distribution of second-order nonlinearity, χ(2) , of waveguide is altered by periodically poling of nonlinear polymer to achieve quasi-phase matching (QPM) between fundamental frequency and second harmonic waves. The simulations show that first-order QPM SHG with high conversion efficiency of η ≍ 7.2% can be achieved in the optimized structure with relatively small input power of 25 mW. This level of improvement is the result of high confinement of SOH waveguide as well as possible fabrication of very small period length in silicon photonics which could not be realized in traditional nonlinear materials such as LiNbO3. Our simulation results show ∼17% efficiency with only 25 mW input power for forward SHG being comparable to the other platforms even with smaller device length and lower pump power. We expect this device will open up its application in the fields of all-optical signal processing, optical (mirrorless) parametric amplification, low-noise detection of mid-IR signal in near-IR windows, and mid-IR sources.

A new mode of contrast in biological second harmonic generation microscopy

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature.

Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development.

We developed a multimodal nonlinear optical (NLO) microscope system by integrating stimulated Raman scattering (SRS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging. The system was used to study the morphological and biochemical characteristics of tibial cartilage in a kinesin-1 (Kif5b) knockout mouse model. The detailed structure of fibrillar collagen in the extracellular matrix of cartilage was visualized by the forward and backward SHG signals, while high resolution imaging of chondrocytes was achieved by capturing endogenous TPEF and SRS signals of the cells. The results demonstrate that collagen fibrils in the superficial surface of the articular cartilage decreased significantly in the absence of Kif5b. The distorted morphology along with accumulated intracellular collagen was observed in the Kif5b-deficient chondrocytes, indicating the critical roles of kinesin-1 in the chondrocyte morphogenesis and collagen secretion. The study shows that multimodal NLO imaging method is an effective approach to investigate early development of cartilage.

Use of Second Harmonic Imaging and Fourier Transformation to Analyze Cartilage Repair in Mice Injected with a Novel Peptide CK2.1

Second harmonic generation (SHG) imaging is a powerful imaging tool that allows to study the distribution of the collagen network in cartilage. This imaging modality has been successfully utilized for visualizing physiologic and pathological changes in tissues and organs. However only recently the quantification of SHG images by Fourier Transform analysis allowed for successful quantification of the collagen network in cartilage. Here, we demonstrate that systemic injection of CK2.1, a novel mimetic peptide based on the Bone Morphogenetic Protein (BMP) receptor type I sequence, induced cartilage formation in mice and reassembly of collagen fibers. Intra-articular injections of slow release HGPCK2.1 demonstrated a robust regeneration of the damaged cartilage tissue and the collagen network. Collection of the forward (aligned) and backward (non-aligned) SHG allowed us to quantify the fibrillar distribution in peptide induced cartilage formation and repair. Our data show that the Forward SHG obtained by the collagen structures were greater compared to Backward SHG in CK2.1 in the cartilage of CK2.1 injected mice. These data suggest the cartilage elasticity to withstand stresses placed on the tissue is increased. Our data provide us insights into the peptides capabilities as a future therapeutic agent that can regenerate the lost cartilage to its native form.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

Purpose: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states.Methods: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly.Results: Our results revealed differences in SHG forward–backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.Conclusions: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Improving liver fibrosis diagnosis based on forward and backward second harmonic generation signals

The correlation of forward second harmonic generation (SHG) signal and backward SHG signal in different liver fibrosis stages was investigated. We found that three features, including the collagen percentage for forward SHG, the collagen percentage for backward SHG, and the average intensity ratio of two kinds of SHG signals, can quantitatively stage liver fibrosis in thioacetamide-induced rat model. We demonstrated that the combination of all three features by using a support vector machine classification algorithm can provide a more accurate prediction than each feature alone in fibrosis diagnosis.

Quantifying the backscattering of second harmonic generation in tissues with confocal multiphoton microscopy

The backward second harmonic generation (SHG) in mouse tissues is studied with a confocal multiphoton microscopy system. The total backward collected SHG (B-SHG) consists of the backward generated SHG and the backward-scattered forward-generated SHG (BS-SHG), which can be modeled by a Gaussian and a uniform distribution, respectively, at the confocal pinhole plane. By varying the pinhole size with a series of collection fibers, the proportion of the BS-SHG to the B-SHG and the proportion of BS-SHG to the forward generated SHG can be obtained. The approach is first validated by Monte Carlo simulation. It is then applied to two types of mouse tissues: mouse tail tendon and Achilles tendon. It is found that the BS-SHG contributes less to the B-SHG for the tail tendon than Achilles tendon with thicknesses of ~300 μm. With the thickness of the Achilles tendon tissue increased to 1000 μm but the focal plane kept at the same depth, as high as ~10% of the total forward SHG is backscattered and collected. The results indicate that BS-SHG may not be the major source of B-SHG in the tail tendon, but it may be the major source in the Achilles tendon. These methods and results provide a noninvasive method and supporting information for investigating the generation mechanism of SHG and help with optimizing backward SHG microscopy and spectroscopy measurements.

Characterizing the morphologic changes in collagen crosslinked–treated corneas by Fourier transform–second harmonic generation imaging

To evaluate the efficacy of using forward second harmonic generation (SHG) and 2-dimensional fast Fourier-transform (2D-FFT) analysis for the label-free characterization and quantification of morphologic changes in the corneal stroma after collagen crosslinking (CXL). Department of Physics, National Taiwan University, Taipei, Taiwan. Experimental study. En face forward SHG imaging and 2D-FFT analysis were performed on ex vivo porcine corneas at the depths of 100, 200, 400, and 800 μm. Morphologic changes in stromal collagen fiber in control, ultraviolet-A (UVA), riboflavin, and riboflavin-UVA treated porcine corneas were assessed. Hematoxylin-eosin staining and Sirius red staining were performed for comparison. Corneas after CXL treatment tended to have collagen fibers that were wavy compared with the linear pattern in normal corneas. Quantitative 2D-FFT analysis of forward SHG images also showed an increase in the standard deviations of the distribution of stromal collagen fiber orientations, which is indicative of the changed pattern of crosslinked stromal collagen fibers. Second harmonic generation imaging showed the morphologic changes in stromal collagen after CXL treatment. The linear collagen fibers in normal corneal stroma became wavy after treatment. With the introduction of 2D-FFT analysis, the morphologic changes can be quantified.

Quantification of lamellar orientation in corneal collagen using second harmonic generation images

Second harmonic generation (SHG) is a well-established optical modality widely used in biomedical optics to image collagen based tissues. The coherent signal of the forward direction SHG produces a high resolution image that can resolve individual fibers (groups of fibrils). In highly ordered collagen lamellae, such as in the corneal stroma, it is important to determine the orientation of the fibers as they contribute significantly to the biomechanics of the tissue. However, due to the crimped structure of the fibers, it is challenging to robustly determine their orientation using an independent computational method, compared to the straight fibers problem. Previous work in the field used the polarization of the fundamental or other techniques involving a more manual selection of the orientation, in order to differentiate between various directions in corneal structures. Yet those lack accuracy and independency. We present a robust independent technique to determine the orientation of the fibers in the corneal structure. The experimental results presented here, taken from different lamellae, demonstrate strongly the correct orientation.

Microscopic second-harmonic generation emission direction in fibrillous collagen type I by quasi-phase-matching theory

Quasiphase matching (QPM) is a widely used theory in crystal to analyze the character of second-harmonic generation (SHG) emitted from it. Based on the structural features of collagen type I, where the constituted fibrils in collagen function as a crystal which has the structure of two-dimensional (2D) quasicrystalline, in this paper, we use the QPM theory on collagen for SHG emission direction study under the excitation of laser light through a microscope. The effects of numerical aperture NA, as well as the structural parameters, such as QPM order (m,l) and collagen period a=d1+d2 associated with the fibrils diameter (d1), packing density and interfibrils structure (d2), etc., on SHG emission angle φ have been investigated. Our theoretical results show that collagen period a has threshold effect on φ to present forward or backward SHG emission and NA has minor influence on this threshold value a. Collagen period of a has more significant influence on SHG emission angle φ when a is smaller than the threshold value. In reality, we realize that diameter of collagen fibrils d1 plays a major role on forward or backward emission of SHG. Here, for example, (we assume d1=a/2), when d1≤95 nm [(m,l)=(1,0)], the backward SHG shows up at any magnitude of NA, while when d1≥150 nm [(m,l)=(1,0)], SHG presents forward emission feature under all circumstances. Between them, SHG emits from forward direction to backward direction as the increase in NA. The QPM order (m,l) has nonlinear impact on SHG emission angle φ and has different degrees of influence on different collagen period a. Our theoretical results are highly consistent with the experiments results demonstrated by other researchers and provide a proper explanation of the phenomenon of appreciable backward SHG signals appearing in collagen type I. Our established theoretical model of applying QPM theory in 2D quasicrystalline fibrils is therefore confirmed to be a suitable model for dealing with SHG in type I collagen.

Quantitative analysis of forward and backward second-harmonic images of collagen fibers using Fourier transform second-harmonic-generation microscopy

Fourier transform second-harmonic generation (SHG) microscopy has been applied to quantitatively compare the information content between SHG images obtained from the forward and backward direction for three tissue types: porcine tendon, sclera, and ear cartilage. Both signal types yield consistent information on the preferred orientation of collagen fibers. For all specimens, the Fourier transform of the forward and backward SHG images produces several overlapping peaks in the magnitude spectrum at various depths into the tissues, indicating that some information present in the forward SHG images can be extracted from the backward SHG images. This study highlights the potential of backward SHG microscopy for medical diagnostics.

Structural Characterization of Edematous Corneas by Forward and Backward Second Harmonic Generation Imaging

The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 μm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.

Second Harmonic Generation Microscopy Characterization of Corneal Edema

The purpose of this study is to investigate the structural features of corneal edema by multiphoton fluorescence and second harmonic generation (SHG) microscopy and the potential of this technique as a clinical, in vivo monitoring technique for cornea disease diagnosis. Corneal edema is a build up of fluid or swelling of the cornea that can produce clouding of the cornea and significant decrease in vision. Since collagen can be induced to generate strong second harmonic generation (SHG) signal, multiphoton excitation provide direct visualization of collagen orientation within corneal stroma.In this study, normal and over-hydrated bovine corneal specimens used in this study were observed to distinguish the structural alteration due to edema. We collected forward and backward SHG signals simultaneously within different depths and also with large region images. From the result, we found that the organization of corneal stroma was not significantly altered, except for the increase of interfibrillar spacing. This structural information provided by multiphoton imaging may help the evaluation of the necessity for full-thickness corneal transplantation in pathological cases.

Selective imaging in second-harmonic-generation microscopy with anisotropic radiation

As a novel modality of optical microscopy, second-harmonic generation (SHG) provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that well-formed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.

Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage

We used the combination of multiphoton autofluorescence (MAF), forward second-harmonic generation (FWSHG), and backward second-harmonic generation (BWSHG) imaging for the qualitative and quantitative characterization of thermal damage of ex vivo bovine cornea. We attempt to characterize the structural alterations by qualitative MAF, FWSHG, and BWSHG imaging in the temperature range of 37 to 90 degrees C. In addition to measuring the absolute changes in the three types of signals at the stromal surface, we also performed image correlation analysis between FWSHG and BWSHG and demonstrate that with increasing thermal damage, image correlation between FWSHG and BWSHG significantly increases. Our results show that while MAF and BWSHG intensities may be used as preliminary indicators of the extent of corneal thermal damage, the most sensitive measures are provided by the decay in FWSHG intensity and the convergence of FWSHG and BWSHG images.

Three‐dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues

In this work we aim to show how it is possible to exploit the second harmonic generation (SHG) signal for producing multimodal microscopic images of biological tissues. SHG microscopy constitutes an important tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architectures. The physical origins of SHG within these tissues are addressed and reported in a comprehensive image gallery. Although SHG is a coherent process, the multiple scattering of tissue samples determines the ability to acquire signal in both backward and forward direction. We discuss here some key elements related to the backward and forward SHG signal in terms of acquisition architecture and related microscopic imaging.

Multimodal nonlinear optical imaging of collagen arrays

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.