Polarization Second Harmonic Generation Research Articles

Categorization of collagen type I and II blend hydrogel using multipolarization SHG imaging with ResNet regression

Previously, the discrimination of collagen types I and II was successfully achieved using peptide pitch angle and anisotropic parameter methods. However, these methods require fitting polarization second harmonic generation (SHG) pixel-wise information into generic mathematical models, revealing inconsistencies in categorizing collagen type I and II blend hydrogels. In this study, a ResNet approach based on multipolarization SHG imaging is proposed for the categorization and regression of collagen type I and II blend hydrogels at 0%, 25%, 50%, 75%, and 100% type II, without the need for prior time-consuming model fitting. A ResNet model, pretrained on 18 progressive polarization SHG images at 10° intervals for each percentage, categorizes the five blended collagen hydrogels with a mean absolute error (MAE) of 0.021, while the model pretrained on nonpolarization images exhibited 0.083 MAE. Moreover, the pretrained models can also generally regress the blend hydrogels at 20%, 40%, 60%, and 80% type II. In conclusion, the multipolarization SHG image-based ResNet analysis demonstrates the potential for an automated approach using deep learning to extract valuable information from the collagen matrix.

Polarization Second Harmonic Generation Discriminates Between Fresh and Aged Starch-Based Adhesives Used in Cultural Heritage

In this work, we report that polarization second harmonic generation (PSHG) microscopy, commonly used in biomedical imaging, can quantitatively discriminate naturally aged from fresh starch-based glues used for conservation or restoration of paintings, works of art on paper, and books. Several samples of fresh and aged (7 years) flour and starch pastes were investigated by use of PSHG. In these types of adhesives, widely used in cultural heritage conservation, second harmonic generation (SHG) contrast originates primarily from the starch granules. It was found that in aged glues, the starch SHG effective orientation (SHG angle, θ) shifts to significantly higher values in comparison to the fresh granules. This shift is attributed to the different degree of granule hydration between fresh and aged adhesives. Thus noninvasive high-resolution nonlinear scattering can be employed to detect and quantify the degree of deterioration of restoration adhesives and to provide guidance toward future conservation treatments.

Bulk quadrupole and interface dipole contribution for second harmonic generation in Si(111)

The second harmonic generation (SHG) response was measured for arbitrarily oriented linear input polarization on Si(111) surfaces in rotational anisotropy experiments. We show for the first time, using the simplified bond hyperpolarizability model (SBHM), that the observed angular shifts of the nonlinear peaks and symmetry features—related to changes in the input polarization—help to identify the corresponding interface dipolar and bulk quadrupolar SHG sources, yielding excellent agreement with the experiment. Additionally, we evaluate for the s-in/p-out (sp) and p-in/p-out (pp)-polarization SHG intensities the contributions from the individual Si bonds. Furthermore, a relation between the four parameters arising from SBHM and six coefficients of the phenomenological SHG theory needed to reproduce experimental data is established.

Molecular Order of Arterial Collagen Using Circular Polarization Second-Harmonic Generation Imaging

Second-harmonic generation (SHG) originates from the interaction between upconverted fields from individual scatterers. This renders SHG microscopy highly sensitive to molecular distribution. Here, we aim to take advantage of the difference in SHG between aligned and partially aligned molecules to probe the degree of molecular order during biomechanical testing, independently of the absolute orientation of the scattering molecules. Toward this goal, we implemented a circular polarization SHG imaging approach and used it to quantify the intensity change associated with collagen fibers straightening in the arterial wall during mechanical stretching. We were able to observe the delayed alignment of collagen fibers during mechanical loading, thus demonstrating a simple method to characterize molecular distribution using intensity information alone.

Label-free mapping of microstructural organisation in self-aligning cellular collagen hydrogels using image correlation spectroscopy

Hydrogels have emerged as promising biomaterials for regenerative medicine. Despite major advances, tissue engineers have faced challenges in studying the complex dynamics of cell-mediated hydrogel remodelling. Second harmonic generation (SHG) microscopy has been a pivotal tool for non-invasive visualization of collagen type I hydrogels. By taking into account the typical polarization SHG effect, we recently proposed an alternative image correlation spectroscopy (ICS) model to quantify characteristics of randomly oriented collagen fibrils. However, fibril alignment is an important feature in many tissues that needs to be monitored for effective assembly of anisotropic tissue constructs. Here we extended our previous approach to include the orientation distribution of fibrils in cellular hydrogels and show the power of this model in two biologically relevant applications. Using a collagen hydrogel contraction assay, we were able to capture cell-induced hydrogel modifications at the microscopic scale and link these to changes in overall gel dimensions over time. After 24h, the collagen density was about 3 times higher than the initial density, which was of the same order as the decrease in hydrogel area. We also showed that the orientation parameters recovered from our automated ICS model match values obtained from manual measurements. Furthermore, regions axial to cellular processes aligned at least 1.5 times faster compared with adjacent zones. Being able to capture minor temporal and spatial changes in hydrogel density and collagen fibril orientation, we demonstrated the sensitivity of this extended ICS model to deconstruct a complex environment and support its potential for tissue engineering research. Statement of significanceIt is generally accepted that looking beyond bulk hydrogel composition is key in understanding the mechanisms that influence the mechanical and biological properties of artificial tissues. In this manuscript, we performed label-free non-invasive imaging and extended a robust automated analysis method to characterize the microstructural organisation of cellular hydrogel systems. We underpin the sensitivity of this technique by capturing minor changes in collagen density and fibril orientation in biologically relevant systems over time. Therefore, we believe that this method is applicable in fundamental cell-matrix research and has high-throughput potential in screening arrays of hydrogel scaffolds, making it an interesting tool for future tissue engineering research.

Second Harmonic Generation Mediated by Aligned Water in Starch Granules.

The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.

Polarization second harmonic generation by image correlation spectroscopy on collagen type I hydrogels

Successful engineering of biomimetic tissue relies on an accurate quantification of the mechanical properties of the selected scaffold. To improve this quantification, typical bulk rheological measurements are often complemented with microscopic techniques, including label-free second harmonic generation (SHG) imaging. Image correlation spectroscopy (ICS) has been applied to obtain quantitative information from SHG images of fibrous scaffolds. However, the typical polarization SHG (P-SHG) effect, which partly defines the shape of the autocorrelation function (ACF), has never been taken into account. Here we propose a new and flexible model to reliably apply ICS to P-SHG images of fibrous structures. By starting from a limited number of straightforward assumptions and by taking into account the P-SHG effect, we were able to cope with the typically observed ACF particularities. Using simulated datasets, the resulting model was thoroughly evaluated and compared with models previously described in the literature. We showed that our new model has no restrictions concerning the fibre length for the density retrieval. For certain length ranges, the model can additionally be used to obtain the average fibre length and the P-SHG related non-zero susceptibility tensor element ratios. From experimental data on collagen type I hydrogels, values of SHG tensor element ratios and fibre thickness were determined which match values reported in the literature, thereby underpinning the validity and applicability of our new model.

Determination of Collagen Nanostructure from Second-Order Susceptibility Tensor Analysis

A model is proposed to describe the polarization dependence of second harmonic generation (SHG) from type I collagen fibrils. The model is based on sum-frequency vibrational spectrum experiments that attribute the molecular origins of collagen second-order susceptibility to the peptide groups in the backbone of the collagen α-helix and the methylene groups in the pyrrolidine rings. Applying our model to a polarization SHG (P-SHG) experiment leads to a predicted collagen I peptide pitch-angle of 45.82° ± 0.46° and methylene pitch-angle of 94.80° ± 0.97°. Compared to a previous model that accounts for only the peptide contribution, our results are more consistent with the x-ray diffraction determination of collagen-like peptide. Application of our model to type II collagen from rat trachea cartilage leads to similar results. The peptide pitch-angle of 45.72° ± 1.17° is similar to that of type I collagen, but a different methylene pitch-angle of 97.87° ± 1.79° was found. Our work demonstrates that far-field P-SHG measurements can be used to extract molecular structural information of collagen fibers.

Estimating the helical pitch angle of amylopectin in starch using polarization secondharmonic generation microscopy

Starch granules are among the brightest natural second harmonic generation (SHG)converters. They basically consist of amylose and amylopectin molecules and the source ofthe SHG signal is still undetermined. In the present study we perform polarizationsensitive SHG (PSHG) imaging of wheat starch granules and we fit the SHGsignal variation of each pixel of the PSHG images into a generalized biophysicalmodel. By assuming that the SHG source molecule is a helix with cylindricalsymmetry along its long axis, the model extracts the helical pitch angle of the SHGsource for every pixel of the image. By displaying the pixel histogram representingthis helical pitch angle we found a highly peaked histogram with maximum atθe = 36.1° and awidth of Δθe = 9.3°. This pitch angle corresponds to the strand of the parallel double helical structure, calledamylopectin (as measured by a small angle x-ray scattering technique). This demonstratesamylopectin (and not amylose) as the source of SHG in starch. According to ourknowledge, this is the first estimation of the amylopectin’s pitch angle in starch usingPSHG.

Easing attosecond generation

A new gating scheme based on polarization control and second-harmonic generation is greatly simplifying the production of isolated attosecond pulses.

“Half-hydration” at the air/water interface revealed by heterodyne-detected electronic sum frequency generation spectroscopy, polarization second harmonic generation, and molecular dynamics simulation

A solute-solvent interaction at the air/water interface was investigated both experimentally and theoretically, by studying a prototypical surface-active polarity indicator molecule, coumarin 110 (C110), adsorbed at the air/water interface with heterodyne-detected electronic sum frequency generation (HD-ESFG) spectroscopy, polarization second harmonic generation (SHG), and a molecular dynamics (MD) simulation. The second-order nonlinear optical susceptibility (chi((2))) tensor elements of C110 at the air/water interface were determined experimentally by HD-ESFG and polarization SHG, and information on "intermediate" polarity sensed by C110 at the interface was obtained by HD-ESFG. An MD simulation and a time-dependent density functional theory calculation were used to theoretically evaluate the chi((2)) tensor elements, which were in good agreement with the experimental results of HD-ESFG and polarization SHG. The microscopic "half-hydration" structure around C110 at the water surface was visualized on the basis of the MD simulation data, with which we can intuitively understand the microscopic origin of the surface activity of C110 and the intermediate polarity sensed by C110 at the air/water interface.

Second harmonic generation χ tensor microscopy for tissue imaging

Important biological molecules such as collagen and myosin are strong second harmonic generators. The polarization dependence of second-harmonic generation (SHG) is a property of the second order susceptibility elements (χ tensor) of the molecules that produce the signal. In this work, we used polarization SHG microscopy to image and map pixel scale polarization anisotropy information of collagen-muscle junction of chicken wing and the dermis of human skin. We show that the variation in the χ tensor components can be displayed as images to show the contrast between different molecular sources of SHG.

Collagen and myosin characterization by orientation field second harmonic microscopy

Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor chi(2) can be determined for each pixel of the image. The ratio rho = chi33/chi15 of the principal components of chi(2) of collagen and myosin was obtained with the same method, and found within the range 1.6-1.8 and 0.5-0.6 respectively. The orientation of the principal axis of chi(2) is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).

Competition of frequency conversion and polarization coupling in periodically poled lithium niobate

In this paper wave-coupling equations including polarization coupling and frequency doubling in periodically poled lithium niobate (PPLN) is proposed. Numerical solutions of the coupling equations show that there is a competition between the polarization coupling and second-harmonic generation in a PPLN under the phase-matching condition. The influences of the external electric field on the polarizations and amplitudes of three interactive waves are studied.

Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation

In imaging anisotropic samples with optical microscopy, a controlled, polarized light source can be used to gain molecular information of fibrous materials such as muscles and collagen fibers. However, the delivery of the polarized excitation light source in a system such as a laser scanning optical microscope often encounters the problem of the polarization ellipticity altering effects of the optical components. Using a half-wave plate and a quarter-wave plate, we demonstrate that the polarization ellipticity altering effect of the dichroic mirror in an epi-illuminated multiphoton laser scanning microscope can be corrected, and that this approach can be used to obtain polarized second-harmonic generation (SHG) images of rat tail tendon and mouse leg muscle. The excitation polarization dependence of the SHG intensity is fitted to determine the ratio of the second-order susceptibility tensor elements associated with type I collagen in the rat tail tendon and myofibril in the mouse leg muscle. Our methodology can be applied to polarized SHG imaging without sample rotation. This approach has great potential for imaging noncentrosymmetric biological samples, providing structural information on the molecular scale in addition to morphological information of tissues.

Analysis of second-harmonic generation microscopy under refractive index mismatch

On the basis of the vector diffraction theory and Green's function method, this paper investigates the effects of refractive index mismatch on second-harmonic generation (SHG) microscopy. The polarization distribution and SHG intensity are calculated as functions of the sample radius and probe depth. The numerical results show that refractive index mismatch can result in peak intensity degradation, increase secondary lobes and extension of second-harmonic polarization distribution. Because of the attenuation of polarization intensity, the detected SHG intensity significantly decreases with increasing probe depth, which can limit the imaging depth of SHG microscopy inside thick samples. Forward SHG intensity decays slowly than backward SHG, due to the combination of extension second-harmonic polarization distribution and strong dependency of forward SHG on sample radius.

Optical Second Harmonic Generation Study of Malachite Green Orientation and Order at the Fused-Silica/Air Interface

Optical second harmonic generation (SHG) studies of malachite green (MG) adsorbed at the fused-silica/air interface have been conducted. The concentration dependence of the second harmonic intensity indicates the formation of ordered monolayers normal to the interface. Polarization SHG experiments have been used to extract the averaged molecular orientation in adjacent molecular layers and to characterize the breadth of the orientation distribution. MG adsorbs in an upright orientation with its molecular plane near normal to the surface. This orientation is consistent with the interaction of the polar surface and the polar dimethylamino substituents of the adsorbate. Molecular orientation in the second monolayer resembles that in the first, but also reflects some additional broadening of the orientation distribution function. The onset of parallel, sandwich-type aggregates has been observed within the first monolayer. The formation of parallel, sandwich-type, intralayer aggregates is distinct from the int...