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The relationship between acoustic parameters and the microstructure of a Cu30Zn brass plate subjected to plastic deformation was evaluated. The plate, previously annealed at 550 °C for 30 min, was cold rolled to reductions ranging from 10% to 70%. Linear ultrasonic measurements were performed on each of the nine specimens, corresponding to the nine different reductions, using the pulse-echo method to record the times of flight of longitudinal waves along the thickness axis. Subsequently, acoustic measurements were conducted to determine the nonlinear parameter ß through second harmonic generation. Microstructural analysis, carried out by X-ray diffraction, Vickers hardness testing, and optical microscopy, revealed an increase in deformation twins, reaching a maximum at 40% thickness reduction. At higher deformations, the microstructure showed the generation and proliferation of shear bands, coinciding with a decrease in the twinning structure and an increase in dislocation density. The longitudinal wave velocity exhibited a 0.9% decrease at 20% deformation, attributed to dislocations and initial twin formation, followed by a continuous increase up to 2% beyond this point, resulting from the combined effects of twinning and shear banding. The nonlinear parameter ß displayed a notable maximum, approximately one order of magnitude greater than its original value, at 40% deformation. This peak correlates with a roughly tenfold increase in twinning fault probability at the same deformation level.

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The first hyperpolarizability of graphene quantum dots (GQDs) suspended in water was determined using the hyper-Rayleigh scattering (HRS) technique. To the best of our knowledge, this is the first application of the HRS technique to characterize GQDs. Two commercial GQDs (Acqua-Cyan and Acqua-Green) with different compositions were studied. The HRS experiments were performed with an excitation laser at 1064 nm. The measured hyperpolarizabilities were (1.0±0.1)×10-27 esu and (0.9±0.1)×10-27 esu for Acqua-Cyan and Acqua-Green, respectively. The results were used to estimate the hyperpolarizability per nanosheet obtained by assuming that each GQD has five nanosheets with 0.3 nm thickness. The two-level model, used to calculate the static hyperpolarizability per nanosheet, provides values of (2.4±0.1)×10-28 esu (Acqua-Cyan) and (0.5±0.1)×10-28 esu (Aqua-Green). The origin of the nonlinearity is discussed on the basis of polarized resolved HRS experiments, and electric quadrupolar behavior with a strong dependence on surface effects. The nontoxic characteristics and order of magnitude indicate that these GQDs may be useful for biological microscopy imaging.

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We calculate the linear and the second harmonic (SH) spin current response of two anisotropic systems with spin-orbit (SO) interaction. General expressions of wide applicability for the these response functions are first derived for a generic two-band Hamiltonian. The first system is a two-dimensional (2D) electron gas in the presence of Rashba andk-linear Dresselhaus SO couplings. The calculations show how narrow or wide the response spectra can be, what is their overall shape and size, and frequency shiftings, depending on which crystal orientation is selected. The quantitative knowing of this makes possible a comparative study for several orientations, which would allow to select a spectrum with particular characteristic. We find that vanishing linear and second order response tensors are achievable under SU(2) symmetry conditions, characterized by a collinear SO vector field. Additional conditions under which specific tensor components vanish are possible, without having such collinearity. Thus, a proper choice of the growth direction and SO strengths allows to select the polarization of the linear and SH spin currents according to the direction of flowing. The second system is an anisotropic 2D free electron gas with anisotropic Rashba interaction, which has been employed to study the optical conductivity of 2D puckered structures with anisotropic energy bands. The presence of mass anisotropy and an energy gap open several distinct scenarios for the allowed optical interband transitions, which manifest in the linear and SH response contrastingly. The linear response displays only out-of-plane spin polarized currents, while the SH spin currents flow with spin orientation lying parallel to the plane of the system strictly. The models illustrate the possibility of the nonlinear spin Hall effect in systems with SO interaction, under the presence or absence of time-reversal symmetry. The results suggest different ways to manipulate the linear and nonlinear optical generation of spin currents which could find spintronic applications.

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Recent studies have shown that higher-order topologies in photonic systems lead to a robust enhancement of light-matter interactions. Moreover, higher-order topological phases have been extended to systems even without a band gap, as in Dirac semimetals. In this work, we propose a procedure to simultaneously generate two distinctive higher-order topological phases with corner states that allow a double resonant effect. This double resonance effect between the higher-order topological phases, was obtained from the design of a photonic structure with the ability to generate a higher-order topological (HOTI) insulator phase in the first bands and a higher-order Dirac half-metal phase (HODSM). Subsequently, using the corner states in both topological phases, we tuned the frequencies of both corner states such that they were separated in frequency by a second harmonic. This idea allowed us to obtain a double resonance effect with ultra-high overlap factors, and a considerable improvement in the nonlinear conversion efficiency. These results show the possibility of producing a second-harmonic generation with unprecedented conversion efficiencies in topological systems with simultaneous HOTI and HODSM phases. Furthermore, since the corner state in the HODSM phase presents an algebraic 1/rdecay, our topological system can be helpful in experiments about the generation of nonlinear Dirac-ligh-matter interactions.

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ABSTRACT Purpose To evaluate the effect of fractional carbon dioxide (CO2) laser treatment using lower power associated with pulse stacking within collagen fibers, using second harmonic generation microscopy and computerized image analysis. Methods Twenty male Wistar rats aging eight weeks were used. Each treatment area received a single-pass CO2 fractional laser with different parameters. The 20 animals were divided into two groups and euthanized after 30 and 60 days. Second harmonic generation images were obtained and program ImageJ was utilized to evaluate the collagen organization within all areas. Collagen anisotropy, entropy and optical density were quantified. Results Increased anisotropy over time was observed in all four areas, but only reached statistical significance (p = 0.0305) when the mildest parameters were used (area four). Entropy decreased over time in all areas, but without significance(p = 0.1779) in area four. Density showed an overtime increase only in area four, but no statistical significance was reached (p = 0.6534). Conclusions When combined, the results obtained in this study regarding anisotropy, entropy and density tend to demonstrate that it is possible to achieve collagen remodeling with the use of lower power levels associated with stacked pulses.

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ABSTRACT Purpose To evaluate the effect of fractional carbon dioxide (CO2) laser treatment using lower power associated with pulse stacking within collagen fibers, using second harmonic generation microscopy and computerized image analysis. Methods Twenty male Wistar rats aging eight weeks were used. Each treatment area received a single-pass CO2 fractional laser with different parameters. The 20 animals were divided into two groups and euthanized after 30 and 60 days. Second harmonic generation images were obtained and program ImageJ was utilized to evaluate the collagen organization within all areas. Collagen anisotropy, entropy and optical density were quantified. Results Increased anisotropy over time was observed in all four areas, but only reached statistical significance (p = 0.0305) when the mildest parameters were used (area four). Entropy decreased over time in all areas, but without significance(p = 0.1779) in area four. Density showed an overtime increase only in area four, but no statistical significance was reached (p = 0.6534). Conclusions When combined, the results obtained in this study regarding anisotropy, entropy and density tend to demonstrate that it is possible to achieve collagen remodeling with the use of lower power levels associated with stacked pulses.

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Purpose: To evaluate the effect of fractional carbon dioxide (CO2 ) laser treatment using lower power associated with pulse stacking within collagen fibers, using second harmonic generation microscopy and computerized image analysis. Methods: Twenty male Wistar rats aging eight weeks were used. Each treatment area received a single-pass CO2 fractional laser with different parameters. The 20 animals were divided into two groups and euthanized after 30 and 60 days. Second harmonic generation images were obtained and program ImageJ was utilized to evaluate the collagen organization within all areas. Collagen anisotropy, entropy and optical density were quantified. Results: Increased anisotropy over time was observed in all four areas, but only reached statistical significance (p = 0.0305) when the mildest parameters were used (area four). Entropy decreased over time in all areas, but without significance (p = 0.1779) in area four. Density showed an overtime increase only in area four, but no statistical significance was reached (p = 0.6534). Conclusion: When combined, the results obtained in this study regarding anisotropy, entropy and density tend to demonstrate that it is possible to achieve collagen remodeling with the use of lower power levels associated with stacked pulses.(AU)

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Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiologyand pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis withglandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary amongindividuals according to the disease progression. The aim of this research was to combine histology with new immune andhistochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples weresubjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specifictechniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were presentin those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods forthe detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order todetect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. Asa result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb andIII (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result wefound specific staining in 15 samples coming from higher endometrial damage using Masson-...(AU)

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Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiologyand pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis withglandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary amongindividuals according to the disease progression. The aim of this research was to combine histology with new immune andhistochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples weresubjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specifictechniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were presentin those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods forthe detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order todetect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. Asa result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb andIII (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result wefound specific staining in 15 samples coming from higher endometrial damage using Masson-...

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INTRODUCTION AND OBJECTIVES: Hepatitis B virus (HBV) might be an etiological factor modulating fat distribution in steatotic livers. We aim to compare hepatic steatosis distribution patterns between NAFLD and FL&CHB patients with second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) method. PATIENTS AND METHODS: 42 patients with NAFLD, 46 with FL&CHB and 55 without steatosis were enrolled in the study. Overall and regional steatosis in liver sections were quantified by SHG/TPEF method. The accuracy of which was validated by pathologist evaluation and magnetic resonance spectroscopy (MRS). Difference in degree of overall and regional steatosis between NAFLD and FL&CHB groups was analyzed by Mann-Whitney U test. Multivariable linear regression analysis was used to model factors contributing to steatosis distribution. RESULTS: The hepatic steatosis measured by SHG/TPEF method was highly correlated with pathologist grading (r=0.83, p<0.001) and MRS measurement (r=0.82, p<0.001). The level of overall steatosis in FL&CHB group is significantly lower than that in NAFLD group (p<0.001). In NAFLD group, periportal region has significantly lower steatosis percentage than lobule region and overall region (p<0.001); while in FL&CHB group there is no difference among regions. The ratio of steatosis at periportal region to lobule region is significantly higher in FL&CHB group than that in NAFLD group (p<0.05). Multivariable linear regression analysis shows that HBV infection is the major contributing factor (ß=0.322, p<0.01). CONCLUSIONS: SHG/TPEF method is an accurate and objective method in hepatic steatosis quantification. By quantifying steatosis in different histological regions, we found steatosis distribution patterns are different between FL&CHB and NAFLD patients.

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Sarcoidosis and tuberculoid leprosy (TL) are prototypes of granulomatous inflammation in dermatology, which embody one of the histopathology limitations in distinguishing some diseases. Recent advances in the use of nonlinear optical microscopy in skin have enabled techniques, such as second-harmonic generation (SHG), to become powerful tools to study the physical and biochemical properties of skin. We use SHG images to analyze the collagen network, to distinguish differences between sarcoidosis and TL granulomas. SHG images obtained from skin biopsies of 33 patients with TL and 24 with sarcoidosis retrospectively were analyzed using first-order statistics (FOS) and second-order statistics, such as gray-level co-occurrence matrix (GLCM). Among the four parameters evaluated (optical density, entropy, contrast, and second angular moment), only contrast demonstrated statistical significance, being higher in sarcoidosis (p = 0.02; 4908.31 versus 2822.17). The results may indicate insufficient differentiating power for most tested FOS and GLCM parameters in classifying sarcoidosis and TL granulomas, when used individually. But in combination with histopathology (H&E and complementary stains, such as silver and fast acid stains), SHG analysis, like contrast, can contribute to distinguishing between these diseases. This study can provide a way to evaluate collagen distribution in granulomatous diseases.

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Second-harmonic generation microscopy represents an important tool to evaluate extracellular matrix collagen structure, which undergoes changes during cancer progression. Thus, it is potentially relevant to assess breast cancer development. We propose the use of second-harmonic generation images of tumor stroma selected on hematoxylin and eosin-stained slides to evaluate the prognostic value of collagen fibers analyses in peri and intratumoral areas in patients diagnosed with invasive ductal breast carcinoma. Quantitative analyses of collagen parameters were performed using ImageJ software. These parameters presented significantly higher values in peri than in intratumoral areas. Higher intratumoral collagen uniformity was associated with high pathological stages and with the presence of axillary lymph node metastasis. In patients with immunohistochemistry-based luminal subtype, higher intratumoral collagen uniformity and quantity were independently associated with poorer relapse-free and overall survival, respectively. A multivariate response recursive partitioning model determined 12.857 and 11.894 as the best cut-offs for intratumoral collagen quantity and uniformity, respectively. These values have shown high sensitivity and specificity to differentiate distinct outcomes. Values of intratumoral collagen quantity and uniformity exceeding the cut-offs were strongly associated with poorer relapse-free and overall survival. Our findings support a promising prognostic value of quantitative evaluation of intratumoral collagen by second-harmonic generation imaging mainly in the luminal subtype breast cancer.

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BACKGROUND: The diagnosis of idiopathic atrophoderma of Pasini and Pierini (IAPP) relies on typical clinical features, particularly distinctive pigmented ovular/round depressed plaques. Histologic examination often reveals no obvious changes, but patterns of collagen distribution, using multiphoton imaging and second harmonic generation can help track hidden details of tissue organization contributing to atrophy. OBJECTIVE: To identify histologic features that distinguish IAPP from unaffected skin. METHODS: Eleven patients were included for conventional analyses. Masson trichrome- and Unna-Tanzer orcein-stained sections were evaluated using automated morphometry. Hematoxylin-eosin-stained sections were analyzed by multiphoton imaging using 2-photon excited fluorescence and second harmonic generation. RESULTS: No abnormalities were found under light microscopy or by automated quantification. Multiphoton imaging revealed no difference in optical density of either collagen or elastic fibers in lesioned and unaffected skin; however, horizontal collagen fiber organization in lesion specimens increased toward the lower dermis, whereas elastic fibers featured greater disorganization within the upper dermis. LIMITATIONS: The low number of patients evaluated. CONCLUSION: The atrophic appearance of IAPP lesions reflects changes in organization, but not in collagen and elastic tissue content. Minute organizational differences that are imperceptible to the experienced pathologist and undetectable by automated analyses were revealed by multiphoton analyses, particularly second harmonic generation, in association with texture analyses.

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Collagen is the most abundant structural protein found in organs and is responsible for providing tissues with structure and function. In order to investigate in canine uteri the potential effect of medroxyprogesterone acetate (MPA) on the changes in collagen deposition were grouped as nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5 mg/kg; i.m.). The amount of collagen was studied in the fold and basal regions of the endometrium and myometrium using second harmonic generation with a two-photon spectral confocal microscope, quantified using ImageJ software with a color segmentation plugin, was expressed as fraction area (%) and analyzed by ANOVA (p<0,05). No differences were observed between groups in the fold (p=0,3995) or base (p=0,7392) of the endometrium and myometrium (p=0,1781). In conclusion, our data demonstrate that two doses of MPA (5 mg/kg; i.m.) do not affect the total collagen deposition in canine uteri undergoing contraceptive treatment.

El colágeno es la proteína estructural más abundante presente en órganos y es responsable de proporcionar la sostén y función a los tejidos. Para investigar en caninos el efecto potencial del acetato de medroxiprogesterona (MPA) sobre cambios en el depósito de colágeno en útero, éstos fueron agrupados como nulíparos (n = 11), multíparos (n = 11) y tratados con MPA (n = 11, nulíparos con dos tratamientos 5 mg/kg, im). El colágeno fue evaluado en el pliegue y regiones basales del endometrio y en miometrio utilizando la Generación de un Segundo Harmónico con un microscopio confocal espectral y dos fotones y cuantificado utilizando el software ImageJ a partir de la segmentación de colores. Los resultados fueron expresados y analizados como fracción de área (%; ANOVA; p<0,05). No se observaron diferencias entre los grupos en el pliegue (p = 0,3995) y base (p=0,7392) del endometrio y tampoco en miometrio (p=0,1781). En conclusión, nuestra evidencia demuestra que dos dosis de MPA (5 mg/kg, i.m.) no afectan el depósito total de colágeno en úteros caninos expuestos a tratamiento anticonceptivo.

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We introduce a plasmonic-semiconductor hybrid nanosystem, consisting of a ZnO nanowire coupled to a gold pentamer oligomer by crossing the hot-spot. It is demonstrated that the hybrid system exhibits a second harmonic (SH) conversion efficiency of ∼3 × 10(-5)%, which is among the highest values for a nanoscale object at optical frequencies reported so far. The SH intensity was found to be ∼1700 times larger than that from the same nanowire excited outside the hot-spot. Placing high nonlinear susceptibility materials precisely in plasmonic confined-field regions to enhance SH generation opens new perspectives for highly efficient light frequency up-conversion on the nanoscale.

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Murine induced colon cancer has been used to demonstrate that Second Harmonic Generation (SHG) microscopy images, combined with Two-Photon Excitation Fluorescence (TPEF) and specific quantization scoring methods allow distinguishing early alterations in colon mucosa. TPEF was used only to identified crypts and submucosa regions, whereas the image analysis was used to get quantitative data (Integrated Intensity and Aspect Ratio scoring) of different cancer stages. The submucosa amount of collagen fibers was significant and their orientation suffering proportional changes with the development of the pathological processes. Both after the fourth and eighth weeks after colon cancer induction, integrated intensity and aspect ratio values have shown significant statistical differences compared with control samples. Thus, SHG microscopy has proved to be a useful quantitative tool to highlight early changes of submucosa and the progression of these through the cancer development.

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In this study we showed that second-harmonic generation (SHG) microscopy combined with precise methods for images evaluation can be used to detect structural changes in the human ovarian stroma. Using a set of scoring methods (alignment of collagen fibers, anisotropy, and correlation), we found significant differences in the distribution and organization of collagen fibers in the stroma component of serous, mucinous, endometrioid and mixed ovarian tumors as compared with normal ovary tissue. This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. Our results indicated that the combination of different image-analysis approaches presented here represent a powerful tool to investigate collagen organization and extracellular matrix remodeling in ovarian tumors.

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