Optical delineation of human malignant melanoma using second harmonic imaging of collagen

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.     

Assessment of the corneal collagen organization after chemical burn using second harmonic generation microscopy

The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.     

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation

In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.     

Characterization of collagen in non-small cell lung carcinoma with second harmonic polarization microscopy

Polarization second harmonic microscopy was used for collagen imaging in human non-small cell lung carcinoma and normal lung tissues ex vivo and revealed significant differences in the nonlinear susceptibility component ratio, demonstrating potential use in cancer diagnosis.OCIS codes: (180.0180) Microscopy, (180.4315) Nonlinear microscopy, (190.4160) Multiharmonic generation     

Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy

Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.OCIS codes: (180.4315) Nonlinear microscopy, (170.3880) Medical and biological imaging, (170.4730) Optical pathology, (170.6935) Tissue characterization     

Quantifying collagen structure in breast biopsies using second-harmonic generation imaging

Quantitative second-harmonic generation imaging is employed to assess stromal collagen in normal, hyperplastic, dysplastic, and malignant breast tissues. The cellular scale organization is quantified using Fourier transform-second harmonic generation imaging (FT-SHG), while the molecular scale organization is quantified using polarization-resolved second-harmonic generation measurements (P-SHG). In the case of FT-SHG, we apply a parameter that quantifies the regularity in collagen fiber orientation and find that malignant tissue contains locally aligned fibers compared to other tissue conditions. Alternatively, using P-SHG we calculate the ratio of tensor elements (d₁₅/d₃₁, d₂₂/d₃₁, and d₃₃/d₃₁) of the second-order susceptibility χ² for collagen fibers in breast biopsies. In particular, d₁₅/d₃₁ shows potential differences across the tissue pathology. We also find that trigonal symmetry (3m) is a more appropriate model to describe collagen fibers in malignant tissues as opposed to the conventionally used hexagonal symmetry (C6). This novel method of targeting collagen fibers using a combination of two quantitative SHG techniques, FT-SHG and P-SHG, holds promise for breast tissue analysis and applications to characterizing cancer in a manner that is compatible with clinical practice.OCIS codes: (180.4315) Nonlinear microscopy, (100.2960) Image analysis     

A novel approach for assessing cardiac fibrosis using label-free second harmonic generation

To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red staining was used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p < 0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts.     

The structural origin of second harmonic generation in fascia

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.     

The structural origin of second harmonic generation in fascia

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.OCIS codes: (180.4315) Nonlinear microscopy, (190.4160) Multiharmonic generation     

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy

We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.     

High-resolution X-ray microtomography for three-dimensional imaging of cardiac progenitor cell homing in infarcted rat hearts

The recent introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathological heart and has opened a search for new therapeutic strategies. Recent published reports have contributed to identifying possible cellular therapy approaches to generate new myocardium, involving transcoronary and intramyocardial injection of progenitor cells. However, one of the limiting factors in the overall interpretation of clinical results obtained by cell therapy is represented by the lack of three-dimensional (3D) high-resolution methods for the visualization of the injected cells and their fate within the myocardium. This work shows that X-ray computed microtomography may offer the unique possibility of detecting, with high definition and resolution and in ex vivo conditions, the 3D spatial distribution of rat cardiac progenitor cells, labelled with iron oxide nanoparticles, inside the infarcted rat heart early after injection. The obtained 3D images represent a very innovative progress as compared to experimental two-dimensional (2D) histological analysis, which requires time-consuming energies for image reconstruction in order to provide the overall distribution of rat clonogenic cells within the heart. Through microtomography, we were able to observe in 3D the presence of these cells within damaged cardiac tissue, with important structural details that are difficult to visualize by conventional bidimensional imaging techniques. This new 3D-imaging approach appears to be an important way to investigate the cellular events involved in cardiac regeneration and represents a promising tool for future clinical applications.     

Quantification of scar collagen texture and prediction of scar development via second harmonic generation images and a generative adversarial network

Widely used for medical analysis, the texture of the human scar tissue is characterized by irregular and extensive types. The quantitative detection and analysis of the scar texture as enabled by image analysis technology is of great significance to clinical practice. However, the existing methods remain disadvantaged by various shortcomings, such as the inability to fully extract the features of texture. Hence, the integration of second harmonic generation (SHG) imaging and deep learning algorithm is proposed in this study. Through combination with Tamura texture features, a regression model of the scar texture can be constructed to develop a novel method of computer-aided diagnosis, which can assist clinical diagnosis. Based on wavelet packet transform (WPT) and generative adversarial network (GAN), the model is trained with scar texture images of different ages. Generalized Boosted Regression Trees (GBRT) is also adopted to perform regression analysis. Then, the extracted features are further used to predict the age of scar. The experimental results obtained by our proposed model are better compared to the previously published methods. It thus contributes to the better understanding of the mechanism behind scar development and possibly the further development of SHG for skin analysis and clinic practice.     

经静脉二次谐波超声造影对正常兔肝造影分期的声学定量分析

目的　利用声学定量技术对正常兔肝的超声造影分期进行探讨。方法　对 10只健康大白兔进行二次谐波超声造影 ,观察经耳缘静脉注射造影剂时 ,门静脉主干血流阻断前后 ,肝组织声学定量 (AD)曲线形态及测值变化 ;及经耳缘静脉和经肠系膜静脉注射造影剂时 ,肝组织AD曲线形态及测值变化。结果　经耳缘静脉注射造影剂 ,正常兔肝组织AD曲线形态呈单峰形 ,近似直角三角形 ,可分为上升段、高峰段和下降段 ,峰值密度(PI)值为 (18.4± 1.7)dB。上升段为肝动脉期 ,高峰段和下降段均为门静脉期 ;门静脉主干血流阻断后 ,肝组织AD曲线形态与阻断前基本相似 ,但峰值略减低 ,其PI值平均减低 2dB。经耳缘静脉注射造影剂后 10～ 15s再经肠系膜静脉注射造影剂 ,肝组织AD曲线呈“驼峰征” ,第二个峰高于第一个峰。结论　正常兔肝声学造影肝动脉期短暂 ,门静脉期长。肝组织回声增强主要由于肝动脉来源造影剂所致 ,门静脉来源造影剂增强幅度低 ,仅起次要作用     

微泡声学造影剂气体构成对二次谐波显像的作用

目的探讨二次谐波成像条件下改变微泡造影剂的气体构成是否能够增强经静脉注射后的心肌显像效果。方法对１０条开胸犬经静脉注射含有不同气体的声振右旋糖酐白蛋白微泡造影剂ＲＡ（空气,ｒｏｏｍａｉｒ）、ＳＦ６（六氟化硫,ｓｕｌｆｕｒｈｅｘａｆｌｕｏｒｉｄｅ）、ＦＸ系列（含有氟碳气体Ｃ３Ｆ８、Ｃ４Ｆ１０、Ｃ５Ｆ１２）。二次谐波显像条件下取左心室短轴切面观,观察心肌造影效果。结果含有氟碳气体的微泡造影剂经静脉注射后二次谐波显像下产生强烈的心肌显影,心肌声学密度分别为（１４．７２±２．７）ｄＢ（ＦＸ３３０）、（１４．３±２．４ｄＢ）（ＦＸ４３０）、（１３．７８±２．２４）ｄＢ（ＦＸ５３０）;氟硫气体仅存在轻微的心肌显像［（６．２４±１．５８）ｄＢ］,但心腔显影强烈［（２９．４８±６．０２）ｄＢ］,空气造影剂经静脉注射后几乎不产生心肌显影［（２．５３±０．７８）ｄＢ］,心腔内造影剂亦较弱［（１１．３±４．９６）ｄＢ］。结论不同气体构成的微泡声学造影剂二次谐波显像效果差异显著。氟碳气体声振右旋糖酐白蛋白是较好的经静脉声学造影剂。     

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.     

正常肝脏间歇二次谐波声学造影的初步研究

目的探讨正常肝组织间歇二次谐波声学造影显像特点.方法20例正常自愿者接受肝脏间歇二次谐波声学造影检查,间歇成像由心电图R波触发,触发间隔为1～5个心动周期.声学造影剂经左前臂浅静脉"弹丸式"注射.结果注射造影剂后15～18 s[平均(16.8±1.5)s]肝动脉显影,实质回声增强.肝动脉血管呈"亮线"状强回声,似"枯树枝状"形态.肝动脉显影后6～10 s[平均(7.9±1.7)s]门静脉显影,肝实质回声再次增强,门脉血管呈"条带"状强回声.显影全过程持续95～126 s[平均(108±7)s].注射造影剂后延迟3min再触发成像,肝实质呈均匀增强回声.结论间歇二次谐波声学造影是检测组织血流的敏感方法,不仅能清楚显示较大血管结构,还能反映微循环血供状况,为临床提供极为丰富的血流信息.     

肝脏VX_2肿瘤声学造影的动态变化时相

目的　观察兔肝脏VX2 肿瘤在二次谐波声学造影中的增强效果及动态变化时相。方法　 1 7只接种VX2 肝肿瘤的新西兰白兔经外周静脉注射氟碳声学造影剂FX5 30 ,采用Sequoia5 1 2超声仪的二次谐波显像观察肿瘤造影增强过程 3min。结果　VX2 肿瘤在声学造影中出现了三个特异性的动态变化时相 ,即动脉相、门脉相和延迟相。动脉相 (造影后 5～ 1 0s)中出现肿瘤血管早期增强 ;门脉相 (造影后 1 0～ 30s)中肝实质逐渐增强 ,肿瘤血管影像减弱 ;延迟相 (造影 30s以后 )肝实质强烈增强 ,VX2 肿瘤呈弱回声病灶。结论　新型声学造影方法可以发现VX2 肿瘤特异性的造影动态变化 ,显著提高肿瘤诊断特异性。     

二次谐波技术在胰腺肿块诊断中的应用

目的 探讨二次谐波技术对胰腺肿块的诊断价值。方法 对47例患者和30例正常胰腺者比较基波与谐波条件下二维及彩色血流特点。结果 谐波条件下不同类型胰腺肿块的二维特征性更加清晰，正常胰腺和肿块周围及内部的血流可得到不同显示。结论 谐波技术对受气体干扰明显、位置较深的胰腺肿块的超声检出和鉴别诊断有重要临床意义。     

Effects of transmit focusing on finite amplitude distortion based second harmonic generation

Generation of the second harmonic signal was studied for finite amplitude distortion based harmonic imaging. Acoustic field amplitudes along the range axis of a fixed focus transducer were measured using a PVDF needle hydrophone. Results indicated that on-axis amplitudes strongly depended on the f-number at both the fundamental and the second harmonic frequencies. Differences of the on-axis amplitudes between the two frequencies were also investigated. To explore the possibility of increasing harmonic generation by extending the depth of focus, a two-focus transducer was employed. Hydrophone measurements, pulse-echo imaging and simulations were performed. Although the increase in harmonic generation depended on specific imaging parameters, the effectiveness of improving the harmonic signal-to-noise ratio (SNR) by increasing the depth of field was clearly demonstrated. Degradation in contrast resolution associated with the two-focus transducer was also evaluated. It was found that the contrast resolution was still significantly better than that of the fundamental image at the same frequency. Results of the study using the two-focus transducers can be generalized to imaging systems with full dynamic transmit focusing capabilities. In other words, it is expected that dynamic transmit focusing can improve the SNR of finite amplitude distortion based second harmonic imaging while improving the contrast resolution over fundamental imaging.     

Chiral imaging of collagen by second-harmonic generation circular dichroism

We provide evidence that the chirality of collagen can give rise to strong second-harmonic generation circular dichroism (SHG-CD) responses in nonlinear microscopy. Although chirality is an intrinsic structural property of collagen, most of the previous studies ignore that property. We demonstrate chiral imaging of individual collagen fibers by using a laser scanning microscope and type-I collagen from pig ligaments. 100% contrast level of SHG-CD is achieved with sub-micrometer spatial resolution. As a new contrast mechanism for imaging chiral structures in bio-tissues, this technique provides information about collagen morphology and three-dimensional orientation of collagen molecules.OCIS codes: (170.3880) Medical and biological imaging, (180.4315) Nonlinear microscopy, (190.2620) Harmonic generation and mixing