Monitoring process of human keloid formation based on second harmonic generation imaging

In this paper, the morphological variation of collagen among the whole dermis from keloid tissue was investigated using second harmonic generation (SHG) microscopy. In the deep dermis of keloids, collagen bundles show apparently regular gap. In the middle dermis, the collagen bundles are randomly oriented and loosely arranged in the pattern of fine mesh while the collagen bundles are organized in a parallel manner in the superficial dermis near the epidermis. The developed parameters COI and BD can be used to further quantitatively describe these changes. Our results demonstrate the potential of SHG microscopy to understand the formation process of human keloid scar at the cellular level through imaging collagen variations in different depth of dermis.     

Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy

The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25 degrees-60 degrees C. We find that the SHG signal decreases rapidly starting at 45 degrees C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57 degrees C and worsens with increasing temperature. At 57 degrees C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60 degrees C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.     

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.     

Examination of Validity of Paraxial Approximation in Second Harmonic Generation Microscopy under Low Numerical Aperture

By using a vectorial approach, the validity of paraxial approximation in second harmonic generation （SHG） microscopy under low numerical aperture （NA） is examined when the sample is a collagen fibril. Due to the larger value of dzzz and tensorial nature of SHG, the component Ez of the focused fieM may have strong effect on the radiation pattern of SHG. Numerical results indicate that when the value of NA exceeds 0.3, the effect of Ez can not be neglected, which results in the invalidation of paraxial approximation in SHG microscopy despite the fact that SHG microscopy is still under low NA focusing.     

Study of van Gieson''s picrofuchsin staining on second-harmonic generation in type I collagen

Second-harmonic generation （SHG） microscopy is a recently developed nonlinear optical imaging modality for imaging tissue structures with submicron resolution and is a potent tool for visualizing pathological effects of diseases. In this letter, we present our investigation on the influence of van Gieson＇s （VG） alcoholic pierofuchsin staining on SHG in type I collagen （from tendon-rich C57BL/6）. Multi-channel imaging and spectra analysis show that the strong SHG signal produced in fresh collagen type I fiber has been greatly suppressed after VG staining, which indicates that staining may induce the structural or characteristic changes of SHG-dependent crystal formed by collagen constituents, such as glycine, proline, and hydroxyproline.     

Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90 degrees in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 theta distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.     

Effects and mechanisms of cold atmospheric plasma on skin wound healing of rats

Cold atmospheric plasma (CAP) has been reported as a novel potential way to promote skin wound healing. The aim of this study was to explore the effects of an atmospheric‐pressure plasma generated in helium gas through dielectric barrier discharge on skin wound healing in rats and its mechanism. A total of 44 adult male Sprague–Dawley rats were divided into the CAP group and the control group after a full‐thickness skin wound model was established. The animals in the CAP group received CAP treatment (driving frequency 17 kHz, root‐mean‐square [rms] values of the discharge voltage 1.33 kV, rms values of the discharge current 10.7 mA, helium flow rate 11 slpm [standard litres per minute]) for 1 min. After that, the size of the healed area of the wound, morphological changes in the wound, wound neovascularization, collagen deposition, and the level of inflammatory factors were recorded. The results showed that compared to that of the control group, the size of the wound area in the CAP group significantly decreased, the contraction and re‐epithelialization of the wounds were obviously speeded up, and the protein level of smooth muscle actin markedly increased. However, the levels of collagen type I A2, collagen type III, and the deposition of collagen bundles did not change. In addition, it was found that the level of inflammatory factors (such as tumour necrosis factor and interleukin‐1β) significantly increased at 24 hr after the creation of the wound. These results suggest that CAP treatment in this condition could promote skin wound healing by accelerating the contraction, re‐epithelialization, neovascularization, and inflammatory response.     

Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

ItisreportedrecentlythatnonlinearopticalphenomenonofSHGandTHGhasbeenobservedinmanybiologicaltissues[16].SHGandTHGhavebeenusedtoperformthethree-dimensionalimaginginlivingtissuesandhaveattractedmuchattentionrecently.TherearemanyadvantagesofusingSHGandTHGtoperformthethree-dimensionalimaginginlivingtissues,suchasnoninvasiveandnophotobleaching,inadditiontotheimagingpropertiesofmulti-photonfluorescenceimaging[7—9].Firstly,unlikeinthesingle-andmulti-photonfluorescenceprocesses,onlyvirtualstat…     

肿瘤细胞DNA样品基于二次谐波发生(SHG)的非线性光谱学成像

二次谐波的产生是一个二阶非线性光学过程,这个过程只发生在中心对称系数不为零的非中心对称区域。利用二次谐波显微技术可以对各种具有内源性信号的生物组织进行无损伤实时成像,如结缔组织的胶原纤维或肌细胞的肌动球蛋白。在生物化学和结构生物学中,虽然DNA是由脱氧核糖核苷酸构成而蛋白质是由氨基酸残基组成,但是DNA和蛋白质大分子高级结构的形成机制是相似的。利用光谱学成像技术对不同DNA样品进行检测,获取DNA样品的SHG信号并进行高解析度成像。这些DNA样品包括基因组DNA溶液、细胞核提取物以及培养细胞的细胞核。实验结果表明在常规条件下可以获得基因组DNA溶液和细胞核提取物的SHG信号,但几乎观测不到来自培养细胞核区的SHG信号。通过在培养基中添加少量无水乙醇(体积比小于5%),可以在培养细胞的细胞核区域检测到SHG信号。推测在培养细胞中乙醇和DNA相互作用引起DNA分子构象发生变化,这些变化可能导致了DNA分子非线性光学性质的改变。     

Second harmonic generation polarization microscopy with tightly focused linearly and radially polarized beams

Second harmonic generation microscopy was conducted on rat-tail tendons with linearly and radially polarized beams. Transverse and axial field components were generated in the focal region through tight focusing of linearly and radially polarized. It was found that the generated SHG signals could not be qualitatively explained with a scalar approximation to the electric field at the focus. Only by accounting for the interactions of the axial and transverse components of the electric field interacting through the nonlinear susceptibility χ⁽²⁾ tensor could the SHG images be explained. For the case of collagen we find that the SHG signal varies as a function of the analyzer angle with a cos² or sin² dependency for linearly polarized beams. For tightly focused radially polarized beams we find that the output SHG is radially polarized after collimation and is independent of the analyzer angle.     

Tomographic Imaging of Collagen Fiber Orientation in Human Tissue Using Depth-Resolved Polarimetry of Second-Harmonic-Generation Light

We propose a nonlinear optical probe method to image the distribution of collagen fiber orientation in human tissue by measuring the polarization of collagen-induced second-harmonic-generation (SHG) light (SHG polarimetry). Depth-resolved SHG polarimetry, with a depth resolution of 14 μm, was used to evaluate the cross-sectional profile of collagen fiber orientation in Achilles tendon and dentin, revealing a characteristic distribution of collagen orientation along the depth direction. We evaluated the two-dimensional (2D) lateral distribution of collagen fiber orientation in back reticular dermis and anklebone by polarization-resolved SHG imaging, and confirmed an appreciable difference in the distribution profiles of the two samples. We further extended the method to a depth-resolved measurement of the three-dimensional (3D) distribution of collagen orientation in anklebone. The proposed system promises to be a powerful tool for in vivo measurement of collagen fiber orientation in human tissue.     

Dorsal Skin Fold Chamber for High Resolution Multiphoton Imaging

The advantages offered by multiphoton microscopy enabled this technique to be applied for in vivo understanding of physiological processes. However, multiphoton intravital microscopy also requires associated technologies to be developed. In this work, we detailed the design of a dorsal skin fold chamber made of titanium alloy that allow high resolution multiphoton fluorescence and second harmonic generation (SHG) microscopy to be achieved. We demonstrate that our apparatus is capable of obtaining high resolution, images of blood vessels and collagen matrix in nude mice. Such an imaging chamber will allow physiological processes to be investigated at high resolution     

A quasi-crystal model of collagen microstructure based on SHG microscopy

<正>Second harmonic generation(SHG) results from molecules which are polarized by an external electric field often provided by an intense laser beam.The polarizability depends on firstly the intrinsic structural properties of the substance and hence the second-order nonlinear susceptibility,and secondly the intensity and polarization direction of the incident light.The polarization characteristics of the beam are therefore of interest.In this letter,we discuss some considerations in SHG microscopy of collagen when the incoming beam is circularly polarized,and present some supporting results as well as a numerical analysis.We propose a quasi-crystal model of collagen microstructure in an effort to further our understanding on this protein.     

Investigation of Anti-Hyaluronidase Treatment on Vocal Fold Wound Healing

SUMMARY: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. Methods: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1,200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice.     

Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.     

Comparative study of laser and LED systems of low intensity applied to tendon healing

The aim of this study was to compare the effects of Low-intensity Laser Therapy (LILT) and Light Emitting Diode Therapy (LEDT) of low intensity on the treatment of lesioned Achilles tendon of rats. The experimental model consisted of a partial mechanical lesion on the right Achilles tendon deep portion of 90 rats. One hour after the lesion, the injured animals received applications of laser/LED (685, 830/630, 880 nm), and the same procedure was repeated at 24-h intervals, for 10 days. The healing process and deposition of collagen were evaluated based on a polarization microscopy analysis of the alignment and organization of collagen bundles, through the birefringence (optical retardation-OR). The results showed a real efficiency of treatments based on LEDT and confirmed that LILT seems to be effective on healing process. Although absence of coherence of LED light, tendon healing treatment with this feature was satisfactory and can certainly replace treatments based on laser light applications. Applications of infrared laser at 830 nm and LED 880 nm were more efficient when the aim is a good organization, aggregation, and alignment of the collagen bundles on tendon healing. However, more research is needed for a safety and more efficient determination of a protocol with LED.     

Refractive index dispersion properties of collagen for microscopic second-harmonic generation

We theoretically simulate second-harmonic generation (SHG) in collagen under linearly polarized focused laser beam. With this model, the effects of numerical aperture (NA) and refractive index dispersion on SHG emission have been investigated. Dispersion properties of collagen are significant under incident wavelength in the visible range. Our results show that the efficient SHG is obtained by controlling the NA, and the higher NA is a necessity when the dispersion effect is considered. Our theoretical simulation results provide useful clues for experimental study of microscopic SHG emission in collagen excited by focused beam.     

In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non-linear phenomena

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image contrast modes on the same microscope. Three-dimensional (3D) reconstructions of TPEF, SHG and THG images were also performed. Moreover, THG imaging technique has been tested as a potential, novel, non-destructive diagnostic tool for monitoring cellular processes in vivo, such as neuronal degeneration.     

Atomic force microscopy investigation of chemically stabilized pericardium tissue

Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.     

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.