Perfectly registered multiphoton and reflectance confocal video rate imaging of in vivo human skin

We present a multimodal in vivo skin imaging instrument that is capable of simultaneously acquiring multiphoton and reflectance confocal images at up to 27 frames per second with 256 × 256 pixel resolution without the use of exogenous contrast agents. A single femtosecond laser excitation source is used for all channels ensuring perfect image registration between the two‐photon fluorescence (TPF), second harmonic generation (SHG), and reflectance confocal microscopy (RCM) images. Images and videos acquired with the system show that the three imaging channels provide complementary information in in vivo human skin measurements. In the epidermis, cell boundaries are clearly seen in the RCM channel, while cytoplasm is better seen in the TPF imaging channel, whereas in the dermis, SHG and TPF channels show collagen bundles and elastin fibers, respectively. The demonstrated fast imaging speed and multimodal imaging capabilities of this MPM/RCM instrument are essential features for future clinical application of this technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Charge contrast imaging of biomaterials in a variable pressure scanning electron microscope

Charge contrast imaging (CCI) is a dynamic phenomenon recently reported in insulating and semiconducting materials imaged with low vacuum or variable pressure scanning electron microscopes (SEM). Data presented in this paper illustrates that CCI can also be applied to biominerals and biological soft-tissues and that useful and unique structural information can be obtained from routine samples. Various resin-embedded samples were considered and example images from several different biomaterials are presented. Due to the diverse nature of samples that appear to exhibit charge contrast, this imaging technique has prospective application in a wide range of biological and biomedical research. This work represents the first application of CCI to biomaterials and in particular, highlights a new method for investigating the formation, structure and growth of biominerals.     

Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

A developed temporal focusing‐based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back‐focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 μm to 1.5 μm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture : TPEF images of the eosin‐stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 μm^?1 spatially modulated illumination at 90° (center) and 0° (right) orientations.

     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

A compact high‐speed full‐field optical coherence microscope for high‐resolution in vivo skin imaging

A compact high‐speed full‐field optical coherence microscope has been developed for high‐resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm³ and a weight of 210 g. Full‐field illumination with low‐coherence light is achieved with a high‐brightness broadband light‐emitting diode. High‐speed full‐field detection is achieved by using part of the image sensor of a high‐dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 μm × 1.2 μm (axial × transverse), over a field of view of 245 × 245 μm². Images of human skin, revealing in‐depth cellular‐level structures, were obtained in vivo and in real‐time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm², but at a reduced depth.      

大豆食心虫触角感器的扫描电镜观察

利用扫描电镜观察了大豆食心虫Leguminivora glycinivorella Matsumura成虫的触角感器的形态和分布。结果表明,大豆食心虫成虫触角呈线型,感器包括毛型感器、耳型感器、刺型感器、腔锥型感器、栓锥形感器、鳞型感器和Bhm氏鬃毛,共7种。雌雄个体之间触角感器的分布特点基本相同,但是种类、数量有差异,存在性二型现象。     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

麻疯树柄细蛾触角及其感器的扫描电镜观察

应用扫描电镜对麻疯树柄细蛾Stomphastis thraustica Meyrick成虫触角的外部形态结构及其感器进行了观察和研究。结果表明,麻疯树柄细蛾成虫触角上存在着5种感器即毛形感器、刺形感器、腔椎形感器、锥形感器和鳞形感器。对各种感器的形态特点进行描述,其中毛形感器数量最多,并分为长毛形和短毛形2种。刺形感器也分为长刺形和短刺形2种。     

瘤胫厕蝇触角感受器的超微结构观察

应用扫描电镜对瘤胫厕蝇Fannia scalaris(Fabricius)成虫触角的外部形态结构及其感受器进行了观察和研究.结果表明,瘤胫厕蝇成虫触角上存在着2类感受器:毛形感受器和锥形感受器,其中毛形感受器数量最多,分为5种.对各种感受器的形态特点进行了描述.     

In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation

This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to approximately 65 μm s^?1. Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.      

Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging

Light‐sheet fluorescence microscopy (LSFM) is a powerful tool for biological studies because it allows for optical sectioning of dynamic samples with superior temporal resolution. However, LSFM using 2 orthogonally co‐aligned objectives requires a special sample geometry, and volumetric imaging speed is limited due to physical sample translation. This paper describes an oblique scanning 2‐photon LSFM (OS‐2P‐LSFM) that eliminates these limitations by using a single objective near the sample and a refractive scanning‐descanning system. This system also provides improved light‐sheet confinement against scattering by using a 2‐photon Bessel beam. The OS‐2P‐LSFM hold promise for studying structural, functional and dynamic aspects of living tissues and organisms because it allows for high‐speed, translation‐free and scattering‐robust 3D imaging of large biological specimens.      

Self‐referenced axial chromatic dispersion measurement in multiphoton microscopy through 2‐color third‐harmonic generation imaging

With tunable excitation light, multiphoton microscopy is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here, we experimentally demonstrate a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2‐color third‐harmonic generation imaging excited by a 2‐color soliton source with tunable wavelength separation. Our technique is self‐referenced, eliminating potential measurement error when 1‐color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2‐color imaging, may open up opportunity for simultaneous imaging of 2 different axial planes.      

近场扫描光学显微镜及其在生物学领域的应用

评述了近场扫描光学显微镜（near-field scanning optical microscopy,NSOM）的仪器构造、工作原理及其在生物学领域的应用成果。对NSOM目前存在的主要问题进行了讨论,并展望了NSOM在该领域的发展潜力。     

激光共聚焦显微镜在微痕定量分析中的应用综述

微痕分析是石器、骨器、牙齿等工艺、功能研究的常用方法,但无论是高倍还是低倍等常用方法,受限于体式光学显微镜、扫描电镜等观察方法,微痕分析通常很难进行原位定量分析,因此微痕分析的定量化是目前学术界遇到的主要问题。近年来随着激光共聚焦显微镜（LSCM）在微痕分析中的应用,微痕定量化在西方学术界得到了新的实践和进展。本文主要介绍激光共聚焦显微镜的原理及其在微痕分析中的应用案例,以期促进微痕分析在中国的进一步发展。     

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.     

Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo

In this study, intravital multiphoton microscopy was used to quantitatively investigate hepatobiliary metabolism in chronic pathologies of the liver. Specifically, through the use of the probe molecule 6‐carboxyfluorescein diacetate, the effects of liver fibrosis, fatty liver, and hepatocellular carcinoma on the metabolic capabilities of mouse liver were investigated. After the acquisition of time‐lapse images, a first order kinetic model was used to calculate rate constant resolved images of various pathologies. It was found that the ability of the liver to metabolically process the probe molecules varies among different pathologies, with liver fibrosis and fatty liver disease negatively impacted the uptake, processing, and excretion of molecules. The approach demonstrated in this work allows the study of the response of hepatic functions to different pathologies in real time and is useful for studying processes such as pharmacokinetics through direct optical imaging.      

屋尘螨成螨形态的扫描电镜观察

【目的】利用扫描电子显微镜（scanning electron microscopy, SEM）观察屋尘螨Dermatophagoides pteronyssinus颚体、 躯体、 外生殖器及足等的形态结构。【方法】从床尘、 枕尘中采集屋尘螨, 分离出雌雄成螨, 在体视显微镜下清洗处理活螨后, 用SEM观察其外部形态特征。【结果】SEM照片显示, 屋尘螨螯肢钳状, 须肢扁平; 体表具细密皮纹, 似指纹状, 纹间距小于2 μm。外生殖器位于腹面正中, 雌螨为产卵孔, 雄螨生殖孔具1对叶状生殖盖。肛门呈纵行裂孔, 雄螨具2个肛吸盘。雌螨足跗节末端各具爪垫1个, 雄螨跗节Ⅳ具2个吸盘。【结论】本研究观察结果为尘螨鉴定提供了更多依据。     

High order DNA structure as inferred by optical fluorimetry and scanning calorimetry

New quantitative insights on the native high order chromatin-DNA structure existing within interphase nuclei are obtained by monitoring the effects of two common well-characterized fixatives, glutaraldehyde and ethanol/acetic acid mixture, at the level of the intranuclear DNA distribution and structures. Reproducible distinct levels of DNA fluorescence intensity and their intranuclear distribution are apparent in unfixed and fixed thymocytes by using DAPI and quantitative optical microscopy based on a charge coupled device. The fluorescent histograms correlated with the calorimetric thermograms on the very same thymocytes fixed and unfixed, establish an unequivocal baseline for the different levels of structural organization of the chromatin within the intact nucleus; namely their number, DNA packing ratio and fiber diameter. A systematic comparison among all the numerous models, being so far proposed for the quinternary and quaternary levels of DNA folding, to identifies the rope or ribbon-like and the chromonema as the ones that best fit with the in situ distribution. This revised version was published online in June 2006 with corrections to the Cover Date.     

The digestive tract of the amberjack Seriola dumerili, Risso: a light and scanning electron microscope study

The histology of the digestive tract of the amberjack ( Seriola dumerili , Risso) was studied using light and scanning electron microscopy. The anterior oesophagus mucosa displays primary and secondary folds lined with a stratified squamous epithelium with fingerprint-like microridges which is substituted, on the top of the oesogaster folds, by a simple columnar epithelium with short microvilli. Only primary folds are present in the stomach. The anterior portion is rich in simple tubular glands, whereas the oesogaster and the pyloric region are devoid of them. Pyloric caeca and anterior and middle intestine mucosa display the same pattern of folding. The dominant cell type is the enterocyte, which exhibits larger and thinner microvilli in the caeca than in the intestine. The columnar epithelium of the rectum is replaced, in the anal sphincter, by a stratified flattened epithelium. Goblet cells are numerous throughout the whole length of the tract with the exception of the initial part of the oesophagus, the oesogaster, the stomach and the anal sphincter. Mucosubstances have been shown to vary in the different regions of the gut: acid mucines are found in the oesophagus, pyloric stomach, caeca, intestine and rectum, whereas neutral mucosubstances dominate in the anterior portion of the stomach. The muscularis is well developed throughout the length of the tract: two layers of striated muscle at the oesophageal level; two layers of smooth muscle in the stomach wall and three at the intestinal level.