Quantification of the second-order nonlinear susceptibility of collagen I using a laser scanning microscope

Characteristic changes in the organization of fibrillar collagen can potentially serve as an early diagnostic marker in various pathological processes. Tissue types containing collagen I can be probed by pulsed high-intensity laser radiation, thereby generating second harmonic light that provides information about the composition and structure at a microscopic level. A technique was developed to determine the essential second harmonic generation (SHG) parameters in a laser scanning microscope setup. A rat-tail tendon frozen section was rotated in the xy-plane with the pulsed laser light propagating along the z-axis. By analyzing the generated second harmonic light in the forward direction with parallel and crossed polarizer relative to the polarization of the excitation laser beam, the second-order nonlinear optical susceptibilities of the collagen fiber were determined. Systematic variations in SHG response between ordered and less ordered structures were recorded and evaluated. A 500 microm-thick z-cut lithiumniobate (LiNbO(3)) was used as reference. The method was applied on frozen sections of malignant melanoma and normal skin tissue. Significant differences were found in the values of d(22), indicating that this parameter has a potential role in differentiating between normal and pathological processes.     

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.     

Detection and diagnosis of oral neoplasia with an optical coherence microscope

The use of high resolution, in vivo optical imaging may offer a clinically useful adjunct to standard histopathologic techniques. A pilot study was performed to investigate the diagnostic capabilities of optical coherence microscopy (OCM) to discriminate between normal and abnormal oral tissue. Our objective is to determine whether OCM, a technique combining the subcellular resolution of confocal microscopy with the coherence gating and heterodyne detection of optical coherence tomography, has the same ability as confocal microscopy to detect morphological changes present in precancers of the epithelium while providing superior penetration depths. We report our results using OCM to characterize the features of normal and neoplastic oral mucosa excised from 13 subjects. Specifically, we use optical coherence and confocal microscopic images obtained from human oral biopsy specimens at various depths from the mucosal surface to examine the optical properties that distinguish normal and neoplastic oral mucosa. An analysis of penetration depths achieved by the OCM and its associated confocal arm found that the OCM consistently imaged more deeply. Extraction of scattering coefficients from reflected nuclear intensity is successful in nonhyperkeratotic layers and shows differentiation between scattering properties of normal and dysplastic epithelium and invasive cancer.     

不同细胞培养皿对激光共聚焦显微镜成像效果的影响

目的 探讨3种不同玻片厚度的激光共聚焦专用细胞培养皿制备的细胞样品在激光共聚焦显微镜(CLSM)下采集的荧光染色图像差异.方法通过活细胞及固定后细胞荧光染色实验,采用激光共聚焦显微镜观察细胞形态及荧光亮度,检测不同条件下成像的平均荧光强度,考察3种厚度玻片(0.085～0.13、0.13～0.16、0.16～0.19m...     

Quantification of optical properties of a breast tumor using random walk theory

For the first time we use a random walk methodology based on time-dependent contrast functions to quantify the optical properties of breast tumors (invasive ductal carcinoma) of two patients. Previously this theoretical approach was successfully applied for analysis of embedded objects in several phantoms. Data analysis was performed on distributions of times of flight for photons transmitted through the breast which were recorded in vivo using a time-domain scanning mammograph at 670 and 785 nm. The size of the tumors, their optical properties, and those of the surrounding tissue were reconstructed at both wavelengths. The tumors showed increased absorption and scattering. From the absorption coefficients at both wavelengths blood oxygen saturation was estimated for the tumors and the surrounding tissue.     

Quantification of breast density with spectral mammography based on a scanned multi-slit photon-counting detector: a feasibility study

A simple and accurate measurement of breast density is crucial for the understanding of its impact in breast cancer risk models. The feasibility to quantify volumetric breast density with a photon-counting spectral mammography system has been investigated using both computer simulations and physical phantom studies. A computer simulation model involved polyenergetic spectra from a tungsten anode x-ray tube and a Si-based photon-counting detector has been evaluated for breast density quantification. The figure-of-merit (FOM), which was defined as the signal-to-noise ratio of the dual energy image with respect to the square root of mean glandular dose, was chosen to optimize the imaging protocols, in terms of tube voltage and splitting energy. A scanning multi-slit photon-counting spectral mammography system has been employed in the experimental study to quantitatively measure breast density using dual energy decomposition with glandular and adipose equivalent phantoms of uniform thickness. Four different phantom studies were designed to evaluate the accuracy of the technique, each of which addressed one specific variable in the phantom configurations, including thickness, density, area and shape. In addition to the standard calibration fitting function used for dual energy decomposition, a modified fitting function has been proposed, which brought the tube voltages used in the imaging tasks as the third variable in dual energy decomposition. For an average sized 4.5 cm thick breast, the FOM was maximized with a tube voltage of 46 kVp and a splitting energy of 24 keV. To be consistent with the tube voltage used in current clinical screening exam (～32 kVp), the optimal splitting energy was proposed to be 22 keV, which offered a FOM greater than 90% of the optimal value. In the experimental investigation, the root-mean-square (RMS) error in breast density quantification for all four phantom studies was estimated to be approximately 1.54% using standard calibration function. The results from the modified fitting function, which integrated the tube voltage as a variable in the calibration, indicated a RMS error of approximately 1.35% for all four studies. The results of the current study suggest that photon-counting spectral mammography systems may potentially be implemented for an accurate quantification of volumetric breast density, with an RMS error of less than 2%, using the proposed dual energy imaging technique.     

Quantification of the wound healing using polarization-sensitive optical coherence tomography

We use polarization-sensitive optical coherence tomography (PS-OCT) to monitor the wound healing process in vitro and in vivo, which are affected by various drugs. Five rabbit subjects are used for in vitro studies and another five are used for in vivo studies. The in vitro studies are conducted to compare the PS-OCT images with histopathology. For each subject, three biopsy lesions are created on each ear: one site is not treated (control); the second site is treated with sphingosylphosphorylcholine, which is expected to promote healing; and the last is administered with tetraacetylphytosphingosine, which negatively affects the healing process. Each site is examined with a PS-OCT system at 1, 4, 7, 10, and 14- days after wound generation. The variations of phase retardation values caused by the collagen morphology changes on wound sites are quantified for all cases. Our results suggest that PS-OCT may be a useful tool for visualization of collagen fiber regeneration and for quantification of various drug effects during the wound healing process.     

Selective imaging of surface fluorescence with very high aperture microscope objectives

Three approaches to selective surface fluorescence detection are described. All three of them depend on the use of extremely high numerical aperture (NA) objectives now commercially available (1.45 NA from Zeiss and Olympus and 1.65 NA from Olympus). The first two approaches are elaborations of "prismless" total internal reflection fluorescence (TIRF), one approach with a laser illumination and the second with arc lamp illumination. The new higher NA objectives are much more suitable for TIRF work on biological cells in culture than are 1.4 NA objectives previously described for prismless TIRF. The third approach is not TIRF at all. It uses the high aperture objective to selectively gather the emission of fluorophores located close enough to the substrate for their near-field energy to be captured by the substrate. Schematic diagrams, experimental demonstrations, and practical suggestions for all these techniques are provided.     

共聚焦激光扫描显微镜技术及相关荧光探针在细胞器研究中的应用

共聚焦激光扫描显微镜 (ｃｏｎｆｏｃａｌｌａｓｅｒｓｃａｎｎｉｎｇｍｉｃｒｏｓｃｏｐｅ,ＣＬＳＭ)是上世纪 80年代发展起来的先进的分子细胞生物学分析仪器。它在荧光显微镜成像基础上加装了激光扫描装置 ,利用计算机进行图像处理 ,把光学成像的分辨率提高了 30 %～ 4 0 % ,是光学显微镜发展史上的重大突破。它通过其独特的成像原理和技术[1] ,使用紫外线或可见光激发荧光探针 ,不仅可观察固定的细胞、组织切片 ,还可以对活细胞的结构、分子、离子等进行实时动态观察和检测 ,在亚细胞水平上观察诸如Ｃａ2 + ,ｐＨ值 ,膜电位等生理信号及…     

The optical density of erythrolabe determined by a new method

1. A new method is described for the determination of optical density by retinal densitometry based on an analysis of stray light.2. The basic assumption used is that the stray light present in retinal densitometry is independent of wave-length. The justification for this assumption is considered.3. The stray light and hence the optical density was determined by a method of successive approximations using a Linc 8 computer.4. The optical density of erythrolabe for the direction of maximum Stiles-Crawford efficiency was determined for five subjects, two deuteranopes, two deuteranomalous and one red-rich normal subject. The mean values for three of the subjects were close to the grand mean value of 0.42, but the other two subjects had considerably higher and lower values respectively.5. The results are in satisfactory agreement with optical density values determined by the self-screening method using retinal densitometry, and also with determinations using microspectrophotometry and psychophysics.6. The relevance of dielectric wave-guide modes in the outer segments is considered.     

Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis

We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that obtained with other commonly used red fluorochromes or by inspection of the reaction product under bright field illumination. Of particular value, the reaction product is unaffected by the denaturing conditions required for the detection of incorporated nuclear BrdU with FITC conjugated anti-BrdU antibody and provides a simple and robust method for the simultaneous detection of cell proliferation and cell surface markers.     

Surface density mapping of natural tissue by a scanning haptic microscope (SHM)

Abstract

To expand the performance capacity of the scanning haptic microscope (SHM) beyond surface mapping microscopy of elastic modulus or topography, surface density mapping of a natural tissue was performed by applying a measurement theory of SHM, in which a frequency change occurs upon contact of the sample surface with the SHM sensor – a microtactile sensor (MTS) that vibrates at a pre-determined constant oscillation frequency. This change was mainly stiffness-dependent at a low oscillation frequency and density-dependent at a high oscillation frequency. Two paragon examples with extremely different densities but similar macroscopic elastic moduli in the range of natural soft tissues were selected: one was agar hydrogels and the other silicon organogels with extremely low (less than 25?mg/cm³) and high densities (ca. 1300?mg/cm³), respectively. Measurements were performed in saline solution near the second-order resonance frequency, which led to the elastic modulus, and near the third-order resonance frequency. There was little difference in the frequency changes between the two resonance frequencies in agar gels. In contrast, in silicone gels, a large frequency change by MTS contact was observed near the third-order resonance frequency, indicating that the frequency change near the third-order resonance frequency reflected changes in both density and elastic modulus. Therefore, a density image of the canine aortic wall was subsequently obtained by subtracting the image observed near the second-order resonance frequency from that near the third-order resonance frequency. The elastin-rich region had a higher density than the collagen-rich region.     

Quantification of cardiac fiber orientation using optical coherence tomography

Heterogeneity in cardiac tissue microstructure is a potential mechanism for the generation and maintenance of arrhythmias. Abnormal changes in fiber orientation increase the likelihood of arrhythmia. We present optical coherence tomography (OCT) as a method to image myofibers in excised intact heart preparations. Three-dimensional (3-D) image sets were gathered from the rabbit right ventricular free wall (RVFW) using a microscope-integrated OCT system. An automated algorithm for fiber orientation quantification in the plane parallel to the wall surface was developed. The algorithm was validated by comparison with manual measurements. Quantifying fiber orientation in the plane parallel to the wall surface from OCT images can be used to help understand the conduction system of the specific sample being imaged.     

基于光学、电子显微镜图像的烧骨组织学变化特征

目的:运用光学、电子显微镜观察烧骨组织形态变化特征,探讨其法医学应用价值.方法:对常压下以200℃～1 000℃焚烧1h的30例成人股骨组织学进行光学显微镜、扫描电子显微镜观察和测量.结果:光学、扫描电子显微镜图像中均可辨别出骨单位、间骨板组织.随温度升高,哈佛氏系统渐模糊,骨板渐融合,中央管、Volkmann氏管周围浓染,颗粒块状物增多,骨小梁不规则.骨细胞渐减少、消失并皱缩为圆形、卵圆形、颗粒状.骨墙厚度由70 μm收缩到46-μm,中央管直径在35～20 μm,哈佛氏系统周长在570～420μm,Volkmann氏管直径在22～17 μm.裂纹出现于400C,并随着温度升高;逐渐增多、增宽,以中央管为中心呈放射状排列,裂宽在10～28 μm.结论:烧骨表面颜色可反映焚烧时的大致温度并可估计骨细胞存留状况.运用LM、SEM图像观察烧骨组织学变化可作为烧骨判定的基础资料.     

B7-H4实时荧光定量PCR检测方法的建立及其在人胃癌组织中的初步应用

目的建立实时荧光定量PCR检测B7-H4的方法并初步应用于人胃癌组织。方法将B7-H4和内参GAPDH的PCR扩增产物经测序鉴定正确后克隆入载体pMD19-T,构建重组质粒标准品,并纯化、定量及梯度稀释,分别建立B7-H4和GAPDH的标准曲线,应用实时荧光定量PCR检测8例人胃癌组织中的B7-H4相对于GAPDH的表达情况。结果 B7-H4的最低检测拷贝数为5.27拷贝,线性范围5.27×101~5.27×107拷贝,标准曲线方程y=–3.1395x+41.805,直线回归相关系数r=0.994904,批间变异系数范围2.39%~3.59%,扩增效率108.2%;GAPDH的最低检测拷贝数为38.6拷贝,线性范围3.86×102~3.86×107拷贝,标准曲线方程y=–3.2436x+41.083,直线回归相关系数r=0.998913,批间变异系数范围2.26%~3.86%,扩增效率103.4%。8例人胃癌组织的B7-H4相对于GAPDHmRNA表达水平在0.044~0.888之间。结论荧光定量PCR检测B7-H4的方法具备较高的敏感性和特异性,且系统有良好的重复性和线性范围。