激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700nm）内的光谱带宽,其最小光谱带宽优于5nm。用488nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1mm。在全光谱和50μm出射狭缝（对应2.5nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和AlexaFluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

基于激光共焦扫描显微镜方法的磨损表面三维数字化描述

表面形貌的精确描述在许多领域诸如材料、生物医学、摩擦学和机器状态监测等领域变得越来越重要。开发了一种基于激光共焦显微镜和图像处理技术的研究磨损表面及表面参数的新方法。首先用B io-rad Rad iance 2000激光共焦显微镜方法获得精确的三维表面形貌,然后用计算机辅助图像分析技术自动计算出表面特征参数。应用示例表明本文所研究的方法是可靠的,能对工程表面的表面粗糙度特征进行精确描述。     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

Measurement of geometrical parameters in cocontinuous polymer blends: 3D versus 2D image analysis

Formulae of stereology are used to estimate 3D geometrical parameters of cocontinuous structures measured from 2D micrographs of polymer blends. 3D images of symmetric and nonsymmetric polymer blends made of fluorescently labelled polystyrene and styrene‐ran‐acrylonitrile copolymer were obtained with laser scanning confocal microscopy. Geometrical parameters of the blend interface, specifically volume fraction, surface area per unit volume (S _V ) and average of local mean curvature were measured directly from the 3D images and compared to the values estimated from analysis of a number of 2D slices combined with stereological relations. When the total length of phase boundary considered in the analysis of the 2D slices (L_Tot ) was at least 6000 times bigger than the characteristic length of the microstructure (S^?1_V ), the standard deviation for all the parameters measured became negligible. However, considerable discrepancies between the average values computed from 3D and 2D images were observed for any value of L_Tot . The mean curvature distribution was also measured from both the 3D images and the 2D slices. The distribution was estimated from the 2D slices but with a width about 2.4 times that of the true value obtained from the 3D images.     

共焦显微镜技术及其应用

共焦显微测量是一种很有前景的技术,具有非接触测量和高精度位移识别能力,广泛应用在芯片加工、高精密仪器制造、生物医学、材料化学、工业检测等领域.其沿轴向位置高精度扫描的二维图像可用于三维重建,然而,扫描的速度限制了图像的采集速率,为了克服这一局限性,研究人员提出了许多方法对传统的共聚焦显微镜系统进行了改进.例如,基于扫描振镜光束扫描型共焦显微镜、基于数字微镜装置的共焦显微镜、差分式扫描共焦显微镜等.本文主要讨论了各种共聚焦显微镜的工作原理、物镜类型、扫描方法、优缺点及应用.随着光学核心部件的升级和各种准确、高效算法的出现,未来共焦显微镜的扫描速度会更快、应用范围更广、分辨率更高.     

Three-dimensional image formation in confocal microscopy

Three-dimensional (3-D) imaging in confocal microscopes is considered in terms of 3-D transfer functions. This leads to an explanation of axial imaging properties. The axial response was observed in both object-scanning and beam-scanning microscopes and the influence of off-axis examination investigated. By simple processing of multi-detector signals, imaging in both the axial and transverse directions can be improved.     

用于激光共聚焦显微镜的光谱扫描机构

随着生物医学技术的发展,组织样本经常被多种荧光标记物标记,需要通过光谱成像的方法区分出样本中不同的成分。本文在共聚焦显微镜基础上,介绍了一种由精密丝杠和步进电机控制的狭缝机构实现光谱成像的方法,讨论了狭缝缝片的具体设计和狭缝运动精度对光谱带宽和波长准确度的影响。     

Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans

The utility of cuticular autofluorescence for the visualization of copepod morphology by means of confocal laser scanning microscopy (CLSM) was examined. Resulting maximum intensity projections give very accurate information on morphology and show even diminutive structures such as small setae in detail. Furthermore, CLSM enables recognition of internal structures and differences in material composition. Optical sections in all layers and along all axes of the specimens can be obtained by CLSM. The facile and rapid preparation method bears no risk of artefacts or damage occurring to the preparations and the visualized specimens can be used for later analyses allowing for the investigation of irreplaceable type specimens or parts of them. These features make CLSM a very effective tool for both taxonomical and ecological studies in small crustaceans; however, the maximum thickness of the specimens is limited to a few hundred micrometers. Three‐dimensional models based on CLSM image stacks allow observation of the preparations from all angles and can permit, improve and speed up studies on functional morphology. The visualization method described has a strong potential to become a future standard technique in aquatic biology due to its advantages over conventional light microscopy and scanning electron microscopy.     

激光共焦扫描显微镜及其应用

介绍了共焦激光显微镜的基本光路、成像原理、关键技术及应用。     

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome

Topological analysis of the three‐dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three‐dimensionally conserved nuclei with a ‘resolution equivalent’, many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR‐ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple‐colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state‐of‐the‐art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non‐random angle distribution.     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.     

Space-multiplexed multifocal nonlinear microscopy

Standard forms of nonlinear microscopy rely on single beam scanning, but the usually weaker signal and the need to image in real-time call for parallelization of the image formation. Since the nonlinear susceptibilities necessitate a comparatively large illumination power, with current laser systems the brightness or field of view of any parallelized nonlinear microscope is limited by the brightness of the laser. For example, by producing an array of high aperture foci, multifocal multiphoton microscopy (MMM) provides real-time, light-efficient three-dimensional fluorescence imaging at high-resolution. The available power limits the degree of parallelization and hence codetermines the field of view. As the utilization of all the laser power is imperative, the focal intensity can be adjusted only through altering the number of foci. This compromises to some extent the flexibility to adjust the focal intensity to benign and effective levels. Here we introduce space-multiplexing (SMX) as a novel option in parallelized nonlinear microscopy, which enables an improved exploitation of the total laser power and facilitates changing the intensity levels in selected regions, without attenuating the total laser power. The basic idea of SMX is to overlap arrays of slightly offset coherent focal fields whose interference modulates the intensity across the sample. For a given degree of parallelization and power, SMX increases the two- and three-photon excited signal of parallelized nonlinear microscopy by a factor of up to 1.5 and 2.5, respectively. To some extent, sensitive regions may be spared out, whereas in regions with weaker nonlinear susceptibilities the intensity is increased. SMX is relevant to all modes of nonlinear microscopy, including parallelized second- and third-harmonic imaging, coherent anti-Stokes Raman scattering, and widefield multiphoton excitation.