In-vivo confocal scanning laser microscopy of the cerebral microcirculation

Confocal scanning laser microscopy (CSLM) was used to study the microcirculation of the brain neocortex in anaesthetized rats. After removal of the dura mater, implantation of a closed cranial window, and intravenous injection of fluorescein, three-dimensional reconstructions of cortical capillaries were performed down to a depth of 250 μm below the pial surface. Using a one-dimensional approach (single line scanning), erythrocyte (negative contrast in fluorescently labelled plasma) and leucocyte (labelled with rhodamine 6 G) velocity and supply rate in cortical capillaries were measured. The effect of CO₂-inhalation on capillary blood flow dynamics was studied. Capillaries were imaged continuously for up to 1 h without changes in flow or fluorescence pattern. However, by increasing the laser power 10–100-fold, aggregate formation was induced and capillaries were occluded, possibly due to damage to vascular endothelium. We conclude that CSLM can be used to study morphological and dynamic aspects of fluorescently labelled subsurface structures in organs of experimental animals.     

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（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm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0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm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Alexa Fluor^®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。     

Microfractography of granitic rocks under confocal scanning laser microscopy

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three-dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures, and the rock-forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

The use of laser scanning confocal microscopy (LSCM) in materials science

Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging.     

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.     

激光共聚焦扫描显微镜的光学设计

为获得较高分辨率的细胞图像,设计了激光共聚焦光学系统。通过较复杂结构物镜实现了照明光路系统和发射光路系统的设计。用Zemax对照明光路和发射光路进行了设计,仿真过程中照明光路的聚焦弥散斑直径小于1μm,照明针孔处的聚焦弥散斑直径小于20μm,发射光路的聚焦弥散斑直径小于20μm,同时照明光路和发射光路的MTF曲线接近衍射极限,达到较理想的情况。     

A study of hydrogenated carbon fibers by scanning electron microscopy and confocal laser scanning microscopy

The hydrogen absorption process is studied in carbonaceous fibers produced from a mixture of methane and hydrogen. The absorption of the hydrogen was examined in two types of fibers, in “as-grown” state and after a process of desorption during an annealing to 1.473 K under vacuum. Later to its production process, the fibers withstand an oxidation in air to 973 K. The fibers were examined by means of scanning electron microscopy (SEM) and confocal microscopy by reflection. Differences in the behavior during the oxidation were observed between the fibers in as-grown state and those subjected to a further annealing. It could be verified that the fibers were really constituted by two different phases. In one of the phases, the storage of the hydrogen absorbed took place, whereas in the other phase there was no alteration. The process of annealing prior to the absorption of the hydrogen has an appreciable effect on the desorption rate of the hydrogen. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 m concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at ?10°C, and individual specimens were frozen to ?70°C at cooling rates of 1, 3, 10 and 30°C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2–7 μm in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.     

Laser scanning confocal microscopy characterization of water repellent distribution in a sandstone pore network

In the present work, we propose the use of the Laser Scanning Confocal Microscopy (LSCM) to determine the effect of water repellents on rock's pore-network configuration and interconnection. The rocks studied are sandstones of Miocene age, a building material that is commonly found in the architectural heritage of Tunisia. The porosity quantitative data of treated and untreated samples, obtained by mercury porosimetry tests, were compared. The results show a slight decrease in total porosity with the water repellent treatment, which reduced both microporosity and macroporosity. This reduction produced a modification in pore size distribution and a shift of the pore access size mode interval toward smaller pore diameters (from the 30-40 microm to the 20-30 microm intervals). The water repellent was observed in SEM images as a continuous film coating grain surfaces; moreover, it was easily visualized in LSCM, by staining the water repellent with Epodye fluorochrome, and the coating thickness was straightforwardly measured (1.5-2 microm). In fact, the combination of mercury intrusion porosimetry data and LSCM observations suggests that the porosity reduction and the shift of the pore diameter mode were mainly due to the general reduction of pore diameters, but also to the plugging of the smallest pores (less than 3-4 microm in diameter) by the water repellent film. Finally, the LSCM technique enabled the reconstruction of 3D views of the water repellent coating film in the pore network, indicating that its distribution was uniform and continuous over the 100 microm thick sample. The LSCM imaging facilitates the integration and interpretation of mercury porosimetry and SEM data.     

Application of confocal scanning laser microscopy in experimental pathology

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.     

基于连续扫描方式的激光共焦扫描显微镜的研制

研发了一套基于连续扫描的激光共焦扫描显微镜(laser confocal scanning microscopy,LCSM)系统。该系统采用工作台连续运动方式实现扫描,提出了利用单次采集的数据滤除随机噪声的方法,避免了多帧取平均对成像速度造成的影响。实现连续扫描的关键在于解决工作台运动与数据采集的同步问题,利用采集卡有限采集模式,合理匹配工作台参数和采集参数,成功解决了这一问题。详细介绍了影响分辨率的因素,通过合理选取探测器针孔直径,取样间隔,确保了实现高分辨率的要求。系统利用Visual C#开发的控制平台,成功地对生物细胞进行了扫描成像。实验结果表明:基于连续扫描的LCSM具有较高的分辨率,生成的显微图像没有任何畸变,并且成像速度有了大幅度提高。     

Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy

Knowledge of the water droplet size distribution of fat spreads is necessary for the development and production of high quality microbiological safe products. Fat spreads are water‐in‐oil emulsions. The water droplet size distribution can be determined by confocal scanning laser microscopy (CSLM) after staining the fat with Nile Red. The profiles of the non‐fluorescent water droplets in the 2D images are identified and measured using image analysis. The ‘true’ water droplet size distribution is calculated from the distribution of the measured profile diameters using a Wicksell transformation of log‐normal distributions. The influence of the fluorescent staining and CSLM parameters on the information were studied. The CSLM method was tested on fat spreads with a fat content ranging from 40% to 80%. The results were compared with those obtained by nuclear magnetic resonance spectroscopy (NMR). The distribution parameters [volume weighed geometric mean diameter (D?_3,3) and the standard deviation (σ) of the logarithm of the droplet diameter] calculated for 80% fat spreads are in good agreement with those obtained by NMR (within ± 7% relative). Small differences were found for 65% fat spreads and large differences were identified for 40% fat spreads. The precision for the determination of the D?_3,3 value by CSLM is worse than that of NMR, even when three images were used to calculate this parameter [3–11% and 1–6% relative standard deviation (RSD), respectively]. The precision for the determination of exp(σ) by CSLM is comparable or better than that of NMR (1–5% and 3–6% RSD, respectively). CSLM proved to be a reliable method for the determination of the water droplet size distribution of margarines (80% fat). The advantage of CSLM compared to NMR is that visual information is given about the water droplet size distribution in the sample.     

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.     

Estimation of the directional distribution of spatial fibre processes using stereology and confocal scanning laser microscopy

Fibrous structures like polymers, glass fibres, muscle fibres and capillaries are important components of materials and tissues. A spatial fibre process is the union of smoothly curved or linear one-dimensional features of finite length, arranged in an unbounded three-dimensional reference space according to some random mechanism. Design-based stereology was combined with confocal scanning laser microscopy to study samples of fibre-reinforced composites, which were considered as realizations of not necessarily isotropic fibre processes. The methods enable the unbiased estimation of the intensity and of the directional distribution of spatial fibre processes from arbitrarily directed pairs of registered parallel optical sections a known distance apart. The directions of fibres sampled by a frame of observation on the reference plane are estimated from the coordinates of the intersection points of the fibres with both optical planes using confocal scanning laser microscopy. The true directional distribution of the fibre process is estimated by weighting each measured direction by the reciprocal of its chance of being sampled, which can be inferred from the data. The concept of complete directional randomness for uniformly and independently distributed spatial directions is introduced. The cumulative distribution function of the angular distances between different directions and other exact relations are derived for complete randomness of vectorial and axial directions. A Monte Carlo method is constructed to test spatial fibre processes, whose fibres have negligibly small curvature, for complete directional randomness. Confocal scanning laser microscopy was used to study the angular distribution of glass fibres in a polymer composite which was subjected to increasing hydrostatic extrusion. The hypothesis of complete directional randomness had to be rejected for all samples with 1% probability of error. The directional distribution was of the bipolar type, with the principal axis directed parallel to the axis of extrusion. Progressive stretching of the material increased the degree of anisotropy of the glass fibres. Although presented for an application in polymer physics, the methods are general and may also be applied in biological investigations.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Reflection across plant cell boundaries in confocal laser scanning microscopy

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.