Confocal laser scanning microscopy of calcium dynamics in living cells.

Confocal laser scanning microscopy (CLSM) is widely used to monitor intracellular calcium levels in living cells loaded with calcium-sensitive fluorophores. This review examines the basic advantages and limitations of CLSM in in vivo imaging analyses of calcium dynamics. The benefits of utilizing ratioed images and dextran-conjugated fluorophores are addressed, and practical aspects of handling confocal datasets are outlined. After considering some relatively new microscopical methods that can be used in conjunction with conventional CLSM, possible future applications of confocal techniques in analyses of intracellular calcium dynamics are discussed.     

Three-dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Confocal laser scanning microscopy of chondrocytes in vitro: Cytoskeletal changes after quinolone treatment

The use of quinolone antibiotics would be significant for chronically diseased children (e.g., cysticfibrosis) as a prophylactic long-term treatment. However, quinolones were shown to cause cartilage damage in experimental animals when administered during certain developmental stages. In the present study, the effect of quinolones on chondrocytes was studied in a cell culture model in order to avoid animal experiments, to investigate the influence of single factors, and to open up the possibility to test human tissue. Chondrocytes were obtained from hipjoint cartilage of 3 to 4-weeks-old rats and cultured in control medium or quinolone-supplemented medium. It was shown that quinolones heavily disturbed adhesion of chondrocytes to the culture dish, accompanied by changes in cell shape and cytoskeletal morphology. Reduction of filamentous actin (stress fibers) and disintegration of vimentin fibers was demonstrated by immunofluorescence and evaluated by con-focal laser scanning microscopy. In contrast, distribution and amount of the adhesion molecule integrin α did not change. Results of the present study indicate that quinolones disturb the adherence mechanism of chondrocytes and lead to cytoskeleton changes.     

Confocal laser scanning microscopy as a tool to determine cyanobacteria biomass in microbial mats

We have developed a method based on confocal laser scanning microscopy for detection and quantification of cyanobacteria from the Ebro Delta microbial mats. Cyanobacteria play a major role as primary producers in microbial mats; it is difficult, however, to apply classical methods to estimate their biomass because they establish strong interactions with detritic particles. The protocol described here allows the localization of individual cells or filaments with micrometre precision without the need to either manipulate or stain the samples. This method is suitable for studying biomass ' in situ ' in microbial mats.     

Confocal laser scanning microscopy for the study of the morphological changes of the postextraction sites

A better understanding of the remodeling process of postextraction sockets is essential in dental treatment planning. The aim of this study was to evaluate whether confocal laser scanning microscopy (CLSM) can be applied to imaging contour changes of postextraction sites, as well as to its quantification with image analysis of obtained three‐dimensional images. This work describes a new application of the CLSM technique. The system used was the OLS3100‐USS, LEXT model (Olympus®). CLSM was used for the surface analysis of the extraction site. The measurements taken with CLSM were: (1) mesio‐distal distance, (2) alveolar ridge thickness, and (3) vestibular and lingual alveolar ridge height. Results of study cast scanning at baseline, 1 and 3 months after tooth extraction, with CLSM are well‐detailed images of postextraction areas. The CLSM technique used in study casts is a valid method to measure the dimensional changes that happen in the edentulous area after tooth extraction. This technique allows the evaluation of changes in mesio‐distal distance, thickness of the alveolar ridge and alveolar ridge height based on the measurements on the alveolar contours. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30, and 100 microns solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances. They exhibited a high fluorescence signal at the level of the myelin sheath. Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z-series of stacks of optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship with the afferent fibers.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Confocal laser scanning microscopy to study formation and properties of polyelectrolyte nanocapsules derived from CdCO3 templates

Three-dimensional confocal laser scanning microscopy (CLSM) was used as an essential investigation method to obtain information about the formation and morphological characteristics of nanocapsules. Nanocapsules are built by layer-by-layer deposition of alternatively charged polyelectrolytes on templates forming nanostructured hollow shells. CLSM is unique in allowing for monitoring of the core dissolution process in real time and for studying nanocapsule functioning in hydrated conditions within a three-dimensional and temporal framework. Since we are also interested in the identification of other possible templates, we briefly report on the use of yeast cells as biocolloidal cores monitored by means of two-photon microscopy. Here we focus our attention on the use of CdCO(3) crystals as template candidates for the preparation of stable capsules. Both cubic and spherical CdCO(3) cores have been produced. Cubic cores exhibit higher monodispersity and smaller size compared to spherical ones. Capsules templated on these cores have a higher surface-to-volume ratio that is valuable for applications related to drug delivery, functional properties of the shells and adsorption of proteins, and other biologically relevant molecules. Microsc. Res. Tech. 59:536-541, 2002.     

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.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 degrees C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton.     

Confocal laser scanning microscopy – an attractive tool for studying the uptake of xenobiotics into plant foliage

The application of confocal laser scanning microscopy (CLSM) to the study of xenobiotic uptake into plant foliage is explored in this paper. Three fluorescent dyes of low molecular weight and contrasting polarities (hydrophilic, moderately lipophilic and lipophilic) were selected to represent foliage‐applied pesticides. These model compounds were applied as droplets to the surfaces of various leaves and/or fruits according to the particular experiment. The transcuticular diffusion behaviour, the compartmentation into epidermal cells and the influence of a surfactant on the uptake of these fluorescent compounds were visualized by CLSM. Distinct differences in diffusion speed across the cuticle and distribution in cell compartments were found between different fluorescent compounds. The presence of a surfactant significantly accelerated the uptake of the moderately lipophilic dye into both thin‐ and thick‐cuticled leaves. The results are discussed in relation to the current knowledge on pesticide uptake and translocation. The advantages and limitations of this technique are highlighted.     

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.     

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。     

Healthy and pre-eclamptic placental basal plate lining cells: quantitative comparisons based on confocal laser scanning microscopy

Immunocytochemical confocal laser scanning microscope images of the monolayer of cells lining the intervillus space at the basal plate of term placentae were analysed using stereology. Immunoreactively-distinct regions of this mosaic layer were measured. In basal plate from healthy pregnancies, trophoblast epithelium occupied 18.91% of the surface area and endothelium 60.81%. In pre-eclampsia the equivalent areas were 15.57% and 67.63%. Acellular fibrinoid covers the remaining area and this component decreases in area in pre-eclampsia. The statistically significant increase in the cellular endothelial compartment may be relevant to the hypertensive pathology of pre-eclampsia as endothelial signalling plays a major role in regulation of blood pressure.     

Confocal laser microscopy and three-dimensional reconstruction of nucleus-associated microtubules in the division plane of vacuolated plant cells

The way in which transvacuolar strands radiating from the cell nucleus reorganize to form the phragmosome, within which division occurs, has been thoroughly studied in epidermal explants of Nautilocalyx lynchii. In recent years it has been established that the movement of the nucleus into the centre of large vacuolated cells such as these, in preparation for division, involves actin filaments. In the present study, the appearance and gradual reorganization of nucleus-associated microtubules (NAMTs) over the premitotic period is described. Epidermal explants fluorescently labelled with anti-tubulin were optically sectioned by confocal scanning laser microscopy, the sections reconstructed by an image processing computer and projected as rotating stereo pairs. This revealed that the NAMTs are a major component of the phragmosome, and that they change from a radiating to a planar distribution concomitantly with the ‘bunching’ of cortical MTs to form the pre-prophase band. The continuity of the two sets of MTs indicates that the band contains newly polymerized microtubules. Other recent studies on the division of vacuolated cells are reviewed and factors affecting the alignment of the division plane are discussed.     

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.     

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。     

Development of laser scanning microscopy using a near ultraviolet laser

We have developed a legitimate fluorescence con-focal scanning microscope (CLSM) using a near ultraviolet (UV) laser. This system has almost no chromatic aberration from the near UV region to the visible region (350–600 nm), and the objectives are designed as water-immersion type. Therefore this system provides the high-quality fluorescence image excited by the near UV laser, and high-quality image of deep points in a sample.