Visualization of silver-enhanced reaction products from protein- and immuno-colloidal gold probes by laser scanning confocal microscopy in leflection mode

We have employed a laser scanning confocal microscope in reflection mode to directly and indirectly visualize sites of deposition of silver-enhanced reaction products from colloidal gold probes. A direct approach was used for the localization of alpha-fetoprotein receptors in human myoblasts by incubating primary cultures with an alpha-fetoprotein-gold conjugate. For an indirect approach, cultured CEM cells, derived from a human T-lymphoma cell line, were incubated with a mouse monoclonal antibody to mature T-cells, followed by a gold-labelled antibody to mouse immunglobulins. Multiple optical sections of each sample were collected by reflection laser scanning confocal microscopy and combined into three-dimensional renderings. A (non-confocal) transmission image was generated of each field for comparative purposes. The increasing use of reflection laser scanning confocal microscopy combined with colloidal gold conjugates as biological markes will probably be of considerable advantage in cytochemical analysis.     

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning following by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.     

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.     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

Principles and practices of laser scanning confocal microscopy

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.     

In situ detection of rhodococci associated with activated sludge foams

Genus-specific 16S rRNA targeted oligonucleotide probes, Rco1 and Rco2, were designed and used to detect rhodococci in activated sludge foam samples by confocal laser scanning microscopy. Pure cultures were used to find the optimal hybridisation conditions which were determined by comparing the mean fluorescent intensities of target and non-target cells from images captured using a confocal laser scanning microscope (CLSM). The combination of fluorescent in situ hybridisation with rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy provides an effective way of detecting rhodococci in environmental samples.     

Three-dimensional structure of living chloroplasts as visualized by confocal scanning laser microscopy

Summary The newly developed confocal scanning laser microscope, together with image processing by computer, has been used to obtain three-dimensional information on the organization of grana in chloroplasts in living plant tissue. Chloroplasts are ideally suited for such studies because their pigments show bright autofluorescence. The high-resolution stereo images bridge a gap between classic light microscopy and electron microscopy. Our preliminary observations on several plant species resemble most the early observations of Strugger (1951: Die Strukturordnung im Chloroplasten. Ber Deutsch Bot Ges 64: 69–83) and suggest that the 3-D technique might well be suitable to solve discrepancies in the interpretation of classical light microscopic and electron microscopic observations.Abbreviations 3-D three dimensional - CSLM confocal scanning laser microscopy - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNA deoxyribonucleic acid     

Imaging aspects of cardiovascular disease at the cell and molecular level

Cell and molecular imaging has a long and distinguished history. Erythrocytes were visualized microscopically by van Leeuwenhoek in 1674, and microscope technology has evolved mightily since the first single-lens instruments, and now incorporates many types that do not use photons of light for image formation. The combination of these instruments with preparations stained with histochemical and immunohistochemical markers has revolutionized imaging by allowing the biochemical identification of components at subcellular resolution. The field of cardiovascular disease has benefited greatly from these advances for the characterization of disease etiologies. In this review, we will highlight and summarize the use of microscopy imaging systems, including light microscopy, electron microscopy, confocal scanning laser microscopy, laser scanning cytometry, laser microdissection, and atomic force microscopy in conjunction with a variety of histochemical techniques in studies aimed at understanding mechanisms underlying cardiovascular diseases at the cell and molecular level.     

Spontaneous and glutamate evoked calcium oscillations in rat hippocampal astrocytes

In this work, we researched spontaneous and glutamate evoked Ca²⁺ oscillations in rat hippocampal astrocytes using confocal laser scanning microscopy and bulk-loading of the Ca²⁺-sensitive dye Oregon Green Bapta 1-AM.     

Distribution of microtubules during the growth of tobacco pollen tubes

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α-, β- and tyrosinated α-tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer-assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.     

Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images. RESULTS: By UV-CLSM, 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures. CONCLUSION: The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.     

Practical Confocal Microscopy and the Evaluation of System Performance

The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-microm-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.     

Integrated fluorescence and transmission electron microscopy

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.     

Association of betaine-homocysteine S-methyltransferase with microtubules

In mammals, betaine of the mitochondrial matrix is used in the cytosol by betaine-homocysteine S-methyltransferase for methionine synthesis. The resulting dimethylglycine is shuttled back into the mitochondrial matrix for further degradation. Nanospray tandem mass spectrometry and N-terminal amino acid sequencing of microtubule-associated proteins from rat liver tubulin revealed that betaine-homocysteine S-methyltransferase is microtubule associated. This was confirmed by confocal laser scanning microscopy of HepG2 cells labeled with betaine-homocysteine S-methyltransferase- and alpha-tubulin-specific monoclonal antibodies. The association of betaine-homocysteine S-methyltransferase with the cytoskeleton may functionally integrate the mitochondrial and cytoplasmic compartments of choline degradation.     

Localization of caveolin-3 in the sinus endothelial cells of the rat spleen

The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.     

用免疫荧光标记对人组织中肥大细胞亚型的分选与形态观察

方法:利用中性蛋白酶成分、特征性酶抗体的免疫荧光染色和流式细胞仪确定分选肥大细胞亚型,以激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒。结果:三种免疫表型被确定:肥大细胞的类胰蛋白酶阳性(MCT)、类糜蛋白酶阴性;类糜蛋白酶阳性(MCC)、类胰蛋白酶阴性和类胰蛋白酶阳性、类糜蛋白酶阳性(MCTC)。肥大细胞内分泌颗粒分散或聚集存在,分泌颗粒突起分泌或以分散的方式释放。分泌颗粒大范围释放后,肥大细胞的形态结构发生了改变。结论:利用肥大细胞的特征性酶抗体、免疫荧光标记和流式细胞仪可将人组织中的肥大细胞分选纯化为三种亚型;以共聚焦显微镜显示肥大细胞含有丰富的分泌颗粒,它说明肥大细胞具备了为人体I型变态反应提供快速反应的物质基础。     

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.     

Optical sectioning microscopy

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.