Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.     

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy

A comparative study of the co-localisation of three different families of neuropeptides, viz. allatostatins of the Y/FXFGL-NH(2) type, Manduca sexta allatostatin (Mas-AS) and allatotropin, in the frontal ganglion of lepidopteran larvae has been carried out by means of immunocytochemistry and confocal laser scanning microscopy. The simultaneous application of three types of fluorochrome-conjugated antibodies reveals triple co-localisation in an anterodorsal pair of neurones in the frontal ganglion of the noctuids Heliothis virescens and Lacanobia oleracea. There is no evidence of differential axonal transport, since all parts of these neurones show complete co-localisation of all three peptides. Prominent axons of the ganglionic neurones project in the recurrent nerve to the foregut and stomodeal valve. Over the crop, lateral and sub-lateral branches follow the course of circular muscle fibres and terminate in varicosities. All three neuropeptides have previously been shown to be myoregulatory on the foregut; the Y/FXFGL-NH(2) allatostatins and Mas-AS are inhibitory, whereas allatotropin is excitatory. The morphological evidence of co-localisation of physiologically antagonistic peptides within the same terminals suggests that an extremely complex mechanism controls the contractile activities of the foregut. A posterodorsal pair of neurones in the frontal ganglion have prominent axons projecting via the frontal connectives to the brain and in the recurrent nerve to the stomodeal valve where extensive branching suggests control over the valve movements. Studies of another noctuid, Spodoptera frugiperda, and the sphingid, M. sexta, show interesting variations in the co-localisation phenomenon.     

血管紧张素Ⅱ对大鼠心肌细胞Ca2+信号的影响

目的:在培养的新生大鼠心肌细胞上,观察AngⅡ对Ca2 信号的影响,探讨其时间和空间形式.方法:以Fluo-4/AM荧光指示剂负载培养的心肌细胞,应用激光共聚焦扫描显微镜观察其变化.结果:在激光共聚焦显微镜下,观察到不论静息或受AngⅡ刺激的心肌细胞,均可见钙波在细胞内及相连的另一细胞间彼此传播的现象.正常心肌细胞内核的荧光强度高于核周与细胞浆,且存在小幅度的钙震荡,AngⅡ(10-6 mol/L)引起心肌细胞的核Ca2 和胞浆Ca2 荧光强度升高的同时,其钙震荡幅度明显升高,外源性一氧化氮(NO)供体硝普钠(10-5mol/L)使钙的周期性震荡消失,荧光强度降低.同时还观察到加入AngⅡ(10-6 mol/L)后在部分心肌细胞膜上的不同部位,出现不能传播的钙闪烁团,在此基础上再加入硝普钠(10-5mol/L),不能取消AngⅡ所致的钙闪烁现象.结论:心肌细胞受AngⅡ刺激,可产生多种钙信号形式如钙闪烁、钙波、钙震荡以及瞬时性钙增高,可能在介导细胞功能的调节中起重要作用.     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of intracellular free calcium ([Ca²⁺]_i) was investigated in indo-1 loaded ventricular myocytes from the rat, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-1 at different temperatures was calibrated both at pH-stat and at α-stat, and the result demonstrated that the α-stat calibration should be preferred. Analysis of the fluorescent image showed a striking increase of [Ca²⁺]_i as well as spontaneous calcium waves in rat cells, indicating an overloaded calcium. In contrast, cardiac myocytes of the ground squirrel were found to keep a constant [Ca²⁺]_i without calcium overload regardless of temperature variation. It is believed that understanding of the mechanisms underlying the intercellular calcium homeostasis of hibemators may lead to solutions of some medical questions.     

Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing α-sarcomeric actin, desmin, β-tubulin, spectrin/α-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of α-sarcomeric actin and spectrin/α-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/α-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas β-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/α-fodrin fibres as well as actin and desmin filaments. Accepted: 28 July 1999     

Determination of diffusion coefficients in biofilms by confocal laser microscopy

Microbial exopolymer may hinder the diffusion of nutrients, antibiotics, and other materials to the cell surface. Studies of diffusion in biofilms have been limited to indirect measurements. This study demonstrated the use of fluorescein and size-fractionated fluor-conjugated dextrans in conjunction with scanning confocal laser microscopy to directly monitor and determine diffusion coefficients within biofilms. The monitoring approaches were simple and, when combined with computerized image collection, allowed assembly of a data set suitable for calculation of one-dimensional diffusion coefficients for biofilm regions. With these techniques, it was shown that regional variability in the mobility of the dextrans occurred within mixed-species biofilms. Some regions exhibited rapid diffusion of all test molecules, while adjacent regions were only penetrated by the lower-molecular-weight compounds. The effective diffusion coefficients (D(e)) determined in a mixed-species biofilm were a function of the molecular radius of the probe (i.e., fluorescein, D(e) = 7.7 x 10 cm s; 4,000 molecular weight, D(e) = 3.1 x 10 cm s; and 2,000,000 molecular weight, D(e) = 0.7 x 10 cm s). These results demonstrated that diffusion in the biofilm was hindered relative to diffusion in the bulk solution. The study indicated that in situ monitoring by scanning laser microscopy is a useful approach for determining the mobility of fluorescently labeled molecules in biofilms, allowing image acquisition, appropriate scales of study, both xy and xz monitoring, and calculation of D(e) values.     

Structural analysis of microparticles by confocal laser scanning microscopy

This study demonstrates the potential of conforcal laser scanning microscopy (CLSM) as a characterization tool for different types of microparticles. Microparticles were prepared by various methods including complex coacervation, spray drying, double emulsion solvent evaporation technique, and ionotropic gelation. Protein drugs and particle wall polymers were covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs were labeled by mixing with a fluorescent marker of similar solubility properties. As was demonstrated in several examples, CLSM allowed visualization of the polymeric particle wall composition and detection of heterogeneous polymer distribution or changes in polymer matrix composition under the influence of the drug. Furthermore, CLSM provides a method for three-dimensional reconstruction and image analysis of the microparticles by imaging several coplanar sections throughout the object. In conclusion, CLSM allows the inspection of internal particle structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the particle wall composition.     

Power and limits of laser scanning confocal microscopy

In confocal microscopy, the object is illuminated and observed so as to rid the resulting image of the light from out-of-focus planes. Imaging may be performed in the reflective or in the fluorescence mode. Confocal microscopy allows accurate and nondestructive optical sectioning in a plane perpendicular or parallel to the optical axis of the microscope. Further digital three-dimensional treatments of the data may be performed so as to visualize the specimen from a variety of angles. Several examples illustrating each of these possibilities are given. Three-dimensional reconstitution of nuclear components using a cubic representation and a ray-tracing based method are also given. Instrumental and experimental factors can introduce some bias into the acquisition of the 3-D data set: self-shadowing effects of thick specimens, spherical aberrations due to the sub-optimum use of the objective lenses and photobleaching processes. This last phenomenon is the one that most heavily hampers the quantitative analysis needed for 3-D reconstruction. We delineate each of these problems and indicate to what extent they can be solved. Some tips are given for the practice of confocal microscope and image recovery: how to determine empirically the thickness of the optical slices, how to deal with extreme contrasts in an image, how to prevent artificial flattening of the specimens. Finally, future prospects in the field are outlined. Particular mention of the use of pulsed lasers is made as they may be an alternative to UV-lasers and a possible means to attenuate photodamage to biological specimens.     

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.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy

Although exocytosis is widely believed to involve granule movement, membrane fusion and the emptying of granule content, direct study of these processes has been difficult in living cells because of the limited resolution of conventional light microscopy. Using video-enhanced microscopy and confocal laser microscopy, we have now studied these processes in living rat parotid and submandibular gland acinar cells. Under a differential interference contrast (DIC) microscope equipped with a CCD camera and a high speed image processor, secretory granules were in general stationary even after secretory stimulation with isoproterenol (IPR). Following IPR stimulation, however, there were abrupt changes in light intensity of secretory granules, and many granules disappeared. Confocal microscopy was then performed to confirm whether the observed changes in granules were related to membrane fusion and content release. For this, cells were perfused with the fluid-phase tracer Lucifer Yellow; confocal images thus obtained clearly demonstrated the appearance of fluorescence in omega-shaped invaginations of the apical plasma membrane which corresponded to the sites at which changes were observed in DIC images. The time sequence analyses of confocal images showed that there was a repetitive appearance and disappearance of omega-shaped fluorescent foci at the apical plasma membrane until most of the granules were depleted. During this time, there did not appear to be any significant expansion of the apical plasma membrane and if endocytic uptake of the tracer occurred, it was below the limit of detection. These observations provide new insights into the exocytotic process in salivary glands and are at variance in some respects with previous interpretations made from electron microscopy.     

Fibronectin in rat heart: a link between cardiac myocytes and collagen

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.     

Chromatin arrangements in intact interphase nuclei examined by laser confocal microscopy

Using a laser confocal microscope, chromatin arrangements in intact interphase nuclei were investigated in four plant species. Chromosomes in these plants have specific segments that can be stained with the fluorescent dye chromomycin A₃ (CMA). We stained centromeres inHordeum vulgare, sub-telomeric regions inSecale cereale, satellites inChrysanthemum multicore, and the satellites and the short arms of chromosomes with satellites inHemerocallis middendorfii. The following points were shown: (1) In mitotic interphase nuclei, the centromere and the telomeres of both arms touched the nuclear membrane and had evident polarity. Some CMA-bodies in sub-telomeric regions do not contact the nuclear membrane. (2) Differentiated nuclei had a non-random construction. Polarity of chromosomes is maintained, however, the chromosomes are far apart from the nuclear membrane. (3) Associations in sub-telomeric regions in the interphase nuclei ofSecale cereale were probably due to the association of heterochromatic regions with identical repeated sequences rather than telomere associlations. (4) In interphase nuclei ofChrysanthemum multicore, satellites fused during interphase.     

Actin microridges characterized by laser scanning confocal and atomic force microscopy

We have characterized the cell surface of zebrafish stratified epithelium using a combined approach of light and atomic force microscopy under conditions which simulate wound healing. Microridges rise on average 100 nm above the surface of living epithelial cells, which correlate to bundles of cytochalasin B-insensitive actin filaments. Time-lapse microscopy revealed the bundles to form a highly dynamic network on the cell surface, in which bundles and junctions were severed and annealed on a time scale of minutes. Atomic force microscopy topographs further indicated that actin bundle junctions identified were of two types: overlaps and integrated end to side T- and Y-junctions. The surface bundle network is found only on the topmost cell layer of the explant, and never on individual locomoting cells. Possible functions of these actin bundles include cell compartmentalization of the cell surface, resistance to mechanical stress, and F-actin storage.     

Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae

In amphibian gastrulae, scanning electron microscopy (SEM) has shown the presence of a network of extracellular fibrils on the inner aspect of the ectoderm layer, which serves as the substratum for migration by the presumptive mesoderm cells. In vitro experiments have shown that the fibril network promotes attachment and migration by mesoderm cells, and probably guides the migration by contact guidance. Filopodia of the migrating cells showed preferential attachment to the fibrils. Use of a colloidal gold probe for SEM immunocytochemistry has shown that fibrils observed by SEM contain fibronectin, probably as a major component. This provides direct evidence that the extracellular matrix containing fibronectin provides the substratum and guides cell migration in morphogenetic movement.