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At least 1,423 records · Page 79Linked to original sources
PubMed1990

Electron microscopy study of the bacteria adherent to the rumen wall in young conventional lambs.

The lamb rumen walls were rapidly colonized by an abundant bacterial population after birth. This colonization was examined by electron microscopy in neonatal conventional lambs. The sequence of establishment of the epimural species during the 3 weeks following birth, and the distribution of bacteria on the different sacs of the rumen, were examined by scanning electron microscopy. The population was very dense and consisted of a limited number of morphological types by 2 days after birth. Three types of rods were dominant at that time. The microflora was more complex 2 weeks later. Observations by transmission electron microscopy of desquamated epithelial cells revealed the presence of adherent bacteria that are surrounded by fibrous carbohydrate coats and sometimes partially enclosed by invaginations of the epithelial cell.

Animals↗
PubMed2004

Three-dimensional electron microscopy at molecular resolution.

Emerging methods in cryo-electron microscopy allow determination of the three-dimensional architectures of objects ranging in size from small proteins to large eukaryotic cells, spanning a size range of more than 12 orders of magnitude. Advances in determining structures by "single particle" microscopy and by "electron tomography" provide exciting opportunities to describe the structures of subcellular assemblies that are either too large or too heterogeneous to be investigated by conventional crystallographic methods. Here, we review selected aspects of progress in structure determination by cryo-electron microscopy at molecular resolution, with a particular emphasis on topics at the interface of single particle and tomographic approaches. The rapid pace of development in this field suggests that comprehensive descriptions of the structures of whole cells and organelles in terms of the spatial arrangements of their molecular components may soon become routine.

Cryoelectron Microscopy↗
PubMed2002

Endoscopic microscopy.

In vivo endoscopic optical microscopy provides a tool to assess tissue architecture and morphology with contrast and resolution similar to that provided by standard histopathology--without need for physical tissue removal. In this article, we focus on optical imaging technologies that have the potential to dramatically improve the detection, prevention, and therapy of epithelial cancers. Epithelial pre-cancers and cancers are associated with a variety of morphologic, architectural, and molecular changes, which currently can be assessed only through invasive, painful biopsy. Optical imaging is ideally suited to detecting cancer-related alterations because it can detect biochemical and morphologic alterations with sub-cellular resolution throughout the entire epithelial thickness. Optical techniques can be implemented non-invasively, in real time, and at low cost to survey the tissue surface at risk. Our manuscript focuses primarily on modalities that currently are the most developed: reflectance confocal microscopy (RCM) and optical coherence tomography (OCT). However, recent advances in fluorescence-based endoscopic microscopy also are reviewed briefly. We discuss the basic principles of these emerging technologies and their current and potential applications in early cancer detection. We also present research activities focused on development of exogenous contrast agents that can enhance the morphological features important for cancer detection and that have the potential to allow vital molecular imaging of cancer-related biomarkers. In conclusion, we discuss future improvements to the technology needed to develop robust clinical devices.

Cell Line, Tumor↗
PubMed2004

Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study.

BACKGROUND: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 microm. METHODS: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. RESULTS: In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E(-/-) mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E(-/-) mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. CONCLUSIONS: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.

Animals↗
PubMed1997

Applications of confocal and fluorescence microscopy.

Fluorescence microscopy has become a powerful tool for both the localization of cellular components in fixed cells, using target-specific fluorescent probes and labeled antibodies, and the fluorescence imaging of ions in single living cells. Despite its markedly lower spatial resolution when compared with electron microscopy, the essentially non-invasive nature of light microscopy provides a unique tool for examining cell behaviour at the level of the single cell bringing new insight into both cellular heterogeneities and cell-cell interactions. Further developments in both fluorescent probes and instrumentation should provide even more powerful tools to probe the mechanisms of cell function, both in vitro and in vivo.

Animals↗
PubMed2004

Two-photon microscopy of cells and tissue.

Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering specimens by way of restricting fluorophore excitation (and thus emission) to the focal plane of the microscope. Spatial confinement of two-photon excitation gives rise to several advantages over single-photon confocal microscopy. First, penetration depth of the excitation beam is increased. Second, because out-of-focus fluorescence is never generated, no pinhole is necessary in the detection path of the microscope, resulting in increased fluorescence collection efficiency. Third, two-photon excitation markedly reduces overall photobleaching and photodamage, resulting in extended viability of biological specimens during long-term imaging. Finally, localized excitation can be used for photolysis of caged compounds in femtoliter volumes and for diffusion measurements by two-photon fluorescence photobleaching recovery. This review aims to provide an overview of the use of two-photon excitation microscopy. Selected applications of this technique will illustrate its excellent suitability to assess cellular and subcellular events in intact, strongly scattering tissue. In particular, its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep within intact, buffer-perfused hearts is demonstrated. Potential applications of two-photon laser scanning microscopy as applied to integrative cardiac physiology are pointed out.

Animals↗
PubMedDate not supplied

Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions.

The ability to determine various functions of genes in an intact host will be an important advance in the postgenomic era. Intravital imaging of gene regulation and the physiological effect of the gene products can play a powerful role in this pursuit. Intravital epifluorescence microscopy has provided powerful insight into gene expression, tissue pH, tissue pO2, angiogenesis, blood vessel permeability, leukocyte-endothelial (L-E) interaction, molecular diffusion, convection and binding, and barriers to the delivery of molecular and cellular medicine. Multiphoton laser scanning microscopy (MPLSM) has recently been applied in vivo to overcome three drawbacks associated with traditional epifluorescence microscopy: (i) limited depth of imaging due to scattering of excitation and emission light; (ii) projection of three-dimensional structures onto a two-dimensional plane; and (iii) phototoxicity. Here, we use MPLSM for the first time to obtain high-resolution images of deep tissue lymphatic vessels and show an increased accuracy in quantifying lymphatic size. We also demonstrate the use of MPLSM to perform accurate calculations of the volume density of angiogenic vessels and discuss how this technique may be used to assess the potential of antiangiogenic treatments. Finally, high-speed MPLSM, applied for the first time in vivo, is used to compare L-E interactions in normal tissue and a rhabdomyosarcoma tumor. Our work demonstrates the potential of MPLSM to noninvasively monitor physiology and pathophysiology both at the tissue and cellular level. Future applications will include the use of MPLSM in combination with fluorescent reporters to give novel insight into the regulation and function of genes.

Animals↗
PubMed2000

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy.

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.

Fluorescent Dyes↗
PubMed1998

Correlative microscopy using FluoroNanogold on ultrathin cryosections. Proof of principle.

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for high-resolution correlation between immunofluorescence and electron microscopy.

Frozen Sections↗
PubMed1999

Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy.

The resolution of conventional light microscopy is limited to approximately 200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of approximately 40 nm. (J Histochem Cytochem 47:471-480, 1999)

Cryoultramicrotomy↗
PubMed1993

Conventional and confocal fluorescence microscopy of collagen fibers in the heart.

The arrangement of collagen fibers has previously been studied with picrosirius red (PSR) staining and brightfield microscopy. We discovered that PSR staining can also be visualized by fluorescence microscopy. PSR-stained collagen was strongly fluorescent using excitation and barrier filters for rhodamine, and distracting background cytoplasmic fluorescence was drastically reduced with phosphomolybdic acid (PMA) treatment before PSR staining. The PMA-PSR fluorescence method was more sensitive than the brightfield PSR or PMA-PSR method, and permitted confocal microscopic study. We applied the method to the study of collagen fiber three-dimensional arrangement in perimysial and endomysial septa of the heart, showing the three-dimensional course of the fibers in stereo views generated by confocal microscopy. The PMA-PSR fluorescence method should be generally useful for accurately determining collagen fiber three-dimensional arrangement, a necessary prelude to mechanical modeling of collagen-reinforced tissues.

Animals↗
PubMed2005

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Antibodies, Monoclonal↗
PubMed1992

Chondrons from articular cartilage. V. Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy.

The pericellular microenvironment around articular cartilage chondrocytes must play a key role in regulating the interaction between the cell and its extracellular matrix. The potential contribution of type VI collagen to this interaction was investigated in this study using isolated canine tibial chondrons embedded in agarose monolayers. The immunohistochemical distribution of an anti-type VI collagen antibody was assessed in these preparations using fluorescence, peroxidase and gold particle probes in combination with light, confocal and transmission electron microscopy. Light and confocal microscopy both showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte with reduced staining in the tail region and the interconnecting segments between adjacent chondrons. Minimal staining was recorded in the territorial and interterritorial matrices. At higher resolution, type VI collagen appeared both as microfibrils and as amorphous deposits that accumulated at the junction of intersecting capsular fibres and microfibrils. Electron microscopy also showed type VI collagen anchored to the chondrocyte membrane at the articular pole of the pericellular capsule and tethered to the radial collagen network through the tail at the basal pole of the capsule. We suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the collagens, proteoglycans and glycoproteins of the pericellular microenvironment. Secondly, specific cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. In this way type VI collagen could provide a close functional interrelationship between the chondrocyte, its pericellular microenvironment and the load bearing extracellular matrix of adult articular cartilage.

Animals↗
PubMed1986

Visualization of the living cytoskeleton by video-enhanced microscopy and digital image processing.

Two steps led to our present-day view of the cytoskeleton as a highly dynamic structure that is actively involved in force generation for various kinds of cell motility and, as a result, is itself often actively moving. The first step was the introduction of video microscopy, especially of the Allen Video Enhanced Contrast-Differential Interference Contrast Microscopy (AVEC-DIC), which allows the visualization of cellular structures in the light microscope that are up to 10 times smaller than the limit of resolution. This enables one to see images of unfixed, unstained, native or purified microtubules and actin bundles, and their interaction with membrane-bound organelles. The second step was the discovery of a system exceptionally well-suited to study microtubule and organelle movements, namely, the extruded axoplasm of the squid giant axon. From this axon the cytoplasm can be extruded free from surrounding plasma membrane, and individual microtubules and organelles can be separated from the bulk axoplasm. The study of these native microtubules by AVEC-DIC microscopy yielded a great number of quite unexpected details of the dynamic behaviour of both the microtubules themselves and the motility associated with them.

Animals↗
PubMed2004

Two-color two-photon 4Pi fluorescence microscopy.

In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.

Equipment Design↗
PubMed2005

Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization.

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.

Artifacts↗
PubMed2006

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.

Aluminum Oxide↗
PubMed1988

Autonomic nerve networks in the rat exocrine pancreas as revealed by scanning and transmission electron microscopy.

The three-dimensional architecture of the autonomic nerve terminals in the rat exocrine pancreas was investigated by scanning electron microscopy using the HCl-digestion method as well as by transmission electron microscopy. Unmyelinated nerves presumed to be autonomic in nature were found in networks covering the outer layers of arterioles and their capillary extensions, with nerve fibers often leaving the capillaries to surround acini in the interacinar spaces. Schwann cells formed scaffolds for axons of the networks. No other distinct type of cells such as the so-called interstitial cells of Cajal were found to be associated with the formation of the networks. Although nerve fibers of the networks were locally in close association with the walls of the blood vessels or with the bases of acinar cells, no specialized axonal contacts with these tissues were found. However, local swellings, presumably varicosities, were observed by scanning electron microscopy on the surface of nerves. These findings suggest that the networks of unmyelinated nerves represent terminal apparatuses of the autonomic nerves in the pancreas. Schwann cells in the terminal networks were characterized by an unusual abundance of cell organelles. These "terminal Schwann cells" well correspond in location and reticular extension to the "interstitial cells of Cajal" as demonstrated by silver impregnation and vital methylene blue staining. The occurrence of well developed Golgi apparatuses, rough endoplasmic reticulum, and numerous ribosomes suggests that the cells are specialized Schwann cells which most likely require high levels of cellular activity in order to maintain their elaborate cytoplasmic processes extending along the terminal networks, and also to sustain the specific functions of axon terminals.

Animals↗