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At least 1,009 records · Page 56Linked to original sources
PubMed2002

Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry.

BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression. In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit. Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus. The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry. METHODS: Four commercial anti-NF-kappaB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells. These antibodies were examined further by flow cytometry on purified nuclei. RESULTS: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-kappaB translocation by confocal microscopy. Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells. The measurement of NF-kappaB translocation by flow cytometry on purified nuclei is a quick and sensitive method. Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells. CONCLUSIONS; Microscopy and flow cytometry are quick and reproducible methods to measure NF-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells.

Animals↗
PubMed2004

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.

Flow Cytometry↗
PubMed2004

Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.

Amyloid fat pad aspiration specimens for cases with a clinical suspicion of amyloid typically are stained with Congo red and examined by brightfield microscopy. Congophilia with apple-green birefringence by polarization microscopy (PM) is considered diagnostic for amyloid. Examination of Congo red-stained slides by fluorescent microscopy (FM) is considered by some to be a more sensitive detection method. In this study, we assessed the utility of this technique in cytopathology archival slides from abdominal fat pad aspirations previously stained with Congo red dye. Seventy-eight cases of abdominal fat pad aspirations collected during the last 5 yr and stained with the Congo red procedure were obtained from archival files. Additionally, 20 adipose tissue material slides prepared from the surgical pathology specimens were examined as controls. One representative smear was examined in each case using FM equipped with rhodamine excitation/absorption (540/570 nm) filters. Relevant clinical information was obtained in all cases. Twelve cases (15.4%) of the 78 fat pad aspiration cases were reported originally as positive by Congo red stain using polarization and apple-green birefringence as diagnostic criteria. On review, four cases were deemed unsatisfactory. By FM examination 29 of the 74 (39.2%) cases were reclassified as positive for amyloid. The results were confirmed by immunohistochemical stain for amyloid P protein and electron microscopy. A number of similar distinct fluorescence and immunohistochemical patterns were recognized in the positive cases. Minimally weak fluorescence in the adipose tissue was observed in the control cases. The use of FM in Congo red-stained fat pad smears can improve the detection of amyloid in cytology preparations.

Adipose Tissue↗
PubMed1993

Standardization and quality control of quantitative microscopy in pathology.

Standardization and quality control of quantitative microscopy techniques are distinct but related concerns. The first deals with the great variety of quantitative methods, measured features, and even response variables used in investigation of biological or clinical processes. The latter deals with reproducibility of results from those investigations across time and test performance sites. Though distinct, efforts for standardization and quality control are inherently interactive. Consensus on standard methods, instrumentation, and data analysis is hard to achieve in fields developing as rapidly as quantitative microscopy. Consensus is possible, however, on the issues that affect test performance and interpretation. For example, issues of specimen type, fixation, processing, and staining affect image cytometry just as they do flow cytometry. Raw data acquisition issues include area sampling rules and fidelity of optical and sensor systems (light wavelength, glare/stray light, lens aberrations, numerical aperture, depth of focus, scan precision, pixel spacing and depth, sensor linearity, and stability). Intermediate data issues are primarily related to image foreground/background segmentation techniques--automated versus manual, object-specific versus field-based. Data reduction and interpretation procedures also provide many roads for divergence from uniformity. Each of these issues must be considered in terms of its effect on comparability and utility of quantitative microscopy results. Quality control for quantitative microscopy is as important as standardization for its use in research programs and with clinical specimens. The sine qua non of quality control is comparison of experimental results against a known "correct" value to estimate accuracy, and against other experimental results to estimate precision.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonic Neoplasms↗
PubMed1994

Variations in mitochondrial ultrastructure and dynamics observed by high resolution scanning electron microscopy (HRSEM).

Rat adrenal cortex was processed for high resolution scanning electron microscopy (HRSEM) to confirm tubular cristae, reported by transmission electron microscopy to be present in cortex mitochondria. Mitochondria in several other tissue and cell types were also observed and their ultrastructure confirmed by using three-dimensional, stereo, high resolution scanning electron microscopy. The mitochondria in rat and human hepatocytes as well as human skin fibroblasts grown in culture contained tubular cristae approximately 30 nanometers in diameter. The fibroblast mitochondria proved to be long, up to 46 micrometers and branching, as compared to those in liver which were spherical in shape. Cold adapted brown fat cells were packed with mitochondria, these containing plate or shelf-like cristae. Branched, rat striated muscle mitochondria were observed to curve around contractile protein filament bundles. The muscle mitochondrial cristae were found to be both tubular and plate-like, within the same mitochondrion. The ratio of tubular cristae to plate-like cristae varied considerably between muscle mitochondria. In order to use ultrastructural changes in mitochondria for differential diagnosis, and because 3D reconstruction of mitochondria based on transmission electron microscopy serial sections is severely limited in resolution, it is imperative to first develop a correct understanding of tissue specific, normal mitochondrial ultrastructure based on three-dimensional, HRSEM methods.

Adipose Tissue↗
PubMed2005

Characterization of Alzheimer paired helical filaments by electron microscopy.

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.

Alzheimer Disease↗
PubMed2006

Virtual biopsy of the joint tissues using near-infrared, reflectance confocal microscopy. A pilot study.

Standard noninvasive imaging techniques applied to joints provide gross morphological features, insufficient for assessing histological detail. On the other hand, biopsying is invasive, time consuming, and may involve unwanted processing artifacts. Near-infrared reflectance confocal microscopy is a technique that allows serial, high-resolution optical sectioning through intact tissues without employing exogenous fluorescent stains. The aim of this work was to evaluate the potential utility of near-infrared reflectance confocal microscopy for providing immediate histological information on meniscus, articular cartilage, epiphyseal plate, bone, muscle, and tendon. Images from near-infrared reflectance confocal microscopy were compared with mirror routine histology sections. Characteristic architectural features were readily visualized in the three dimensions of space. Additionally, the use of experimental contrast agents highlighted the localization of nuclei. Limitations include penetration depth and minor optical artifacts. In conclusion, near-infrared reflectance confocal microscopy is a useful technique for immediate, nondestructive, serial "virtual" sectioning through intact tissues, being thus a potential adjunct to current imaging techniques in orthopedics.

Animals↗
PubMed1994

Studies on bromobenzene-induced hepatotoxicity using in vivo MR microscopy with surgically implanted RF coils.

Using surgically implanted RF coils at 300 MHz, three-dimensional microscopic MR images of rat liver were obtained in vivo to follow the development of pathology induced by bromobenzene exposure. Formalin fixed specimens of liver from these animals were also imaged using in vitro MR microscopy, followed by conventional optical microscopy. All MR images were acquired using a spin-warp pulse sequence with TR = 950 ms and TE = 23 ms. The in vivo images were reconstructed as 256(2) x 32 arrays with a voxel size of (50 microns)2 x 219 microns, while the in vitro images were reconstructed as 256(2) x 128 arrays, giving an isotropic resolution at (39 microns)3. Based on results from six animals, we have found in all animals exposed to bromobenzene, image intensity decreased in specific hepatic tissue regions. These regions were well correlated to low signal intensity areas observed in in vitro MR images at higher resolution. Conventional optical microscopy indicated that the low signal intensity regions corresponded to areas of necrosis. The decrease in signal intensity is consistent with increased local diffusion coefficients as a result of necrosis. This study demonstrates that MR microscopy with implanted RF coils can be successfully used to follow tissue pathological changes in living tissues.

Animals↗
PubMedDate not supplied

Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope.

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.

Animals↗
PubMed1993

Electron microscopy visualization of oligonucleotide binding to duplex DNA via triplex formation.

Using biotinylated oligonucleotides and streptavidin as a marker, we have visualized, with the help of electron microscopy, the triplex formation. We used the natural homopurine-homopyrimidine sequence from human papillomavirus 16 cloned within a plasmid. Under conditions favouring the formation of pyrimidine-purine-pyrimidine triplex the corresponding pyrimidine oligonucleotide formed a complex with the insert and this complex was detected by electron microscopy. Similarly, under conditions favouring the formation of pyrimidine-purine-purine triplex the corresponding purine oligonucleotide formed a stable complex detected by electron microscopy. In both cases the complexes we observed exhibited remarkable sequence specificity. Near 80% of DNA molecules carried the streptavidin marker in the correct position and very few cases of non-specific binding were detected. We conclude that the triplex mode of recognition may provide very efficient sequence-specific markers for electron microscopy of DNA.

Base Sequence↗
PubMed1997

Three-dimensional structure of NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt reconstruction.

NADH-dehydrogenase (Complex I) is the first complex of the mitochondrial respiratory chain. It is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear DNA. The whole complex was isolated in detergent from the fungus Neurospora crassa. It is very stable in its isolated form and was analysed as such by electron microscopy. Its mass, determined by dark-field scanning electron microscopy was estimated as 1.12 MDa. The complex was imaged by transmission electron microscopy, by negative staining and by cryo-electron microscopy. A three-dimensional model, with a resolution estimated at 35 A, was calculated from images of negatively stained complexes by the random conical tilt reconstruction technique. This model confirms the general L-shape of the molecule, with arms of equal length and corroborates the hypothesis of a subdivision of the whole complex into three functional domains. Immuno-labelling of the 49 kDA subunit of the peripheral arm allowed its localization within the complex. This is a first step in the subunit mapping of Complex I and the understanding of its activity.

Animals↗
PubMed1999

Static curvature and flexibility measurements of DNA with microscopy. A simple renormalization method, its assessment by experiment and simulation.

We present the derivation of equations based on statistical polymer chain analysis and a method to quantify the average angle value of intrinsic bends and the local flexibility at a given locus on DNA fragments imaged by electron microscopy. DNA fragments of n base-pairs are considered as stiff chains of n jointed unit rigid rods. If the DNA fragments are composed of two branches A0Am and A0Bn, with, respectively, m and n base-pairs, where the standard deviations of the angle formed by two consecutive base-pairs are uniform over each branch, respectively, sigmathetaA and sigmathetaB, we show that the standard deviation of the angle AmA0Bn is: [formula: see text] where sigmatheta0 is the standard deviation of the angle at locus A0. This equation is established for small angular deviations by analysis of DNA at different scales and the validity of the methodology is controlled with the computation of the reduced chi2 statistical test. The length of the DNA fragments must be of the order of, or below, the persistence length, as determined by sets of statistics from computer simulations of DNA fragments. This is verified experimentally by a detailed analysis of the digitized contours of homogeneous linear 139 base-pair DNA fragments observed by electron microscopy. The images are compared to the reconstruction of DNA fragments from the measurements. The value found, sigma0=4.6 degrees/bp, is consistent with the well-accepted value for DNA in a plane. We discuss the relationship between the standard deviation of the measured angles and the flexibility at the base-pair level. This method is useful to quantify directly from microscopy techniques, such as electron or scanning force microscopy, the true bending angle, either intrinsic or induced by a ligand, and its associated flexibility at a given locus in any small DNA fragment.

Base Pairing↗
PubMed1999

Three-dimensional imaging by deconvolution microscopy.

Deconvolution is a computational method used to reduce out-of-focus fluorescence in three-dimensional (3D) microscope images. It can be applied in principle to any type of microscope image but has most often been used to improve images from conventional fluorescence microscopes. Compared to other forms of 3D light microscopy, like confocal microscopy, the advantage of deconvolution microscopy is that it can be accomplished at very low light levels, thus enabling multiple focal-plane imaging of light-sensitive living specimens over long time periods. Here we discuss the principles of deconvolution microscopy, describe different computational approaches for deconvolution, and discuss interpretation of deconvolved images with a particular emphasis on what artifacts may arise.

Artifacts↗
PubMed2006

Near-field scanning optical microscopy in cell biology and cytogenetics.

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.

Animals↗
PubMed1976

Interaction of rabbit sperm and egg. Examination of the same cryofractured specimens with scanning and transmission electron microscopy.

Rabbit ova fertilized in vitro were prepared for scanning electron microscopy by ethanol-cryofracturing and critical-point drying methods and were also embedded and sectioned for transmission electron microscopy. Study of a region of interaction between sperm and zona pellucida with scanning electron microscopy reveals the latter to be composed of a complex network of fibers interspersed with numerous pores. Transmission electron microscopy of the same region reveals a "typical" homogeneous composition of the zona pellucida. Ultrastructural observations of thin sections passing through the region of sperm-egg interactions or through other regions of the ovum or its investments reveals very little methodological distortion of the various intracellular organelles or matrix. Application of the procedures described provides not only an elucidation of surface detail but also reveals intracellular cytoplasmic information about the same specimen during in vitro fertilization.

Animals↗
PubMed1980

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus). IV. Light and electron microscopy of synapsis and nucleolar development by silver staining.

Synaptonemal complexes (SCs), X and Y axes, and various nucleolar structures stain preferentially with silver in surface microspread preparations and are analyzable by both light and electron microscopy. Central elements, kinetochore region material and nuclear annuli which stain with ethanolic phosphotungstic acid are seldom visible after silver staining. SCs can be characterized by length measurements equally well in light and electron micrographs, from which stages of pachytene can also be determined by differentiation of the axes of the XY pair. By electron microscopy, the lateral elements appear as single strands at zygotene and early pachytene, then become double in a plane perpendicular to that of the SC and appear denser and thicker until late pachytene when they become progressively more attenuated and again appear single. These transitions are difficult to explain in terms of separation of associated chromatids. Identification of various silver stained bodies as nucleoli is supported by their orange-red fluorescence with acridine orange. SCs, X and Y axes and associated sex body material are, with a few exceptions, virtually indistinguishable from the background yellow-green fluorescence of the chromatin. Comet-shaped nucleolar bodies are regularly associated with five (in one animal) or six (in two animals) SCs; their positions along particular SCs identifiable by relative lengths indicate these bodies to be expressions of nucleolus organizer regions. They first appear at leptotene in association with unpaired axes and undergo progressive changes through late pachytene, at which time they redistribute their contents coincident with disappearance of the SCs. A characteristic nucleolar double dense body appears at zygotene; unlike the comet-shaped nucleoli, it is unassociated with other nuclear structures, and is assumed to arise from coalescence of previously existing smaller dense bodies. - The silver staining method described is remarkable for the speed and simplicity with which large numbers of spermatocyte nuclei are obtainable for light and electron microscopy. The fidelity of the light microscopic counterpart of the electrom microscopic image has been directly assessed at different stages of pachytene. For cytogenetic analysis, critical information often lies beyond the limits of light optical resolution; the correlated electron microscopy required for verification is easily obtained with this method.

Animals↗
PubMed1993

Changes of the Na/K ATPase activity in the cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride: histochemical demonstration with light and electron microscopy.

Since inorganic mercury salts only poorly penetrate the cerebral microvascular endothelial cells comprising the blood-brain barrier (BBB), their neurotoxicity may be predicted to result from interference with BBB transport enzymes. In the present study, we tested the effect of mercuric chloride (HgCl2) on Na+/K+ ATPase activity, a key enzyme involved in the ion transport in and out of the brain. Routine histochemical staining in conjunction with light and electron microscopy was used to evaluate the changes in the Na+/K+ ATPase activity in cerebral cortical microvessels of rats who received a single intraperitoneal injection of 6 mg/kg HgCl2. At 1 h after HgCl2 administration, light microscopy revealed uniform reduction of the Na+/K+ ATPase reaction in all cortical layers. Electron microscopy confirmed the enzyme reaction to be very weak to completely absent in both the luminal and abluminal endothelial cell membranes, and the luminal plasmalemma showed invaginations and pinocytic vesicles indicative of changes in its transport functions. The enzyme inhibition coincided with, and was likely to contribute to, profound perivascular swelling, involving mainly the astrocytic endfeet. The enzyme activity showed a partial recovery 18 h after HgCl2 treatment, mainly in cortical layers II and III. After 5 days, the recovery of the enzyme activity appeared complete as observed by light and electron microscopy. The recovery of the microvascular Na+/K+ ATPase coincided with the appearance of a strongly positive Na+/K+ ATPase reaction in the adjacent astrocytic processes and with the diminution of perivascular swelling.

Animals↗
PubMed1995

High-resolution light microscopy for interpretation of renal biopsies.

Routine transmission electron microscopy is recommended for all renal biopsies. However, most of the diagnostic lesions in renal pathology are 0.2 micron or larger, and are therefore visible by light microscopy. These lesions are difficult to evaluate in paraffin sections due to problems inherent in the method itself. Full utilization of the resolving power of the light microscope or high-resolution light microscopy is possible with resin (plastic) histotechnology. The replacement of conventional histotechnology by epoxy histotechnology allows the visualization of most subcellular structures needed for accurate diagnosis in renal pathology. Electron microscopy remains a very helpful tool for selected morphological problems.

Biopsy↗