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PubMed1998

Electron microscopy in tumor diagnosis: indications for its use in the immunohistochemical era.

The electron microscope reveals much more information on tumor cell structure than can be obtained with the light microscope, and some of the data are useful in diagnostic studies. In this review, major contributions of electron microscopy in the main tumor categories are discussed. To select between immunocytochemistry and electron microscopy, the probable contributions of each in the context of the particular case must be assessed. Usually, electron microscopy will only be requested after a battery of immunostaining procedures has been performed and found to be insufficient, but there are occasions when ulstrastructural study is logically the first choice after routine light microscopy. It is worth taking tissue for possible electron microscopy from any tumor that is known or anticipated to be a diagnostic problem.

Biomarkers, Tumor↗
PubMed2000

In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy.

OBJECTIVE: To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. DESIGN: Observational case series PARTICIPANTS: Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. METHODS: Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. MAIN OUTCOME MEASURES: The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowman's layer, stromal keratocytes, and endothelium was analyzed. RESULTS: The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowman's layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. CONCLUSIONS: In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.

Adult↗
PubMed1999

Confocal microscopy in Bowman and stromal corneal dystrophies.

OBJECTIVE: To use confocal microscopy to demonstrate the similarity among three autosomal-dominant corneal dystrophies and the diversity of the deposit patterns within a single dystrophy. DESIGN: A prospective, comparative case series. PARTICIPANTS: Twenty patients (40 eyes) from 10 families suffering from Bowman or stromal dystrophy agreed to take part: 3 with Reis-Bückler dystrophy, 12 with granular dystrophy, and 5 with lattice type-I dystrophy. Of these, nine had recurrence in their grafts or after phototherapeutic keratectomy before the confocal examination. The confocal images of affected corneas were compared with those of ten normal control eyes (ten subjects). INTERVENTION: All patients were examined by slit-lamp biomicroscopy. Confocal microscopy was performed with Achroplan 40x/numeric aperture (NA) = 0.75 and 63x/NA = 0.9 water immersion objectives. Image analysis was used to identify the corneal epithelial and stromal deposits correlated with each disorder. MAIN OUTCOMES MEASURES: Selected images of the corneal layers were evaluated qualitatively for the size, shape, light scattering, and reflection of the deposits. RESULTS: Slit-lamp biomicroscopy showed stromal involvement in all affected eyes. Confocal microscopy identified epithelial deposits in 30% of the eyes and stromal deposits in all eyes. The deposits within the epithelium were revealed more clearly with the 63x/NA = 0.9 objective (higher numeric aperture). Some of the confocal findings near the Bowman layer were common for all three dystrophies. Normal control eyes showed no epithelial or stromal deposits, either by biomicroscopy or confocal microscopy. CONCLUSIONS: Confocal microscopy provides an in vivo evaluation of the deposits in the cornea, with a higher resolution than biomicroscopy. The confocal findings common to the three dystrophies may agree with previous hypotheses of the same genetic origin. It may be a useful adjunct to slit-lamp biomicroscopy, particularly when histopathologic studies cannot be performed.

Adolescent↗
PubMed1999

Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy.

The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.

Adult↗
PubMed2000

A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells.

The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.

Antigens, CD↗
PubMed2000

Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue.

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Algorithms↗
PubMed2001

4Pi-confocal microscopy of live cells.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

Escherichia coli↗
PubMed1994

Immunoelectron microscopy of different forms of glomerulonephritis in routine biopsy material.

Renal biopsies were investigated of patients with IgA or membraneous glomerulonephritis or with systemic lupus erythematosus by light microscopy, electron microscopy, light microscopic immunohistology and by immunoelectron microscopy using the post-embedding technique applied to LR-White embedded tissue. Aim of the study was to explore whether immunoelectron microscopy is reproduced on routine biopsy material and in accordance with light microscopic immunohistological findings. The study shows that immunoelectron microscopy can be applied to routine biopsy material and gives reproducible results. The applied method proved to be reliable, and, hence, routine biopsy material may be used for further studies concerning subcellular mechanisms in immunocomplex deposition and removal.

Biopsy, Needle↗
PubMed2002

Corneal endothelial cell density and pachymetry measured by contact and noncontact specular microscopy.

PURPOSE: To determine the endothelial cell density and thickness of normal human and postkeratoplasty corneas with contact specular microscopy and to compare these measurements with those obtained by noncontact specular microscopy. SETTING: Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany. METHODS: The central corneal endothelial cell density and thickness were determined in 65 healthy eyes of 39 patients with a mean age of 71 years +/- 12 (SD) and in 50 corneal grafts of 41 patients with a mean age 53 +/- 17 years using noncontact (Topcon SP-2000P, Topcon Corp.) and contact (EM-1000, Tomey) specular microscopes. Appropriate conversion factors were used for accurate cell count comparison. RESULTS: The mean cell count of the normal corneas was 2445 +/- 425 cells/mm(2) measured by noncontact specular microscopy and 2471 +/- 393 cells/mm(2) measured by contact specular microscopy (P =.70). After penetrating keratoplasty, the mean cell density was 1610 +/- 499 cells/mm(2) and 1584 +/- 469 cells/mm(2), respectively (P =.88). Significantly lower thickness was measured with the noncontact specular microscope than by contact pachymetry in normal eyes (543 +/- 46 micro m and 642 +/- 42 micro m, respectively) and postkeratoplasty eyes (538 +/- 61 micro m and 627 +/- 48 micro m, respectively) (P <.0001). CONCLUSION: To determine endothelial cell density, contact and noncontact specular microscopy may be used interchangeably. However, for the combined measurement of endothelial cell density and pachymetry, the use of the same specular microscope is recommended for long-term patient follow-up.

Adult↗
PubMed2003

Using fluctuation microscopy to characterize structural order in metallic glasses.

We have used fluctuation microscopy to reveal the presence of structural order on length scales of 1-2 nm in metallic glasses. We compare results of fluctuation microscopy measurements with high resolution transmission electron microscopy and electron diffraction observations on a series of metallic glass samples with differing degrees of structural order. The agreement between the fluctuation microscopy results and those of the other techniques is good. In particular, we show that the technique used to make thin specimens for electron microscopy affects the structure of the metallic glass, with ion thinning inducing more structural order than electro-polishing. We also show that relatively minor changes in the composition of the alloy can have a significant effect on the medium-range order; this increased order is correlated with changes in mechanical behavior.

Crystallization↗
PubMedDate not supplied

Imaging of anisotropic cellulose suspensions using environmental scanning electron microscopy.

The effect of concentration on anisotropic phase behavior of acid-hydrolyzed cellulose suspensions has been examined using conventional polarizing microscopy and the novel technique of environmental scanning electron microscopy (ESEM). Microcrystalline cellulose dispersed in water formed biphasic suspensions in a narrow concentration range, 4-12 wt % for a suspension pH of 4, where the upper and lower phases were isotropic and anisotropic (chiral nematic), respectively. It is known from previous work that within the biphasic regime total suspension concentration affects only the volume fractions of the two phases, not phase concentration or interfacial packing. As the total suspension concentration surpassed the upper critical limit (c), however, a single anisotropic phase of increasing concentration was observed. It was evident from polarizing microscopy that the chiral nematic pitch of the anisotropic phase decreased with increasing concentration, which has been attributed to a reduction in the electrostatic double layer thickness of the individual rods, thus increasing intermolecular interactions. Chiral nematic textures were also visible using ESEM. This technique has the advantage of studying individual rod orientation within the liquid crystalline phase as it permits the high resolution of electron microscopy to be applied to hydrated samples in their natural state. To our knowledge this is the first time such lyotropic systems have been observed using electron microscopy.

Anisotropy↗
PubMedDate not supplied

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports.

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

Colloids↗
PubMed1997

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy.

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.

Coloring Agents↗
PubMed2005

In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy.

In vivo reflectance confocal microscopy is a novel technique for the noninvasive study and diagnosis of the skin. The aim of this study was to describe and characterize the cytological and architectural aspects of cell clusters in melanocytic lesions observed by confocal microscopy, and to correlate them with routine histopathology. A total of 55 melanocytic lesions comprising 20 melanomas, 25 acquired nevi and 10 Spitz nevi were studied by means of reflectance confocal microscopy, dermoscopy and routine histopathology. Three different types of cell clusters at confocal microscopy observation (dense, sparse cell and cerebriform clusters) were identified and correlated with histopathology. Dense clusters appeared characteristic for benign lesions, although present in 13 out of 20 melanomas. Sparse cell clusters were more frequently observable in melanomas, but also sporadically present in one Spitz nevus. Moreover, cerebriform clusters were exclusively observed in five out of 20 melanomas. Confocal microscopy allowed the in vivo characterization of aspects of melanocytic nests and their exact correlation with histopathology.

Humans↗
PubMed1997

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle.

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.

Animals↗
PubMed2006

Urinary biochemistry and microscopy in septic acute renal failure: a systematic review.

BACKGROUND: Biochemistry and microscopy of urine are widely published diagnostic activities in patients with acute renal failure (ARF). However, their scientific basis in patients with septic ARF has not been assessed systematically. METHODS: We performed a systematic review of MEDLINE, EMBASE, CINHAL, and PubMed databases and bibliographies of retrieved articles for all studies describing urinary biochemistry, indices, and microscopy in patients with septic ARF. RESULTS: We identified 27 articles (1,432 patients). Because of substantial heterogeneity, no formal quantitative analysis could be performed. Urinary biochemistry or derived indices were reported in 24 articles (89%), and microscopy, in 7 articles (26%). The majority were small single-center reports and had serious limitations. For example, only 52% of patients were septic, only 54% of patients had ARF, many studies failed to include a control group, time from diagnosis of sepsis or ARF to measure of urinary tests was variable, and there were numerous potential confounders. Urinary sodium, fractional excretion of sodium, urinary-plasma creatinine ratio, urinary osmolality, urinary-plasma osmolality ratio, and serum urea-creatinine ratio showed variable and inconsistent results. Low-molecular-weight proteinuria was described in only 22% of articles. A few reports of urinary microscopy described muddy brown/epithelial cell casts and renal tubular cells in patients with septic ARF, whereas others described normal urinary sediment. CONCLUSION: The scientific basis for the use of urinary biochemistry, indices, and microscopy in patients with septic ARF is weak. More research is required to describe their accuracy, pattern, and time course in patients with septic ARF.

Acute Kidney Injury↗
PubMed1986

[Light and electron microscopy changes in the endometrium caused by the administration of a norgestimate-containing oral contraceptive (Cilest)].

Endometrium morphology has been analysed by means of light microscopy, scanning and transmission microscopy in patients before and during treatment with a norgestimate containing low dose combined pill (Cilest). Endometrium biopsies were taken after 1 (N = 3), 2 (N = 3), 3 (N = 7), 5 (N = 1) and 6 (N = 4) OC cycles. Light microscopy of the endometrium obtained during the OC-free pretreatment cycles shows a regular secretory transformed endometrium. During the first OC cycles of Cilest treatment slight growth retardation of endometrial glands was observed. Endometrium after 3 to 6 OC cycles showed an increasing delay of the growth of endometrial glands in terms of an abortive secretion. Morphometric studies revealed a retardation of the development of endometrial glands and an "arrest of secretion". The degree of proliferation varied slightly; a general delay between the date of the menstrual cycle and endometrial dating was evident. Using scanning electron microscopy the endometrium presented mostly a regular surface corresponding to the midcycle or a late proliferative phase up to the early secretory phase. Infiltrative dysplasia or inflammatory changes as well as local proliferations could not be detected. In transmission electron microscopy with semi-thin and ultrathin slices, stroma, structure of glands and surface appeared to be normal. Furthermore, no time delay between the endometrium and the day of menstrual cycle was observed. During treatment with a norgestimate containing low dose combined pill, only slight changes of the endometrium in terms of a growth retardation of endometrial glands were seen in the first 6 treatment cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraceptives, Oral, Combined↗
PubMed2003

Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy.

BACKGROUND: Pollen from early-flowering trees (eg, birch, alder, hazel) represent major seasonal allergen sources. The effects of rain on the release of allergens from tree pollen has thus far not been studied at the ultrastructural level. OBJECTIVE: This study was designed to investigate the effects of rain on the morphology of pollens from early-flowering trees and of potential rain-induced mechanisms of allergen release. METHODS: Freshly collected pollen grains (birch, alder, and hazel) were exposed under controlled conditions to rainwater. Changes of pollen morphology and the release of allergens were analyzed by scanning electron microscopy. The release of allergen-bearing submicronic particles was studied by field emission scanning electron microscopy and transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for the major allergens. RESULTS: Scanning electron microscopy showed that freshly isolated pollen grains from birch, alder, and hazel have abortive germination in rainwater. Abortive pollen germination is characterized by the formation of short pollen tubes, which rupture at their tips and release micronic and submicronic particles containing major allergens. Immunogold transmission electron microscopy provided evidence that the allergens are transported through the pollen tubes during germination. CONCLUSIONS: Rainwater-induced release of allergen-bearing submicronic particles from abortively germinated tree pollens may represent a mechanism of allergen release, with important implications on the induction of asthma as well as on current methods for measuring environmental allergen exposure.

Allergens↗