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PubMed2004

Molecular expressions: exploring the world of optics and microscopy. http://microscopy.fsu.edu.

Our knowledge of the structure, dynamics and physiology of a cell has increased significantly in the last ten years through the emergence of new optical imaging modalities such as optical sectioning microscopy, computer- enhanced video microscopy and laser-scanning microscopy. These techniques together with the use of genetically engineered fluorophores have helped scientists visualize the 3-dimensional dynamic processes of living cells. However as powerful as these imaging tools are, they can often be difficult to understand and fully utilize. Below I will discuss my favorite website: The Molecular Expressions Web Site that endeavors to present the power of microscopy to its visitors. The Molecular Expressions group does a remarkable job of not only clearly presenting the principles behind these techniques in a manner approachable by lay and scientific audiences alike but also provides representative data from each as well.

Cell Physiological Phenomena↗
PubMed2005

Web-based virtual microscopy in teaching and standardizing Gleason grading.

Gleason grading forms the basis of prognostic and therapeutic assessment in prostatic carcinoma despite its subjective nature and substantial interobserver variation. The accuracy of Gleason grading can be improved by the use of educational tools such as reference images. However, conventional microscopy images are of limited educational value because it is neither possible to view the sample at different magnifications nor to navigate into different areas of the specimen. This limitation can be overcome by the use of virtual microscopy, which allows viewing entire digitized microscope slides. We created an interactive Web site ( www.webmicroscope.net/gleason ) featuring a comprehensive set of prostatic needle biopsies as virtual slides, which can be viewed with a standard Web browser (Internet Explorer or Netscape). To evaluate the validity of Web-based virtual microscopy for Gleason grading, an experienced uropathologist (TK) scored a series of 62 biopsies from the original glass slides and 6 weeks later from virtual slides on the Web site using an ordinary desktop computer. The intraobserver agreement was excellent, with identical Gleason scores found in 48 of the 62 cases ( kappa = 0.73). The 14 remaining scores differed only by 1 point on the Gleason scale (2-10). The virtual slides were viewed by 2 other uropathologists (PM and HH), with interobserver kappa coefficients ranging from 0.55 to 0.62, which is within the range of previously reported studies using glass slides. The 3 uropathologists' Gleason scores were included as reference scores on the Web site, which now serves as a publicly open platform for self-testing and learning of Gleason grading. We conclude that Web-based virtual microscopy is a promising new tool for teaching and standardizing Gleason grading.

Biopsy, Needle↗
PubMed2006

Analysis of intraocular lens surface properties with atomic force microscopy.

PURPOSE: To analyze the surface optics of 4 currently available intraocular lenses (IOLs) with atomic force microscopy. SETTING: Licryl Laboratory, University of Calabria, Rende, Italy. METHODS: The surface roughness and topography of poly(methyl methacrylate) (PMMA), silicone, hydrophobic, and hydrophilic acrylic IOLs were evaluated with atomic force microscopy in contact mode. The analysis was performed in a liquid environment using cantilevers with a 0.01 Newtonw/meter nominal elastic constant. Measurements were made over areas of 10 microm2 on different locations of the posterior optic surface of the IOL. RESULTS: Atomic force microscopy permitted high-resolution imaging of IOL optic surface characteristics. Surface topography showed different features with respect to the lens biomaterial. The root-mean-square roughness of the IOL optic surface was significantly different between lenses of various materials (P < .001). The hydrophobic acrylic and silicone IOLs had the lowest mean surface roughness, 3.8 nm +/- 0.2 (SD) and 4.0 +/- 0.5 nm, respectively, and the 2 PMMA IOLs had the highest mean surface roughness, 6.6 +/- 0.3 nm and 7.0 +/- 0.6 nm. CONCLUSIONS: Atomic force microscopy was effective and accurate in analyzing IOL optics. The surface topography of IOLs may vary with different manufacturing processes.

Acrylic Resins↗
PubMed2004

XMIPP: a new generation of an open-source image processing package for electron microscopy.

X-windows based microscopy image processing package (Xmipp) is a specialized suit of image processing programs, primarily aimed at obtaining the 3D reconstruction of biological specimens from large sets of projection images acquired by transmission electron microscopy. This public-domain software package was introduced to the electron microscopy field eight years ago, and since then it has changed drastically. New methodologies for the analysis of single-particle projection images have been added to classification, contrast transfer function correction, angular assignment, 3D reconstruction, reconstruction of crystals, etc. In addition, the package has been extended with functionalities for 2D crystal and electron tomography data. Furthermore, its current implementation in C++, with a highly modular design of well-documented data structures and functions, offers a convenient environment for the development of novel algorithms. In this paper, we present a general overview of a new generation of Xmipp that has been re-engineered to maximize flexibility and modularity, potentially facilitating its integration in future standardization efforts in the field. Moreover, by focusing on those developments that distinguish Xmipp from other packages available, we illustrate its added value to the electron microscopy community.

Algorithms↗
PubMed2007

In vivo laser confocal microscopy findings for Bowman's layer dystrophies (Thiel-Behnke and Reis-Bücklers corneal dystrophies).

OBJECTIVE: To investigate microstructures in patients with genetically confirmed Bowman's layer dystrophies (Thiel-Behnke or Reis-Bücklers corneal dystrophy) using an in vivo laser scanning confocal microscope. DESIGN: Single-center, prospective, comparative small case series. PARTICIPANTS: Two patients from one pedigree (a 29-year-old woman and 58-year-old man) with Thiel-Behnke corneal dystrophy (Arg555Gln [R555Q] heterozygous missense mutation of human transforming growth factor beta-induced [TGFBI] gene) and 3 patients from one pedigree (a 70-year-old woman, 58-year-old man, and 14-year old man) with Reis-Bücklers corneal dystrophy (Arg124Leu [R124L] heterozygous missense mutation of the TGFBI gene) were examined. Two patients with Reis-Bücklers corneal dystrophy exhibited recurrence after corneal transplantation. TESTING: All patients were examined by slit-lamp biomicroscopy. The center and the peripheral cornea of both eyes also were examined by in vivo laser scanning confocal microscopey. Image analysis was used to identify the corneal epithelial and stromal deposits correlated with each disorder. MAIN OUTCOME MEASURES: Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the deposits. RESULTS: In each dystrophy, distinct characteristic deposits were observed in the epithelium and Bowman's layer, respectively, by in vivo laser scanning confocal microscopy. In Thiel-Behnke corneal dystrophy, the deposits in the epithelial basal cell layer showed homogeneous reflectivity with round edges accompanying dark shadows. In contrast, deposits in Reis-Bücklers corneal dystrophy in the same cell layer showed extremely high reflectivity from small granular materials without any shadows in all cases. In each dystrophy, Bowman's layer was replaced totally with pathological materials; the reflectivity of those materials is much higher in Reis-Bücklers corneal dystrophy than in Thiel-Behnke corneal dystrophy. CONCLUSIONS: In vivo laser scanning confocal microscopy is capable of identifying in vivo corneal microstructural changes related to Thiel-Behnke and Reis-Bückler corneal dystrophy with a higher resolution than is available with slit-lamp biomicroscopy or in vivo white-light confocal microscopy. As a result, this device may enable differentiation of Thiel-Behnke and Reis-Bücklers corneal dystrophy in vivo. In vivo laser scanning confocal microscopy also may be a valuable tool for further research into the corneal dystrophies, especially to follow the natural course.

Adolescent↗
PubMed2004

X-ray microanalysis of biological specimens by high voltage electron microscopy.

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200-400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory. X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV. From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300-400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.

Animals↗
PubMed2005

Combining electron microscopy and comparative protein structure modeling.

Recently, advances have been made in methods and applications that integrate electron microscopy density maps and comparative modeling to produce atomic structures of macromolecular assemblies. Electron microscopy can benefit from comparative modeling through the fitting of comparative models into electron microscopy density maps. Also, comparative modeling can benefit from electron microscopy through the use of intermediate-resolution density maps in fold recognition, template selection and sequence-structure alignment.

Cryoelectron Microscopy↗
PubMed2004

Acoustic properties of aortic aneurysm obtained with scanning acoustic microscopy.

In aortic aneurysm tissues, macrophages and their secretion of matrix metalloproteinases (MMPs) are playing important role for tissue degeneration. Some studies have shown that weakening of the mechanical properties of the degenerated tissues may progress the expansion of the aneurysm. However, actual measurement of the mechanical properties has not been investigated at microscopic level. The objective of the present study is to assess the mechanical properties of aortic aneurysm tissues by measuring acoustic properties by scanning acoustic microscopy (SAM). Twenty-one cases of aortic aneurysm including renal and common iliac aneurysm tissues were surgically excised. Each tissue was fixed by 4% formaldehyde and the specimens were treated as (1) picrosirius red staining for normal and polarized light microscopy, (2) CD68 staining for macrophage detection, and (3) no staining for acoustic microscopy. A specially developed SAM system operating in the frequency range of 100-200 MHz, was employed in the measurement. Images of amplitude and phase are obtained in a field of 2x2 mm. The intima was mainly consisted of degenerated collagen without polarization of picrosirius red staining. Macrophages stained by CD68 were observed near the degenerated collagen fibers. The sound speed was 1567 m/s in the intima, 1576 m/s in the media, 1640 m/s in the adventitia, respectively. Infiltration of macrophages showed higher values of attenuation and sound speed than the surrounding tissues. The sound speed of the intima was significantly lower than our previous measurement of atherosclerotic aorta without aneurismal change. As the tissue elasticity is closely correlated with the sound speed, the elasticity of the intima was considered to be lower in aneurysm tissues. This mechanical weakness may contribute to the expansion of the diameter of the aneurysm. Acoustic microscopy provided important data for assessing tissue mechanical properties of abdominal aneurysm.

Acoustics↗
PubMed2002

Rapid visualization at high resolution of pathogens by atomic force microscopy: structural studies of herpes simplex virus-1.

A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles. Atomic force microscopy can thus be used as a diagnostic tool to provide a rapid way to obtain high-resolution images of human pathogens from crude preparations. It is a useful technique that complements X-ray-based structure determination, cryo-electron microscopy techniques, and optical microscopies in the field of molecular pathogenesis.

DNA, Viral↗
PubMed1988

Cholangiovenous reflux pathways as defined by corrosion casting and scanning electron microscopy.

Using corrosion casting and scanning electron microscopy of the rat biliary tree, we investigated the site and size of the pathways that allow bacteria to reflux from bile to blood. Nonobstructed rat biliary trees were injected retrograde with methylmethacrylate resin at a constant rate of 0.04 ml/min to volumes of 40, 60, 80, 120, 160, and 200 microliters. The infusion pressure was monitored and a pressure-volume curve was constructed. After polymerization and corrosion in 30 percent potassium hydroxide, the casts were examined with scanning electron microscopy. In addition, to identify the size of the reflux pathways, ceramic particles of 150 A, 1.7 mu, or 10 mu were added to the resin, and the studies were repeated. Finally, intact livers with casted biliary trees were processed and studied by scanning electron microscopy without corrosion. Scanning electron microscopy demonstrated fine anatomic detail of the cholangiovenous reflux pathway. At 40 microliters (20 cm water pressure), normal biliary radicals were filled. Between 40 and 80 microliters (20 to 50 cm water pressure), the cast material refluxed from the bile ductules into the spaces of Mall and Disse and then into the hepatic sinusoids. Filling of sinusoids continued at volumes between 80 to 160 microliters, and filling of collecting veins was seen above 160 microliters. Particles of 1.7 mu and smaller readily refluxed, but there was no sinusoidal reflux of casting material that contained particles of 10 mu. Casting without corrosion showed that the liver parenchyma remained intact. There was no evidence of reflux across hepatocytes. This study shows that cholangiovenous reflux occurs directly from bile ductules through the spaces of Mall and Disse into the hepatic sinusoids. The pathways measure between 1.7 and 10 mu. Since this is the path of least resistance, it may be of greater importance in the reflux of bacteria and toxins than other high-resistance pathways, for example, biliary canaliculi, tight junctions, or hepatocytes.

Animals↗
PubMed1990

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

Animals↗
PubMed1995

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.

Fluorescein↗
PubMed1998

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy.

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.

Cholesterol↗
PubMed1983

Scanning electron microscopy of intestinal metaplasia of the human stomach.

Biopsy samples of gastric mucosa were obtained at endoscopy from patients suspected of having intestinal metaplasia. These samples were prepared for light microscopy and scanning electron microscopy. Observations of the luminal surface of the gastric mucosa in the scanning electron microscope revealed distinct topographic differences in the intestinalized and nonintestinalized regions. The latter area showed features consistent with those described in chronic gastritis, i.e., the exaggeration of the "cobblestone relief" appearance of the luminal surface of the stomach and the presence of numerous short, stubby, bulbous microvilli which project from the free border of the surface mucous cells toward the lumen of the stomach. The intestinalized region revealed a plush microvillous (striated) border periodically interrupted by apical openings of goblet cells. The scanning electron microscope is a useful research aid for examination of cell membrane expressions of the surface cells of the gastric mucosa not totally appreciated by light microscopy and transmission electron microscopy.

Endoscopy↗
PubMed2003

A solid-phase method for evaluation of gold conjugate used in quantitative detection of antigen by immunogold-labeling electron microscopy.

Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly desirable. ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron microscopy. The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used for the final observed and measured signal. In this work, we demonstrate a simple and rapid, solid-phase method in ELISA format that is also suitable for evaluation and optimization of the gold conjugate. We have demonstrated the utility of this technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold-labeling transmission electron microscopy (TEM) of cell sections. The sensitivity of detection and quantitation of antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal. Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this reagent for immunogold localization and quantification of antigens by TEM.

Antigens↗
PubMedDate not supplied

A comparison of phase contrast microscopy and an immunofluorescence test for the detection of Giardia spp. in faecal specimens from cattle and wild rodents.

As part of a study on the presence of Giardia cysts in water, a commercial direct immunofluorescence (DIF) test for the cysts was evaluated and compared to phase contrast microscopy. Forty faecal samples were collected from cattle; 31 contained Giardia. All 31 samples were positive by DIF whereas only 17 were detected by microscopy. The detected Giardia cysts were identified as belonging to the G. duodenalis group. 216 faecal samples were collected from wild rodents (Apodemus flavicollis, A. sylvaticus and Clethrionomys glareolus); 103 contained Giardia. Cysts were detected in 97 samples by DIF and in 57 by microscopy; 51 samples were positive by both methods. Nineteen rodents harboured cysts which morphologically resembled G. duodenalis. Specific identification was possible only by phase contrast microscopy, but the results suggested that DIF was superior for the detection of Giardia cysts in faeces of the animals tested.

Animals↗
PubMed1982

Gluten-sensitive enteropathy: sequence of villous regrowth as viewed by scanning electron microscopy.

Small-intestinal biopsy specimens from five children with gluten-sensitive enteropathy (GSE) were studied by scanning electron microscopy (SEM) before dietary treatment and sequentially after removal of gluten from the the diet. Compared with biopsy specimens from patients without evidence of villous atrophy, these specimens showed marked architectural changes. In patients with total villous atrophy observed by light microscopy, absent villi and prominent crypts were observed by SEM. Patients with subtotal villous atrophy had the first stages of villous formation characterized by semicircular ridge formation. Biopsy specimens obtained from the five patients with GSE after dietary treatment demonstrated a sequence of changes characterizing villous repair. The earliest change consisted of semicircular elevations of cells around the crypts. Unequal cellular repletion around the crypts and migration of the cells led to the formation of parallel ridges. Increased cell proliferation resulted in thickening and twisting of the ridges to form convoluted ridges. Increased complexity of the ridges appears to lead to the formation of cerebriform ridges, and septation at various points in the ridges results in the formation of mitten-like villi. In biopsy specimens from patients with villous atrophy from other causes, similar reparative changes were seen by SEM. Scanning electron microscopy is a useful adjunct to the light microscopic and dissecting microscopic assessment of small intestine biopsy specimens, and may be more sensitive tan light microscopy in documenting early morphologic response to dietary treatment for gluten-sensitive enteropathy.

Adolescent↗
PubMed1988

Adenocarcinoma of the lung: a comparative diagnostic study using light and electron microscopy.

Several techniques for diagnosing adenocarcinoma of the lung are commonly available, but the frequency of their use and diagnostic sensitivity may vary. Twenty cases of primary lung adenocarcinoma obtained at surgery were studied by the following four routine techniques: light microscopy (LM) using hematoxylin-eosin (H&E) stain, mucicarmine stain, and PAS-diastase stain, and electron microscopy (EM). Three observers independently determined the positivity (0 [none], +/- [equivocal], 1 + [slight], 2 + [moderate], 3 + [marked]) of each of these cases for lumen formation in H&E-stained sections (LM lumens), intracytoplasmic (cytoplasmic mucicarmine) or intraluminal (luminal mucicarmine) mucicarmine, intracytoplasmic (cytoplasmic PAS) or intraluminal (luminal PAS) PAS-diastase, and lumen formation (EM lumens) or microvilli (EM microvilli) on electron microscopy. Comparative matching of these seven microscopic determinants (using Wilcoxon signed-rank test) demonstrated significant (P less than .01) sensitivity of EM microvilli over EM lumens, EM microvilli over luminal mucicarmine, cytoplasmic PAS over luminal mucicarmine, EM microvilli over cytoplasmic mucicarmine, cytoplasmic PAS over cytoplasmic mucicarmine, and EM microvilli over LM lumens, and a significant (P less than .05) sensitivity of cytoplasmic PAS over LM lumens, EM microvilli over luminal PAS, luminal PAS over luminal mucicarmine, and cytoplasmic PAS over EM lumens. Friedman's nonparametric test (P less than .05) indicated a significant difference among the microscopic determinants. The most sensitive was EM microvilli (mean rank score, 5.17) followed by cytoplasmic PAS (4.77), luminal PAS (4.02), cytoplasmic mucicarmine (3.62), LM lumens (3.52), EM lumens (3.47), and luminal mucicarmine (3.40). However, each of the diagnostic techniques had case examples positive for one, but not for the others, indicating that maximum yield of adenocarcinoma diagnoses will be obtained by performing all four techniques (H&E, mucicarmine, PAS-diastase, and electron microscopy.

Adenocarcinoma↗