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PubMed1983

Transmission electron microscopy and serial sections for light microscopy from the same block in routine renal pathology.

Routine serial sections for light microscopy (LM) from one sample and transmission electron microscopy (TEM) from selected areas of another sample are current procedures in renal pathology. At the present time, there are methods available to perform high-resolution light microscopy (HRLM) and TEM from the same block. However, this technology is not directly applicable or convenient for routine renal biopsies. Most methods presently available to obtain TEM studies from different areas of the same large HRLM block use reembedding methods, which considerably delay TEM studies, or mesa technics that fragment and waste the original block. Present methods to obtain serial sections from plastic blocks have been used in plant and animal morphology and need to be modified for renal pathology. In the present article, the authors present a technic that produces immediate TEM sections from different areas of the same HRLM block. This method does not produce fragmentation or waste of the original block, and TEM sections are obtained within two hours after large HRLM sections are screened. The latter sections are mounted serially on a regular microscopic slide on 8-15 separate wells painted with water-repellent ink. The methods are simple and produce large HRLM and TEM sections in 24 hours, avoiding separate samples for LM and TEM studies.

Biopsy, Needle↗

PubMed2000

Specular microscopy, confocal microscopy, and ultrasound biomicroscopy: diagnostic tools of the past quarter century.

PURPOSE: To identify, characterize, and illustrate the most important past and future potential contributions of specular, confocal, and ultrasound biomicroscopy to clinical diagnosis and research applications in the cornea from the past 25 years. METHODS: Specular microscopy, in vivo tandem scanning confocal microscopy (TSCM), scanning slit confocal microscopy (SSCM), and high-frequency ultrasound biomicroscopy are examined. RESULTS AND CONCLUSIONS: This review demonstrates the abilities and limitations of three powerful new in vivo imaging modalities to resolve the cellular and structural layers of the cornea temporally and spatially in three or four dimensions, (x, y, z, t). Clinical pathological processes such as inflammation. infection, wound healing, toxicity, embryonic development, differentiation, and disease, which previously could be studied only under static ex vivo conditions, can now be dynamically evaluated over time. Thus, with continued development and application in vivo, noninvasive microscopic techniques should provide exciting new insights into understanding the structure and function of not only the eye, but also other multicellular organ systems in health and disease. These new imaging paradigms are in the first rank of advances in medical science in the past quarter century.

Cornea↗

PubMed1982

One very rare and one new tracheal tumour found by electron microscopy: glomus tumour and acinic cell tumour resembling carcinoid tumours by light microscopy.

Tracheal tumours were removed surgically from two patients and diagnosed as carcinoid tumours by routine light microscopy. At a later date, electron microscopy was performed on stored tumour tissue and no neurosecretory granules were found in either case. One showed features of a glomus tumour and the other of an acinic cell tumour. Only two glomus tumours appear to have been reported previously in the trachea, and no acinic cell tumours. Electron microscopy is thus sometimes of great assistance in diagnosing accurately unusual tumours of the lower respiratory tract.

Carcinoid Tumor↗

PubMed1999

Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

Animals↗

PubMed1985

Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

Adenocarcinoma↗

PubMed1996

Scanning electron microscopy of fragmentary marker chromosomes observed by light microscopy.

To study the fine structure of fragmentary marker chromosomes, we performed scanning electron microscopy (SEM) on samples isolated from two carriers (Case 1: 46, XY/47, XY, +mar/48, XY, +mar, +mar; Case 2: 47, XY, +mar). In both cases, light microscopic observation revealed that marker chromosomes lacked a centromere and were fragmented in appearance. However, SEM observation of the metaphasic cells in both cases showed three variations. One variation was a structure that seemed to be metacentric, another was a structure that seemed to be submetacentric, and the remaining one was essentially fragmentary. However, neither the usual chromatid nor centromere formations were observed in the metacentric-like and submetacentric-like structures, even when both cases were observed by SEM. Moreover, the marker chromosomes of the boy of Case I, who suffered from various clinical troubles, included a greater population of metacentric-like or submetacentric-like structures than of essentially fragmentary structures. The marker chromosomes of the fetus of Case 2, who suffered from no clinical problems, included a much greater population of essentially fragmentary structures than metacentric-like or submetacentric like structures. Therefore, SEM observation of fragmentary marker chromosomes that are visible on light microscopy might be used to define specific structures. Moreover, SEM observation might provide clinical criteria relating to the pathogenesis of fragmentary marker chromosomes found on light microscopy.

Abnormalities, Multiple↗

PubMed1996

Swift transformation and locomotion of polymorphonuclear leukocytes and microglia as observed by VEC-DIC microscopy (video microscopy).

The detailed assembly used by us for video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy (video microscopy is first described. Employing such video microscopy, we then examined the morphological changes occurring during locomotion and activation processes of polymorphonuclear leukocytes (PMNL) and microglia at an almost electron microscopic magnification. Upon contacting the substratum, PMNL transformed into a polarized ameboid shape and crawled extending pseudopodia, as has been well documented previously. The PMNL sometimes displayed a peculiar locomotion as if they were stepping on "tiny legs", or sliding on a treadmill of cell membrane. Cultured microglia were observed to exist in 4 forms; ramified, reactive, villous, and ameboid. Microglia in the reactive form pivoted, circled and crawled on the astroglial cell layer using their transparent lamellipodia with no morphological changes in their cell body. Unlike PMNL, reactive microglia exhibited no agitated movements of their intracellular organelles, including granules and cytosol, during locomotion. Lamellipodia on the undersurface of the cell body touching the cell layer adhesively, appeared to serve as the locomotive apparatus. When activated, both floating PMNL and microglia of villous form assumed an ameboid shape within a few seconds. Microglia occasionally swam in the medium waving their lamellipodia towards a target object (e.g. zymosan A particles), remodelling to an amorphous ameboid form and covering up the target. We attempt to discuss such swift morphological changes from the standpoint of thermodynamic potential of Gibbs free energy which is stored within the cells.

Cell Movement↗

PubMed2002

Imaging of fluorine and boron from fluorinated boronophenylalanine in the same cell at organelle resolution by correlative ion microscopy and confocal laser scanning microscopy.

PURPOSE: There is a clear need for a technique that provides subcellular locations of fluorine and boron atoms from fluorinated neutron capture agents because positron emission tomography is being tested as a tool for providing tumor boron concentrations in boron neutron capture therapy. EXPERIMENTAL DESIGN: Ion microscopy was used in combination with confocal laser scanning microscopy to investigate the subcellular locations of fluorine and boron from fluorinated p-boronophenylalanine (F-BPA) in human glioblastoma T98G cells. The fluorinated compound was also compared with p-boronophenylalanine (BPA) for delivery of boron after a clinically relevant 6-h exposure. Mitochondria were identified by rhodamine 123 labeling. A strict cryogenic sample preparation was used, and measurements were made in fractured freeze-dried cells. RESULTS: The nucleus, a perinuclear mitochondria-rich cytoplasmic region, and the remaining cytoplasm were the three subcellular regions identified in individual T98G cells. In cells treated with F-BPA, the mitochondria-rich perinuclear cytoplasmic region exhibited significantly lower fluorine and boron signals than the remaining cytoplasm and the nuclei. Ion microscopy observations revealed a nearly 1:1 distribution of fluorine and boron in subcellular compartments. Quantitative subcellular observations indicated that there was no significant difference in boron delivery to subcellular compartments between the F-BPA and nonfluorinated BPA. CONCLUSIONS: These observations provide the first direct evidence that fluorine and boron from fluorinated BPA are cocompartmentalized in cells and that the fluorinated compound is as efficient for boron delivery as the nonfluorinated BPA at a clinically relevant time point. These observations provide strong support for the use of F-BPA in positron emission tomography biodistribution studies for boron neutron capture therapy.

Boron↗

PubMed2002

Study of gastrointestinal stromal tumors by light microscopy, electron microscopy and immunohistochemistry.

OBJECTIVE: To investigate the morphological features, immunohistochemical speciality of the gastrointestinal stromal tumors (GISTs), and its histogenesis as well. METHODS: The morphologic characteristics of GISTs were studied in 65 cases using light microscopy and 17 cases by electron microscopy. The expression of c-kit (CD117), CD34, vimentin, SMA, actin, desmin, S-100 and MBP were detected in all the cases with EnVision trade mark staining. RESULTS: Among 65 cases of GISTs, 46 were spindle cell type, 6 epithelioid cell type and 13 mixture type, equivalent to 85.5% (65 of 76) of all of the mesenchymal tumors of gastrointestinal tract admitted in the same period. The epithelioid cell type tumors composed of mainly the epithelioid cells, predominantly short spindle, oval or round in pattern, with an overall eosinophilic cytoplasm by hematoxylin-eosin stain. Focal cytoplasmic vacuolization was often seen. Sometimes signet-ring like cells and cells with a clear cytoplasm were seen in the epithelioid stromal tumor. The tumor cells arranged in interlacing fascicles forming whorls or sometimes cell clusters. Electronic microscopy showed the presence of interdigitating cell processes, in some areas, synapse-like structure and numerous desmosome-like junctions as well as a few gap junctions and small round neurosecretory granules. There were also abundant intermediate filaments and thin filaments of actin-type with longitudinal condensations (dense bodies). All of the 65 stromal tumors were strongly positive for vimentin (100%), 61 out of 65 tumors positive for CD117 (c-kit) (93.8%) and 51 out of 65 positive for CD34 (78.5%). Some cases also expressed SMA, actin, S-100 and MBP. CONCLUSIONS: GISTs were the most frequent mesenchymal tumor seen in the gastrointestinal tract. Under light microscope, the morphology of stromal tumors looks sometimes like a leiomyoma and Schwannoma. The application of immunohistochemical markers (particularly CD117 and CD34) and ultrastructural study are considered necessary for the differential diagnosis. GISTs may originate from the pluripotential precursor cells like the interstitial cells of Cajal.

Gastrointestinal Neoplasms↗

PubMed2003

A new method to correlate histology with biomechanical properties in urethral tissue. An in-vitro study using light microscopy and scanning acoustic microscopy.

When surveying the classical biomechanical theory of flow and resistance, the passive elastic properties of the urethra seems to be important for the transport of urine though the urethra. The aim of this study was to show that scanning acoustic microscopy (SAM) is a suitable methodology for investigating elastic properties of the urethra, and that it can be used to correlate elastic properties to histological areas. One 40 kg female pig and one 2 kg male rabbit comprised the material. A SAM2000 was used at a working frequency of 1000 MHz. Sections of nominal 3 micrometer thickness fixed urethral tissue were prepared for SAM and stained for light microscopy. The histological layers of the urethra were evident in the SAM image, and showed highly variable values of elastic properties. The layers seen with SAM correlated well with those seen with light microscopy. In conclusion, we have provided the first images of the microelastic properties of the urethra and correlated them to histology.

Animals↗

PubMed1985

A new approach for studying semithin sections of human pathological material: intermicroscopic correlation between light microscopy and scanning electron microscopy.

In order to obtain useful and complete information on the study of pathological material, we observed by scanning electron microscopy (SEM) the same semithin sections observed by light microscopy (LM). For this purpose, the specimen must have, at the same time, chromatic and electron dense characteristics. We thus developed different specimen preparation methods, subjecting the semithin sections to specific polychromatic staining with high atomic number (Z) elements, to monochromatic staining followed by routine contrasting with uranyl acetate and lead citrate, and to specific cytochemical and immunocytochemical procedures. The specimens were examined in sequence by LM, by SEM equipped with secondary electron, backscattered electron, transmitted electron detectors and by scanning transmission electron microscopy (S(T)EM).

Actins↗

PubMed1993

Biological microscopy: the emergence of digital microscopy.

The past few years have seen a tremendous renaissance in biological optical microscopy, mainly as a result of the application of digital image processing and video imaging techniques. We review recent developments in microscopy that are permitting unprecedented views of biological structure and function.

Animals↗

PubMed2004

Study of the lignin model compound supramolecular structure by combination of near-field scanning optical microscopy and atomic force microscopy.

In this paper, we present a nanoscale study of the supramolecular structure of the dehydrogenate polymer (ZL-DHP) lignin model compound. The combination of near-field scanning optical microscopy (NSOM or SNOM) and atomic force microscopy (AFM) has been utilized to explore physicochemical properties of the lignin model compound on a scale ranging from individual macromolecules to globular supramolecular assemblies. By utilizing NSOM in transmission mode, the optical inhomogeneity in the lignin supramolecular structure has been observed for the first time. In particular, the transmission-mode NSOM images reveal a combination of hollow and layered supramolecular globular structure in the lignin model compound. Through the paired use of TappingMode and pulsed-mode AFM, we have also confirmed the existence of regions with different rheological properties on the single lignin model compound supramolecular assembly.

Alcohols↗

PubMed2006

Combined phase-sensitive acoustic microscopy and confocal laser scanning microscopy.

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.

3T3 Cells↗

PubMed2005

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone.

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.

Animals↗

PubMed2006

High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy.

Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component alpha-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.

Cell Cycle↗

PubMed2002

Atomic force microscopy and magnetic force microscopy study of model colloids.

Atomic force microscopy (AFM) is used to study the size, shape, and polydispersity of a variety of magnetic and nonmagnetic model colloids, previously imaged by transmission electron microscopy (TEM) only. Both height and phase images are analyzed and special attention is given to 3D morphology and softness of particles, as well as structures and presence of secondary components in the colloid, difficult to investigate with TEM. Several methods of tip characterization followed by deconvolution were applied in order to improve the accuracy of lateral diameter determination. In the case of magnetite particles dispersed in conventional ferrofluids, we explore both experimentally and theoretically the possibility of using magnetic force microscopy (MFM). We propose and discuss several models which allow to estimate the magnetic moment of a single domain superparamagnetic sphere using MFM, which cannot be done with other techniques; alternatively the tip magnetization can be determined.

Journal Article↗

PubMed1999

An effective method for suspicious cases in urinary cytodiagnosis by the combined use of scanning and transmission electron microscopy following light microscopy.

In urinary cytodiagnosis, pathological characterization of atypical cells is sometimes difficult because cells in urine samples tend to be degenerated. To overcome this problem, we adopted serial examination under light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Five patients with transitional cell carcinoma primarily developed in the bladder, three patients with dysplastic cells, and one patient with metastatic and infiltrative colorectal carcinoma were subjects of the present study. Sample cells were smeared on a film slide especially suitable for serial use (a film slide used in X-ray diagnosis), fixed in 1% glutaraldehyde (GA) for 30 min, stained with Papanicolaou (Pap) staining, and immediately processed for fixation in 2% paraformal-dehyde (PA) and 2.5% GA. Slides were postfixed in 0.5% osmium tetroxide. Then, cell samples were subjected to a series of observations under LM, SEM, and TEM. One of three patients in whom dysplastic cells were suggested under LM was finally diagnosed as transitional cell carcinoma by subsequent examination under SEM followed by TEM. Thus, serial use of LM, SEM, and TEM proved useful in discriminating transitional cell carcinoma (grade 1) from dysplastic cells and also in distinguishing metastatic tumor cells from primary tumor cells in urine samples.

Journal Article↗