Transmission and scanning electron microscopic study of the same cytologic material

The same cytologic material was successively examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After the SEM examination, the specimens were rehydrated for a long period of time to allow the penetration of Epon 812 into the cells. The TEM examination showed the cell organelles to be comparatively well preserved. These consecutively performed LM-SEM-TEM examinations provided useful information on cytologic subjects, especially concerning the origin of the cells.     

"Repair cells" in equine uterine cytologic and histologic specimens

Cells resembling those known as "repair cells" in gynecologic cytology specimens from women were identified in uterine cytology specimens from infertile mares treated with antibiotics using indwelling uterine catheters. This prompted a study of the effect on the equine uterus of indwelling catheterization without antibiotic infusion, using light microscopic examination of cytologic and biopsy specimens and electron microscopic examination of biopsy specimens. Cytologic and biopsy specimens had features within normal limits at the start of the study. Following five days of indwelling catheterization, neutrophils were present in both cytologic and biopsy specimens. In cytologic specimens, numerous groups of "repair cells" were present; similar cells in biopsy specimens indicated this was a focal reaction. The large nuclei and prominent nucleoli of the "repair cells" suggested cellular proliferation or regeneration. However, this was contradicted by the ultrastructural sparsity of ribosomes, endoplasmic reticulum, Golgi apparatus and mitochondria. Inflammation and "repair cells" were not present in cytologic or biopsy specimens collected 40 days after the start of the study. Although these cells may be a component of a repair process, our results support the hypothesis that "repair cells" in human and equine gynecologic cytology specimens are injured, rather than regenerating, cells. The term dysphaneroplastic (Greek: "abnormal cytosol development") is proposed to describe these cells since the cytoplasm does not reflect the features of cellular activity suggested by the nuclear appearance.     

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

Transmission and Scanning Electron Microscopy of Cell Monolayers Grown on Polymethylpentene Coverslips

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized. separated from the coverslip by liquid nitrogen immersion, and sectioned either “en face” or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

The jaw apparatus of the Cretaceous ammonite Reesidites

Jaws are preserved within the body chambers of three specimens of a collignoniceratid ammonite Reesidites minimus (Hayasaka and Fukada) from the Upper liuoniaq of Hokkaido, Japan. Light microscopic and SEM observations of sections indicate that both upper and lower jaws consist mainly of a thick, double-walled chitinous lamella with a beak-like anterior projection. The outer chitinous lamella of the lower jaw is covered by a thick calcareous layer. The jaw apparatus of this species morphologically resembles aptychus-type jaws of Jurassic ammonites, but is distinguished by the presence of an anterior beak-like projection with serrated ridges and grooves in the lower jaw. These observations strongly suggest a biting ability in this species.     

The exfoliating cervical epithelial surface in dysplasia, carcinoma in situ and invasive squamous carcinoma. I. Scanning electron microscopic study.

The exfoliating epithelial surface in cone specimens of the human cervix with histologically normal epithelium, squamous metaplasia, dysplasia, carcinoma in situ and in cervical biopsies with invasive squamous carcinoma was studied by scanning electron-microscopy (SEM). A cobblestone-like surface with anisovillosis seems to herald the appearance of malignant cells on the exfoliating epithelial surface. Post-scanning histologic examination served as a diagnostic reference for the SEM findings.     

Preparation techniques for the electron microscopy of granular sludge from an anaerobic digester

Several fixation and dehydration techniques for scanning and transmission electron microscopy of glycocalyx and microbial populations within granules from an upflow anaerobic sludge blanket digester purifying a brewery wastewater were compared. Sputter-cryo and freeze-drying techniques prior to scanning electron microscopy (SEM) allowed viewing of the glycocalyx. In contrast standard fixation and dehydration techniques were suitable for examination of underlying microbial populations by both SEM and transmission electron microscopy.     

The Influence of Specimen Preparation on Artefacts in Scanning Electron Microscopy of Respiratory Cilia

The effects of various handling procedures used in preparing specimens of human and pig respiratory mucosa for scanning electron microscopy (SEM) were studied. Using five different washing methods, the percentage area of mucosa covered with extracellular material varied from 1.5 to 53.1%. The best results were achieved when specimens were washed by gently inverting a sample 30 times in a container filled with physiological saline. Fixation and drying of the surface layer caused disorientation of cilia and made examination difficult. Mechanical damage caused loss of cilia and rupture of the cell membrane. For SEM of respiratory cilia it is important to wash the specimen in saline before fixation and to use biopsy forceps as little as possible.     

Benign squamous cells with pleomorphic microvilli in urine or bladder washings

Exfoliated cells in catheterized urine or bladder washings from 40 patients were observed by light microscopy (LM) and by scanning electron microscopy (SEM). The specimens from seven of these patients (six postmenopausal females and one 85-year-old male) contained squamous cells with pleomorphic microvilli (PMV) on their surfaces. Four of these cases had no bladder lesions by cystoscopic examination. Three patients had recurrent papillary transitional-cell carcinoma of the bladder, and the cytologic specimens from two of them contained transitional cells with PMV. The distinction between squamous and transitional cell is readily made by SEM, based primarily on cell shape and thickness. The presence of PMV on otherwise-benign-appearing squamous cells in urine or bladder washing specimens may be a source of confusion in the interpretation of SEM findings. The presence of PMV on exfoliated squamous cells in cytologic material from the human urinary tract does not seem to have the same diagnostic and prognostic significance as the presence of PMV on transitional cells.     

原子力显微镜在染色体研究中的应用

原子力显微镜(Atomic force microscopy, AFM)是一种具有超高分辨率的显微成像仪器, 可在空气、真空和液体环境下对样本的表面结构进行实时观察。文章介绍了AFM的工作原理, AFM相对于其他种类显微镜在观察生物样本方面的显著优势, 并综述了AFM在染色体研究中的应用和进展。     

Standardization of fine needle aspiration cytology of the breast – comparison of Auto Cyto Fix and conventional smears

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Standardization of fine needle aspiration cytology of the breast -- comparison of Auto Cyto Fix and conventional smears.

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Reliable diagnosis of Trichosomoides crassicauda in the urinary bladder of the rat

Two reliable methods are described for identifying infection of laboratory rats with the nematode Trichosomoides crassicauda. The first is a rapid method where cryostat sections of the rat urinary bladder are stained with acridine orange and viewed under a fluorescence microscope. The second involves the stabilization of the bladder surface prior to examination using scanning electron microscopy (SEM).     

Improved coating and fixation methods for scanning electron microscope autoradiography

A simple apparatus for emulsion coating is described. The apparatus is inexpensive and easily assembled in a standard glass shop. Emulsion coating for scanning electron microscope autoradiography with this apparatus consistently yields uniform layers. When used in conjunction with newly described fixation methods, this new approach produces reliable autoradiographs of undamaged specimens.     

Scanning electron microscopy of heart muscle freeze-dried from dimethylsulfoxide for simultaneous demonstration of cell morphology and microvascular function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 +/- 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

Scanning Electron Microscopy of Heart Muscle Freeze-Dried from Dimethylsulfoxide for Simultaneous Demonstration of Cell Morphology and Microvascular Function

Scanning electron microscopy was used to examine cryofracture surfaces of ventricular myocardium from glutaraldehyde fixed rat and rabbit hearts subjected to intravascular injection of polymerizing acrylic resin. This allowed simultaneous observation of morphological features of cardiac muscle cells and the functional state of their associated small blood vessels. Because the resin injected to identify capillaries accessible to flow might be soluble in commonly used tissue dehydrating agents, alternative preparation methods using the cryoprotectants dimethylsulfoxide (DMSO) and glycerol were investigated. Provided a high performance backscattered electron detector and simple environmental cell were used to abolish specimen charging and circumvent potential instrument contamination, immersion in 2.82 M DMSO for 12 hr prior to cryofracture and freeze-drying gave the best results. The SEM appearance of specimens dehydrated in this way differed little from that of specimens prepared by ethanol dehydration and freeze-drying or by acetone dehydration and critical-point drying. Tissue shrinkage was 26.5 ± 9.4%, comparable to that found after standard methods using solvent dehydration and critical-point drying.     

An apparatus for the simultaneous demineralization of fifty-four specimens.

An apparatus designed to demineralize 54 specimens simultaneously is described. A drum with built-in specimen holders rotates continuously through a bath of acid, allowing a free exchange of demineralizing fluid over the specimens. Individual specimens can be easily introduced or withdrawn from the apparatus without disturbing others.     

The antenna cleaner gland in Messor rufitarsis (hymenoptera, formicidae)

TEM and SEM analysis of the tibio-tarsal antennal cleaner in Messor rufitarsis reveals a pore region on the surface of the basitarsus. These pores are originating from a cluster of highly prismatic epidermal cells that fill the anterior hemolymph space of the leg almost completely. Within the cytoplasma of the cells, basal labyrinth, nucleus/rough endoplasmic reticulum, 'secondary lysosomes', a lot of smooth endoplasmic reticulum and distal microvilli regions can be distinguished indicating that the cells are 'class I' gland cells. Their secretion appears to be transported to the antennal cleaner apparatus via a subcuticular space and pores. It is suggested that the secretion might be used for cleaning of the antenna and/or chemical communication. SEM examination of six additional species indicates that an antennal cleaner gland might be present throughout the ants.     

An Apparatus for the Simultaneous Demineralization of Fifty-Four Specimens

An apparatus designed to demineralize 54 specimens simultaneously is described. A drum with built-in specimen holders rotates continuously through a bath of acid, allowing a free exchange of demineralizing fluid over the specimens. Individual specimens can be easily introduced or withdrawn from the apparatus without disturbing others.     

Aspiration cytology from the pouch of Douglas at hysteroscopy

Eighty-seven fine needle aspiration cytological samples from 29 patients suffering from irregular perimenopausal uterine bleeding were evaluated. Aspiration cytology was performed prior to hysteroscopy, after distension of the uterine cavity and finally after uterine curettage. In this paper, cytological examination of fluid from the pelvic content in women with benign endometrial findings is compared with that of patients with adenocarcinoma. Endometrial curettage influenced the cell content of the cytologica specimens.