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At least 19 recordsLinked to original sources

Cryostat microtomy of lung tissue in an expanded state.

A technique is reported for cryostat sectioning of lung tissue in an expanded state for use in viral immunofluorescence studies. A 1:2 mixture of O.C.T. embedding compound and phosphate-buffered saline is injected intratracheally into fresh lung tissue. The lung tissue is frozen in liquid nitrogen and sectioned with a cryostat. Compared to other published reports of lung sectioning for immunofluorescence microscopy, this method has the advantages of being easy and quick, maintaining the lung sections in an expanded rather than collapsed state and avoiding contact with chemicals potentially capable of altering sensitive viral antigen.

Animals

Postembedding adjuvants in routine microtomy of paraffin-processed tissues.

Soaking the exposed face of routine paraffin-processed tissue blocks from human autopsies in solutions containing either 60% ethyl alcohol-glycerol 9:1; glycerol-aniline 9:1; 5% aqueous phenol; 5% aqueous Photo-flo, or 10% aqueous ammonium hydroxide for 2--4 hours at 0--4 C resulted in greater ease of ribboning and overall improvement of slide quality in the majority of sections compared to the results with the same blocks soaked only in water at 0--4 C. The use of postembedding adjuvants, especially 10% aqueous ammonium hydroxide, presents a simple, convenient, inexpensive means of increasing section quality with routine as well as refractory paraffin-embedded tissue without sacrificing tissue adhesion, staining qualities or cellular detail.

Aniline Compounds

Methods for improved light microscope microtomy.

Experiments undertaken to improve the ease and quality of preparing tissue for light microscopy have resulted in methods for routine production of high quality serial paraffin and glycol methacrylate sections in the 1 to 4 micrometer range using a standard rotary microtome. These methods involve sectioning with mechanically-broken Ralph-type knives, inspected for quality before mounting with double-stick tape on a holder-fluid trough in which the ribbon floats over an immersed slide and sections are mounted by lowering the fluid level in the trough. Specimen return stroke retraction, necessary for quality sectioning of plastic or with a fluid trough, is inexpensively provided by an attachable specimen holder that fits into the rotary microtome's chuck and, actuated by the microtome mechanisms, retracts the specimen during each return stroke. Descriptions of four instruments: a mechanical breaker for Ralph knives, a knife edge inspection device, an attachable specimen retractor for rotary microtomes, and a section collecting-mounting device as well as details of their use are included. Differences in cutting speed and cutting cycle cadence critical to quality sectioning with different embedments are detailed. Factors important in choosing the best embedment and most appropriate section thickness as well as choosing between dry or wet sectioning methods are also considered.

Fixatives

Changes in alkaline phosphatase activity in periosteal cells in healing fractures.

The quantitative changes in alkaline phosphatase activity in the periosteal cells close to the fracture in rat metatarsal bones has been measured during the first 5 days postfracture. This study has been made possible by two technological advances, firstly the development of cryostat microtomy for cutting unfixed, undemineralised bone, and secondly the use of scanning and integrating microdensitometry for quantifying the activity in each periosteal cell. The results showed a loss of alkaline phosphatase activity close to the fracture site, with activity rising to normal values 0.8-1.0 mm from the site. No alkaline phosphatase activity was found in the cells which proliferated from the periosteum. It is suggested that reduced glutathione could cause such inhibition.

Alkaline Phosphatase

Origin of artifactual quantitation of electrolytes in microprobe analysis of frozen sections of erythrocytes.

Frozen sections of erythrocytes have been used to validate microprobe X-ray analysis of diffusible elements in biological samples. At this meeting last year we reported that intracellular Na concentrations measured by microprobe were much higher than those measured by bulk chemical methods. It was suggested that this might be due to the movement of extracellular material over the cells by microtomy. We now present evidence that such results are better explained by electron scattering within the sample during analysis. This evidence includes observations that the excess measured Na varies directly with section thickness and inversely with accelerating voltage. At 80 kV accelerating voltage there is excellent agreement between microprobe and chemical analysis. It may be concluded that specimen preparation techniques such as used in our laboratory are satisfactory for reliably localizing diffusible elements in small volumes, but that instrumental factors can seriously affect their quantitation.

Electron Probe Microanalysis

A freeze-etching and replication study of wall deposition in elongating plant cells.

The architecture of the expanding wall of mung bean hypocotyl (Phaseolus aureus) and collenchyma of celery (Apium graveolens) was examined using freeze-etching without any cryoprotectant, and surface-replication of frozen-ground and air-dried specimens. The polylamellated organization of the wall was seen. Freeze-etching clearly visualized, within one single fracture plans, the intermediate strata in which the microfibril orientation gradually changes between the main transverse and longitudinal directions. They corresponded to the bow-shaped arcs seen with surface replication and conventional microtomy. The organization of newly-formed microfibrils (periplasmic microfibrils) was seen by their imprints on the plasmalemma. When they were being deposited the microfibrils were loose and sinuous though tightly packed, rigid and parallel on the further layers. Therefore it seems that the fibrillogenesis and the spatial orientation of the microfibrils are two subsequent steps. The role of the periplasm in controlling the three-dimensional arrangement of the wall is emphasized.

Cell Membrane

Lobular carcinoma of the breast in situ. Are we too radical in its treatment?

Twenty-four patients (average age, 46 years) with 29 instances of lobular carcinoma in situ of the breast have been treated from 1952 to 1971 at the Henry Ford Hospital (incidence, 1%). Six patients had bilateral lesions, one synchronous and 5 metachronous. The initial complaint in 23 of 24 patients was a mass in the breast. Diagnosis was based on permanent histologic section as mammorgraphy and frozen section analysis were inconclusive. Treatment consisted of radical mastectomy in six, modified radical mastectomy in five, and simple mastectomy in 20. All lymph nodes recovered showed no metastatic disease. All patients are alive and well with no evidence of disease. Based on our experience, we recommend a simple mastectomy for treatment of lobular carcinoma in situ with contraleteral bipsy examination in those instances when clinical or roentgenographic evidence suggests a pathologic process.

Adult

Microphotometric determination of enzyme activities in cryostat sections by the gel film technique.

Use of the gel film technique in microphotometric determinations of enzyme activity is described. The microscope photometer is computer-controlled. It is programmed to deal with repetitive measurements at up to 12 selected positions within a tissue section and to evaluate recorded reaction rates statistically. Films of polyacrylamide gel with entrapped glucose-6-phosphate dehydrogenase are used as a model to demonstrate the correlation between local enzyme activity and the microphotometrically determined reaction rate. Enzyme activities at different positions in the same tissue section are determined and compared. Activity profiles of five enzymes (glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, NAD-dependent tetrazolium reductase) in the liver are presented and show non-uniform intra-acinar distribution patterns. These results are interpreted in the light of the metabolic zonation of the hepatic acinus. Further applications of the method are discussed.

Animals

Three-dimensional architecture of the golgi apparatus in Sertoli cells of the rat.

Glutaraldehyde-fixed testes were stained "en bloc" with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 micron) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (+/- 7 degrees). At low magnification, in thick sections (0.5-3 micron) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the "saccular region" corresponding to the conventional stack of saccules and the "intersaccular connecting region" made up of anastomotic tubules which bridge adjacent stacks. In the saccurlar regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off" from the pile. The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.

Animals

Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.

The appearance and migration of mesenchymal cushion tissue within the truncus arteriosus of the normal 2.5 to 6-day chick embryo heart was surveyed systemically with the light microscope. Series of cross-sections taken from replicate hearts at successive developmental stages allowed comparison of the following qualitative and quantitative aspects of early truncal morphogenesis. Mesenchyme within the truncus was derived from two distinct sources. The first mesenchyme appeared to migrate caudally into the cardiac jelly of the distal truncus from the nearby aortic arch region, coincident with slowing of the anterior elongation of the heart tube (Hamburger-Hamilton Stage 17-18). A second, separate mesenchymal population, derived from endocardium, began to fill the conus and proximal truncus in a radial direction, coicident with expansion of the bulbs cordis (Stage 12-19). The measured kinetics of relative cell numbers, distributions, and mitotic indices suggest substantial contributions from both sources. By Stage 26, the conotruncal region was filled with mesenchyme, which then condensed to form the anlagen of three future structures: the semilunar valves, the aorticopulmonary septum, and the tunica media of the great arteries.

Animals

Scanning electron microscopy of frozen sections: a method for studying selected planes in small specimens.

A procedure for studying small biological specimens with the SEM, using frozen sections of fixed material is described. Controlled fixation schedules and convenient handling of sections insure optimal preservation of cell shape and size. The greatest advantage of the method presented is to allow scanning through any desired plane of the specimen, even in those planes parallel to an epithelial face. SEM of frozen sections is thus helpful for the study of cell distribution and organization within a tissue.

Animals

Frozen section- frozen attitudes.

Patients with breast cancer who have clinically negative axillae, tumors 2 cm in diameter or less, and tumors with a moderate or low degree of anaplasticity are candidates for treatment by local excision and irradiation. A definitive histologic diagnosis should be made in all cases of breast cancer before a final treatment decision is made. This can be accomplished by needle or excisional biopsy. After a histologic diagnosis is established, the decision regarding treatment can be arrived at with the patient enering into the process. Those patients requiring mastectomy will be better able to cope with this, knowing why the operation was considered best for them. The elimination of frozen section diagnosis will free the patient from one of the great fears of the current treatment of breast cancer- subjecting herself to biopsy without knowing what the outcome will be. It will free the surgeon from the rigid sequence of frozen section followed by mastectomy and allow him to begin to rationally assign patients with proper criteria to conservative treatment, reserving mastectomy for those who do not meet these criteria.

Adult

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy.

The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20 A-ca. 62 A and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40 A is seen in preparations lightly treated by OsO4-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.

Animals

Electron microprobe investigations into the process of hard tissue formation.

The electron microprobe microanalyser has been used to measure the concentrations of Ca, P and S in the predentine of young rat incisors. The specimens were prepared as alcohol embedded ultrathin sections, unfixed vacuum embedded dry cut ultrathin sections and as thin cryostat sections. The results show the influence of preparation on the measured compositions and indicate that Ca is tightly bound to the matrix, whereas P can be easily washed out. Measurements along the dentine-predentine border demonstrated zones of Ca enrichment, the average size of which suggests that the zones could be the prestages of calcospherites. A mineralisation mechanism is discussed in which the high Ca concentration activates pyrophosphosphatase or ATPase before the onset of nucleation.

Animals

Tubular structures associated with intracytoplasmic spirochetes.

When tissues of spirochete-infected brine shrimp (Artemia salina) were examined by electron microscopy, tubular structures were often seen closely associated with intracytoplasmic spirochete profiles. These tubules were never observed in association with spirochetes found in the haemocoeal or other extracellular spaces. Since the trilaminar layer comprising the wall of the tubules was identical in ultrastructure to the outer envelope of the spirochete, these structures were interpreted to be finger-like extensions of the spirochete outer envelope.

Animals