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PubMed2004

Optical methods and sensors for in situ histology in surgery and endoscopy.

For endoscopic in situ histology, cellular structures have to be imaged non-invasively with sufficient resolution and contrast. Confocal, multi-photon or coherence-gated microscopy are able to reject multiply scattered photons and thus provide sufficient contrast at microscopic resolution. They are the basis for imaging methods such as reflection microscopy, fluorescence microscopy, autofluorescence microscopy, optical coherence microscopy and tomography and multi-photon microscopy, which will be explained and compared. Finally, current concepts for miniaturized optical sensors aiming to implement these imaging methods endoscopically will be presented.

Journal Article↗
PubMed2005

Stochastic cell.

Accumulating experimental evidence of stochasticity, self-organization and abrupt non-linear transitions underlying the dynamics of cellular structure and function is increasingly more consistent with the concepts and models of phase transitions, critical phenomena and non-linear thermodynamics rather than with the conventional clockwork description of the cell. The novel emerging image of the stochastic cell suggests that familiar and convenient classico-mechanical interpretations may be limiting our ability to understand the behavior of biological systems and calls for active exploration of alternative interpretational frameworks.

Cell Physiological Phenomena↗
PubMedDate not supplied

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.

Animals↗
PubMed1985

Identification of two lysosomal membrane glycoproteins.

Two murine lysosome-associated membrane proteins, LAMP-1 of 105,000-115,000 D and LAMP-2 of 100,000-110,000 D, have been identified by monoclonal antibodies that bind specifically to lysosomal membranes. Both glycoproteins were distinguished as integral membrane components solubilized by detergent solutions but not by various chaotropic agents. The lysosome localization was demonstrated by indirect immunofluorescent staining, co-localization of the antigen to sites of acridine orange uptake, and immunoelectron microscopy. Antibody binding was predominantly located at the limiting lysosomal membrane, distinctly separated from colloidal gold-labeled alpha-2-macroglobulin accumulated in the lumen during prolonged incubation. LAMP-1 and LAMP-2 also appeared to be present in low concentrations on Golgi trans-elements but were not detected in receptosomes marked by the presence of newly endocytosed alpha-2-macroglobulin, or in other cellular structures. LAMP-1 and LAMP-2 were distinguished as different molecules by two-dimensional gel analysis, 125I-tryptic peptide mapping, and sequential immunoprecipitations of 125I-labeled cell extracts. Both glycoproteins were synthesized as a precursor protein of approximately 90,000 D, and showed a marked heterogeneity of apparent molecular weight expression in different cell lines. LAMP-2 was closely related or identical to the macrophage antigen, MAC-3, as indicated by antibody adsorption and tryptic peptide mapping. It is postulated that these glycoproteins, as major protein constituents of the lysosomal membrane, have important roles in lysosomal structure and function.

Animals↗
PubMed1986

Molecular mobility and nucleocytoplasmic flux in hepatoma cells.

Fluorescence microphotolysis (photobleaching) was used to measure, in single polyethylene glycol-induced polykaryons of hepatoma tissue culture cells, nucleocytoplasmic flux and intracellular mobility for a series of dextrans ranging in molecular mass from 3 to 150 kD and for bovine serum albumin. For the dextrans, the cytoplasmic and the nucleoplasmic translational diffusion coefficients amounted to approximately 9 and approximately 15%, respectively, of the value in dilute buffer. The diffusion coefficients depended inversely on molecular radius, suggesting that diffusion was dominated by viscosity effects. By application of the Stokes-Einstein equation, cytoplasmic and nucleoplasmic viscosities were derived to be 6.6 and 8.1 cP, respectively, at 23 degrees C. Between 10 and 37 degrees C nucleoplasmic diffusion coefficients increased by approximately 45-85%, whereas cytoplasmic diffusion coefficients were virtually independent of temperature. In contrast to that of the dextrans, diffusion of bovine serum albumin was more restricted. In the cytoplasm the diffusion coefficient was approximately 1.5% of the value in dilute buffer; in the nucleus albumin was largely immobile. This indicated that albumin mobility is dominated by association with immobile cellular structures. Nucleocytoplasmic flux of dextrans depended inversely on molecular mass with an exclusion limit between 17 and 41 kD. This agrees with previous measurements on primary hepatocytes (Peters, R., 1984, EMBO [Eur. Mol. Biol. Organ.] J. 3:1831-1836), suggesting that in both cell types the nuclear envelope has properties of a molecular sieve with a functional pore radius of approximately 55 A.

Animals↗
PubMed1986

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells.

A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.

Animals↗
PubMed1986

Formation and alignment of Z lines in living chick myotubes microinjected with rhodamine-labeled alpha-actinin.

We have used fluorescence analogue cytochemistry in conjunction with time lapse recording to study the dynamics of alpha-actinin, a major component of the Z line, during myofibrillogenesis. Rhodamine-labeled alpha-actinin microinjected into living cultured chick skeletal myotubes became localized in discrete cellular structures within 1 h and remained specifically associated with structures for up to 4 d, allowing individual identified structures to be followed during development. In the most immature cells used, alpha-actinin was found in diffuse aggregates, some of which displayed sarcomeric periodicity. Aggregates were observed to coalesce into better defined structures (Z bands) that were approximately 1.0-micron wide. Z bands condensed into narrow, more intensely fluorescent Z lines in 4-48 h. During this period, Z lines grew laterally, primarily by the addition of small beads of alpha-actinin to existing Z lines or by the merging of small Z lines. In more mature cells, alpha-actinin added to Z lines without going through a visible intermediary structure. Mean sarcomere length did not change significantly during the stages examined, although the variability of sarcomere length did decrease markedly over time for identified sets of sarcomeres. At early stages, myofibrils frequently shifted position in both the longitudinal and lateral directions. Neighboring myofibrils were frequently associated for one or more sarcomeres sporadically along their length, such that the intervening sarcomeres were often misaligned. Associations between myofibrils were often transitory. Shifts in myofibril location in conjunction with the formation, breaking, and reformation of lateral associations between myofibrils facilitated the alignment of Z lines through a trial and error process.

Actinin↗
PubMed1994

Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell.

Cell motility is dependent upon the reorganization of the cellular cytoskeleton. Actin filaments form the major component of the cytoskeleton and respond rapidly to serum growth factors. We have previously shown that myoblasts sort the two cytoskeletal beta- and gamma-actin isoform mRNAs to different intracellular regions and that only beta-actin mRNA was associated with peripheral regions of cell motility (Hill, M.A. and P. Gunning. 1993. J. Cell Biol. 122: 825-832). We now show by in situ hybridization that 3T3 fibroblasts similarly sort actin isoform mRNAs and that peripheral beta-actin mRNA is regulated by serum. In the absence of serum, we could not detect beta-actin mRNA at the periphery. Addition of serum rapidly redistributed beta-actin mRNA to the periphery. gamma-actin mRNA distribution was not altered by serum addition at any time. Both proteins, as identified by immunochemistry with isoform-specific antibodies, were found in similar cellular structures. Serum-stimulated cell motility is mediated through the GTPase signal transduction pathway. We find that an RNA-binding protein, p62, that is part of this pathway, displays a localization pattern similar to beta-actin mRNA. Our results suggest a new biological mechanism which integrates signal transduction with the supply of an architectural component required for membrane remodeling. We propose that active transport of beta-actin mRNA to regions of cell motility is one possible objective of these signal transduction pathways.

3T3 Cells↗
PubMed1963

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS. II. ENZYMATIC AND OTHER HYDROLYTIC TREATMENTS.

The effects of proteolytic enzymes, ribonuclease, and deoxyribonuclease upon a fibrous component of chick embryo mitochondria, which was previously shown to have many fixation and staining properties characteristic of the bacterial nucleoplasm, are reported. Pepsin digestion of formaldehyde-fixed tissues removed the membranes and matrices of mitochondria, but a pepsin-resistant fibrous material remained which was heavily stained by uranyl and lead ions. Experiments on a DNA "model system" showed that DNA treated with osmium tetroxide can be depolymerized by deoxyribonuclease. Zinc ions strongly inhibited the depolymerization of DNA. Digestion of osmium tetroxide-fixed tissues (fixed only briefly) with deoxyribonuclease for 1 hour greatly reduced the Feulgen staining of the nuclei, and after 4 hours the Feulgen reaction was completely abolished. The reduction and the disappearance of the Feulgen reaction in nuclei was paralleled by partial to complete digestion of the mitochondrial fibers in the regions studied (after 1 and 4 hours, respectively), without any other obvious changes in cellular structures. When deoxyribonuclease was inhibited by the addition of zinc ions, the nuclear Feulgen reaction was not diminished, nor were the mitochondrial fibers removed. Buffer control incubations for deoxyribonuclease and ribonuclease did not alter the structure or staining properties of the mitochondrial fibers, nor did incubation with ribonuclease. The latter reaction digested the cytoplasmic and nucleolar ribosomes after a 4-hour incubation period, in parallel with the abolishment of toluidine blue staining. The results contribute further evidence that these mitochondria contain deoxyribonucleic acid.

Animals↗
PubMed1968

Studies on the posterior silk gland of the silkworm, Bombyx mori. I. Growth of posterior silk gland cells and biosynthesis of fibroin during the fifth larval instar.

Growth of the posterior silk gland and biosynthesis of fibroin during the fifth larval instar of the silkworm, Bombyx mori, have been studied. In accordance with the exponential increase in the wet weight of the gland, the amounts of DNA, RNA, protein, and lipids per animal increased rapidly in the early stage of the fifth instar (0-96 hr). Biosynthesis of fibroin, on the contrary, mainly proceeds in the later stage of the fifth instar (120-192 hr). Electron microscopical observations have shown that, in the very early stage (0-12 hr), a number of free ribosomes exist in the cytoplasm. Rough endoplasmic reticulum (ER) with closely spaced cisternae was also observed. Then rough ER starts to proliferate rapidly, and at the same time lamellar ER is rapidly or gradually transformed into vesicular or tubular forms. In the later stage of the fifth instar (120-192 hr), the cytoplasm is mostly filled with tubular or vesicular ER. Golgi vacuoles, free vacuoles (fibroin globules), and mitochondria are also observed. It is concluded that in the early stage of the fifth instar the cellular structures necessary for the biosynthesis of fibroin are rapidly formed, while in the later stage the biosynthesis of fibroin proceeds at a maximum rate and utilizes these structures.

Animals↗
PubMed1958

Metachromasy: an experimental and theoretical reevaluation.

Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.

Coloring Agents↗
PubMed1977

Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.

Actins↗
PubMed1981

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens.

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross-sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.

Animals↗
PubMed1983

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Amnion↗
PubMed1984

Intracellular water and the cytomatrix: some methods of study and current views.

The extent to which the properties of water in cells are like those of water in dilute aqueous solutions is a question of broad significance to cell biology. A detailed answer is not available at present, although evidence is accumulating that the properties of at least a large fraction of intracellular water are altered by interactions with cell ultrastructure, notably the cytomatrix. That and related evidence also suggests that the properties, composition, and activities of the "aqueous cytoplasm" of intact cells bear little resemblance to those of the "cytosol" obtained by cell fractionation. This paper will consider some of the evidence for these possibilities and some of their potential consequences with regard to cellular structure and function.

Animals↗
PubMed1984

Interactions between glycolytic enzymes and components of the cytomatrix.

Evidence is provided that enzymes absorb to cellular structures in a wide range of tissues. In particular, the interactions between glycolytic enzymes and the microfilaments of the cytoplasm are described. The relevance of these interactions to the compartmentation of carbohydrate metabolism is discussed. Examples are given of the variations in degree of binding during alteration of tissue metabolism and, for individual glycolytic enzymes, during fetal development and differentiation. Overall, these data support the concept that metabolic activities in the cytoplasm have an organized structure. Just as the structural elements of the cytosolic compartment have evolved with the capacity to assemble and disassemble in response to the changing requirements of the organism, so the metabolic elements appear to have evolved a parallel system that provides for the appropriate positioning of an energy-producing sequence in relation to the specific, dynamic requirements of the cytoskeleton.

Actins↗
PubMed1984

Reorganization of actin filament bundles in living fibroblasts.

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image-intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells. Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells.

Actins↗
PubMed1984

Axonal tubulin and axonal microtubules: biochemical evidence for cold stability.

Nerve extracts containing tubulin labeled by axonal transport were analyzed by electrophoresis and differential extraction. We found that a substantial fraction of the tubulin in the axons of the retinal ganglion cell of guinea pigs is not solubilized by conventional methods for preparation of microtubules from whole brain. In two-dimensional polyacrylamide gel electrophoresis this cold-insoluble tubulin was biochemically distinct from tubulin obtained from whole brain microtubules prepared by cold cycling. Cleveland peptide maps also indicated some differences between the cold-extractable and cold-insoluble tubulins. The demonstration of cold-insoluble tubulin that is specifically axonal in origin permits consideration of the physiological role of cold-insoluble tubulin in a specific cellular structure. It appears likely that much of this material is in the form of cold-stable microtubules. We propose that the physiological role of cold-insoluble tubulin in the axon may be associated with the regulation of the axonal microtubule complexes in neurons.

Animals↗