[Direct pictures by means of negative phase contrast].
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Humans↗
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[Demonstration of single elements of the endoplasmic reticulum in the living state].
By means of a special selective preparation technique, it is possible to investigate in thin sections, by electron microscopy, areas of a cell that have been observed in the living state, by phase-contrast microscopy, up to the time of fixation. Structures recorded in the living state can thus be compared to structures seen in electron micrographs. In cells of the fungus Polystictus versicolor, aggregates of membrane systems as well as single cisternae with a diameter of approximately 200 to 300 A can be detected with phase optics. It can be shown, by calculation, that these structures, which are far below the limit of resolution of the light optical system, give enough contrast to be discernible by phase optics. Thus a basis is provided for observing the dynamics of membrane systems which perhaps may contribute to the analysis of the functional significance of these cell components.
Basidiomycota↗
Supramolecular ordering of DNA in the cholesteric liquid crystalline phase: an ultrastructural study.
Aqueous solutions of 146-base pair DNA fragments form a cholesteric liquid crystalline phase in the range of about 160-290 mg/ml. We present a structural analysis of this phase by comparing the data obtained from polarizing and electron microscopy. This phase shows multiple aspects or "textures" which are presented and interpreted. They mainly depend on the orientation of the structure relative to the observation plane and on the nature, distribution, and amount of defects present in the phase. These defects are then analyzed with the two methods, and the molecular orientations can be defined precisely in their core. The biological interest of such structural analyses is discussed in relation with the understanding of chromatin structure and function.
Biophysical Phenomena↗
Pulsed growth of fungal hyphal tips.
Somatic fungal hyphae are generally assumed to elongate at steady linear rates when grown under constant environmental conditions with ample nutrients. However, patterns of pulsed hyphal elongation were detected during apparent steady growth of hyphal tips in fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and Saprolegnia ferax; Zygomycetes, Gilbertella persicaria; Deuteromycetes, Trichoderma viride; Ascomycetes, Neurospora crassa and Fusarium culmorum; Basidiomycetes, Rhizoctonia solani). Growing hyphal tips were recorded with video-enhanced phase-contrast microscopy at high magnification, and digital images were measured at very short time intervals (1-5 s). In all fungi tested, the hyphal elongation rate was never perfectly steady but fluctuated continuously with alternating periods of fast and slow growth at more or less regular intervals. Pulsed growth was observed in fungi differing in cell diameter, overall growth rate, taxonomic position, and presence and pattern of Spitzenkörper organization, suggesting that this is a general phenomenon. Frequency and amplitude of the pulses varied among the test organisms. T. viride and N. crassa showed the most frequent pulses (average of 13-14 per min), and F. culmorum the least frequent (2.7 per min). Average pulse amplitude varied from 0.012 microns/s for F. culmorum to 0.068 microns/s for G. persicaria. In F. culmorum and T. viride, the fast phase of the growth pulses was correlated with the merger of satellite Spitzenkörper with the main Spitzenkörper. These findings are consistent with a causal relationship between fluctuations in the overall rate of secretory vesicle delivery/discharge at the hyphal apex and the fluctuations in hyphal elongation rate.
Activity Cycles↗
Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.
Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.
Animals↗
Shuttling of CTP:Phosphocholine cytidylyltransferase between the nucleus and endoplasmic reticulum accompanies the wave of phosphatidylcholine synthesis during the G(0) --> G(1) transition.
The transition from quiescence (G(0)) into the cell division cycle is marked by accelerated phospholipid turnover. We examined the rates of phosphatidylcholine (PC) synthesis and the activity, membrane affinity, and intracellular localization of the rate-limiting enzyme in the synthesis of PC, CTP:phosphocholine cytidylyltransferase (CT) during this transition. The addition of serum to quiescent IIC9 fibroblasts resulted in a wave of PC synthesis beginning at approximately 10 min, peaking at approximately 3 h with a >10-fold increase in rate, and declining to near basal rates by 10 h. CT activity, monitored in situ, was elevated approximately 3-fold between 1 and 2 h postserum. Neither CT mass nor its phosphorylation state changed during the surge in PC synthesis and CT activity. On the other hand, the ratio of particulate/soluble CT surged and then receded in concert with the wave of PC synthesis. During quiescence, CT was confined to the nucleus, as assessed by indirect immunofluorescence. Within 10 min after serum stimulation, a portion of the CT fluorescence appeared in the cytoplasm, where it intensified until approximately 4 h postserum. Thereafter, the cytoplasmic CT signal waned, while the nuclear signal increased, and by 8 h CT was once again predominantly nuclear. The dynamics of CT's apparent translocation in and out of the nucleus paralleled the wave of PC synthesis and the solubility changes of CT. Cytoplasmic CT co-localized with BiP, a resident endoplasmic reticulum protein, in a double labeling experiment. These data suggest that the wave of PC synthesis that accompanies the G(0) --> G(1) transition is regulated by the coordinated changes in CT activity, membrane affinity, and intracellular distribution. We describe for the first time a redistribution of CT from the nucleus to the ER that correlates with an activation of the enzyme. We propose that this movement is required for the stimulation of PC synthesis during entry into the cell cycle.
Biological Transport↗
Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells.
Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.
Animals↗
Harmonic phase-dispersion microscope with a Mach-Zehnder interferometer.
Harmonic phase-dispersion microscopy (PDM) is a new imaging technique in which contrast is provided by differences in refractive index at two harmonically related wavelengths. We report a new configuration of the harmonic phase-dispersion microscope in a Mach-Zehnder geometry as an instrument for imaging biological samples. Several improvements on the earlier design are demonstrated, including a single-pass configuration and acousto-optic modulators for generating the heterodyne signals without mechanical arm scanning. We demonstrate quantitative phase-dispersion images of test structures and biological samples.
Equipment Design↗
Fine structure of early human embryos frozen with 1,2 propanediol.
Previous studies by a French group (Fertil Steril 44:645-651, 1985) have shown that two- to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268-272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.
Blastomeres↗
An evaluation of the precision and accuracy of the direct transfer method for the analysis of asbestos fibers with comparison to the NIOSH method.
Transmission electron microscopy (TEM) of asbestos on polycarbonate membrane filters prepared using the direct transfer (DT) method is used as a reference for determining the precision and accuracy of phase contrast microscopy (PCM) of the same filters and for determining the correlation between DT-PCM and the NIOSH method of asbestos analysis. Replicate aliquots of an aqueous suspension of chrysotile were filtered through either polycarbonate or cellulosic membrane filters. The polycarbonate filters were prepared using the DT method and analyzed using TEM and PCM; the cellulosic filters were treated and analyzed using the NIOSH method. Compared to the results of the TEM examination of the polycarbonate filters, about 50% of the fibers longer than 5 micron were detected using DT-PCM, which was more than 3 times the result obtained using the NIOSH method. The improvement in accuracy using DT-PCM is attributed to the increased visibility of thin fibers (diameters less than 0.1 micron). The largest variation in results was found to be due to the fiber distribution on the filters. This factor may be eliminated from interlaboratory comparisons, etc. by the use of pre-designated and well-defined fields-of-view.
Asbestos↗
Comparison of the results of asbestos fibre dust counts in lung tissue obtained by analytical electron microscopy and light microscopy.
The efficiency of the light microscope with that of the electron microscope in detecting asbestos fibres in human lung tissue was computed. Necropsy material from 55 patients who had died from asbestos related diseases was analysed independently by phase contrast microscopy and electron microscopy. As expected the number of fibres identified using electron microscopy was higher than that identified by light microscopy. By adjusting the electron allow for the limited resolving power of the light microscope, however, a significant correlation of the number of fibres identified using the two methods was obtained. The best correlation was found with specimens containing crocidolite (correlation coefficient 0.79) and amosite (correlation coefficient 0.74), while chrysotile gave a much lower correlation (correlation coefficient 0.15). The cumulated fibre diameter distribution obtained using the electron microscope suggests that the light microscope is able to visualise only 5% of crocidolite, 26.5% of amosite, and 0.14% of chrysotile present in lung tissue. Therefore, although it is possible, using the electron microscope, to predict the asbestos fibre count that would be obtained by light microscopy, the reserve prediction cannot be made: it is impossible to determine the proportion of the various asbestos mineral types using the light microscope.
Asbestos↗
Improved spatial and temporal resolution in an apparatus for time-lapse phase contrast ciné micrography of cells in vitro.
An apparatus for time-lapse cinemicrography of living animal cells in vitro is described. It has an increased information capacity in comparison with conventional types of equipment in that successful combination of the highest possible spatial resolution with phase contrast microscopy and an improved temporal resolution provided by flash light illumination has been achieved. The interval between exposures of 1/200 s approximately can be reduced to 1/4s using a negative phase-contrast objective of NA 1 . 3. Negative phase-contrast also appeared to be the best technique for imaging of tiny cell surface structures. Thus the new apparatus is suitable for the study of the patterns of cell surface motility in vitro.
Animals↗
Dome formation by oral epithelia in vitro.
Multicellular, cystic structures, termed domes, have been described previously in epithelia cultured from various tissues that have a known transport or secretory function in vivo and in vitro. We report for the first time dome formation in cells cultured from "covering" and "rest" epithelia of oral tissues: porcine gingival and alveolar mucosa epithelium and epithelial rests of Malassez. As demonstrated by light- and electron microscopy, the morphology of the domes varied with the location of their lumen and the number of cells or cell layers involved in their structure. Sequential observations using phase contrast microscopy and time lapse cinematography of living cultures showed that the domes were dynamic structures with expansion-collapse cycles of between 30 min and 17 h duration. Dome formation in oral epithelia was stimulated by dibutyryl cyclic AMP (dbcAMP, 10(-3) to 10(-6) M) and abolished by ouabain (10(-10) M), an inhibitor of sodium transport. The morphological features and the dynamic nature of domes found in oral epithelia, and their dbcAMP and ouabain responsiveness are similar to those demonstrated previously in several other epithelia that have a known transport function in vivo and in vitro. Such fluid transport is not thought to be a property of oral epithelia in vivo. Our data, however, suggest a similar function of these epithelia cultured in vitro, and perhaps in pathological cyst formation in vivo.
Animals↗
Diagnosis of glomerular haematuria: role of dysmorphic red cell, G1 cell and bright-field microscopy.
Differentiation between glomerular and non-glomerular haematuria by observation of the changes in red cell morphology using phase-contrast microscopy is a well established technique. However, the method is not widely accepted in clinical practice because of controversy regarding the minimum percentage of dysmorphic red cells required to diagnose glomerular aetiology, as well as the need for specialized microscopes. Recently, a glomerular-specific morphological alteration of red cells has been described, which has the form of a doughnut shape with one or more blebs and which is termed the "G1" cell. In the present double-blind prospective study 250 urine samples were examined without any knowledge of diagnosis. Haematuria was detected in 122 cases. The type of haematuria was characterized by counting dysmorphic cells and G1 cells separately, in each case using a phase-contrast microscope as well as an ordinary bright-field microscope with and without staining of urinary sediments. The results were later correlated with the confirmed diagnosis. The study showed that the G1 cell is more specific than the dysmorphic cell for the diagnosis of glomerular haematuria. Evaluation of both dysmorphic red cells and G1 cells can be done using bright-field microscopy with 100% specificity and sensitivities of 82 and 100%, respectively. It has been concluded that the ordinary bright-field microscope can be used for the diagnosis of glomerular haematuria with an efficiency similar to that of a phase-contrast microscope.
Adolescent↗
Real time scanning force microscopy observation of a structural phase transition in self-assembled alkanethiols.
We present here a new approach based on high resolution scanning force microscopy that permits molecular determination and frictional discrimination between coexisting molecular configurations of alkanethiols on Au(111). The method has allowed visualizing in real time a phase transition between two differently ordered self-assembled configurations, which spontaneously takes place with time at constant coverage and under ambient conditions. Frictional force measurements indicate that the molecular rearrangement observed occurs via an order-disorder-order process.
Alkanes↗
Morphology of renal parenchymal culture in collagen gels.
Explants of rabbit renal parenchyma have been grown in primary tissue culture suspended within hydrated collagen gels. Light and phase contrast microscopic analysis of the first 17 days in culture is described. Pieces of NZW rabbit renal parenchyma were suspended in collagen gels and bathed in supplemented RPMI 1640 medium and incubated at 37 degrees C in 5% CO2 in air. Tubules demonstrated a fine granularity by phase contrast microscopy and glomeruli appeared as red spheres. Blebs formed at the sides and ends of the explant and a monolayer outgrowth of tightly packed polygonal cells occurred from day 4. Histologically an immediate phase of necrosis was followed by regeneration whereby tubules became lined with a confluent epithelium composed of a single layer of flat to cuboidal-shaped cells sitting on an intact tubular basement membrane (TBM). Intraluminal casts of organized cellular debris as well as material presumed to be Tamm Horsfall protein were present. Glomeruli demonstrated collapsed capillary loops. The interstitium became widened by eosinophilic material. The tissue surface contained epithelial cells arranged in places into sac-like structures enclosing a space.
Animals↗
Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.
The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.
Blotting, Western↗
Maturation of equine epididymal spermatozoa.
Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothreitol (DTT), or SDS+DDT was evaluated by phase-contrast and electron microscopy. The percentage of eosinophilic spermatozoa did not differ among regions of the epididymis and was not increased by cold shock. Cooling ejaculated spermatozoa to 0 C in 2 minutes increased (P < 0.01) the occurrence of eosinophilia (32% vs 73%). Spermatozoa released from the caput or proximal corpus epididymidis into 0.154 M NaCl were not progressively motile; only 4% of the spermatozoa from the distal corpus were motile Cauda epididymal and ejaculated spermatozoa exhibited similar motility (41% vs 47%). Stability of chromatin was greater in spermatozoa from the distal corpus than those from the caput epididymis. Chromatin of spermatozoa from the distal corpus was resistant to 7.5 minutes of SDS+DTT treatment, whereas virtually all spermatozoal nuclei from the caput were decondensed by SDS alone. Tail organelles of spermatozoa acquired stability between the proximal corpus and the cauda epididymidis. All tail organelles of spermatozoa from the proximal corpus were dissolved by 7.5 minutes of SDS treatment, whereas tail organelles of distal corpus epididymal spermatozoa had dissolved after 7.5 minutes of SDS+DTT treatment. Stability of tail organelles in cauda epididymal and ejaculated spermatozoa was similar. Seemingly, equine spermatozoa are infertile until they enter the cauda epididymidis.
Animals↗