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Optical phase contrast measurement of ultrasonic fields.

This report describes an optical phase contrast imaging technique for the measurement of wide bandwidth ultrasound fields in water. In this method, a collimated optical wavefront (lambda(l) = 810 nm) impinges on a wide bandwidth ultrasound pulse. The method requires that refractive index perturbations induced by the ultrasound field be sufficiently small. Specifically, on exit from the acoustic field, the phase of the optical wavefront must be proportional to the ray sum of local density taken in the direction of propagation of the incident optical wave. A similar restriction is placed on the dimensions of the ultrasound pulse. Repeated measurement of this phase as the ultrasound field is rotated through 180 degrees about an axis normal to the direction of propagation of the incident optical wave generates the Radon transform of the ultrasonically induced refractive index perturbation. Standard tomographic reconstruction techniques are used to reconstruct the full three-dimensional refractive index perturbation. A simple two-lens imaging system and an optical signal processing element from phase contrast microscopy provide a method of directly measuring an affine function of the desired optical phase for small optical phase shifts. The piezo- and elasto-optic coefficients (the first partial derivatives of refractive index with respect to density and pressure) relate refractive index to density and pressure via a linear model. The optical measurement method described in this paper provides a direct, quantitative measurement of the piezo- and elasto-optic coefficients (from the density or pressure fields).

Journal Article↗

Spindle microtubules: thermodynamics of in vivo assembly and role in chromosome movement.

In this paper I have presented results of experiments in which spindle microtubules were depolymerized by hydrostatic pressure, in order to examine the Inoué dynamic equilibrium concept of spindle assembly and the possible role of microtubule depolymerization-polymerization in the movement of chromosomes. Using a newly developed optical hydrostatic pressure chamber, I investigated with polarization microscopy the quantitative effects of pressure on the polymerization of spindle microtubules and, with phase contrast microscopy, the relationship of pressure-induced spindle microtubule depolymerization to chromosome movement in living cells. From results of earlier experiments, principally those of Inoué et al. with low temperature and colchicine as microtubule-depolymerizing agents, and from results of my own research, I have concluded that: (1) spindle fiber microtubules are sensitive to depolymerization by pressure (3000-7000 psi), spindle microtubules do exist in a labile equilibrium with a pool of subunits, and the Inoué simple equilibrium model does predict changes in spindle microtubule assembly at metaphase induced by pressure; (2) the stability of microtubules depends on the number of "attached ends;" (3) the longest interpolar microtubules and the longest chromosomal fiber microtubules regulate the spindle interpolar length and the chromosome-to-pole positions; (4) chromosome velocity is independent of the number of spindle microtubules, as well as of the drag force of the chromosomes; (5) the chromosomal fiber microtubules transmit the forces between the poles and between the chromosomes and the poles; and (6) polymerization of microtubules does produce pushing forces and, if controlled microtubule depolymerization does not actually produce pulling forces, at least it governs the velocity of chromosome-to-pole movement.

Animals↗

Regional influences of the fallopian tubes on the rate of boar sperm capacitation in surgically inseminated gilts.

Aliquots of ejaculated boar semen containing known numbers of spermatozoa were deposited into the caudal isthmus or rostral ampulla of the Fallopian tubes of gilts at, or immediately after, ovulation to assess regional influences on the rate of capacitation. Eggs were recovered during a second intervention 4, 5, 6 or 7 h after surgical insemination and were examined by phase-contrast microscopy. Results were obtained from ten animals in each of the 4-, 5- and 6-h groups and from eight animals in the 7-h group. With two exceptions, fertilized eggs were not recovered until 6 h after insemination into the isthmus, the proportion (45.6%) being significantly greater than the corresponding figure (1.4%) for ampullary insemination (P < 0.001). Similarly, the proportion of fertilized eggs recovered 7 h after insemination into the isthmus (58.7%) was significantly greater than after ampullary insemination (21.9%; P < 0.01). Numbers of spermatozoa associated with the zona pellucida remained low in all these instances, with mean figures per egg ranging from 0.3 to 3.8. Insemination into the isthmus gave a 1-2 h advantage in fertilization compared with insemination into the ampulla. Although relative rates of sperm cell progression to the site of fertilization may have contributed to this, there is strong evidence that rates of capacitation differ significantly in the respective portions of the Fallopian tube. Therefore, attention was focused on: (1) the viscous glycoprotein secretion in the caudal isthmus acting to remove seminal plasma from the sperm surface; and (2) the phase of sperm head binding to the isthmus epithelium. Gradients in local endocrine modulation by the adjacent ovary offer one explanation for the functional specialization of different regions of the Fallopian tubes.

Animals↗

[Corpuscular pollutants: the problem of particles and fibers (optic microscopy)].

The "Clinica del Lavoro" had already begun work on dust pollution in the working environment in the mid-1930's but was only able to study the problem more in depth at the beginning of the 1950's when the Industrial Hygiene Laboratory was established under the fortunate direction of Professor Nicola Zurlo. The first research studies in that period were aimed at the correct choice of the most suitable sampling equipment and analytical methods for assessment of the extent of such types of pollution. The results achieved in these initial studies, the experience acquired in the field and further, more detailed research enabled Professor Zurlo to develop and perfect new types of sampling instruments that were more appropriate to the goals it was desired to achieve and these instruments are still widely used to-day. The paper also considers the evolution of analytical methods using the optical microscope for determination of the concentration of particles and fibres, most of which were developed and perfected in our Laboratory, up to the present time. It is stressed that phase contrast microscopy is still indispensable for dust analysis and still offers ample scope for research in the field of artificial fibres that have recently been put on the market, even though the electron microscope is now the only acceptable solution for research in living environments.

Air Pollutants↗

Observations on the musculature and isolated muscle fibres of the liver fluke, Fasciola hepatica.

The liver fluke, Fasciola hepatica relies on a well-developed muscular system, not only for attachment, but for many aspects of its biology. Despite this, little is known about the system beyond the gross organization of the main somatic muscle layers. In the present study, a range of techniques have been applied to F. hepatica in order to understand more about various aspects of muscle organization, biochemistry (in terms of muscle proteins) and identity of isolated muscle fibres. Scanning electron microscopy has provided a direct visualization in situ of the somatic muscle layers and the organization of the muscle fibres within the ventral sucker. The muscle bundles contributing to the main somatic muscle layers are made up of up to 10 individual muscle fibres. Phalloidin staining for actin, in conjunction with confocal microscopy, confirmed the presence of 2 main somatic muscle layers (outer circular, inner longitudinal), beneath which lies a third layer of oblique muscle fibres. The use of propidium iodide in combination with phalloidin staining for actin demonstrated that the cell bodies associated with the 2 main somatic muscle layers are situated beneath the longitudinal muscle layer and are connected to their respective muscle fibres by short cytoplasmic processes. Myosin immunoreactivity was demonstrated in the somatic muscle layers and in the muscle layers surrounding various organ systems within the fluke. Double labelling for actin and myosin confirmed the co-localization of the 2 muscle proteins in the muscle fibres of the ventral sucker. Muscle fibres from the somatic muscle layers and the ventral sucker have been isolated and images obtained with phase-contrast microscopy and scanning electron microscopy. The muscle fibres contain actin and myosin, but lack a nucleus, the connection with the cell body having been broken during the isolation procedure.

Actins↗

Parameter-optimized digital holographic microscope for high-resolution living-cell analysis.

A parameter-optimized off-axis setup for digital holographic microscopy is presented for simultaneous, high-resolution, full-field quantitative amplitude and quantitative phase-contrast microscopy and the detection of changes in optical path length in transparent objects, such as undyed living cells. Numerical reconstruction with the described nondiffractive reconstruction method, which suppresses the zero order and the twin image, requires a mathematical model of the phase-difference distribution between the object wave and the reference wave in the hologram plane. Therefore an automated algorithm is explained that determines the parameters of the mathematical model by carrying out the discrete Fresnel transform. Furthermore the relationship between the axial position of the object and the reconstruction distance, which is required for optimization of the lateral resolution of the holographic images, is derived. The lateral and the axial resolutions of the system are discussed and quantified by application to technical objects and to living cells.

Algorithms↗

Isolation, characterization, and mapping of Bacillus subtilis 168 germination mutants.

After mutagenesis with nitrosoguanidine, germination mutants of Bacillus subtilis 168 were selected by killing, with heat, spores that germinated at 42 C and collecting survivors at 30 C. The germination properties of nine mutants variously affected in amino acid biosynthesis and sugar utilization were studied in detail. They were divided into two groups: (i) Ger-ALA mutants, failed to germinate in 10 mM L-alanine but germinated in complex media (some of these mutants were temperature sensitive); (ii) Ger-PAB mutants, germinated poorly, even in complex media, suggesting that they were blocked in important germination functions. All the mutants failed to germinate in L-alpha-amino-n-butyrate or L-valine (including temperature-sensitive mutants only at the restrictive temperature) showing that there is a step necessary for germination affected by all three acids. The mutants had normal growth rates, indicating that the defective gene products were specific for germination functions. These defects were not identified. Eight of the mutants were mapped by transduction with phage PBS-1. The recombinants were scored either by observations, by microscopy of phase darkening of the spores, or by a plate test involving the reduction of tetrazolium by heated colonies of spores. Five of the mutations, of at least three phenotypes, were between thr-5 and cysB3 away from all the sporulation markers that have been previously mapped. A linked ald (alanine dehydrogenase) locus was on the other side of thr-5. The other Ger markers were located in at least two additional positions. Auxotrophic strains that were used for mapping germinated normally, but germination of the Ger mutants differed slightly in different genetic backgrounds.

Bacillus subtilis↗

Digital holographic microscopy with dual-wavelength phase unwrapping.

We apply the techniques of digital holography to obtain microscopic three-dimensional images of biological cells. The optical system is capable of microscopic holography with diffraction-limited resolution by projecting a magnified image of a microscopic hologram plane onto a CCD plane. Two-wavelength phase-imaging digital holography is applied to produce unwrapped phase images of biological cells. The method of three-wavelength phase imaging is proposed to extend the axial range and reduce the effect of phase noise. These results demonstrate the effectiveness of digital holography in high-resolution biological microscopy.

Algorithms↗

Structural conformation of in vitro and in vivo aged orthodontic elastomeric modules.

The objectives of this study were to investigate the structural characteristics of open and closed elastomeric modules in the as-received condition, and following (a) 24-hour in vitro loading determined as a 50 per cent extension relative to their original length; (b) 24-hour intra-oral exposure; (c) 3-week intra-oral exposure. Ten specimens of each type (open and closed) of the three brands of elastic chains were included in the study (total of 60). All specimens were subjected to (a) optical transmission microscopy utilizing phase contrast and polarized light modes, (b) micro MIR FT-IR spectroscopy, and (c) scanning electron microscopy and electron probe X-ray microanalysis. Stretched elastomers demonstrated a honeycomb pattern of filament detachment corresponding to strained areas. The chains possessing a well-differentiated inter-modular link presented higher strain concentration gradients located in the link. Phase contrast imaging revealed that the stress adsorption mechanism of these materials involved macromolecular chain orientation and elongation co-axially to the applied load. The specimens retrieved after 24 hours had acquired a proteinaceous film rich in alcohol groups with minimal evidence of phosphate mineralization, while the 3-week retrieved specimens presented precipitation of Ca- and P-forming calcium phosphates. The high protein content of the biofilm organized on the surface of these materials, as well as the calcification pattern found, were similar to a non-specific mechanism of film adsorption of biomaterials exposed to body fluids. The results have clinical implications for aspects of retraction control through sliding mechanics with the use of elastomeric ligatures, and the potential detrimental effects on dental and periodontal tissues such as decalcification and gingival inflammation, respectively. In addition, the experimental protocols adopted for the in vitro study of the force decay of elastomerics should be modified to include parameters pertinent to adsorption phenomena and calcification processes documented to occur in vivo.

Biofilms↗

Autonomic receptor characterization in cultured adult rat cardiac myocytes: morphologic-functional correlation.

Adult calcium tolerant rat ventricular myocytes were maintained under serum-free culture conditions for five days. beta-adrenergic and muscarinic cholinergic receptor expression was assessed by radioligand binding determinations using 125I-iodocyanopindolol (ICYP) and [3H]-quinuclidinyl benzilate (QNB), respectively. The binding data were correlated with myocyte structural integrity and contractile responsiveness to norepinephrine (NE). During the 5 days in primary culture, beta-adrenergic and muscarinic cholinergic receptor binding capacity diminished Bmax = 17.1 to 9.2 fmol/mg protein and Bmax = 169.0 to 26.6 fmol/mg protein, respectively. The affinity of both autonomic receptors was unaltered during the period of observation. The majority of isolated myocytes were viable (65 to 85%) and remained rod-shaped for 5 days as assessed by phase contrast microscopy. Up to 2 days in vitro the rod-shaped myocytes appeared ultrastructurally similar to their in vivo counterparts and displayed intact nuclei and the usual complement of cellular organelles. From day 3, phase contrast as well as transmission electron microscopy revealed a progressive increase in autophagic vacuoles consisting primarily of disrupted mitochondria. The number of myocytes that contracted in response to norepinephrine (NE) decreased from 57.2 to 2.3% by day 5. These data indicate that adult rat cardiac myocytes maintained in serum free culture for 5 days, express beta-adrenergic and muscarinic cholinergic receptors. There is a rapid decline (50%) in muscarinic cholinergic receptor number and contractile response to NE by day 2. However, the decrease in beta-receptor Bmax by day two is insufficient to explain the severe loss of cell responsiveness to NE. This functional loss may be related, at least in part, to the ultrastructural abnormalities that are first evident at day 2 in culture. Thus, short-term myocyte cultures that retain phenotypic and physiologic characteristics of in vivo cardiac myocytes could provide a useful in vitro system for exploring pharmacologic-functional interactions in the myocardium.

Animals↗

SV-40 virus-induced neoplastic transformation of hamster brain cells in vitro. Studies with scanning and transmission electron microscopy.

Neoplastic transformation of one-day-old hamster brain cells was produced by infection with SV-40 virus and verified by phase contrast microscopy, growth in semisolid media, and intracranial tumor production after inoculation of the cells into other one-day-old hamsters. Transformed cells were studied by transmission and scanning electron microscopy. The numerous alterations in cell surface structure and in nuclear and cytoplasmic organization suggest a marked increase in cell metabolism and in the rate of mitosis and cell division. Cilia with a nine-to-zero pattern of microtubule doublets were present in cells with intermediate size filaments which stained for glial fibrillary acidic protein. The findings indicate that infection of one-day-old hamster brain cells in culture by SV-40 virus results in their transformation to a neoplastic state and the transformed cells are differentiating neoplastic astrocytes.

Animals↗

Comparison of phase-contrast and fluorescence digital autofocus for scanning microscopy.

Reliable autofocus is required to obtain accurate measurements of fluorescent stained cellular components from a system capable of scanning multiple microscope fields. Autofocus could be performed directly with fluorescence images, but due to photobleaching and destructive fluorescence by-products, it is best to minimize fluorescence exposure for photosensitive specimens and live cells. This exposure problem could be completely avoided by using phase-contrast microscopy, implemented through the same optics as fluorescence microscopy. The purpose of this work was to evaluate functions for both phase-contrast and fluorescence autofocus and determine the suitability of phase-contrast autofocus for fluorescence microscopy. Eleven autofocus functions were independently evaluated for fluorescence and phase-contrast microscopy. The most suitable functions were then chosen from these and phase-contrast and fluorescence autofocus were compared on scans each comprising more than 1,000 microscope fields. Autofocus standard deviation (S.D.) of better than 100 nm was achieved for both phase contrast and fluorescence. There was a measurable difference between the best focus positions in the two modes, but the difference was constant enough to be measured and corrected, suggesting the possibility of using phase contrast to predict best focus in fluorescence microscopy. The scanning experiments also showed that autofocus can be performed at least as fast as 0.25 s/field without loss of precision.

3T3 Cells↗

Changes in network topology during the replication of kinetoplast DNA.

Kinetoplast DNA of Crithidia fasciculata is a network containing several thousand topologically interlocked DNA minicircles. In the prereplicative Form I network, each of the 5000 minicircles is intact and linked to an average of three neighbors (i.e. the minicircle valence is 3). Replication involves the release of minicircles from the interior of the network, the synthesis of nicked or gapped progeny minicircles and the attachment of the progeny to the network periphery. The ultimate result is a Form II network of 10,000 nicked or gapped minicircles. Our measurements of minicircle valence and density, and the network's surface area, revealed striking changes in network topology during replication. During the S phase, the peripheral newly replicated minicircles have a density twice that of minicircles in Form I networks, which suggests that the valence might be as high as 6. Most of the holes in the central region that occur from the removal of intact minicircles are repaired so that the central density and valence remain the same, as in prereplicative networks. When minicircle replication is complete at the end of the S phase, the isolated network has the surface area of a prereplicative network, despite having twice the number of minicircles. During the G2 phase, the Form II network undergoes a remodeling in which the area doubles and the valence is reduced to 3. Finally, the interruptions in the minicircles are repaired and the double-sized network splits in two.

Animals↗

Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells.

Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and necrotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo.

Antigen Presentation↗

Extracellular matrix regulates apoptosis in human neutrophils.

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.

Apoptosis↗

[Quartz crystals in the skin as a cause of progressive systemic scleroderma].

There is a close correlation between the systemic sclerosis and the exposure to crystalline silica in the GDR. The silica content was investigated in the skin of the patients. Silica particles were found using phase-contrast and polarizing microscopy. The size of the crystals varied from 1 to 90 micrometer. Below a silica content of 10% in the dust it is unlikely that systemic sclerosis is developing.

Biopsy↗