Interference Contrast Technique Research Articles

Calibration of optical tweezers with differential interference contrast signals

A comparison of different calibration methods for optical tweezers with the differential interference contrast (DIC) technique was performed to establish the uses and the advantages of each method. A detailed experimental and theoretical analysis of each method was performed with emphasis on the anisotropy involved in the DIC technique and the noise components in the detection. Finally, a time of flight method that permits the reconstruction of the optical potential well was demonstrated.

Quantitative optical phase microscopy

We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.

Surface and Interfacial Roughness of Polished and Oxidized Silicon Wafers Evaluated by an Infrared Differential Interference Contrast Technique

An infrared differential interference contrast method using a YLF laser (1.3 μm) was used to evaluate the surface roughness of polished silicon wafers and wafers oxidized at 700, 900, and 1000°C. For polished wafers, the root‐mean‐square optical phase shifts (Δϕrms) from this method exhibit a nonlinear relation with the average roughness, Ra, obtained by a conventional laser interferometry technique. The surface roughness of the thermally oxidized wafers is found to have a nonmonotonic trend that evolves with time. This trend is also dependent on the oxidation temperature.

Reporter genes and fluorescent probes for studying the colonisation of biofilms in a drinking water supply line by enteric bacteria

Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system. Escherichia coli, taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene. The resulting transformant, PRB1, was introduced into the chemostat. After 7 and 13 days, an E. coli strain with an anaerobically induced Lac + phenotype was present in the biofilm. Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E. coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of β-galactosidase from pnirB.

Reporter genes and fluorescent probes for studying the colonisation of biofilms in a drinking water supply line by enteric bacteria.

Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system. Escherichia coli, taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene. The resulting transformant, PRB1, was introduced into the chemostat. After 7 and 13 days, an E. coli strain with an anaerobically induced Lac+ phenotype was present in the biofilm. Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E. coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of beta-galactosidase from pnirB.

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using H Ep-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the H Ep-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100°C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of H Ep-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to H Ep-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.

Phänoembryoiogie der Hochgebirgspflanzen Saxifraga oppositifolia und Cerastium uniflorum

Summary The morphological and temporal course of embryogenesis and seed development of Saxifraga oppositifolia and Cerastium uniflorum during the entire growing season were studied in their natural high-mountain habitat and were related to the sequence of rcproductive phenology. At short intervals, buds, flowers and fruits were collected from the same individual. Thc cleared ovules and immature seeds were examined and measured microscopically using the interference contrast technique. S. oppositifolia blooms as soon as the snow has melted. Immediately before the flowers open, the ovules are in the post-meiotic state and are not ready for fertilization, until the flowcrs are fully open. After fertilization there is a delay of 2 — 3 weeks before development of the helobial endosperm begins. The embryogeny is of the Caryophyllad type. In all, the differentiation and ripening of the seeds take a total of 11 weeks. C. uniflorum flowers in summer on the new shoots approximately a month after the snow has melted. The campylotropic ovules are already prepared for fertilization when the flowers open, and continue their development without delay after fertilization. Embryogenesis proceeds according to the Caryophyllad pattern. The storage tissue is a perisperm, the endosperm being restricted to a single layer surrounding the embryo. After 5-6 weeks the embryonic organs are developed; growth of the embryo continues for a further 2 wecks. The specific reproduction period, i.e. the time elapsing between fertilization and [mal differentiation of the seeds, amounts to 70-80 days in S. oppositifolia and about 40 days in C. uniflorum. S. oppositifolia can come into flower very early because the flower buds are almost completely differentiated in the previous year; in the spring, the plant's rich stores oflipids are available for flowering and formation of new shoots. Fruit sct coincides with expansion of the shoots and roots. In C. uniflorum , however, the assimilatory organs have first to develop before they can deliver the energy needed for flowering and the rapid development of seeds. If in C. uniflorum the vegetative phase before onset of the reproductive period is included, both species require 10-11 weeks of favourable weather for their annual seed production.

Immunogold and fluorescein immunolabelling of Legionella pneumophila within an aquatic biofilm visualized by using episcopic differential interference contrast microscopy.

Biofilms containing diverse microflora were developed in tap water on glass and polybutylene surfaces. Legionella pneumophila within the biofilms was labelled with monoclonal antibodies and visualized with immunogold or fluorescein isothiocyanate conjugates. Development of a differential interference contrast technique in an episcopic mode enabled simultaneous visualization of the total biofilm flora and gold-labelled legionellae. The legionellae occurred in microcolonies within the biofilm in the absence of amoebae, suggesting that the bacterial consortium was supplying sufficient nutrients to enable legionellae to grow extracellularly within the biofilm.

Influence of the deposition gas mixture on the structure and failure modes of diamondlike carbon films

Diamondlike carbon films have been deposited from binary mixtures of 1,3 butadiene and hydrogen or argon in a radio-frequency parallel plate plasma reactor. An infrared spectroscopic analysis revealed some of the effects of the diluents (hydrogen and argon) on the bonding characteristics of the deposited films. The film properties are a strong function of the deposition gas mixture. Hydrogen and argon both increase the diamondlike behavior. However, there is a trade-off between enhanced diamondlike characteristics and a tendency to delaminate or crack due to excessive stress and poor adhesion. The failure modes and stress relief forms have been imaged by the Nomarski interference contrast technique. Two new stress relief forms have been observed.

The composition of metal phases in Bruderheim (L6) and implications for the thermal histories of ordinary chondrites —Erratum

The compositions and textures of metal phases in Bruderheim have been examined in detail using Nomarski differential interference contrast techniques, backscattered electron imaging and quantitative electron microprobe analysis for Fe, Ni and Co. The results clearly demonstrate substantial inter- and intra-grain compositional variations, and that these persisted through the high-temperature thermal event which largely homogenised the silicate phases forming the bulk of the meteorite. These results are difficult to reconcile with prograde thermal metamorphism. Instead, accretion at high temperatures is envisaged with homogenisation of silicates occurring at these temperatures. Relatively sparse metal grains were largely unaffected at these temperatures because they did not form three-dimensional networks (and hence lattice diffusion of metallic Fe and Ni was ineffective), and because, at the temperature of accretion, most grains were present as homogeneous particles of the γ phase. The complexities now apparent in metal grains were established first during the decline of temperature and retrograde metamorphism after accretion and then later during relatively low-temperature burial metamorphism in asteroidal bodies. The reliability and significance of cooling rate calculations based on measurements of Ni concentrations in metal grains of such meteorites are questioned.

The composition of metal phases in Bruderheim (L6) and implications for the thermal histories of ordinary chondrites

The compositions and textures of metal phases in Bruderheim have been examined in detail using Nomarski differential interference contrast techniques, backscattered electron imaging and quantitative electron microprobe analysis for Fe, Ni and Co. The results clearly demonstrate substantial inter- and intra-grain compositional variations, and that these persisted through the high-temperature thermal event which largely homogenised the silicate phases forming the bulk of the meteorite. These results are difficult to reconcile with prograde thermal metamorphism. Instead, accretion at high temperatures is envisaged with homogenisation of silicates occurring at these temperatures. Relatively sparse metal grains were largely unaffected at these temperatures because they did not form three-dimensional networks (and hence lattice diffusion of metallic Fe and Ni was ineffective), and because, at the temperature of accretion, most grains were present as homogeneous particles of the phase. The complexities now apparent in metal grains were established first during the decline of temperature and retrograde metamorphism after accretion and then later during relatively low-temperature burial metamorphism in asteroidal bodies. The reliability and significance of cooling rate calculations based on measurements of Ni concentrations in metal grains of such meteorites are questioned.

Landmarks in biological light microscopy

SUMMARYThe pioneering seventeenth‐century work of Hooke (using the compound microscope) and Leeuwenhoek (using the simple microscope) was followed in the eighteenth century by a period of comparative stagnation until the early nineteenth‐century work of Lister (who evolved the principle of aplanatic focal points in lens design). The founding of the Microscopical Society of London in 1839 reflected the increasing importance of the microscope as a scientific instrument and its involvement in specimen preparation techniques, cell theory and bacteriology is described.The pursuit of increased resolving power—in particular the development of apochromatic lenses by Abbe and Zeiss and enhanced contrast techniques—polarized fight, u.v. microscopy, u.v. absorption and fluorescence, phase contrast and the differential interference contrast techniques, etc., are discussed. The story of microscopy in the twentieth century is one of interlocking discoveries and advances and the techniques of epi‐illumination in scanning fluorescence microscopy, video acquisition and analyses of images in digital form and confocal scanning microscopy are surveyed. It is concluded that these techniques have opened up whole new areas for the future development of light microscopy.

Diazepam induces abnormal mitosis in the early Drosophila embryo

Drosophila embryos, because of their high proportion of dividing nuclei, offer many advantages for the study of the mitotic cycle. In the present study we combined immunofluorescence with interference contrast techniques to follow centrosome and spindle behavior in embryos exposed to diazepam during the first stages of development. Exposure to 100 μg/ml of diazepam produced polyploid and aneuploid figures resulting from the unusual fusion of one or more adjacent spindles. Diazepam also causes the inhibition of centrosome shifting and induces the formation of monopolar spindles during the metaphase-anaphase transition.

Diazepam induces abnormal mitosis in the early Drosophila embryo

Drosophila embryos, because of their high proportion of dividing nuclei, offer many advantages for the study of the mitotic cycle. In the present study we combined immunofluorescence with interference contrast techniques to follow centrosome and spindle behavior in embryos exposed to diazepam during the first stages of development. Exposure to 100 micrograms/ml of diazepam produced polyploid and aneuploid figures resulting from the unusual fusion of one or more adjacent spindles. Diazepam also causes the inhibition of centrosome shifting and induces the formation of monopolar spindles during the metaphase-anaphase transition.

Observation of Bloch wall fine structures on iron whiskers by a high-resolution interference contrast technique

The use of interference contrast equipment for Bitter pattern studies on iron whiskers results in a significant improvement in the resolution power and contrast transference of the optical microscope. Details of domain wall topology and internal wall structure are revealed. A discussion of the technique and some experimental results are presented.

A combined study of the dynamical properties of dislocations in molybdenum single crystals by etch pitting and interference contrast techniques

Special features of the motion and multiplication of dislocations in Mo single crystals at 300 K are investigated comparitively by etch pitting of dislocations and by observing the surface slip steps in the Nomarski differential interference contrast. It is shown that an existing surface film has a decisive influence on the dynamical properties of dislocations emerging through the surface. The limits of applicability of the studied methods for performing direct measurements of the dislocation mobility in b.c.c. metals are discussed. Die Bewegung und Vervielfachung von Versetzungen in Mo-Einkristallen bei 300 K werden unter sucht bei vergleichsweiser Anwendung der Versetzungsätztechnik und der Beobachtung von Oberflächengleitstufen im Nomarski-Differential-Interferenz-Kontrast. Es wird gezeigt, daß die Anwesenheit eines Oberflächenfilmes die dynamischen Eigenschaften der Versetzungen, die an der Oberfläche enden, entscheidend beeinflußt. Die Anwendungsgrenzen der benutzten Methoden für die direkte Messung der Versetzungsbeweglichkeit in k.r.z.-Metallen werden diskutiert.

Investigation of bubble stray field effect on the domain wall structure of the overlay bars by interference contrast technique

The effect of bubble stray field on the magnetization distribution of a permalloy overlay bar is photographed by Interference Contrast Technique (ICT) in here and the results are compared to the experimental data available from the magneto-optical Kerr effect measurements. The 180° domain wall deformation and the resultant magnetic domain patterns due to a travelling bubble under an I bar is shown. The magnetization distribution curves along the I bar due to different positions of the bubble under the I bar are plotted and compared qualitatively to the similar curves obtained by continuum model. The magnetic field well under an I bar due to a trapped bubble is also calculated.

Calculation and analysis of the forces on ferro-fluid particles on the surface of a Permalloy overlay bar

The formation of the ferro-fluid particles on the domain walls of a Permalloy overlay bar is due to the forces exerted on each particle by the stray field of the bar. Domain wall observation by Interference Contrast Technique reveals that the ferro-fluid particles do not form at some parts of the 180° domain walls. A further investigation by normal illumination technique has shown in fact that the particles are repelled from these areas as they look brighter than the grayish background. By calculation of the magnetic force from magnetic stress tensor and employing the domain wall model for an overlay bar, it is shown that a magnetization reversal inside the 180° domain wall is responsible for repelling the ferro-fluid particles. Inside the volume of an overlay bar, the forces exerted on the magnetization inside a 90° domain wall is also calculated and it is shown that these walls rotate under the torques applied by these forces until the resultant torque becomes zero. The force lies along the wall at this stage.

Simple methods for observing cortical granules in living mammalian eggs

Simple light microscopic methods for routine observation and checking cortical granules of vital mammalian eggs will be described. By using interference contrast and dark field techniques, even small cortical granules can easily be examined. These methods are time-saving compared to the usual ones using an electron microscope. They make distribution studies possible without difficulties.

A Modified Technique to Study Leaf Epidermis in Triticeae

A rapid modified method is described to study stomata and other epidermal features without elaborate processing. The preparations are semipermanent and permit spatial relationship studies of both leaf surfaces from the same field. The cleared preparations are optically sectioned with brightfield or Nomarski interference contrast techniques without staining for contrast. The technique permits superior tissue resolution, clarity, minimal distortion of sections, and preservation of trichomes. The method is applicable to all species of Triticeae.