Interference Contrast Microscopy Research Articles

Reflection Interference Contrast Microscopy Combined with Scanning Force Microscopy Verifies the Nature of Protein-Ligand Interaction Force Measurements

The integration of a stand-alone scanning force microscope (SFM) scanner with a reflection interference contrast microscope (RICM) makes it possible to measure directly the separation distance between the SFM probe and the sample surface. The SFM-RICM combination, when applied to the force measurements between ligand-derivatized SFM probe and a protein receptor-derivatized surface, showed that the anomalous force discontinuities often observed for such interacting pairs were indeed a real behavior characteristic of a particular experimental configuration. Apart from small discrepancies due to transient damping, commercially available cantilevers did behave in an ideal mechanical fashion, thus indicating that protein-ligand unbinding events were occurring at distances much larger than their maximum extended length. This external verification of separation distance requires a closer examination of the physical events occurring upon detachment of the surfaces. An alternative interpretation of such force measurements is proposed here in which the protein and/or ligand immobilization chemistry is called into question.

Microscopic differential interference contrast image processing by line integration (LID) and deconvolution

Differential interference contrast (DIC) microscopy is the preferred imaging mode for studying live unstained cells and embryos. This is mainly attributed to the optical sectioning capability which makes DIC suitable for three-dimensional microscopy. However, DIC images are not convenient for standard computerized image interpretation. We present here a processing algorithm that converts DIC images into a form which is much more amenable for such analysis. The algorithm is based on computational inversion of the directional gradient performed optically by DIC, namely path integration (line integrated DIC, or LID). LID images relate to the refractive index of the specimen, and are inherently positive in value, thus many powerful algorithms developed for fluorescent images can be applied to them. The method is demonstrated here for identifying and tracking nuclei in developing zebrafish embryos, and for reconstructing the shape of live leukocytes.

Postinfection development of Polymyxa graminis in roots of Triticum aestivum

Differential interference contrast, laser confocal, scanning electron and transmission electron microscopy were used to study developmental anatomy of sporangial and sporogenic plasmodia, zoosporangia, sporosori and resting spores of Polymyxa graminis in roots of Triticum aestivum grown in infested field soil. Anatomical properties of each stage were described, including the transition from wall-less plasmodia to zoosporangial initials, development of mature zoosporangia with multi-lobed compartments separated by septa and accompanied by exit tubes, and the formation of zoospores within zoosporangia, for the zoosporangial stage. Sporogenic plasmodia, which were larger than sporangial plasmodia, underwent rapid, total cleavage to form resting spores aggregated into sporosori of discoid or other flattened shapes. The cell walls of mature resting spores in sporosori contained up to five distinct layers. Numerous electron opaque storage bodies were present in resting spores; many of them migrated to just beneath the plasma membrane and cell wall during spore maturation.

Growth and characterization of Ge 1− xSi x ( x⩽10 at%) single crystals

Abstract GeSi single crystals in the range Si⩽10 at% have been grown on Ge 〈1 1 1〉-seed crystals by the vertical Bridgman method in a radiation heated mirror furnace. The diameter of the crystals was 9 mm, their length about 30–40 mm. Axial and radial macrosegregation have been measured by energy dispersive analysis by X-ray (EDAX), microsegregation was investigated using interference contrast microscopy. The average EPD is in the range 6×10 4 –1×10 5 cm −2 and is enhanced at the seed/crystal interface and in the part grown in the vicinity of the container wall. Measurements with a Bartels five crystal diffractometer of the 〈1 1 1〉 peak resulted in FWHM (full-width at half-maximum) values of 20 to 40 arcsec, compared to the theoretical value of 17 arcsec. The FWHM values of the bound excitons, determined by photoluminescence, are in the range 2.5–4.5 meV, which are the best values reported hitherto, and show a linear enlargement with increasing silicon concentration. The segregation coefficients of Ga, In, P, As, and Sb fit well to a linear interpolation between the Ge and Si values.

Measurement-based evaluation of optical pathlength distributions reconstructed from simulated differential interference contrast images.

Recently a method was presented for reconstructing optical pathlength distributions (OPDs) from images of weak phase objects obtained by a conventional differential interference contrast (DIC) microscope. A potential application of this technique is the determination of the mass of biological objects: by integrating the optical pathlength over the projected surface of the image of an object, a measure of the dry mass, i.e. the total mass of all solid constituents present in the object, is obtained. To assess the possibilities of DIC microscopy for this application, simulations were performed on computer-generated DIC images of objects of various sizes, shapes and orientation angles. After reconstructing the OPDs from these images, the integrated optical pathlength of each of the test objects was determined, and compared with the expected results. The parameter settings used in the reconstruction algorithm were found to be very important in obtaining a reliable measurement. Using optimal parameter settings, errors in the integrated OPD could be limited to a few per cent for circular objects within the investigated size range. For non-circular objects, however, the orientation angle of the object relative to the lateral shift was found to influence the measured values. Ellipses with their long axes perpendicular to the shift direction had a significantly higher integrated OPD than ellipses orientated parallel to the shift. By adjusting the reconstruction parameters the effect could be limited, but complete elimination of the artefact was not possible within the parameter range investigated.

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

PH and Ion-Triggered Volume Response of Anionic Hydrogel Microspheres.

Micrometer-sized (4-7 µm diameter) poly(methacrylic acid) (PMAA) hydrogel microspheres were synthesized by precipitation polymerization. Individual microspheres were held in a micropipet and visualized by interference contrast microscopy. They were characterized with regard to their mass, density, water content, electrophoretic mobility, and apparent pKa. Equilibrium changes in volume were measured as functions of the pH and NaCl concentration of the suspending solution. The maximum reduction in the microsphere equilibrium volume (Vrmax) at pH 3.0 was 0.28, where Vr was the ratio of the microsphere volume at the test pH to its volume at pH 6.6. A Donnan-based thermodynamic model, modified to include counterion binding because of the high fixed charge density in the microspheres (3.0 M), was applied to determine the difference in the ion concentration between the interior and exterior of the gel. The ion concentration differences (which were related to the osmotic pressure) predicted by the model were proportional to the microsphere equilibrium volume with changing pH and salt concentration. This supported the hypothesis that the equilibrium volume of the microspheres was set by a force balance between the osmotic pressure and the elasticity of the hydrogel matrix. Microspheres changed from their maximum equilibrium volume at pH 6.6 to their minimum equilibrium volume at pH 3.0 in 300 ms. This indicated that diffusion of the polymer matrix and not diffusion of ions into and out of the microsphere was the rate-limiting factor in determining a microsphere's swelling rate.

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis.

We have taken advantage of the optical transparency of zebrafish embryos to investigate the patterns of cell division, movement and shape during early stages of development of the central nervous system. The surface-most epiblast cells of gastrula and neurula stage embryos were imaged and analysed using a computer-based, time-lapse acquisition system attached to a differential interference contrast (DIC) microscope. We find that the onset of gastrulation is accompanied by major changes in cell behaviour. Cells collect into a cohesive sheet, apparently losing independent motility and integrating their behaviour to move coherently over the yolk in a direction that is the result of two influences: towards the vegetal pole in the movements of epiboly and towards the dorsal midline in convergent movements that strengthen throughout gastrulation. Coincidentally, the plane of cell division becomes aligned to the surface plane of the embryo and oriented in the anterior-posterior (AP) direction. These behaviours begin at the blastoderm margin and propagate in a gradient towards the animal pole. Later in gastrulation, cells undergo increasingly mediolateral-directed elongation and autonomous convergence movements towards the dorsal midline leading to an enormous extension of the neural axis. Around the equator and along the dorsal midline of the gastrula, persistent AP orientation of divisions suggests that a common mechanism may be involved but that neither oriented cell movements nor shape can account for this alignment. When the neural plate begins to differentiate, there is a gradual transition in the direction of cell division from AP to the mediolateral circumference (ML). ML divisions occur in both the ventral epidermis and dorsal neural plate. In the neural plate, ML becomes the predominant orientation of division during neural keel and nerve rod stages and, from late neural keel stage, divisions are concentrated at the dorsal midline and generate bilateral progeny (C. Papan and J. A. Campos-Ortega (1994) Roux's Arch. Dev. Biol. 203, 178-186). Coincidentally, cells on the ventral surface also orient their divisions in the ML direction, cleaving perpendicular to the direction in which they are elongated. The ML alignment of epidermal divisions is well correlated with cell shape but ML divisions within the neuroepithelium appear to be better correlated with changes in tissue morphology associated with neurulation.

Phenomenological investigation of inhomogeneities in Nd3+-doped Ba2NaNb5O15 single-crystal fibres grown by the laser-heated pedestal growth technique

Inhomogeneities in Ba2NaNb5O15 (BNN) single-crystal fibres undoped or doped with 1–5 at% Nd3+ and grown by the laser-heated pedestal growth technique were investigated by energy-dispersive X-ray microprobe analysis (EDXA), second-harmonic generation (SHG) measurements and interference contrast microscopy after chemical etching. The observed internal morphology of BNN fibres was correlated to SHG and EDXA measurements and involves convective flow instabilities in the molten zone and possibly unsteady interface kinetics. From this study, it appears that for a doping concentration equal to 3 at% Nd3+, no growth striations nor any kind of inhomogeneity could be detected, indicating, as also shown by differential thermal analysis, that this mixture congruently melts, ensuring very-high-optical-quality crystals are obtained as no cracks nor microtwins are also present for this amount of neodymium. © 1998 Chapman & Hall

The effects of the morphological response of Enterobacteriaceae to cephalosporins on PAE and CERT.

PAE and CERT values of cefaclor, loracarbef and cefuroxime (0.1-100 x MIC) were established for 8 E. coli, 3 K. pneumoniae and 2 P. mirabilis isolates. Cell enumeration was by impedance (IMP) monitoring in combination with either bioluminescence (BIOL) or viable counting (VC). Morphology was determined by interference contrast microscopy. After 2 h exposure to cefaclor, loracarbef and cefuroxime; concentration-dependent differences in counts were seen by BIOL and VC, varying from a mean value of 0.07 x log10 after exposing the P. mirabilis isolates to 0.1 x MIC cefaclor to a mean value of 2.24 x log10 after exposing the E. coli strains to 100 x MIC cefuroxime. Higher concentrations gave rise to fragile morphological forms including spheroplasts and lower concentrations to less fragile forms such as long bacilli. The longest PAE and CERT values were obtained after exposing the E. coli strains to 100 x MIC cefaclor with mean values of 4.07 and 4.87 h, respectively. Corresponding values were PAE and CERT values of 2.17 and 2.60 h for 100 x cefuroxime and 3.45 and 2.91 h for 100 x MIC loracarbef. By the Student's t-test, PAE values determined by IMP/BIOL and IMP/VC were found to be significantly different, whereas CERT values were found not to be significantly different. PAE and CERT are concentration dependent and vary with specific antibiotic/organism combinations.

Abnormal spermatogenesis in the common liver fluke (Fasciola sp.) from Japan and Korea.

Diploid and triploid specimens of Japanese and Korean Fasciola sp. showed abnormality in their spermatogenesis. Live germ cells obtained from the testes were observed under a differential interference contrast microscope. In the stages from spermatogonium to spermatid, the cells combined together at the central cytoplasmic bridge during a series of divisions. One spermatogonium becomes a cell group of 8 primary spermatocytes through 3 mitoses. Until the primary spermatocyte stage, cells are divided in a uniform manner. In most of the diploid specimens, the primary spermatocytes are irregularly divided into non-uniform secondary spermatocytes, however, some specimens perform a regular division. In the majority of triploid flukes, the primary spermatocytes are divided in a regular pattern, but some of the specimens perform an irregular division. The non-uniform spermatids do not perform a spermiogenesis. In the diploid specimens, no spermatozoa were found that were produced by spermiogenesis. Whereas in the triploid specimens, some spermatids distributed uniformly on the surface went through a spermiogenesis. We observed some moving spermatozoa in one triploid specimen. The spermatozoa possibly retain their normal reproductive function.

CNS neuroblasts migrate in three-dimensional gels with extracellular matrix containing laminin

The effect of dibutyryl cyclic AMP (dbcAMP) on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95±1.01 μm/s (n=55) to 2.66±1.26 μm/s (n=58) and from 1.94±0.85 (n=57) to 2.39±0.93 (n=57). Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport.

Modulatory effect of prostaglandin E 2 on axonal transport in cultured mouse dorsal root ganglion cells: Involvement of cyclic AMP/protein kinase a cascade

The effect of dibutyryl cyclic AMP (dbcAMP) on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95±1.01 μm/s (n=55) to 2.66±1.26 μm/s (n=58) and from 1.94±0.85 (n=57) to 2.39±0.93 (n=57). Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport.

Mitotic activity in wheat shoot apical meristems: effect of dissection to expose the apical dome

An efficient method, less laborious than histological procedures, is described to screen relatively large numbers of shoot apices for mitotic activity. Mitotic activity of shoot apices of Triticum aestivum L. was observed by differential interference contrast (DIC) microscopy of apices infiltrated with a clearing fluid (chloral hydrate/phenol/lactic acid/dibutylphthalate/benzyl benzoate). Serial optical sections were viewed through entire vegetative apical domes and floral primordia. In vegetative shoots, mitotic cells were observed throughout the light and dark cycles of plants maintained in either 8 or 16 h photoperiods. Mitotic activity was lower in the dark phase and increased through the light cycle in both photoperiods. Cells in L1 and L2 layers at the summit of the apex were mitotically active and contributed to the developing shoot and floral structures. Thus, cells in L2 at the summit of vegetative apices are valid targets for transformation leading subsequently to modified germ line cells. Dissections to expose apices for DNA delivery inhibited mitotic activity; recovery periods greater than 48 h would be needed for restoration of normal activity. This suggests that a period of recovery from dissection would be beneficial for attempts at integrative transformation of apical cells.

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope.

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.

Direct visualization and characterization of stable microtubules from the neurites of cultured dorsal root ganglion cells

We tested the stability of microtubules (MTs) in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under observation with a video-enhanced differential interference contrast (DIC) microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be MTs by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be MTs by specific immunostaining with anti-tubulin antibody. A significant number of MTs in the established neurites of 6-day-old cultures remained longer than 10 min after membrane removal while MTs in the Schwann cell processes or in the distal regions of the growth cone-bearing neurites of 3-day-old cultures disappeared within 2 min. A population of very stable MTs persisting longer than 30 min was also found specifically in the 6-day-old cultures. Association with other structures or bundling seemed to stabilize the MTs to some degree. The most stable MTs, however, were not associated with some structure along the length but were mainly anchored at points, suggesting that specific point attachments may be another important mechanism operating in MT stabilization. The present method is thus capable of directly demonstrating the unusual stability of neuritic MTs, and provides a new system for further investigation on the mechanism of stabilization.

Projecting two-axis nanometer scale displacement of microscopic beads onto a quadrant photodetector with a laser beam

A quadrant photodetector has been added onto a custom differential interference contrast microscope to measure two-axis displacement of silica beads with a noise ⩽0.1 nm/√Hz below 150 Hz. A diode-pumped solid-state laser is incorporated to function not only as a light source for displacement detection but also as an optical trap for motility studies of protein motors. This system is designed for studies in which the motility needs to be simultaneously monitored along both axes at nm accuracy.

Using microdensitometry to correlate cell morphology with the nuclear cycle in Ustilago maydis

Nuclear cycle events are usually studied using 3H-thymidine, which cells take up from culture medium and incorporate into DNA. Because fungi do not incorporate exogenously supplied thymidine, other methods must be used for these organisms. The experiments described here utilized nuclear staining and computer-assisted microdensitometry. Vegetative Ustilago maydis cells were taken from liquid cultures at various points along the growth curve, attached to microscope slides, stained with 4′,6-diamidino-2-phenylindole, and viewed using Differential Interference Contrast and epifluorescence microscopy. Digital images of fluorescing nuclei were captured and inverted (negated) so that densitometry software could be used to determine the relative DNA content of the nuclei. When nuclei were grouped by relative density, two peaks presumably corresponding to groups of nuclei before (1C) and after (2C) DNA synthesis were observed. Correlating nuclear density with cell morphology showed that cells complete DNA synthesis before beginning to form buds. The presence of a number of unbudded cells with the 1C amount of DNA indicated that there was a clearly defined G1 period. During early log phase, approximately half of the cells in a culture were in G1. As the growth rate slowed, the percentage of cells in G1 increased dramatically. This indicates that the length of time spent in G1 varies with growth conditions in U. maydis. When cells from late log phase populations were subcultured into fresh medium, a lag period preceded resumption of cell growth. In addition, a significant percentage of these cells underwent a round of nuclear division and formed a central septum prior to bud formation. This resulted in a population of binucleate cells that budded asynchronously at both poles.

Meiotic Anomalies in Hybrids between Wheat and Apomictic Elymus rectisetus (Nees in Lehm.) A. Löve & Connor

Four B111 hybrids (2n = 9x = 63, ABDStStYYWW) were obtained between wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) and a diplosporic and 2n‐pollen‐producing Elymus rectisetus (2n = 6x 42, StStYYWW) accession. We determined whether apomeiosis and 2n pollen formation, which occur in the male parent, also occur in the Bill hybrids. Pistils were cleared for UV microscopy of callose and recleared for cytological observations using interference contrast microscopy. Callose fluorescence from most megaspore mother cells (MMCs) was typical of sexual species. These were classified as sexual. Abnormally elongated MMCs with walls devoid of callose were observed in 4.7% of pistils collected at meiotic prophase. These traits are typical of many apomeiotic MMCs in E. rectisetus. Asynapsis followed by mitosis in pollen mother cells (PMCs) consistently preceded unreduced pollen formation in E. rectisetus. This was not observed in the B11l hybrids, though the following meiotic anomalies were observed: (i) asynapsis, aggregation of chromosomes to several poles, and multiple divisions to form four to eight unbalanced microspores, and (ii) incomplete synapsis, formation of anaphase I laggards, and a second division resulting in four unbalanced microspores with micronuclei. The wheat haplome enforced PMC meiotic pairing (albeit abnormal) among homologous E. rectisetus chromosomes and generally inhibited apomeiosis, also by enforcing meiosis. Diplospory may be caused by the expression of embryo sac signals from one genome precociously expressed with megasporogenesis signals from another. If this is correct, the successful induction of apomixis in wheat may require the transfer of alien gene cassettes that confer appropriate degrees of reproductive asynchrony.