High-resolution Differential Interference Contrast Research Articles

The micro and ultrastructural anatomy of bone spicules found in the osteochondral junction of bovine patellae with early joint degeneration.

The structural changes in the tissues of the osteochondral junction are a topic of interest, especially considering how bone changes are involved in the initiation and progression of osteoarthritis (OA). Our research group has previously demonstrated that at the cement line boundary between the zone of calcified cartilage (ZCC) and the subchondral bone, in mature bovine patellae with early OA, there are numerous bone spicules that have emerged from the underlying bone. These spicules contain a central vascular canal and a bone cuff. In this study, we use high-resolution differential interference contrast optical microscopy and scanning electron microscopy to compare the cartilage-bone junction of three groups of mature bovine patellae showing healthy to mild to moderately degenerate cartilage. The ZCC and bone junction was carefully examined to estimate the frequency of marrow spaces, bone spicules and fully formed bone bulges. The results reveal that bone spicules are associated with all grades of cartilage tissue studied, with the most occurring in the intermediate stages of tissue health. The micro and ultrastructure of the bone spicule are consistent with that of an osteon, especially those found in compression zones in long bones. Also considering the coexistence of marrow spaces and fully formed bone, this study suggests that these bone spicules arise similar to the formation of osteons in the bone remodelling process. The significance of this conclusion is in the way researchers approach the bone formation issue in the early degenerative joint. Instead of endochondral ossification, we propose that bone formation in OA is more akin to a combination of primary bone remodelling and de novo bone formation.

The plastic nervous system of Nemertodermatida

Nemertodermatida are microscopic marine worms likely to be the sister group to acoels, forming with them the earliest extant branch of bilaterian animals, although their phylogenetic position is debated. The nervous system of Flagellophora cf. apelti, Sterreria spp. and Nemertoderma cf. westbladi has been investigated by immunohistochemistry and confocal microscopy using anti-tubulin, anti-5-HT and anti-FMRFamide antibodies. The nervous system of F. cf. apelti is composed of a large neuropile and a loose brain at the level of the statocysts with several nerve fibres surrounding them and innervating the broom organ. Sterreria spp. shows a commissural-like brain and several neurite bundles going frontad and caudad from this. At the level of the statocysts there is also a thicker aggregation of immunoreactive fibres. The nervous system of N. cf. westbladi consists of a nerve ring lying outside the body wall musculature at the level of the statocyst and a pair of ventro-lateral neurite bundles, from which extend thinner fibres innervating the ventral side of the animal. Numerous bottle-shaped glands were observed, innervated by fibres starting both from the brain and the neurite bundles. The nervous system of the nemertodermatids studied to date displays no common pattern; instead, there is considerable plasticity in the general morphology of the nervous system. In addition, the musculature of Sterreria spp. has been studied by phalloidin staining. It shows diagonal muscles in the anterior quarter of the body and a simple orthogonal grid in the posterior three quarters, being simpler than that of the other nemertodermatids. High-resolution differential interference contrast microscopy permitted to better visualize some morphological characters of the species studied, such as statocysts, sperm and glands and, in combination with anti-tubulin staining, describe in detail the broom organ in F. cf. apelti. Finally, we note an apparent absence of innervation of the gut in Nemertodermatida similar to the condition in Xenoturbella.

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo's Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain's slices in a customized glass-bottom plate containing Dulbecco's Modified Eagle's Medium (DMEM)/High-Glucose at 37 °C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Morphologic and mitochondrial characterization of human spermatogenic cells dispersed in wet preparation for testicular sperm extraction: establishment of a microscopic diagram of developing human spermatogenic cells

The successful identification of spermatozoa at microdissection testicular sperm extraction is critical and can depend on the judgment of experienced reproductive clinicians. Therefore, we characterized human spermatogenic cells based on stage-specific mitochondrial location and morphologic change by using a vital mitochondrion-specific fluorescent probe, Mitotracker, and stage-specific antibodies to make a useful microscopic diagram.

A Comparative Study of Embedded and Anesthetized Zebrafish in vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction.

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin-transferrin-selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell-matrix constructs. In a medium containing 1% each of serum and insulin-transferrin-selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell-matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils.

Dynamics structures of mechanosensory cilia

BackgroundNon‐motile primary cilia have been proposed to be mechanosensory organelles in chondrocytes, cholangiocytes, osteocytes, renal epithelial, pancreatic epithelial, endocardial, and vascular endothelial cells. We report here the dynamic structure of the biophysical property of cilia.Methods and ResultsUsing formvar as a flexible substrate, we record ciliary behavior for a period of time with a high resolution differential interference contrast microscopy. Under static conditions in the absence of fluid flow, we can observe that each well‐developed cilium contains a bulb in the middle of the ciliary shaft. In a less‐developed cilium, the bulb is less likely to be present. However, bending of cilium by mechanical fluid flow could induce the appearance of the bulb or bulbs within one cilium. Furthermore, fluid flow tends to dynamically move the bulb to the tip of the cilium.ConclusionWe propose that the dynamics of ciliary bulb is mechanically regulated. Ciliary bulb may therefore have sensory function within mechano‐ciliary structure.

Compact high-resolution differential interference contrast soft x-ray microscopy

We demonstrate high-resolution x-ray differential interference contrast (DIC) in a compact soft x-ray microscope. Phase contrast imaging is enabled by the use of a diffractive optical element objective which is matched to the coherence conditions in the microscope setup. The performance of the diffractive optical element objective is evaluated in comparison with a normal zone plate by imaging of a nickel siemens star pattern and linear grating test objects. Images obtained with the DIC optic exhibit typical DIC enhancement in addition to the normal absorption contrast. Contrast transfer functions based on modulation measurements in the obtained images show that the DIC optic gives a significant increase in contrast without reducing the spatial resolution. The phase contrast operation mode now available for our compact soft x-ray microscope will be a useful tool for future studies of samples with low absorption contrast.

High-resolution differential interference contrast X-ray zone plates: Design and fabrication

Differential interference contrast is a potentially powerful technique for contrast enhancement in soft X-ray microscopy. We describe the design and fabrication of single-element diffractive optical elements suitable as objectives for high-resolution differential interference contrast microscopy in the water-window spectral range. A one-dimensional pattern calculation followed by an extension to two dimensions results in a pattern resolution of 1 nm, which is well below fabrication accuracy. The same fabrication process as for normal zone plates is applicable, but special care must be taken when converting the calculated pattern to a code for e-beam lithography.

Intralamellar relationships within the collagenous architecture of the annulus fibrosus imaged in its fully hydrated state

The anisotropic, inhomogeneous, multiply collagenous architecture of the annulus reflects the complex pattern of mainly tensile stresses developed in this region of the disc during normal function. Structural and mechanical responses of fully hydrated in-plane sections taken from within single lamellae of the outer annulus of healthy bovine caudal discs have been investigated using a micromechanical technique in combination with simultaneous high-resolution differential interference contrast optical imaging. Responses both along and across (i.e. transverse to) the primary direction of the mono-array of collagen fibres were studied. Stretching along the alignment direction revealed a biomechanical response consistent with the behaviour of an array whose overall strength is governed primarily by the strength of embedding of the fibres in the vertebral endplates, rather than from interfibre cohesion along their length. The mono-aligned array, even when lacerated, is highly resistant to any further tearing across the alignment direction. Although not visible in the relaxed mono-arrays, transverse stretching revealed a highly complex set of interconnecting structures embodying hierarchical relationships not previously revealed. It is suggested that these structures might play an important role in the containment under pressure of the nuclear contents. The dramatic differences in rupture behaviour observed along vs. across the primary fibre direction are consistent with the known clinical consequences arising from varying degrees of annular wall damage, and might also explain various types of disc herniation. The lamellar architecture of the healthy disc revealed by this investigation provides an important reference framework for exploring structural changes associated with disc trauma and degeneration.

Cyclic generation of wall slip at the exit of plane Couette flow

Sharkskin-type melt fracture is generally produced by a discontinuous boundary condition at a die exit. In order to investigate this flow instability, a shear flow having a negative pressure gradient at the exit was generated by a wedge-shaped slider. A high-resolution differential interference contrast microscope was used for simultaneous observation of the inside and outside of the flow cell, which employed linear PDMS. At the cell exit, the spatial distribution and the temporal change in velocity were measured under sharkskin-like instability by use of a particle tracking method and laser Doppler velocimetry. A slip flow and a stick flow appeared alternately at the exit of the sliding plate; the slip flow is associated with cavity penetration from the cell exit. This inner instability was found to be associated with the formation of valleys and ridges on the outer free surface. At the cell exit, a slip flow appeared with a valley, and a stick flow appeared with a ridge. The penetration length of the cavity increases with sliding-plate speed, and this increase in penetration length induces flow stagnation upstream of the cavity. This stagnation relaxes high shear upstream of the cavity and suppresses the increase in penetration length of the cavity. Except for incipient instability, the penetration length has a linear relation with the wavelength of the outer ridges. A close relationship is found to exist between free surface roughness and inner flow instability.

The endoplasmic reticulum--structure and function (Bristol, September 20-22, 1999).

The intention of this meeting, organised under the auspices of the British Society for Cell Biology (BSCB), was to bring together researchers with a range of specific interests, and a shared fascination with the endoplasmic reticulum. There were sessions on ‘ER structure, remodelling and dynamics’, ‘Receptors:channels’, ‘ER and calcium’ and ‘Protein synthesis and chaperones’ as well as poster sessions and ample opportunity for discussion. Thus, whilst the meeting itself generated lively and informative interactions between individuals from different research backgrounds, this report will focus on those talks most closely related to aspects of trafficking. A more broad ranging overview of the meeting is to be published elsewhere (Molecular Membrane Biology). Viki Allan opened the meeting by showing some high resolution Differential Interference Contrast (DIC) microscopy movies of ER membranes (from Xenopus egg extracts) moving on microtubules. She went on to discuss interactions between the ER and microtubules and the role of various motor proteins in this system, with dynein being the motor driving ER movement towards the minus end of microtubules (a process which is regulated by the reversible phosphorylation of the light intermediate chain of dynein). In

On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff.

Elongation factor‐1α stabilizes microtubules in a calcium/calmodulin‐dependent manner

Elongation factor-1α (EF-1α), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of EF-1α (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1α modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1α, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1α significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1α, occur. In addition, calcium/calmodulin (Ca2+/CaM), which regulates the ability of EF-1α to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1α on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1α was never observed. These data support the hypothesis that EF-1α modulates the dynamic behavior of microtubules assembled in vitro in a Ca2+/CaM-dependent manner. Cell Motil. Cytoskeleton 41:168–180, 1998. © 1998 Wiley-Liss, Inc.

Elongation factor-1alpha stabilizes microtubules in a calcium/calmodulin-dependent manner.

Elongation factor-1alpha (EF-1alpha), a highly conserved protein named for its role in protein translation, is also a microtubule-associated protein (MAP). We used high-resolution differential interference contrast microscopy to quantify the effect of substoichiometric amounts of EF-1alpha (isolated from Daucus carota) on the dynamic instability of microtubules assembled in vitro from either animal or plant tubulin. EF-1alpha modulates the dynamic behavior of microtubules assembled from either tubulin source, resulting in longer and more persistent microtubules. EF-1alpha, at a 1:20 molar ratio to tubulin, significantly (P < 0.05) reduces the frequency of catastrophe threefold and decreases shortening velocities almost twofold for microtubules assembled from animal tubulin. For microtubules assembled from plant tubulin, substoichiometric amounts of EF-1alpha significantly (P < 0.05) suppress the frequency of catastrophe greater than twofold and causes an almost threefold reduction in shortening velocities. Elongation velocities increase almost twofold and rescues, which are not observed in the absence of EF-1alpha, occur. In addition, calcium/calmodulin (Ca2+/CaM), which regulates the ability of EF-1alpha to bundle taxol-stabilized microtubules in vitro, also modulates the effect of EF-1alpha on the dynamic behavior of microtubules assembled in vitro from animal tubulin. Microtubule severing in the presence of EF-1alpha was never observed. These data support the hypothesis that EF-1alpha modulates the dynamic behavior of microtubules assembled in vitro in a Ca2+/CaM-dependent manner.

Imaging green fluorescent protein fusion proteins in Saccharomyces cerevisiae

Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria[1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein–GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90–120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 μm-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein–GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].

A Differential Interference Contrast-Based Light Microscopic System for Laser Microsurgery and Optical Trapping of Selected Chromosomes during Mitosis In Vivo

Laser microsurgery and laser-generated optical force traps (optical tweezers) are both valuable light microscopic-based approaches for studying intra- and extracellular motility processes, including chromosome segregation during mitosis. Here we describe a system in use in our laboratory that allows living cells to be followed by high-resolution differential interference contrast (DIC) video-enhanced time-lapse light microscopy while selected mitotic organelles and spindle components are subjected to laser microsurgery and/or manipulation with an optical force trap. This system couples the output from two different Neodymium-YAG lasers to the same inverted light microscope equipped with both phase-contrast and de Senarmont compensation DIC optics, a motorized stage, and a high-resolution low-light-level CCD camera. Unlike similar systems using phase-contrast optics, our DIC-based system can image living cells in thin optical sections without contamination due to phase halos or out-of-focus object information. These advantages greatly facilitate laser-based light microscopic studies on mitotic organelles and components, including spindle poles (centrosomes) and kinetochores, which are at or below the resolution limit of the light microscope and buried within a large complex structure. When used in conjunction with image processing and high-resolution object-tracking techniques, our system provides new information on the roles that kinetochores and spindle microtubules play during chromosome segregation in plant and animal cells.

Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion.

Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.

Activation of Salivary Secretion: Coupling of Cell Volume and [Ca 2+ ] i in Single Cells

High-resolution differential interference contrast microscopy and digital imaging of the fluorescent calcium indicator dye fura-2 were performed simultaneously in single rat salivary gland acinar cells to examine the effects of muscarinic stimulation on cell volume and cytoplasmic calcium concentration ([Ca2+]i). Agonist stimulation of fluid secretion is initially associated with a rapid tenfold increase in [Ca2+]i as well as a substantial cell shrinkage. Subsequent changes of cell volume in the continued presence of agonist are tightly coupled to dynamic levels of [Ca2+]i, even during [Ca2+]i oscillations. Experiments with Ca2+ chelators and ionophores showed that physiological elevations of [Ca2+]i are necessary and sufficient to cause changes in cell volume. The relation between [Ca2+]i and cell volume suggests that the latter reflects the secretory state of the acinar cell. Agonist-induced changes in [Ca2+]i, by modulating specific ion permeabilities, result in solute movement into or out of the cell. The resultant cell volume changes may be important in modulating salivary secretion.

Correlative Video-Enhanced Light Microscopy High-Resolution TEM

Allen video-enhanced contrast, differential interference-contrast microscopy (AVEC-DIC) combines high resolution differential interference contrast microscopy of the Zeiss IM35 microscope with a Hamamatsu video camera to obtain images with increased magnification, resolution, contrast and visibility. Through this combination and by capitalizing on light diffraction phenomena, it is possible to visualize subcellular organelles, particles, and structures which are an order of magnitude smaller (0.01 μm) than the normal limits of resolution with light microscopy (0.2 μm). The use of gold “finder” grids mounted on glass coverslips provides the mechanism by which cells after being observed in the video system can be located in the electron microscope. Such a correlative approach allows static ultrastructural features to be associated with cellular dynamics as observed and recorded by video. When the electron microscopy is done using either IVEM, HVEM or conventional TEM at 120 keV and the images recorded as stereo pair micrographs, the AVEC-DIC spatial data can be directly compared to high resolution 3-D data obtained by EM.