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Superoxide anions (O2-) generated by rabbit neutrophils were detected and quantified by a video microscope equipped with a photon-counting camera. One count obtained by this system was equivalent to 59 amol of O2-. Maximum O2- production was observed at 6-8 min after stimulation and was estimated as 1.9 fmol/min per cell on the average.
Video and digital image processing have been used to amplify the contrast of light microscopic images, making it possible to observe in real time the diffraction images of cell structures 10 times smaller than the Raleigh resolution limit of 0.2 micron. In this paper we discuss how quantitative analysis of diffraction images can be used to extract information about motion or structure at the nanometer level. This issue is considered in the context of a method for tracking the motion of kinesin-coated beads on microtubules with 1-2 nm precision (Gelles et al.:Nature 331:450-453, 1988).
We described the initial events in the interaction between an oligodendrocyte process and an axon in culture utilizing video time-lapse microscopy. Myelination of an axon by the lamellipodium of an oligodendrocyte was achieved in several steps of cellular process development and coordinated interaction between axon and oligodendrocyte. The initial stage of contact included the formation of a lamellipodium process at the end of an oligodendrocyte process. It appeared that this process contacted the axon several times and was then retracted, and that the filopodia and lamellipodium underwent morphological changes prior to the onset of the myelination. In the second stage, the lamellipodium appeared to thicken and anchor to the axon. Finally, when rippling of the lamellipodial ruffling occurred, the angle between the anchoring filopodium and the axon changed depending on the direction of lamellipodial movement, and the lamellipodium, which was folded in layers, wrapped around the axon like a transverse wave in one motion as observed on the video screen. Thereafter, the lamellipodium assumed a "bursting" form within minutes in real time. This is the first comprehensive overview of how an oligodendrocyte plasma membrane wraps around an axon to form myelin.
The present paper describes in detail the complex movement of the protozoon Tritrichomonas foetus. By the use of analogue and digital video techniques, we were able to analyze frame by frame the beatings of the anterior flagella and discuss their role in the movement of the cell. We also measured the productive displacement of the cell during one flagellar beating cycle. The obtained data were digitally improved and compared to analogue quantifications. It is shown that during 1 s of recorded movement, T. foetus performs 4 complete anterior flagella beating cycles (with active-like and recovery-like beatings). In each cycle the cell swims +/- 6.5 microns forwards, after the recovery of +/- 1.5 microns of receded movement. These observations led us to conclude that the estimated average speed of T. foetus is 25 microns/s, and that all flagella participate in the cell movement. The recurrent flagellum continuously contribute to the forward movement of the protozoon. The cell also performs rotational movements. The obtained results led us to suggest a model for the movement of T.foetus.
The validity of intraoperative frozen section diagnosis (IFSD) via telepathology between Hokkaido University School of Medicine (HUSM) and a remote hospital was evaluated. Video-microscopic images of hematoxylin-eosin (HE)-stained frozen sections were transmitted by a cytoscreener via the Pathtran 1,000 system and a telephone line to a color monitor at HUSM (120 km from the submitting hospital) with a resolution of 512 x 480 pixel matrix. The pathologists at HUSM made diagnoses on the received images. Of the 59 cases, there was diagnostic concordance between telepathology and permanent paraffin sections in 19 malignant, 36 benign, and one borderline case. The latter was a case of atypical ductal hyperplasia of the breast. The telepathology diagnostic modality was inconclusive in three cases, two of which were benign and one malignant by paraffin section examination. These results provide evidence for the diagnostic adequacy of video-microscopic images, the interpretation of which compared favorably with that of conventional frozen sections. Although the need for IFSD is increasing, the proportion of hospital pathologists accepting this modality is not. For the appropriate use of telepathology, it is necessary that more pertinent personnel, especially cytoscreeners and pathologists receive a thorough training and become familiar with the system. Telepathology offers pathologists a diagnostic modality that responds to the needs of physicians and serves to enhance the pathologist's position in health care services.
Early embryonic development involves complex events such as the regulation of cell division and the establishment of embryonic polarity. To identify genes involved in these events, we collected four-dimensional time-lapse video recordings of the first three cell divisions and analysed terminal phenotypes after RNA interference of 147 embryonic lethal genes previously identified in a systematic screen of Caenorhabditis elegans chromosome I. Over half gave defects in early processes such as meiosis, the assembly or position of the first mitotic spindle, cytokinesis, and proper nuclear positioning. For some phenotypic classes, the majority of genes are involved in a shared biochemical process. In addition, we identified loss-of-function phenotypes for genes of unknown function, but for which homologues exist in other organisms, shedding light on the function of these uncharacterized genes. When applied to the whole genome, this approach should identify the vast majority of genes required for early cell processes, paving the way for a greatly improved understanding of these processes and their regulation at the molecular level.
Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations.
Modification of rectified Nomarski differential interference contrast optics (Nikon) and the epi-illumination system (Nikon IGS-cube) improved the detection of colloidal gold particles with analog video enhanced microscopy. Immuno-gold labelled microtubules of Haemanthus endosperm are visualized at a level of detection unmatched in conventional light microscopy. Single gold, or gold silver enhanced particles in suspension viewed with the modified epi-illumination after pressure injection into cells, are well distinguished from other granular cell components. Immuno-gold has also been detected on the surface of chromosomes and the nuclear envelopes in cells during the rapid experimental disassembly of microtubules. Thus, under certain conditions tubulin in a form other than microtubules may be detected. Practical applications of this "optical stain" for non fading immuno-gold 5-40 nm markers are discussed.
In situ surface modification techniques to improve the blood compatibility of blood contacting surfaces of medical devices have been developed by the authors. The techniques include heparin immobilization and sulfonated polymer grafting onto a polyurethane (PU) surface by using either ozone oxidation or photo reaction. These modified PUs were evaluated using an epifluorescent video microscope combined with a parallel plate flow cell. The epifluorescent video microscope system measured the amount of platelet coverage on the PU surfaces using whole human blood containing mepacrine labeled platelets perfused at a wall shear rate of 100 sec-1 for 20 min. Platelet activation and complement activation were also measured. Both immobilized heparin and sulfonated PUs showed significantly lower levels of platelet adhesion than the control PU. The platelet activation levels of these modified PUs also correspond to the results of the platelet adhesion. As for complement activation, heparin the immobilized surface showed the least complement activation, while sulfonated PU and the control PU showed higher levels of complement activation. In situ surface modification techniques, which use either ozone oxidation or photo reaction, are useful in a variety of medical devices even of a complex design, such as membrane oxygenators or artificial hearts.
For the digital processing of microscopical images, mosaicking is a prerequisite if the specimen is larger than the camera field at the necessary magnification. This study investigates the possibilities and limitations of fully automated mosaicking on a desktop computer. Cross-correlation-based frame registration was performed with high reliability if the video frames were edge-enhanced before matched filtering, and also in a reasonable time since the search for matches was restricted to pairs of consecutive frames. An environment for routine mosaicking was developed and implemented in a widely used desktop image-processing program. The software developed during this study has been released to the public domain.
The behavior of white and red blood cells, platelets, and circulating injected particles is one of the most studied areas of physiology. Most methods used to analyze the circulatory patterns of cells are time consuming. We describe a system named CellTrack, designed for fully automated tracking of circulating cells and micro-particles and retrieval of their behavioral characteristics. The task of automated blood cell tracking in vessels from in vivo video is particularly challenging because of the blood cells' nonrigid shapes, the instability inherent in in vivo videos, the abundance of moving objects and their frequent superposition. To tackle this, the CellTrack system operates on two levels: first, a global processing module extracts vessel borders and center lines based on color and temporal patterns. This enables the computation of the approximate direction of the blood flow in each vessel. Second, a local processing module extracts the locations and velocities of circulating cells. This is performed by artificial neural network classifiers that are designed to detect specific types of blood cells and micro-particles. The motion correspondence problem is then resolved by a novel algorithm that incorporates both the local and the global information. The system has been tested on a series of in vivo color video recordings of rat mesentery. Our results show that the synergy between the global and local information enables CellTrack to overcome many of the difficulties inherent in tracking methods that rely solely on local information. A comparison was made between manual measurements and the automatically extracted measurements of leukocytes and fluorescent microspheres circulatory velocities. This comparison revealed an accuracy of 97%. CellTrack also enabled a much larger volume of sampling in a fraction of time compared to the manual measurements. All these results suggest that our method can in fact constitute a reliable replacement for manual extraction of blood flow characteristics from in vivo videos.
In patients with atopic dermatitis (AD), it is debatable whether clinically normal-appearing skin is equal to non-atopic normal skin. The aim of this study was to quantitatively evaluate the characteristics of normal-appearing skin of AD. We examined the value of skin surface morphological changes using a new, simple, computer-assisted method with a video microscope. We also investigated the physiological function as represented by transepidermal water loss (TEWL) levels in 44 patients with AD and 15 normal controls. The morphological changes were represented by a variation coefficient score that reflected the irregularity of skin ridges, named the surface irregularity index (SII). There were significant differences between the normal-appearing skin of AD and non-atopic normal skin in both SII (P<0.001) and in TEWL (P<0.01). Especially for the SII, there were significant differences between AD subgroups subdivided by peripheral blood eosinophil count (Eo), serum lactate dehydrogenase level, and clinical score. TEWL values were significantly higher in the high-Eo AD group (n=15) than in the low-Eo AD group (n=29) (P<0.05). These findings indicate that clinically normal-appearing skin of AD patients with high disease activity differs from non-atopic normal skin in both surface morphology and physiology and that these changes reflect the current disease activity.
An intriguing characteristic of the ontogenic development of the cerebral vasculature is the rapid differentiation of the neonatal leptomeningeal vascular plexus into the mature, adult network form. The physiological and cellular mechanisms of this cerebrovascular remodeling process are unclear. The objective of this work was to determine and correlate changes in vascular density, network pattern and flow velocity in leptomeningeal microvessels of the rat during postnatal development in vivo. To this end, microvascular diameter, segment length, and vascular density of reconstructed leptomeningeal networks were measured from video-recordings of the microcirculation visualized through a cranial window in 0-15-day-old Sprague-Dawley rats. The velocity of erythrocytes in the microvessels was measured by frame to frame tracking of fluorescently labeled red blood cells. We found that surface vascular density (total vessel length per area), node density and segment density (object per area) decreased significantly by the second week after birth. Anastomosing vascular polygons, characteristic to newborn networks, became less numerous and larger in diameter during the postnatal 2-week period, indicating progressive rarefaction of the networks. Vessel diameter and red cell velocity showed transient increases at 1.5 weeks. The velocity/diameter ratio (V/D), an index of wall shear rate, increased by the age of 1.5 weeks and remained unchanged afterwards. There was a negative correlation between V/D and diameter at 1 week; this relationship was reversed to a positive correlation at 2 weeks. We conclude that postnatal remodeling of the leptomeningeal vascular network is associated with rarefaction and an adaptation of vessel caliber to wall shear rate. These changes may contribute to arterio-venous differentiation and redistribution of blood flow from the superficial to the intracortical vasculature in the developing brain.
Conventional in vitro studies of pepsinogen secretion have measured secretion into the bulk medium and have demonstrated the critical role of Ca2+ in the process. The present study was undertaken to obtain further details of the process of secretion and its relation to Ca2+ changes over very short time periods. The relation between Ca2+ mobilization and exocytosis in an isolated individual peptic cell of the bullfrog was investigated by a method to measure both intracellular Ca2+ ([Ca2+]i), using a fluorescent Ca2+ indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 x 10(-7) M) and bethanechol (3.2 x 10(-4) M) caused a rapid increase in [Ca2+]i (initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak, [Ca2+]i was maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated [Ca2+]i and the related exocytosis were eliminated by the depletion of extracellular Ca2+. Low concentrations of bombesin (3.2 x 10(-10) M) and bethanechol (3.2 x 10(-7) M) caused sustained low-amplitude Ca2+ oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that 1) cellular response differs between high and low concentrations of stimulus, 2) there is a close relation between [Ca2+]i and exocytosis, 3) exocytosis follows elevation of [Ca2+]i by 14-45 s (n = 6), and 4) there is a significant positive correlation between the peak [Ca2+]i and the number of exocytoses.
BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.
The Optical Triplicator produces three copies of a portion of a microscopic image and places them side-by-side on the face of a video image tube, so that all three images can be viewed simultaneously in each video frame. The Optical Triplicator was used in an intravital microscopy assembly to obtain simultaneous images of a microvessel at three visible wavelengths selected to enable the accurate determination of oxygen saturation in microvessels of the hamster retractor muscle. An image processing system was used to obtain light intensity and optical density from video recordings made using the triplicator. Lumenal oxygen saturation profiles were determined using the measured intensity values and a published three wavelength photometric method.
The objectives of this study were to develop a method to quantitate the displacement and strain fields within articular cartilage during equilibrium confined compression, and to use the method to determine the variation of the equilibrium confined compression modulus with depth from the articular surface in bovine cartilage. The method made use of fluorescently labeled chondrocyte nuclei as intrinsic fiducial markers. Articular cartilage was harvested from the patellofemoral groove of adult bovines and trimmed to rectangular blocks 5 mm long, 0.76 mm wide, and 500 microns deep with the articular surface intact. Test specimens were stained with the DNA binding dye Hoechst 33258, placed in a custom confined compression chamber, and viewed with an epifluorescence microscope equipped for video image acquisition. Image processing was used to localize fluorescing chondrocyte nuclei in uncompressed and compressed (approximately 17%) specimens, allowing determination of the intra-tissue displacement profile. Strain was determined as the slope of linear regression fits of the displacement data in four sequential 125-microns-thick layers. Equilibrium strains varied 6.1-fold from the articular surface through 500 microns of cartilage depth, with the greatest compressive strain in the superficial 125-microns layer and the least compressive strain in the two deepest 125-microns layers. Thus, the four successive 125-microns layers have moduli that are 0.44 (superficial), 1.07, 2.39, and 2.67 (deep) times the apparent modulus for a 500-microns thick cartilage sample assumed to be homogeneous.
Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2 x 10(3) microm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2 x 10(3) to 0 microm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.