Biomedical subjects
Publications and source records attributed to W B Amos.
A series of simple tests have been used to measure the performance of flat-bed film scanners suitable for digitisation of electron micrographs. Two of the film scanners evaluated are commercially available and one has been constructed in the laboratory paying special attention to the needs of the electron microscopist. The tests may be useful for others.
A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.
In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long-established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain-free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.
BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.
In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.
We present a three-dimensional (3D) map, reconstructed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtubules (MTs) in ice. We compare it with the tubulin in six 3D maps of MTs decorated with motor domains, three from frozen MTs decorated with kinesin or ncd in the tightly bound AMP-PNP state, and three from negatively stained MTs decorated with kinesin in different nucleotide states. The comparison confirms that kinesin and ncd bind to identical sites and interact with both monomers of a tubulin dimer. Maps of specimens in negative stain and in ice are similar except that the protein in the top half of a motor domain appears denser in negative stain. The interactions have only a small effect on tubulin structure; the outward appearance is unchanged, but there seems to be a small internal rearrangement. The relative polarity of undecorated and decorated MTs is evident from their 3D structures. This agrees with the absolute polarities indicated by the orientations of motors in decorated specimens and by polar superposition patterns calculated for undecorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria.
A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.
This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.
Individual microtubules can be visualised by confocal microscopy in reflection mode; when associated with a glass surface, they show up as black lines against the bright reflection from the surface. The high contrast imaging allows details of the behaviour of sliding microtubules to be studied easily. Taxol-stabilised microtubules sliding over kinesin-coated surfaces are normally straight, but can bend into tight loops if the leading end sticks to the surface. Some remain curved after release and move in circles. In such cases, the microtubule lattice must have become stably deformed. Electron microscopy of microtubules fixed during sliding shows no gross rearrangement of the subunit lattice and indicates that microtubule bending is mainly achieved by increased twisting of the longitudinal protofilaments around the microtubule.
Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.
Antibodies directed against three regions of tau, ubiquitin, and B-amyloid were used in a histologic study of neurofibrillary degeneration in Alzheimer's disease to distinguish two populations of neurofibrillary tangles. Intracellular tangles were immunolabeled exclusively by two antibodies raised against antigens in the fuzzy coat of the paired helical filament (PHF). Extracellular tangles were distinguished by selective immunolabeling with a monoclonal antibody raised against antigens in the PHF core. This was associated with removal of the fuzzy coat and exposure of PHF-core epitopes. In the transition from intracellular to extracellular compartments in vivo, tangles appeared to undergo changes similar to protease digestion in vitro. The transition was associated with the appearance of amyloid immunoreactivity. These findings suggest that tangle degradation occurs in a series of distinct stages, including ubiquitination of some unknown molecule, a change in tau immunoreactivity, and partial proteolysis of tangle-bound tau in extracellular tangles.
Demembranated Xenopus sperm nuclei were induced to replicate synchronously in a low-speed supernatant (LSS) of Xenopus eggs by preincubation in a high-speed supernatant (HSS). DNA replication was observed by incorporation of [alpha-32P]dATP, BrdUTP or biotin-dUTP. Biotin-dUTP incorporation, visualised with fluorescent streptavidin, reveals a striking pattern of replication foci throughout replicating nuclei. We show that this represents a precursor to the bright uniform fluorescence seen later. Confocal microscopic analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100-300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells. Foci are evenly distributed throughout the nuclei and are not concentrated at or near the nuclear envelope. Complete replication of each nucleus occurs in an average time of only one hour in this system. Hence we calculate that there must be at least 300-1000 replication forks together in each cluster. Furthermore, pulse labelling at later times in the period of replication reveals a similar pattern of foci indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of DNA replication.
A wide variety of specimens has been examined with our apparatus, a commerical version of which is being manufactured by Bio-Rad/Lasersharp. The advantages expected of a confocal system have been realised in practice, the most striking advantage being the exclusion of glare from out-of-focus structures. This has made it possible to image cytological details in unflattened cells and intact tissues that were previously inaccessible to the light microscope.
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Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.
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Chemically resistant 2-3 nm filaments with a high alpha-helical content, isolated from sea-urchin sperm flagellar doublet microtubules, consist of proteins that have been named tektins. Polyclonal affinity-purified antibodies to tektins labelled sperm tails all along their lengths, as shown by indirect immunofluorescence microscopy, provided the specimens were not too well fixed. Results obtained for unfixed specimens studied by immunoelectron microscopy suggested the tektins are normally masked by tubulin. A monoclonal anti-tektin antibody labelled bare tektin filaments at longitudinal intervals of approximately 48 nm, which fits in well with the 96 nm longitudinal repeat of axonemes. We discuss a possible scheme for the regular interaction of tubulin monomers with an alpha-helical coiled coil.
Affinity-purified antibodies against Strongylocentrotus purpuratus sperm flagellar tektin polypeptides have been tested for cross-reactivity with microtubules isolated from various sources, using indirect immunofluorescent staining and antibody binding to nitrocellulose replicas of SDS polyacrylamide gels. The antitektins reacted with sperm tail axonemes from four genera of sea urchins and with cilia from sea urchin embryos. Antibody binding was observed only if the specimens were prefixed by methods that would not preserve them well at an ultrastructural level. However, even after such fixation regimes, no antibody binding was detected to cytoplasmic microtubule arrays in the same embryos, to mitotic spindles isolated from sea urchin eggs or to gill cilia from a mollusc. We conclude that, if tektins are present in sea urchin egg cytoplasmic microtubules, they are sufficiently different from the sperm tektins to have no common strongly antigenic determinants.