VANQUIS, a system for the interactive semantic content analysis and spatio-temporal query by content of videos.

Using the video metadata descriptors and data model defined in the accompanying paper (Shotton, D. M. et al. (2002) A metadata classification schema for semantic content analysis of videos. J. Microsc. 205, 33-42), we discuss how analysis of the content of scientific videos, and subsequent query by content of the resulting semantic metadata, can be enhanced by the use of an object-relational database. We illustrate this by describing VANQUIS, a Web-based prototype video analysis and query interface system for the interactive spatio-temporal analysis and subsequent query by content of videos. Using VANQUIS to generate standard SQL (structured query language) statements that address complex data types stored in an object-relational database, relationships between characters and events contained within and between videos can be identified, and the appropriate video segments containing these characters and events can be retrieved for viewing. We give examples of analysis and query implementation by using VANQUIS to analyse a biological microscopy video, and discuss the wider potential of this methodology for the analysis and query by content of videos containing more general subject matter.

A metadata classification schema for semantic content analysis of videos.

Simple ancillary metadata, such as those encompassed by the 15 elements of the Dublin Core, may be sufficient and entirely appropriate for basic coarse-granularity cross-domain resource discovery. However, they are insufficient and inappropriate for content description of complex data types such as videos, which require more detailed relational models. We propose a metadata classification schema for the characterization of items and events in videos that permits subsequent query by content. Following MPEG-7 nomenclature, metadata intrinsic to the information content of the video are defined as either structural or semantic, where structural metadata are numerical feature primitives produced by analysing the colour, shape, texture, structure and motion within the video frames, whereas semantic metadata describe the locations and timings of individual items and particular actions or events in the video, and are thus of higher information value. In this paper, the semantic metadata required to describe the visual information content of videos are defined and classified into four distinct classes: Media Entities; Content Items; Events; and Supplementary Items, and three types of property tables are defined: Identity Tables; Spatio-Temporal Position Tables; and Event Tables, in which these metadata may be stored in a relational database.

Cytotoxic T lymphocytes can induce a condemned state and synchronous post-mitotic apoptosis of daughter target cells.

We have used time-lapse video microscopy to study cytotoxic T lymphocyte (CTL)-mediated apoptosis of LDb fibroblast target cells at different phases of the cell cycle. When aphidicolin-synchronized target cells were exposed to the CTL clone F5, apoptosis occurred with similar morphology during G1, S/G2 and M phase, showing that apoptosis and mitosis are not mutually exclusive cellular events. Interestingly, following normal mitosis of target cells that had been previously contacted by CTL, pairs of daughter cells would occasionally undergo apoptosis within minutes of each other. Such synchronous post-mitotic apoptosis was also observed when using mitotically unsynchronized target cells, and also when using d11S T cell hybridomas as alternative Fas- (CD95-) based effector cells, even if these effectors were physically washed away after an initial period of co-incubation with the target cells. Our observations show that cytotoxic cells can induce a condemned state in pre-mitotic target cells, which can be inherited by both daughter cells, leading to their synchronous apoptosis after mitosis.

Video on the Internet: An introduction to the digital encoding, compression, and transmission of moving image data.

In this paper, we seek to provide an introduction to the fast-moving field of digital video on the Internet, from the viewpoint of the biological microscopist who might wish to store or access videos, for instance in image databases such as the BioImage Database (http://www.bioimage.org). We describe and evaluate the principal methods used for encoding and compressing moving image data for digital storage and transmission over the Internet, which involve compromises between compression efficiency and retention of image fidelity, and describe the existing alternate software technologies for downloading or streaming compressed digitized videos using a Web browser. We report the results of experiments on video microscopy recordings and three-dimensional confocal animations of biological specimens to evaluate the compression efficiencies of the principal video compression-decompression algorithms (codecs) and to document the artefacts associated with each of them. Because MPEG-1 gives very high compression while yet retaining reasonable image quality, these studies lead us to recommend that video databases should store both a high-resolution original version of each video, ideally either uncompressed or losslessly compressed, and a separate edited and highly compressed MPEG-1 preview version that can be rapidly downloaded for interactive viewing by the database user.

Organising multi-dimensional biological image information: the BioImage Database.

Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www. bioimage.org and http://www-embl.bioimage.org

Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity.

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after "browsing" their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous "boiling" of the cytoplasm and blebbing of the plasma membrane) for 10-20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5-LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity.

Structural analysis of the CD5 antigen--expression, disulphide bond analysis and physical characterisation of CD5 scavenger receptor superfamily domain 1.

CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.

Robert Feulgen Prize Lecture 1995. Electronic light microscopy: present capabilities and future prospects.

Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies.

The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.

Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages.

Macrophage subpopulations in the mouse express a lectin-like receptor, sialoadhesin (originally named sheep erythrocyte receptor, SER), which selectively recognizes sialoglycoconjugates and is likely to be involved in cellular interactions of stromal macrophages in haematopoietic and lymphoid tissues. In this report we describe the purification and ligand specificity of sialoadhesin isolated from mouse spleen. Purified sialoadhesin, a glycoprotein of 185 kd apparent Mr, agglutinated sheep or human erythrocytes at nanomolar concentrations in a sialic acid-dependent manner. Low angle shadowing and electron microscopy showed that sialoadhesin consisted of a globular head region of approximately 9 nm and an extended tail of approximately 35 nm. To investigate the specificity for sialic acid, we studied the interaction of sialoadhesin with derivatized human erythrocytes, glycoproteins, and glycolipids. In conclusion, sialoadhesin specifically recognizes the oligosaccharide sequence Neu5Ac alpha 2----3Gal beta 1----3GalNAc in either sialoglycoproteins or gangliosides. These findings imply that specific sialoglycoconjugates carrying this structure may be involved in cellular interactions between stromal macrophages and subpopulations of haematopoietic cells and lymphocytes.

The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.

Effects of capping on the non-ionic detergent solubility of rat thymocyte glycoproteins.

The non-ionic detergent solubility of surface antigens during capping, following their cross-linking by specific antibodies, was investigated for three rat thymocyte glycoproteins, the leucocyte-common antigen (L-CA), the leucocyte sialoglycoprotein (LSGP) and Thy-1, using a combination of immunofluorescence microscopy, covalent surface radiolabeling and quantitative analysis using a radiolabeled antibody. Prior to the addition of cross-linking antibody, both L-CA and LSGP were soluble in the non-ionic detergent Triton X-100, while Thy-1 was largely insoluble. Addition of sufficient antibody to induce capping led to a significant reduction in the solubility of L-CA and LSGP, even prior to warming to induce capping of the antigen under investigation. Subsequent capping did not increase the amount of insoluble antigen, suggesting that ligand binding, rather than the process of capping itself, is sufficient to cause this partial Triton insolubility. These results indicate the formation of an association between these liganded glycoproteins and the cell's detergent-insoluble cytoskeleton. In contrast, the Thy-1 antigen became progressively more extractable by Triton X-100 as the antigen was capped, suggesting that the insertion of the Thy-1 lipid tail into the plasma membrane was progressively more easily perturbed by the non-ionic detergent during the capping process. The reciprocal maintenance of solubility of either LSGP or L-CA during the capping of the other shows that the majority of the 180 kDa thymocyte L-CA molecules play no role in the mechanism of capping of LSGP. Furthermore, immunoprecipitates of solubilized L-CA contained no detectable amounts of the cytoskeletal protein fodrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy.

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.

Cytoskeletal involvement in the sequential capping of rat thymocyte surface glycoproteins.

The independent capping of the three major rat thymocyte glycoproteins, the leucocyte-common (L-C) antigen, the leucocyte sialoglycoprotein (LSGP) and Thy-1, was investigated using specific monoclonal antibodies. The capping of each antigen did not require redistribution of the other major surface glycoproteins, and was accompanied by a partial co-capping of the cytoskeletal proteins fodrin and actin, but not of tubulin. A study of the ability of a cell that already possesses one glycoprotein cap to cap a second different glycoprotein showed that this was possible in all cases to varying degrees, the second cap always forming at the same position on the cell surface as the first. Colchicine failed to perturb this observed sequential capping polarity, indicating that microtubules did not direct this second capping event.

Video-enhanced light microscopy and its applications in cell biology.

The combination of novel optical microscopic techniques with advanced video and digital image-processing technology now permits dramatic improvements in the quality of light-microscope images. Such video-enhanced light microscopy has lead to a renaissance in the applications of the light microscope for the study of living cells in two important areas: the intensification of faint fluorescence images, permitting observation of fluorescently labelled cells under conditions of very low illuminating intensity; and the enhancement of extremely low contrast images generated by minute cellular structures, so that these may be clearly seen and their normal intracellular movements recorded. Application of both these aspects of video-enhanced light microscopy have recently led to major discoveries concerning the functioning of the living cell. In this review I discuss the equipment, procedures and image-processing principles employed in these applications, and describe and illustrate some of the spectacular results that have recently been obtained.

Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody against slow myosin.

Existing methods for the ultrastructural identification of fibre types in human skeletal muscle are fallible. This has prompted us to develop a reliable immunoelectron microscopic approach for the identification of human skeletal muscle fibre types. Here we report the unambiguous electron microscopic identification of human type 1 muscle fibres, achieved by combining cryoultramicrotomy with colloidal gold immunocytochemical labelling, using a monoclonal antibody (N0Q7.5.4D) which is specific for the heavy chain of the slow myosin isoform of human skeletal muscle. This method for the identification of muscle fibre types and determination of myosin isoform distributions may have important applications in the ultrastructural study of pathological muscle and in the analysis of myofibrillar assembly during myogenesis.

Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles.

We have used light microscopic histomorphometry to quantify the developmental histopathological changes induced by muscular dystrophy in the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse. We find that this X-linked disease exhibits early fibre necrosis with foci of invasive cells, clustering of affected fibres, hyaline fibres, and, in the mixed soleus muscle, a progressive increase in the proportion of type 1 fibres, the mdx soleus containing 58 +/- 5% type 1 fibres by 26 weeks, compared with 27 +/- 4% in control C57BL/10 ScSn mice. This increase is not due to atrophy or slow axon reinnervation of type 2 fibres. Although only 5% of all original fibres survive by 26 weeks in the EDL, the diseased mdx fibres are continuously and successfully replaced by new fibres with internal nuclei, the affected mice thus avoiding the end-stage histopathology and physical disability characteristic of the X-linked human Duchenne and Emery-Dreifuss muscular dystrophies. Homozygous mdx mice share the life expectancy of normal C57BL/10 mice and appear behaviourly normal. The mdx mouse is therefore an excellent mammalian model in which to study the processes of muscle fibre degeneration and regeneration.

Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoproteins: evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping.

Two major rat thymocyte surface glycoproteins, the leucocyte-common (L-C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX-1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing. TRITC-phalloidin staining of the isolated caps demonstrated the presence of F-actin within these structures, and lectin-affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L-C antigen and LSGP caps, but absent from the detergent-insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross-linked glycoproteins with the cytoskeleton during capping.

Electron-microscopic studies of the CA antigen, epitectin.

Epitectin, the mucin-like glycoprotein defined by the monoclonal antibodies CA1, CA2 and CA3, has been examined by electron microscopy to determine its shape and size. It appears to be a single extended strand with a mean length of about 270 nm. The antibodies CA1 and CA2 appear to bind preferentially to a terminal site on the epitectin molecule.

Quantitative freeze-fracture studies of membrane changes in chicken muscular dystrophy.

Muscular dystrophy induces extensive changes in the patterning of sarcolemmal caveolae of fast-twitch fibers from the chicken posterior latissimus dorsi (PLD) muscle, which in healthy fibers are arranged in striking bands over the myofibrillar I-bands. In dystrophic fibers the caveolae lack this patterned arrangement, and instead are dispersed over the entire sarcolemma, are irregular in shape, and are more numerous in older birds. Quantitative analysis of these differences provides three independent numerical indices of the dystrophic state and suggests that constraints responsible for normal patterning are lost in diseased fibers. These observations support theories that defects of the muscle plasma membrane are important for dystrophic pathogenesis. In contrast, the sarcolemma of slow tonic fibers from anterior latissimus dorsi (ALD) and metapatagialis latissimus dorsi (MLD) muscles have randomly dispersed caveolae whose appearance and distribution are unaffected by the disease.