Moss et al. (2005) describe, in a recent paper, a filter that they use to detect lines. We noticed that the wavelet on which this filter is based is a difference of uniform filters. This filter is an approximation to the second-derivative operator, which is commonly implemented as the Laplace of Gaussian (or Marr-Hildreth) operator. We have compared Moss' filter with (1) the Laplace of Gaussian operator, (2) an approximation of the Laplace of Gaussian using uniform filters and (3) a few common noise reduction filters. The Laplace-like operators detect lines by suppressing image features both larger and smaller than the filter size. The noise reduction filters only suppress image features smaller than the filter size. By estimating the signal-to-noise ratio and mean square difference of the filtered results, we found that the filter proposed by Moss et al. does not outperform the Laplace of Gaussian operator. We also found that for images with extreme noise content, line detection filters perform better than the noise reduction filters when trying to enhance line structures. In less extreme cases of noise, the standard noise reduction filters perform significantly better than both the Laplace of Gaussian and Moss' filter.

The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently stained nuclear protein NuMA in different mammary phenotypes obtained using 3D cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from 3D confocal images. Prominent features of fluorescently stained NuMA were detected by using a previously undescribed local bright feature analysis technique, and their normalized spatial density was calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features when nonneoplastic cells underwent phenotypically normal acinar morphogenesis. Conversely, we did not detect any reorganization of NuMA during formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating nonneoplastic from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues.

The Berkeley Drosophila Transcription Network Project (BDTNP) has developed a suite of methods that support quantitative, computational analysis of threedimensional (3D) gene expression patterns with cellular resolution in early Drosophila embryos, aiming at a more in-depth understanding of gene regulatory networks. We describe a new tool, called PointCloudXplore (PCX), that supports effective 3D gene expression data exploration. PCX is a visualization tool that uses the established visualization techniques of multiple views, brushing, and linking to support the analysis of high-dimensional datasets that describe many genes’ expression. Each of the views in PointCloudXplore shows a different gene expression data property. Brushing is used to select and emphasize data associated with defined subsets of embryo cells within a view. Linking is used to show in additional views the expression data for a group of cells that have first been highlighted as a brush in a single view, allowing further data subset properties to be determined. In PCX, physical views of the data are linked to abstract data displays such as parallel coordinates. Physical views show the spatial relationships between different genes’ expression patterns within an embryo. Abstract gene expression data displays on the other hand allow for an analysis of relationships between different genes directly in the gene expression space. We discuss on parallel coordinates as one example abstract data view currently available in PCX. We have developed several extensions to standard parallel coordinates to facilitate brushing and the visualization of 3D gene expression data.

A polymerizable composition suitable for moulding under heat and/or pressure comprises a polyvinyl halide, e.g. chloride, bromide or iodide or a copolymer of a vinyl halide with vinyl acetate, propionate or butyrate, plasticized with a mixture of (1) an unsaturated alkyd resin made from an alpha-unsaturated alpha-beta polycarboxylic acid and a polyhydric alcohol, e.g. diethylene glycol maleate resin; (2) an allyl or substituted allyl polyester of an aromatic polycarboxylic acid copolymerizable with the alkyd resin, e.g. diallyl phthalate; (3) a non-polymerizable plasticizer for the vinyl halide resin and optionally a polymerization catalyst. The alkyd resin may be made from acids such as maleic, monohalomaleic, fumaric, monohalofumaric, citraconic, mesaconic, acetylene dicarboxylic, aconitic, and itaconic acid, and alcohols such as ethylene glycol, di-, tri- and tetra-ethylene glycols, propylene glycol, trimethylene glycol, thiodiglycol, glycerine, and pentaerythritol. The polyester may be made from allyl, methallyl, 2-chlorallyl or 2-phenyl allyl alcohols and an acid such as tetrachlorophthalic, 4-chlorophthalic, phthalic, benzoyl phthalic, terephthalic, isophthalic, benzophenone-2,4 dicarboxylic or naphthalic acids. Specified plasticizers are tricresyl phosphate, dibutyl sebacate, dibenzyl sebacate, dioctyl phthalate, dibutyl phthalate, dioctyl sebacate and isophorone. Catalysts mentioned are benzoyl, acetyl, lauryl, stearyl, barium, and sodium peroxides, ozone, ozonides, perborates, persulphates, perchlorates, t-butyl perbenzoate and ditertiary butyl perphthalate. The compositions may include fillers, pigments, dyes, opacifiers, mould lubricants and colour stabilizers and may be made into sheets and used in insulated electrical conductors and cables.

Quantitative analysis of spatial and temporal concurrent responses of multiple markers in 3-dimensional cell cultures is hampered by the routine mode of sequential image acquisition, measurement and analysis of specific targets. A system was developed for detailed analysis of multi-dimensional, time-sequence responses and in order to relate features in novel and meaningful ways that will further our understanding of basic biology. Optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating as described by Lanni. The automated microscopy system can image multicellular structures and track dynamic events, and is equipped for simultaneous/ sequential imaging of multiple fluorescent markers. Computer-controlled perfusion of external stimuli into the culture system allows (i) real-time observations of multiple cellular responses and (ii) automatic and intelligent adjustment of experimental parameters. This creates a feedback loop in real-time that directs desired responses in a given experiment. On-line image analysis routines provide cell-by-cell measurement results through segmentation and feature extraction (i.e. intensity, localization, etc.), and quantitation of meta-features such as dynamic responses of cells or correlations between different cells. Off-line image and data analysis is used to derive models of the processes involved, which will deepen the understanding of the basic biology.

The development of an animal embryo is orchestrated by a network of genetically determined, temporal and spatial gene expression patterns that determine the animals final form. To understand such networks, we are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within pregastrula Drosophila. Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy. Innovative steps have been made which allow the DNA-image to be automatically segmented to produce a morphological mask of the individual nuclear boundaries. For each stage of development an average morphology is chosen to which images from different embryo are compared. The morphological mask is then used to quantify gene-product on a per nuclei basis. What results is an atlas of the relative amount of the specific gene product expressed within the nucleus of every cell in the embryo at the various stages of development. We are creating a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. Our goal is to uncover the rules determining how patterns of gene expression are generated.

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology., deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.

Advancements in image analysis shave recently made it possible to segment the cells and nuclei, of a wide variety of tissues, from 3D images collected using fluorescence confocal microscopy. This has made it possible to analyze the spatial organization of individual cells and nuclei within the natural tissue context. We present here a spatial statistical method which examines an arbitrary 3D distribution of cells of two different types and determines the probability that the cells are randomly mixed, cells of one type are clustered, or cells of different types are preferentially associated. Beginning with a segmented 3D image of cells, the Voronoi diagram is calculated to indicate the nearest neighbor relationships of the cells. Then, in a test image of the same topology, cells are randomly assigned a type in the same proportions as in the actual specimen and the ratio of cells with nearest neighbors of the same type versus the other types is calculated. Repetition of this random assignment is used to generate a distribution function which is specific for the tissue image. Comparison of the ratios for the actual sample to this distribution assigns probabilities for the conditions defined above. The technique is being used to analyze the organization of genetically normal versus abnormal cells in cancer tissue.

While advances in science and technology have increased options for treating breast cancer, current social trends have changed the way people deal with this disease.Women in the United States are no longer simply passive patients, but rather they are survivors, advocates and activists who are speaking up for themselves and speaking out for issues relevant to the treatment and prevention of breast cancer.As the discoveries of basic science have been translated to better clinical treatment, a new sense of hope has emerged.Quality of life now shares the spotlight with quantity of life as breast cancer has shifted from an acute to a chronic condition and as the numbers of long-term survivors increase.While this new population tends to have more optimistic expectations for survival, they are also expressing concerns about issues affecting their lives through and beyond treatment.These issues include, but are not limited to, such concerns as efficient and accurate diagnosis, the complexity of treatment decisions, access to quality cancer care, informed consent, privacy issues, availability of supportive care treatments, and effective communication skills, especially with their physicians.Survivors are also concerned about the impact of their disease on spouses and family, on fertility and sexuality issues, on their employment and (in the USA) insurability, and on their long-term survival.The identification of these increasing issues has given rise to a consumer movement that encourages a shift away from powerless victim to empowered survivor.Historically, breast cancer advocates asked for increased educational and supportive care resources.As the survivorship movement matured, new responsibilities and differing agendas arose amongst these groups.Some organizations defined their mission as one that would raise funds to support scientific research.Others felt compelled to raise awareness about early detection and treatment, controversial environmental issues, and prevention or risk reduction.A few organizations later entered the more political arenas and began lobbying for issues related to health care delivery, clinical trials access, and quality cancer care.Meanwhile, these many and varied missions are all helping to define an international agenda for breast cancer research and care, to guarantee the inclusion of consumer voices in most levels of decision-making, and to create partnerships between patients with breast cancer and the professionals who care for them.

A mechanically stable association between the membrane bilayer and the underlying membrane associated skeleton is important for maintaining the integrity of the plasma membrane. This is particularly true for red blood cells, which must maintain their physical integrity and deformability within the dynamic environment of the vasculature. To probe the molecular mechanisms that account for membrane stability, we have combined a magnetic force transducer with fluorescence imaged microdeformation to measure the forces required to separate the membrane bilayer by pulling thin lipid cylinders, or tethers, off of the cell surface and observed the response of integral membrane proteins to bilayer separation. The results from these experiments indicate that, over a period of up to 30 minutes, the tethering force decreases until it reaches an equilibrium value of 54.7/spl plusmn/8.3 pN while the surface density of fluorescently labeled integral proteins on the cell body is increased after tether formation.

Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate–anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-αAQP1) in the undeformed red cell membrane is 66±10% and the average lateral diffusion coefficient is (3.1±0.5)×10−11cm2/s. F-αAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-αAQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, ∼50% of the aspirated F-αAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-αAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6×10−10cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin-based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.

Confocal microscopy is revealing associations between the internal organization of the nucleus and tissue architecture and function. Such associations may exist which are too subtle or complex for visual observation or quantitative analysis may be required, for example as input data to mathematical modeling of cellular processes. In these circumstances, it is necessary to perform the analysis using computer algorithms. We have developed 3D image analysis (IA) algorithms for segmenting nuclei from within intact tissue specimens, measuring the structure of the nuclei and for segmenting specifically- labeled punctate entities within nuclei. In this study we developed algorithms for measuring the spatial organization of the two copies of a specific DNA locus (labeled using fluorescence in situ hybridization (FISH) ) inside diploid nuclei and with respect to the nuclear organization of the tissue. For each segmented nucleus, its center of mass (CoM) was determined, which informed us about its position in the tissue.

The human erythrocyte membrane comprises many different biological macromolecules arranged into a cohesive two dimensional structure. It has long been a model system for studying cell membrane component interactions and their related underlying cell biology. Erythrocyte membrane rigidity emanates from its hexagonal semi-solid cytoskeletal network of spectrin polymers joined at junctional complexes by globular proteins. The network supports a 2D fluid double layer of lipid in which is dissolved a large array of receptor proteins some of which completely span the lipid bilayer and link to the underlying cytoskeletal network. To study membrane component - component interactions, we have used the technique of fluorescence imaged microdeformation (1). This technique combines fluorescence labeling of specific membrane components, single cell microdeformation and state-of-the-art image collection and analysis to map the distribution of labeled components on the surface of the cell.