Computer-aided Microscopy Research Articles

Imaging Chromosome Separation in Mouse Oocytes by Responsive 3D Confocal Timelapse Microscopy.

Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.

Fractalkine/CX3CL1 protects striatal neurons from synergistic morphine and HIV-1 Tat-induced dendritic losses and death

BackgroundFractalkine/CX3CL1 and its cognate receptor CX3CR1 are abundantly expressed in the CNS. Fractalkine is an unusual C-X3-C motif chemokine that is important in neuron-microglial communication, a co-receptor for HIV infection, and can be neuroprotective. To assess the effects of fractalkine on opiate-HIV interactive neurotoxicity, wild-type murine striatal neurons were co-cultured with mixed glia from the striata of wild-type or Cx3cr1 knockout mice ± HIV-1 Tat and/or morphine. Time-lapse digital images were continuously recorded at 20 min intervals for up to 72 h using computer-aided microscopy to track the same cells repeatedly.ResultsCo-exposure to Tat and morphine caused synergistic increases in neuron death, dendritic pruning, and microglial motility as previously reported. Exogenous fractalkine prevented synergistic Tat and morphine-induced dendritic losses and neuron death even though the inflammatory mediator TNF-α remained significantly elevated. Antibody blockade of CX3CR1 mimicked the toxic effects of morphine plus Tat, but did not add to their toxicity; while fractalkine failed to protect wild-type neurons co-cultured with Cx3cr1-/--null glia against morphine and Tat toxicity. Exogenous fractalkine also normalized microglial motility, which is elevated by Tat and morphine co-exposure, presumably limiting microglial surveillance that may lead to toxic effects on neurons. Fractalkine immunofluorescence was expressed in neurons and to a lesser extent by other cell types, whereas CX3CR1 immunoreactivity or GFP fluorescence in cells cultured from the striatum of Cx3cr1-/- (Cx3cr1GFP/GFP) mice were associated with microglia. Immunoblotting shows that fractalkine levels were unchanged following Tat and/or morphine exposure and there was no increase in released fractalkine as determined by ELISA. By contrast, CX3CR1 protein levels were markedly downregulated.ConclusionsThe results suggest that deficits in fractalkine-CX3CR1 signaling contribute to the synergistic neurotoxic effects of opioids and Tat. Importantly, exogenous fractalkine can selectively protect neurons from the injurious effects of chronic opioid-HIV-1 Tat co-exposure, and this suggests a potential therapeutic course for neuroAIDS. Although the cellular mechanisms underlying neuroprotection are not certain, findings that exogenous fractalkine reduces microglial motility and fails to protect neurons co-cultured with Cx3cr1-/- mixed glia suggest that fractalkine may act by interfering with toxic microglial-neuron interactions.

Observer Variability in the Interpretation of HER2/neu Immunohistochemical Expression With Unaided and Computer-Aided Digital Microscopy

Observer variability in digital microscopy and the effect of computer-aided digital microscopy are underexamined areas in need of further research, considering the increasing use and future role of digital imaging in pathology. A reduction in observer variability using computer aids could enhance the statistical power of studies designed to determine the utility of new biomarkers and accelerate their incorporation in clinical practice. To quantify interobserver and intraobserver variability in immunohistochemical analysis of HER2/neu with digital microscopy and computer-aided digital microscopy, and to test the hypothesis that observer agreement in the quantitative assessment of HER2/neu immunohistochemical expression is increased with the use of computer-aided microscopy. A set of 335 digital microscopy images extracted from 64 breast cancer tissue slides stained with a HER2 antibody, were read by 14 observers in 2 reading modes: the unaided mode and the computer-aided mode. In the unaided mode, HER2 images were displayed on a calibrated color monitor with no other information, whereas in the computer-aided mode, observers were shown a HER2 image along with a corresponding feature plot showing computer-extracted values of membrane staining intensity and membrane completeness for the particular image under examination and, at the same time, mean feature values of the different HER2 categories. In both modes, observers were asked to provide a continuous score of HER2 expression. Agreement analysis performed on the output of the study showed significant improvement in both interobserver and intraobserver agreement when the computer-aided reading mode was used to evaluate preselected image fields. The role of computer-aided digital microscopy in reducing observer variability in immunohistochemistry is promising.

The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time

BackgroundTumor-associated macrophages (TAMs) play an important role in growth, progression and metastasis of tumors. In non-small cell lung cancer (NSCLC), TAMs' anti-tumor or pro-tumor role is not determined. Macrophages are polarized into M1 (with anti-tumor function) and M2 (with pro-tumor function) forms. This study was conducted to determine whether the M1 and M2 macrophage densities in NSCLC are associated with patient's survival time.MethodsFifty patients with an average of 1-year survival (short survival group) and 50 patients with an average of 5-year survival (long survival group) were included in this retrospective study. Paraffin-embedded NSCLC specimens and their clinicopathological data including up to 8-year follow-up information were used. Immunohistochemical double-staining of CD68/HLA-DR (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded fashion. The M1 and M2 macrophage densities in the tumor islets, stroma, or islets and stroma were determined using computer-aided microscopy. Correlation of the macrophage densities and patient's survival time was analyzed using the Statistical Package for the Social Sciences.ResultsApproximately 70% of TAMs were M2 macrophages and the remaining 30% were M1 macrophages in NSCLC. The M2 macrophage densities (approximately 78 to 113 per mm2) in the tumor islets, stroma, or islets and stroma were not significantly different between the long survival and short survival groups. The M1 macrophage densities in the tumor islets (approximately 70/mm2) and stroma (approximately 34/mm2) of the long survival group were significantly higher than the M1 macrophage densities in the tumor islets (approximately 7/mm2) and stroma (13/mm2) of the short survival group (P < 0.001 and P < 0.05, respectively). The M2 macrophage densities were not associated with patient's survival time. The M1 macrophage densities in the tumor islets, stroma, or islets and stroma were positively associated with patient's survival time in a univariate analysis (P < 0.01 or 0.001). In a multivariate Cox proportional hazards analysis, the M1 macrophage density in the tumor islets was an independent predictor of patient's survival time.ConclusionsThe M1 macrophage density in the tumor islets is an independent predictor of survival time in NSCLC patients.

The Role of Computer-Aided 3-Dimensional Analytic Tools and Virtual Microscopy in the Investigation of Radiologic-Pathologic Correlation

To cope with recent advances in radiologic imaging technology, a corresponding method for pathomorphologic demonstration should be developed to promote better understanding of radiologic-pathologic correlation. We attempted to obtain gross and microscopic images by using a 3-dimensional analytic tool and virtual microscopy and to link these images with multidetector computed tomography images. Surgically resected specimens were sliced to a thickness of 3 mm, and the digital images of each slice were 3-dimensionally reconstructed with RATOC TRI/3D SRF II software. Histology slides were digitized by using virtual microscopy with an Olympus VS-100. We obtained clear gross pathologic images in arbitrary cut sections of organs, and it was possible to rotate these 3-dimensional images at any angle. Furthermore, we created synchronous cut-section movies of computed tomography and gross pathologic images. Subsequently, we switched from these cut-section movies to virtual microscopy images by clicking on the hyperlink button to observe radiologic-pathologic correlation.

The viability assessment of ethanol-producing yeast by computer-aided fluorescence microscopy

Vital staining of the ethanol-producing yeast Saccharomyces cerevisiae with ethidium bromide and DAPI allows intact and damaged cells to be differentiated by fluorescence microscopy. A computer image analysis procedure has been developed for the automatic determination of the relative number of damaged cells using ImageJ software (National Institute of Health, United States; http://rsb.info.nih.gov./ij/). A good correlation has been found between the viability rates determined by the plate count method and the relative numbers of intact cells assessed by the developed procedure in the dry-yeast preparations rehydrated under various conditions.

Surface analyses of archaeological objects - Some new perspectives with Laser Scanning Microscopy

The development of computer-aided microscopy opens new opportunities for the documentation of anthropogenious surface modification of archaeological finds. Laser Scanning Microscopes contribute significantly to advances in quantifying the surface topography of objects, as is shown here on two kinds of human modified finds : The first example deals with cutmarks on animal bones, shown here on some very remarkable objects from the Middle Pleistocene site of Bilzingsleben. The main focus is on bones with regular engravings associated with intentional activity of early humans. Characterising the cuts this way reveals new indications of the amazing regularity, probably on every bone produced by the same cutting edge of a tool. Surface modifications of cutting edges on flint tools were also investigated, in order to differentiate possible usewear from sedimentological alteration. Measurment of roughness becomes possible within well-defined technical parameters. Given the technical potential of LSM there very promising data-based systems for quantifying modifications on both lithic artefacts and bones can be developed.

Digital Microscopy Imaging in Drug Discovery and Development

Digital microscopy, the integration of digital and microscopy technologies, was initiated for quantitative microscopic image analysis, but it is now for almost all microscopy applications. During the past decade, with the advance of digital technologies, digital microscopy imaging is becoming an indispensable technology in drug discovery.We started establishing state-of-the-art digital microscopy imaging for drug discovery with the investigation of bioimaging applications at our US research site. Our results shown that all the top 5 bioimaging needs require computer-aided microscopy. Based on this investigation and our review of the microscopy imaging applications in the pharmaceutical industry, we determined four directions for microscopy in drug discovery: multidimensional/multimodal microscopy, digitalization, automation, and bioimage informatics.Multidimensional/multimodal microscopy imaging is required by the nature of biological research, which is fundamental in drug discovery. From genomic imaging to pathology observation, we require biological details and compound activities at the levels from subcellular organelles to organ tissues, from cellular signaling to anatomical locations of compounds.

Computer-aided epiluminescence microscopy of pigmented skin lesions: the value of clinical data for the classification process.

Early melanoma is often difficult to differentiate from benign pigmented skin lesions (PSLs). Digital epiluminescence microscopy (DELM) and automated image analysis could represent possible aids for inexperienced clinicians. We designed an automated computerized image analysis system that has the potential for use as an additional tool for the differentiation of melanoma from dysplastic naevi and common naevi. The PC-based pilot system was attached to a common DELM system as the image source. Digital images of PSLs were automatically segmented and a panel of 107 morphological parameters were measured. Additionally, seven clinical parameters were evaluated and used as an additional source of information. Neural networks were then trained to distinguish melanoma from benign PSLs. One class of networks was trained solely based on the morphometric features, whereas the second class of networks was trained on the combination of morphometric and clinical features. The automatic segmentation algorithm was correct in 96% of cases. Using three-way receiver operating characteristic (ROC) analysis, for networks trained solely on morphometric features the volume under surface (VUS) was 0.617 (SD 0.036). The performance was significantly better for networks trained on the combination of both morphometric and clinical features (VUS = 0.682, SD 0.035). In a dichotomous model, distinguishing benign lesion (common naevi + dysplastic naevi) from melanoma, the area under the curve (AUC) from two-way ROC analysis was 0.942 (SD 0.018) for networks trained solely on morphometric features and 0.968 (SD 0.012) for those trained on the combination of clinical and morphometric data (P= NS). Automated feature extraction from PSLs and the training of neural networks as classifiers has thus shown satisfactory performance in a large scale experiment. The addition of clinical data significantly increases the diagnostic performance for distinguishing three classes of lesions (i.e. common naevi, dysplastic naevi and melanoma). Such integrated systems hold promise as a decision aid for the diagnosis of PSLs.

Entorhinal cortex of the rat: Topographic organization of the cells of origin of the perforant path projection to the dentate gyrus

By using three-dimensional computer reconstruction techniques and the production of two-dimensional unfolded maps, we analyzed the topographic organization of projections from the entorhinal cortex of the rat to the dentate gyrus. The retrograde tracers, Fast blue and Diamidino yellow, were injected at all septotemporal levels of the dentate gyrus, and the distribution of retrogradely labeled layer II cells in the entorhinal cortex was plotted by using computer-aided microscopy systems. Discrete injections of fluorescent dyes into the dentate gyrus labeled bands of layer II neurons in the entorhinal cortex that covered approximately 45% of its surface area. Injections confined to the septal half of the dentate gyrus resulted in a band that occupied the most lateral and caudomedial portions of the entorhinal cortex. Although there were subtle changes in the density of labeled cells in this region, essentially the same region of cells was labeled after any injection into the septal half of the dentate gyrus. Injections into mid-septotemporal levels of the dentate gyrus (50-75% of the distance from the septal pole) led to a distinctly different pattern of retrograde labeling. A more medial portion of the lateral entorhinal cortex and a more rostral portion of the medial entorhinal area were labeled in these cases. Another change in entorhinal labeling occurred when the injection involved the most temporal quarter of the dentate gyrus. Injections into this area led to a constrained region of entorhinal labeling that included the most medial portion of the lateral entorhinal area and the most rostral portion of the medial entorhinal area. Although the domains of cells projecting to septal, mid-septotemporal, and temporal levels of the dentate gyrus were not entirely segregated, there was relatively little overlap of the three populations of neurons. These data raise the possibility that different portions of the entorhinal-hippocampal circuit are capable of semiautonomous information processing, at least at the stage of input to the dentate gyrus.

A Trend Towards Computer Aided Microscopy

Abstract As the dawn of the next millennium approaches, the light microscope enters its fifth century of use, while the personal computer barely enters its third decade. When Antony van Leeuwenhoek peered through his self made hand crafted microscope and documented the first observations of bacteria and cells, much of microbiology and cell biology was born. When the personal computer became a tool in modern science, molecular cell biologists were well into the age of human genetic engineering. While the rise and acceptance into scientific importance of the microscope and the personal computer represent vastly different periods of sophistication in modem science, the combination of these two important scientific tools for computer aided microscopy (CAM) clearly provides bold uncharted potentials and opportunities for medicine, biomedical research, and industry in the future century to come.

Statistical analysis of spatial patterns of the heliozoanActinophrys sol

Cell-to-cell interaction and spatial distribution of the heliozoanActinophrys sol was analyzed with computer-aided video microscopy. By means of goodness-of-fit statistics (χ2 analysis) and a quadrat-count analysis (Iδ-curve analysis), the spatial point pattern of the cells was shown to be of regular distribution, which implies that a regulating mechanism is operating to encourage an even spatial distribution of the cell centers ofActinophrys. An attempt was further made to define a unified model which fitsActinophrys cell distribution observed at different cell densities. For this purpose, the fitting of a parameterized potential function φ (r)=(σ/r)12 was carried out, wherer is the distance between cell centers of two neighboring cells. The scaling parameter a was estimated from the maximum likelihood procedure for obtaining the best fit for the data, which was found to be a decreasing function of the cell density; we obtained σ = 0.44 mm at a low cell density (0.5 cell/mm2) and σ=0.10 mm at the highest cell density (6.5 cells/mm2). These results suggest that (1) the possible nearest distance between two neighboring cells is primarily defined by the axopodial length, and (2) at lower cell densities,Actinophrys can recognize the presence of distant neighboring cells by some unknown means.

Four-fold aperture synthesis, three-fold astigmatism, and two-fold resolution improvement: Computer-aided microscopy comes of age

Virtually no electron microscopes are now without internal computer control at least, and externalcomputer support continues to play a more important part every year, fuelled by the hundred-fold increase in computing power over the past five years and the continuing if slow advance in the availability of CCD camera systems for TEM. High resolution electron microscopy is within sight of becoming a well-defined, reproducible, technique in which the variability will be restricted at last to the specimen under investigation. The choice of computer systems has narrowed to Macs, Windows on PCs, or Unixworkstations; the future may be with Windows NT.Image restoration (recovering the specimen exit plane wavefunction) is again the subject of considerable interest as a route to higher resolution, in parallel with biprism holography, and incoherent imaging and ptychography in STEM; and the inverse scattering problem (deducing atomic positions from the scattered wave leaving the specimen) is under active investigation.

Integrated spatial and orientation analysis of quartz c-axes by computer-aided microscopy

A new method for the complete determination of quartz c-axis orientations is proposed. Its primary aim is to integrate the analysis of crystallographic orientations and the spatial distribution of the lattice orientations within the fabric in the form of high resolution AVA (Achsenverteilungsanalyse). The method is based on standard mineral optics and image analysis techniques. It uses a slightly modified polarizing microscope, a surveillance camera and a computer (a universal stage is not necessary). The method has been developed for quartz but can be extended to the analysis of other uniaxial minerals. The primary results of the analysis consist of three logical images which represent: (a) the azimuth and (b) the inclination of the c-axes, and (c) the goodness-of-fit of the analysis at every pixel. Thus, the absolute c-axis orientation is defined at each point of the picture, and can be displayed in colour-coded form (= AVA image). From the azimuth and inclination images, further results are derived. Transferring the orientations to a stereogram, a volume-weighted pole figure is obtained. By calculating the angular mismatch between pixels, i.e. by performing a gradient-filtering in orientation space, high- and low-angle grain boundaries are discriminated. As a consequence, image segmentation, i.e. the identification of grains and grain boundaries, is based on c-axis orientation and not on visual bias. Simultaneous visualization of microstructure and crystallographic orientation greatly facilitates the analysis of deformation fabrics.

Our treatment philosophy of gliomas of the anterior visual pathways

Therapeutic strategy and clinical course of 28 low grade gliomas of the anterior visual tract were analyzed and compared with results of quantitative microscopic examination. Children operated on between 1978 and 1987 were divided into four groups according to the tumor site. DNA microfluorometry and image analysis of bioptic material were used for an objective differentiation between morphologically almost identical gliomas classified as pilocytic astrocytomas (grade I). Six cases of posttraumatic gliosis were examined as controls. Results were tested with factor analysis and by multiple comparison procedures. Significant differences between quantitative parameters of tumors located along the visual pathway were found with the use of computer-aided microscopy. These results corresponded with the clinical experience and patient's outcome. A radical surgical intervention (without additional oncologic therapy) was recommended for patients with intraorbital gliomas which revealed favorable quantitative microscopic features.

Computer-based image analysis of cartilage differentiation in embryonic limb bud micromass cultures.

A computer imaging system was used to analyse the effects of benzamide (BAM), sodium butyrate and dimethyl sulphoxide (DMSO) on chondrogenesis in culture. Embryonic chick limb bud cells were used as a source of undifferentiated mesenchyme, and cultures were prepared using a micromass culture technique. The degree of chondrogenesis is directly related to the amount of Alcian blue staining. Standard assays include either manual tally of individual cartilage nodules, which is both tedious and time-consuming, or extracting bound dye for spectrophotometric analysis, which obscures individual variations because it requires pooled samples. In the present study, low-magnification images of individual micromass colonies were converted into derived images based on their percentage transmittance relative to the background. These derived images were analysed for relative degree of chondrogenesis, using area and integrated optical density (IOD) as descriptors. While both types of data were useful for ranking the degree of chondrogenesis in culture, IOD was the preferred descriptor because of its accuracy over a wide range of threshold values. The area and IOD data both demonstrate that BAM produces marked enhancement of chondrogenesis in culture, sodium butyrate causes marked inhibition and DMSO produces mixed results (enhancement at the high dose, inhibition at the low dose). While the present study demonstrates the usefulness of computer-aided microscopy for analysis of low-magnification images, the same descriptors (area and IOD) should be useful in quantifying data from a variety of objects (cells, nuclei, etc.) which can be stained in a selected, quantitative fashion.

Direct measurements of cell number using computer-aided video microscopy

Quantitative studies in cell culture require accurate measurements of cell density and kinetics. We have developed a direct, rapid, and noninvasive method for measuring cell number in monolayer culture. Using computer-aided video microscopy, cell number was measured without detaching or chemically destroying the cells, thereby allowing sequential measurements in the same cell population. Cell number measured by computer-aided microscopy closely correlated with hemocytometer counts and determinations of total cell protein. For high-density monolayers of mesenchymal cells, however, staining was required for accurate counts. Unlike other techniques for measuring cell density, computer-aided microscopy was especially accurate in medium- to low-density cultures (<6000 cells/cm 2). In addition, we applied this technique to the construction of separate proliferation curves for glomerular mesangial and vascular endothelial cells in coculture. These measurements by cell type in coculture are impossible using conventional methods for determining cell number.

Computer-aided microscopy in medicine: introduction to the 15 August 1987 issue of Applied Optics

New research and development opportunities and advances are evident in the multidisciplinary field of computer-aided microscopy for cell and tissue imaging and analysis. This growth applies to both the development of new and improved scientific methods as well as to the utilization of these techniques to investigate significant biomedical applications. Much of the core and supporting technology in this work involves optical and visual processes. Consequently, progress may be facilitated by stimulating the interaction between the optical scientists/engineers and the applications-oriented investigators. This 15 Aug. 1987 issue of Applied Optics presents a heterogeneous collection of papers in three sections: Scanning microscopy and related optical methodsfor cell and tissue imaging; Image cytometry/histometry systems, methods, and standards; and Biomedical applications of image cytometry and histometry. Since the purposes of this issue are to present a representative international cross-section of current research results and to provide direction and assistance to optical scientists and engineers who are interested in becoming more active in this field, this Introduction also documents relevant peer-reviewed journals and sponsoring societies, recommended didactic reading, and scheduled professional events.

Vitamin A-induced suppression/enhancement of protein glycosylation and neurulation.

Glycoconjugates play major roles in many cellular functions, e.g. cell migration and cell-to-cell adherence, which are involved in neurulation. The maternal administration of vitamin A on gestation day 8.5 and 9.0 resulted in a high percentage of primary and secondary neurulation defects in gestation day 12 mouse embryos. The neuroepithelium of normal and abnormal embryos was analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and one-dimensional Western blots using concanavalin A (Con A) and peroxidase-conjugated wheat germ agglutinin (WGA) lectins. In vitamin A abnormal embryos, WGA binding was decreased to glycoproteins with apparent molecular weights of 15,000 and 30,000 daltons on Western blots, whereas in vitamin A normal embryos, WGA binding was increased to these glycoproteins on Western blots. Computer-aided fluorescence microscopy using fluorescein isothiocyanate (FITC)-conjugated lectins on 1-micron araldite plastic sections indicated a decrease in FITC-WGA binding to the free surface of nonneurulated neuroepithelium. These results suggest: (1) vitamin A administration may have induced a suppression of WGA-binding carbohydrate residues on 15,000- and 30,000-dalton glycoproteins in abnormal embryos, and (2) modification in the type, amount, and distribution of glycoconjugates may provide a basis for the cellular mechanisms of abnormal development of the neural tube.

Measurement of striated muscle fibre diameters using interactive computer-aided microscopy.

We have measured muscle fibre diameters using two methods of interactive computer-aided microscopy. They are simple to perform, reproducible and more convenient than manual methods of measurement. The technique is of general application to histological measurement.