Biomedical subjects

J W Sedat

Publications and source records attributed to J W Sedat.

At least 19 recordsLinked to original sources

PubMed1992

Automated microscopy for electron tomography.

Instrumentation and methodology for the automatic collection of tomographic tilt series data for the three-dimensional reconstruction of single particles is described. The system consists of a Philips EM 430 TEM, with a Gatan 673 cooled slow-scan CCD camera and a Philips C400 microscope computer control unit attached. The procedure for data collection includes direct digital recording of the images on the CCD camera and the automatic measurement and correction of (a) image shifts resulting from tilting the specimen, (b) variation of defocus and (c) the eucentric height position of the specimen. Experiments are described illustrating the possibilities and limitations of automatic data collection. Data collection at a magnification of 30k shows that the exposure time of the specimen to the beam is reduced by a factor of 10-100 compared to manual operation of the TEM.

Artifacts↗

PubMed1992

An extended view of nuclear lamin structure, function, and dynamics.

Molecularly-based studies of nuclear lamins have progressed at a rapid rate in the last decade. However, we still have no answer to the most important question: what are the functions of lamins? In this review we describe recent experiments which challenge traditional views of lamin function and structure. These surprising results indicate that much lamin functionality remains to be discovered, and that more global approaches to lamin structure and function are especially appropriate at this time.

Animals↗

PubMed1991

Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy.

The first event of Drosophila gastrulation is the formation of the ventral furrow. This process, which leads to the invagination of the mesoderm, is a classical example of epithelial folding. To understand better the cellular changes and dynamics of furrow formation, we examined living Drosophila embryos using three-dimensional time-lapse microscopy. By injecting fluorescent markers that visualize cell outlines and nuclei, we monitored changes in cell shapes and nuclear positions. We find that the ventral furrow invaginates in two phases. During the first 'preparatory' phase, many prospective furrow cells in apparently random positions gradually begin to change shape, but the curvature of the epithelium hardly changes. In the second phase, when a critical number of cells have begun to change shape, the furrow suddenly invaginates. Our results suggest that furrow formation does not result from an ordered wave of cell shape changes, contrary to a model for epithelial invagination in which a wave of apical contractions causes invagination. Instead, it appears that cells change their shape independently, in a stochastic manner, and the sum of these individual changes alters the curvature of the whole epithelium.

Animals↗

PubMed1991

Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena.

Cellular events are accomplished by the coordinated interactions of cellular components within the three-dimensional context of a cell. Simultaneous observation of multiple components in three dimensions can be essential for understanding such interactions. Toward this end, we have developed a computerized microscope workstation capable of recording three-dimensional images of multiple cellular components in fixed and living cells. All aspects of microscope control, data collection, image processing and analysis can be performed on the one workstation. In this report, we describe the components and capabilities of this integrated system. In addition, we discuss some general problems of multiple-wavelength, three-dimensional imaging and our application of this technology to the analysis of chromosome organization in Drosophila melanogaster. Three-dimensional imaging of fixed embryos stained by indirect immunofluorescence has revealed the structural organization of chromosomes, microtubules, and the nuclear lamins. Imaging of living embryos injected with fluorescently labelled proteins has confirmed and extended these results by allowing the study of these structures throughout the cell cycle. The combination of the molecular specificity of fluorescence microscopy and the three-dimensional structural information obtained by our workstation has provided novel insights into the dynamic aspects of chromosome behavior during the cell cycle. We believe this system has many important applications in the study of the molecular basis of cellular events.

Animals↗

PubMed1990

Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery.

Antibodies directed against nuclear envelope lamin proteins have been used in conjunction with three-dimensional light and electron microscope methodologies to determine the spatial organization of lamins in diploid interphase nuclei and to relate this organization to the positions of chromatin in the nuclear periphery. Using Drosophila early embryos, Drosophila Kc cells, and human HeLa cells, it is qualitatively and quantitatively observed that lamins are organized as a highly discontinuous, apparently fibrillar network that leaves large voids in the nuclear periphery containing little or no lamin. Using fluorescence microscopy to compare and quantitate the relationship between chromatin and the lamin network, it is found that although there is a strong tendency for the most peripheral chromatin to be positioned directly underneath a lamin fiber, only a small fraction of the chromatin in the nuclear periphery is sufficiently close to a lamin fiber to possibly be in direct contact.

Animals↗

PubMed1990

Determination of three-dimensional imaging properties of a light microscope system. Partial confocal behavior in epifluorescence microscopy.

We have determined the three-dimensional image-forming properties of an epifluorescence microscope for use in obtaining very high resolution three-dimensional images of biological structures by image processing methods. Three-dimensional microscopic data is collected as a series of two-dimensional images recorded at different focal planes. Each of these images contains not only in-focus information from the region around the focal plane, but also out-of-focus contributions from the remainder of the specimen. Once the imaging properties of the microscope system are characterized, powerful image processing methods can be utilized to remove the out-of-focus information and to correct for image distortions. Although theoretical calculations for the behavior of an aberration-free microscope system are available, the properties of real lenses under the conditions used for biological observation are often far from an ideal. For this reason, we have directly determined the image-forming properties of an epifluorescence microscope under conditions relevant to biological observations. Through-focus series of a point object (fluorescently-coated microspheres) were recorded on a charge-coupled device image detector. From these images, the three-dimensional point spread function and its Fourier transform, the optical transfer function, were derived. There were significant differences between the experimental results and the theoretical models which have important implications for image processing. The discrepancies can be explained by imperfections of the microscope system, nonideal observation conditions, and partial confocal effects found to occur with epifluorescence illumination. Understanding the optical behavior of the microscope system has indicated how to optimize specimen preparation, data collection, and processing protocols to obtain significantly improved images.

Microscopy, Fluorescence↗

PubMed1990

Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

Animals↗

PubMed1989

Focal points for chromosome condensation and decondensation revealed by three-dimensional in vivo time-lapse microscopy.

Although the dynamic behaviour of chromosomes has been extensively studied in their condensed state during mitosis, chromosome behaviour during the transition to and from interphase has not been well documented. Previous electron microscopic studies suggest that chromosomes condense in a non-uniform fashion at the nuclear periphery. But chromosome condensation is a complicated and dynamic process and requires continuous observation in living tissues to be fully understood. Using a recently developed three-dimensional time-lapse fluorescence microscopy technique, we have observed chromosomes as they relax from telophase, through interphase, until their condensation at the next prophase. This technique has been improved to produce higher-resolution images by implementing new stereographic projection and computational processing protocols. These studies have revealed that chromosomal regions on the nuclear envelope, distinct from the centromeres and telomeres, serve as foci for the decondensation and condensation of diploid chromosomes. The relative positions of the late decondensation sites at the beginning of interphase appear to correspond to the early condensation sites at the subsequent prophase.

Animals↗

PubMed1989

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro.

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126 +/- 36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 micron thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20-30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.

Animals↗

PubMed1989

Tilted view reconstruction in optical microscopy. Three-dimensional reconstruction of Drosophila melanogaster embryo nuclei.

The resolution along the optical axis (z) is much less than the in-plane resolution in any current optical microscope, conventional or otherwise. We have used mutually tilted, through-focal section views of the same object to provide a solution to this problem. A tilting specimen stage was constructed for an optical microscope, which with the use of a coverslip-free water immersion lens, allowed the collection of data sets from intact Drosophila melanogaster embryos at viewing directions up to 90 degrees apart. We have devised an image processing scheme to determine the relative tilt, translation, and sampling parameters of the different data sets. This involves the use of a modified phase cross-correlation function, which produces a very sharp maximum. Finally the data sets are merged using figure-of-merit and local area scaling techniques borrowed from x-ray protein crystallography. We demonstrate the application of this technique to data sets from a metaphase plate in an embryo of Drosophila melanogaster. As expected, the merged reconstruction combined the highest resolution available in the individual data sets. As estimated from the Fourier transform, the final resolution is 0.25 microns in x and y and 0.4 microns in z. In the final reconstruction all ten chromosome arms can be easily delineated; this was not possible in the individual data sets. Within many of the arms the two individual chromatids can be seen. In some cases the chromatids are wrapped around each other helically, in others they lie alongside each other in a parallel arrangement.

Animals↗

PubMed1989

Solid-state imagers for microscopy.

Solid-state imagers offer a variety of options to the modern optical microscopist. Photodiode arrays, charge injection devices, and charge-coupled devices are the three basic types of solid-state imagers available for research imaging. The charge-coupled device (CCD) is the solid-state imager of choice, because of its superior characteristics and its widespread acceptance in the research environment. The performance characteristics of the CCD are well documented and understood, having been quantified by many experimenters, especially in the physical sciences. CCDs exhibit dynamic ranges up to 50,000:1, very high quantum efficiency, and ultralow noise. The camera system in which a device is used, however, dictates the overall performance which can be achieved. The CCD imaging system as it applies to cell biology is discussed in detail in Hiraoka et al. (1987). A video camera operating at 30 frames/second does not provide the resolution, low noise, dynamic range, and linearity of a slow scan, cooled camera operating at 1 frame/second. Conversely, a slow scan camera does not offer the user the facility to resolve rapidly changing events in real time. The analogy between an 8-mm movie camera (video camera) and a 35-mm snapshot camera (slow scan cooled camera) is a useful one to emphasize the different character of these two electronic imaging systems. These two basic camera systems both employ CCD imagers, and each has very different, but complementary, characteristics. No one camera system can address the wide variety of imaging problems which face the modern microscopist. The user of this new generation of instrumentation must decide which system best fits the problem at hand.

Calibration↗

PubMed1989

Fluorescence microscopy in three dimensions.

The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before. Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity. High-quality, cooled charge-coupled devices have recently become available. Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope. Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing. It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information. The impact of modern video microscopy techniques (Allen, 1985; Inoué, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy. Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated. Cells are intrinsically three-dimensional. This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied. In either case, image processing techniques can be used to rapidly provide the desired representation of the data. In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information. The most straightforward of these methods uses only information from adjacent focal planes to correct the central one. This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time. In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen. This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus. A variation on this method allows efficient calculation of stereo pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos.

One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.

Animals↗

PubMed1989

The three-dimensional organization of polytene nuclei in male Drosophila melanogaster with compound XY or ring X chromosomes.

The three-dimensional organization of polytene chromosomes within nuclei containing rearranged X chromosomes was examined in male Drosophila melanogaster. Salivary glands of third instar larvae containing either an inverted X chromosome (YSX.YL, In(1)EN/O) or a ring X chromosome (R(1) 2/BSYy+) were fixed, embedded, and serially sectioned. The nuclei in contiguous groups of cells were modeled and analyzed. We find that for both genotypes the three-dimensional behavior at each euchromatic locus is independent of the orientation of the chromosome on which it resides, independent of the behavior of loci not closely linked to it, and not similar in neighboring cells. The preference for right-handed chromosome coiling noted in previous studies is shown to be independent of homologous pairing. However, a relation between the extent of chromosome curvature and the handedness of chromosome coiling is present only in homologously paired chromosomes. The attached-XY chromosome has two previously undescribed behaviors: a nearly invariant association of the euchromatic side of the proximal heterochromatin/euchromatin junction with the nucleolus and a frequent failure of this site to attach to the chromocenter. The relative chromosome arm positions are often similar in several neighboring cells. The size of these patches of cells, assuming that they represent clones, indicates that such arrangements are at best quasi-stable: they may be maintained over at least one, but less than four, cell divisions. The observed nuclear organization in salivary glands is inconsistent with the idea that position in the polytene nucleus plays a major role in the normal genetic regulation of euchromatic loci.

Animals↗

PubMed1988

Precise determination of the molecular limits of a polytene chromosome band: regulatory sequences for the Notch gene are in the interband.

We have aligned the molecular map of the Notch locus to the cytological features of the salivary gland polytene chromosomes of D. melanogaster in order to determine the interphase chromatin structure of this gene. Using high-resolution in situ hybridization and computer-aided optical microscope data collection and image analysis, we have determined that the coding portions and introns of the Notch gene, which is not expressed in this tissue, are all contained within the polytene chromosome band 3C7. The portion of the Notch gene that resides 5' to the start of transcription lies in an open chromatin conformation, the interband between bands 3C6 and 3C7. Our data are most consistent with condensation of the chromosomal DNA into 30 nm fibers in this polytene band.

Animals↗

PubMed1988

Three-dimensional light microscopy of diploid Drosophila chromosomes.

Fluorescence microscopy, uniquely, provides the ability to examine specific components within intact, even living, cells. Unfortunately, high-resolution conventional fluorescence microscopy is intrinsically a two-dimensional technique and performs poorly with specimens thicker than about 0.5 micron. Probing the spatial organization of components within cells has required the development of new methods optimized for three-dimensional data collection, processing, display, and interpretation. Our interest in understanding the relationship between chromosome structure and function has led us to develop the necessary methodology for exploring cell structures in three dimensions. It is now possible to determine directly the three-dimensional spatial organization of diploid chromosomes within intact nuclei throughout most of the mitotic the cell cycle.

Animals↗

PubMed1988

Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene.

A cDNA clone encoding a portion of Drosophila nuclear lamins Dm1 and Dm2 has been identified by screening a lambda-gt11 cDNA expression library using Drosophila lamin-specific monoclonal antibodies. Two different developmentally regulated mRNA species were identified by Northern blot analysis using the initial cDNA as a probe, and full-length cDNA clones, apparently corresponding to each message, have been isolated. In vitro transcription of both full-length cDNA clones in a pT7 transcription vector followed by in vitro translation in wheat germ lysate suggests that both clones encode lamin Dm0, the polypeptide precursor of lamins Dm1 and Dm2. Nucleotide sequence analyses confirm the impression that both cDNA clones code for the identical polypeptide, which is highly homologous with human lamins A and C as well as with mammalian intermediate filament proteins. The two clones differ in their 3'-untranslated regions. In situ hybridization of lamin cDNA clones to Drosophila polytene chromosomes shows only a single locus of hybridization at or near position 25F on the left arm of chromosome 2. Southern blot analyses of genomic DNA are consistent with the notion that a single or only a few highly similar genes encoding Drosophila nuclear lamin Dm0 exist in the genome.

Amino Acid Sequence↗

PubMed1987

The use of a charge-coupled device for quantitative optical microscopy of biological structures.

The properties of a charge-coupled device (CCD) and its application to the high-resolution analysis of biological structures by optical microscopy are described. The CCD, with its high resolution, high sensitivity, wide dynamic range, photometric accuracy, and geometric stability, can provide data of such high quality that quantitative analysis on two- and three-dimensional microscopic images is possible. For example, the three-dimensional imaging properties of an epifluorescence microscope have been quantitatively determined with the CCD. This description of the imaging properties of the microscope, and the high-quality image data provided by the CCD, allow sophisticated computational image processing methods to be used that greatly improve the effective resolution obtainable for biological structures. Image processing techniques revealed fine substructures in Drosophila embryonic diploid chromosomes in two and three dimensions. The same approach can be extended to structures as small as yeast chromosomes or to other problems in structural cell biology.

Animals↗