The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures

Hiraoka, Yasushi; Sedat, John W.; Agard, David A.

doi:10.1126/science.3116667

Cited by 291 publications

(132 citation statements)

References 22 publications

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“…Image stacks of thirty-two 0.2 μm optical sections were collected and deconvolved using software from ISee Imaging (Raleigh, NC), which uses a constrained iterative procedure and a predetermined point spread function [20]. Colocalization of EGFP-rab7 with Alexa594-labeled β 2 ARs was quantified using Metamorph (Universal Imaging Corp., Downingtown, PA).…”

Section: Quantification Of Imaging Analysismentioning

confidence: 99%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

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Phosphatidylinositol 3-kinase inhibitors have been shown to affect the endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β 2 -adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β 2 -adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β 2 -adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β 2 -adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β 2 -adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

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Section: Quantification Of Imaging Analysismentioning

confidence: 99%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

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“…The three-dimensional morphology was assessed by confocal laser scanning microscopy (our unpublished data). Best results were obtained with a modification of a procedure described by Manuelidis (1985) (Arndt -Jovin et al 1985, Hiraoka 1987 or by sections generated with a laser scanning device in which out of focus fluorescence is reduced by means of a confocal diaphragm in the emission pathway (Cremer & Cremer 1978, Brakenhoff et al 1979. In general, confocal microscopy yields a z-axis resolution of ~ 0.5 pm, whereas the resolution in the x-and y-axes is about 0.2 pm (Jovin & Arndt-Jovin 1989, Shotton 1989, Van der Voort & Brakenhoff 1990).…”

Section: ---R1mentioning

confidence: 99%

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

et al. 1993

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The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescence in situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocallaser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional networklike compartment, termed the interchromosome domain (ICO) compartment, in wh ich transcription and splicing of mRNA occurs.

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“…The digitizing device either captures a broad part of the (visible) light, such as with a single-CCD camera or a multispectral image by using an array of detectors, a 3CCD color camera, or more channels for spectral imaging (23,24).…”

Section: Imaging Technologymentioning

confidence: 99%

Extracting quantitative information from tissue—An industrial perspective

Osta¹

2006

Cytometry Pt A

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The focus of this article is to provide an overview of the current technologies for the pharmaceutical and biotech industry. Disease processes express themselves in the functional and structural disturbance of cellular systems. Cells and their metabolites constitute the building blocks of tissues and entire organisms. Studying the spatial and temporal phenotype of disease processes in tissues at the cellular level reveals a multitude of information about the progress and status of a disease. Detailed exploration of tissues by slide‐based cytometry is an important source of information about disease processes. Technological and analytical advances allow us to shed a new light on tissues and to come to a better understanding of the complexity of disease processes. Dealing with complex multidimensional datasets from tissue samples requires an advanced approach to image processing and data management. The increase in computing power and the continuing research into imaging algorithms allow us to improve the exploration of the data content of tissues. © 2006 International Society for Analytical Cytology

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The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures

Cited by 291 publications

References 22 publications

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

Extracting quantitative information from tissue—An industrial perspective

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