The use of polarization analysis in the quantification of fluorescent emission: general principles

Entwistle, Alan; Noble, Mark

doi:10.1111/j.1365-2818.1992.tb01491.x

Cited by 20 publications

(21 citation statements)

References 14 publications

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“…The high density of the dye per substrate may cause fluorescence quenching (Förster and König 1957). This was clearly demonstrated on biological specimens, applying a variety of widely used fluorochromes (Entwistle and Noble 1992).…”

Section: Introductionmentioning

confidence: 85%

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Erenpreisa¹,

Freivalds²,

Roach

et al. 1997

Histochemistry and Cell Biology

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Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause--the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells.

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Section: Introductionmentioning

confidence: 85%

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Erenpreisa¹,

Freivalds²,

Roach

et al. 1997

Histochemistry and Cell Biology

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“…Thc finished images were the average of the images collected with 100 scans of the laser where the samples were illuminated with the 488-nm line of the Ar ion laser and the fluorescent emission filtered with a 540 f 30-nm bandpass filter placed before the pinhole. Immediately following collection, a small bias added to the signal to ensure linearity of response (Entwistle and Noble, 1992) was subtracted and the images saved as the unprocessed images.…”

Section: Confocal Microscopymentioning

confidence: 99%

“…Under these conditions, when the pinhole diameter was set to approximately coincide with the first minimum of the Airy disk of the focus, > 99% of all pixels had grey scale levels within 10% of the mean and > 85% within 5% of the mean when a fluorescein film (2 mM) was examined with the laser set to an intensity of M 0.184 nW s pixel-' scan-' and the image averaged over 20 scans. The Zo value for the vertical point spread function, measured by examining scattered light from 5-nm gold particles (gift of J. Chandler, Bio-cell, Cardiff, UK; Entwistle and Noble, 1990) was 0.6-0.9 m throughout the field and 0.7 -0.8 m over > 80% of the field (Entwistle and Noble, 1992).…”

Section: Confocal Microscopymentioning

confidence: 99%

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Tenascin: a modulator of cell growth

End

Panayotou

Entwistle

et al. 1992

European Journal of Biochemistry

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The large, multidomain extracellular matrix protein tenascin displays a markedly restricted tissue distribution during embryogenesis and remains present only in a few adult tissues. The protein is re‐expressed, however, during wound healing and in the stroma of malignant tumours. While a variety of studies have dealt with the important role of tenascin in the development of neural and non‐neural tissues, there is growing evidence that tenascin expression may be associated with proliferation of cells lining these tissues. The presence of repeating domains in tenascin similar to those in epidermal growth factor prompted us to investigate the ability of tenascin to modulate the growth of different cell types. Tenascin was actually found to be mitogenic for several cell types. This mitogenic activity, however, appears to be associated with a region in the fibronectin type III domains. The mitogenic mechanism is clearly distinct from pathways used by peptide growth factors such as epidermal growth factor and platelet‐derived growth factor, which activate the intrinsic tyrosine kinase activity of their cell‐surface receptors. However, we show that this large extracellular matrix molecule is efficiently internalised and may be processed by responding cells.

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“…These step sizes are too large to permit the accurate determination of such small 2 0 .~ values. Alternative means of examining the axial spread function are to measure the point spread function of emission from gold particles 2-20 nm in diameter (Entwistle & Noble, 1992a) or fluorescent beads c. 40 nm in diameter (Shaw & Rawlins, 1991) where the measured Zo.5 values for the point spread functions are a little larger (0.6-0.8 pm). However, these objects were not considered further as obtaining the relevant information over the whole field of view on a routine basis is unacceptably time consuming.…”

Section: Introductionmentioning

confidence: 99%

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

Entwistle

Noble

1994

Journal of Microscopy

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Summary To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.

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The use of polarization analysis in the quantification of fluorescent emission: general principles

Cited by 20 publications

References 14 publications

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

Tenascin: a modulator of cell growth

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

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