摘要:

SUMMARYFluorescent emission and fluorophore concentration are only linearly related below particular concentrations of fluorophore. Theoretically, analysis of fluorescent polarization might allow identification of situations in which local concentrations of fluorophore are above the range of linear response. Using a confocal scanning laser microscope, we demonstrate that progressive depolarization of fluorescent emission from fluorescein and fluorescein analogues occurs over the concentration range where linearity is lost. Critically, depolarization of emission is first seen at concentrations of fluorophore slightly below those at which linearity is lost. Thus, polarization analysis can be used to determine whether the local concentration of fluorophore is such that quantitative analysis can be carried out.

ISSN:0022-2720    

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

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth‐dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emissio

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01492.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYCell structures, particularly cytoskeletal elements, of tobacco (Nicotiana tabacum L. Var. Maryland Mammoth) leaf tissues were studied after high‐pressure freezing and freeze substitution. In general, the freezing quality was superb and all organelles including microtubules, single microfilaments and microtubule‐associated microfilaments were well preserved. However, when compared with those frozen with a propane jet freezer, the microfilament bundles in high‐pressure frozen cells often had a frayed or loosened appearance. We suggest that such an appearance is an artefact, presumably caused by the high pressure developed prior to fre

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01493.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe three‐dimensional (3‐D) transfer function is a useful concept for describing image formation in confocal scanning microscopy. From it we can derive the corresponding 2‐D transfer function for in‐focus imaging. In confocal transmission this can be derived analytically. The 1‐D transfer function for on‐axis imaging, which can be expressed in an analytical form even for confocal fluorescence with differing wavelengths of excitation and fluorescence, can be derived from the 3‐D transfer function. The 2‐D transfer function for in‐focus imaging in confocal fluorescence microscopy with a finite‐sized detector is also presented, which is shown to exhibit sign changes and can therefore result in reversal

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01494.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA number of simple, unbiased and efficient methods are now available in stereology for estimating the number and size of arbitrary particles or voids in a material, with the only assumption that particles must be identifiable on serial sections or confocal planes through the material. In recent years, these methods have been developed and applied mainly in a biomedical context: this paper reviews and illustrates them with the aid of a metallographic example, namely the pore population of a sand‐cast aluminium alloy. Our goal is to convey the fact that stereology is sampling in three dimensions, and therefore its principles remain valid and applicable in no matter what context. The disector, the selector, and an indirect method to estimate the distance between two parallel planes of polish are thereby illustrated. It is also shown how to split the error variance of the estimator of the pore volume fraction (‘porosity’) into the three components due to blocks, sections within blocks and systematic point counting on sec

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01495.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA double‐disector method for obtaining estimates of the number of particles inside other particles, illustrated by an estimation of the average cell number in a glomerulus, has been developed. The method is suitable for paraffin‐embedded tissue because it does not require knowledge about the section thickness. Most importantly, the estimate is absolutely unaffected by tissue shrinkage. The average number of cells in a human glomerulus is 2850 whereas in rats it is

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01496.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe very simple and strong principle of vertical sections devised by Baddeleyet al. has been a major advance in stereology when any kind of anisotropy is present in the specimen under study. On the other hand, some important stereological estimators still require isotropic, uniform random sections. This paper deals with a simple technique for embedding specimens in rubber moulds with spherical cavities. After the embedding, any handling of the resulting sphere independent of the specimen will induce isotropy of the final histological sections.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01497.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe selector was used to make an unbiased estimation of nuclear size variability in one benign naevocellular skin tumour and one cutaneous malignant melanoma. The results showed that the estimates obtained using the selector were comparable to those obtained using the more time consuming Cavalieri‐disector approach. Employing ‘optical sections’, the selector was found to be between five and ten times more efficient than the Cavalieri‐disector method when using physical s

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01498.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Book reviewed in this article:Eighth Pfefferkorn Conference on Fundamental Electron and Ion Beam Inter‐actions with Solids for Microscopy, Microanalysis, and Microlithography (Scanning Microscopy Supplement no. 4). Edited by Jorgen Schou, Pieter Kruit and Dale E. Newbury.Introduction to Flow Cytometry. By James V. Watso

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01499.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY