首页> 外文期刊>Journal of Microscopy >Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.
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Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.

机译:减少荧光显微镜中自发荧光干扰作用的技术:改进动脉组织中磺基若丹明B标记白蛋白的检测。

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摘要

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 degrees C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.
机译:通过将数字成像荧光显微镜应用于就地固定的动脉部分来测量循环磺基若丹明B标记的白蛋白向动脉壁的运输,其灵敏度受到示踪荧光水平低和自体荧光水平高的限制,从而限制了灵敏度。组织。描述了提高这些比率的三种尝试。首先,溶液中的示踪剂光谱和动脉自发荧光光谱用于设计新型的若丹明样染料的显微镜滤光片。通过激发极少使用的水银弧光灯的黄线并检测斯托克斯位移小至15 nm的窄发射带,示踪荧光与自发荧光的比率增加了三倍。第二,研究了不同固定剂的作用。使用模型系统,已确认Karnovsky的固定剂具有良好的示踪剂固定性,但可提高自发荧光,而不含固定剂的缓冲液的自发荧光性低,但不保留示踪剂。进一步发现,迄今被认为是白蛋白的固定剂较弱的简单的甲醛基固定剂,固定了示踪剂和戊二醛基固定剂,同时其自发荧光水平可与仅使用缓冲液时的水平相当。因此,它们具有出色的示踪荧光与自发荧光比率。第三,发现将样品温度降低50摄氏度会使示踪荧光强度增加30%,而自发荧光不受影响。这些数据可能与使用其他组织和荧光示踪剂的显微镜研究有关。

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