期刊
JOURNAL OF MICROSCOPY
卷 216, 期 -, 页码 15-24出版社
WILEY
DOI: 10.1111/j.0022-2720.2004.01390.x
关键词
fluorescence microscopy; fluorescence photobleaching; shading correction
类别
A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non-uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach-rate-related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.
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