Measurement of the width of thin, cylindrical, transparent objects by phase contrast light microscopy6
作者:
Peter Knight,
Nick Parsons,
期刊:
Journal of Microscopy
(WILEY Available online 1985)
卷期:
Volume 137,
issue 1
页码: 65-74
ISSN:0022-2720
年代: 1985
DOI:10.1111/j.1365-2818.1985.tb02562.x
出版商: Blackwell Publishing Ltd
关键词: Phase contrast microscope;electron microscope;glass fibres;cylindrical objects;size measurements;measurement errors;calibration procedure;scanning microdensitometer;myofibrils;bacteria
数据来源: WILEY
摘要:
SUMMARYMeasurement of the width of a thin, cylindrical, transparent object by phase contrast light microscopy has been frustrated by the absence of an established relationship between the true width of the object and its apparent width in the phase contrast image. We have solved this problem by devising a simple method by which individual glass fibres may be measured using both phase contrast light microscopy and electron microscopy. Using this method we have constructed calibration curves relating the diameter measured by phase contrast microscopy to the real diameter of the fibres. These curves are linear in the range 0.10‐2.5 μm real diameter, with slopes close to unity and intercepts of about 0.2 μm. Thus widths of such objects are overestimated. The precise value of the intercept is related to the overall numerical aperture of the optical system. Each calibration curve permits the true width of a cylindrical object to be estimated by phase contrast microscopy with an accuracy of better than ±0.05 μm. We have found that greater precision is obtained by taking measurements of light micrographs subjectively using a microcomparator rather than objectively using a microdensito
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