Theoretical versus experimental resolution in optical microscopy

KOZUBEK, Michal. Theoretical versus experimental resolution in optical microscopy. Microscopy research and technique. USA: Wiley-Liss, 2001, vol. 53, No 2, p. 157-166. ISSN 1059-910X. Available from: https://doi.org/10.1002/jemt.1080.

Basic information

Original name

Theoretical versus experimental resolution in optical microscopy

Authors

KOZUBEK, Michal

Edition

Microscopy research and technique, USA, Wiley-Liss, 2001, 1059-910X

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Other information

Language

English

Type of outcome

Article in a journal

Field of Study

20200 2.2 Electrical engineering, Electronic engineering, Information engineering

Country of publisher

United States of America

Confidentiality degree

is not subject to a state or trade secret

References:

URL

Impact factor

Impact factor: 2.165

RIV identification code

RIV/00216224:14330/01:00004204

Organization unit

Faculty of Informatics

DOI

https://doi.org/10.1002/jemt.1080

UT WoS

000168393000008

Keywords in English

resolution; confocal microscopy; wide-field microscopy; image processing; fluorescent bead

Tags

cbia-web, confocal microscopy, fluorescent bead, image processing, RESOLUTION, wide-field microscopy

Tags

International impact, Reviewed

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Abstract

In the original language

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 mum, 0.2 mum, and 0.5 mum) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.

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Links

GA202/99/P008, research and development project	Name: Využití analýzy obrazu při studiu struktury interfázního jádra Investor: Czech Science Foundation, Image analysis in the study of interphase nucleus structure
MSM 143300002, plan (intention)	Name: Využití počítačové analýzy obrazu v optické mikroskopii Investor: Ministry of Education, Youth and Sports of the CR, Application of computer image analysis in optical microscopy
VS97031, research and development project	Name: Využití analýzy obrazu při studiu mechanismů vzniku, v diagnostice a pro prevenci závažných onemocnění člověka Investor: Ministry of Education, Youth and Sports of the CR, Image analysis in the study of mechanisms of induction, in diagnosis and for prevention of deleterious human diseases