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

Basic information

• Original name: Theoretical versus experimental resolution in optical microscopy
• Authors: KOZUBEK, Michal (203 Czech Republic, guarantor, belonging to the institution).
• Edition: Microscopy research and technique, USA, Wiley-Liss, 2001, 1059-910X.

Other information

• Original 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
• WWW: URL
• Impact factor: Impact factor: 2.165
• RIV identification code: RIV/00216224:14330/01:00004204
• Organization unit: Faculty of Informatics
• Doi: http://dx.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

Abstract

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.

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