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Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion
Umeå University, Faculty of Science and Technology, Physics.
Umeå University, Faculty of Science and Technology, Applied Physics and Electronics.ORCID iD: 0000-0002-9031-4331
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Science and Technology, Physics.
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2004 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, Vol. 19, no 11, 1429-1437 p.Article in journal (Refereed) Published
Abstract [en]

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle’s displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads

Place, publisher, year, edition, pages
2004. Vol. 19, no 11, 1429-1437 p.
Keyword [en]
Force measurements, Optical tweezers, Bacterial adhesion
Identifiers
URN: urn:nbn:se:umu:diva-2747DOI: 10.1016/j.bios.2003.12.029OAI: oai:DiVA.org:umu-2747DiVA: diva2:141006
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2016-05-27Bibliographically approved
In thesis
1. Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles
Open this publication in new window or tab >>Construction of force measuring optical tweezers instrumentation and investigations of biophysical properties of bacterial adhesion organelles
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Optical tweezers are a technique in which microscopic-sized particles, including living cells and bacteria, can be non-intrusively trapped with high accuracy solely using focused light. The technique has therefore become a powerful tool in the field of biophysics. Optical tweezers thereby provide outstanding manipulation possibilities of cells as well as semi-transparent materials, both non-invasively and non-destructively, in biological systems. In addition, optical tweezers can measure minute forces (< 10-12 N), probe molecular interactions and their energy landscapes, and apply both static and dynamic forces in biological systems in a controlled manner. The assessment of intermolecular forces with force measuring optical tweezers, and thereby the biomechanical structure of biological objects, has therefore considerably facilitated our understanding of interactions and structures of biological systems.

Adhesive bacterial organelles, so called pili, mediate adhesion to host cells and are therefore crucial for the initial bacterial-cell contact. Thus, they serve as an important virulence factor. The investigation of pili, both their biogenesis and their expected in vivo properties, brings information that can be of importance for the design of new drugs to prevent bacterial infections, which is crucial in the era of increased bacterial resistance towards antibiotics.

In this thesis, an experimental setup of a force measuring optical tweezers system and the results of a number of biomechanical investigations of adhesive bacterial organelles are presented. Force measuring optical tweezers have been used to characterize three different types of adhesive organelles under various conditions, P, type 1, and S pili, which all are expressed by uropathogenic Escherichia coli. A quantitative biophysical force-extension model, built upon the structure and force response, has been developed. It is found, that this model describes the biomechanical properties for all three pili in an excellent way. Various parameters in their energy landscape, e.g., bond lengths and transition barrier heights, are assessed and the difference in behavior is compared. The work has resulted in a method that in a swift way allows us to probe different types of pili with high force and high spatial resolution, which has provided an enhanced understanding of the biomechanical function of these pili.

Abstract [sv]

Optisk pincett är en teknik i vilken mikrometerstora objekt, inkluderande levande celler och bakterier, beröringsfritt kan fångas och förflyttas med hög noggrannhet enbart med hjälp av ljus. Den optiska pincetten har därmed blivit ett kraftfullt verktyg inom biofysiken, som möjliggör enastående precisions-manipulering av celler och semi-transparenta objekt. Dessutom kan denna manipulation göras intracellulärt, dvs. utan att fysiskt öppna eller penetrera cellernas membran. Den optiska pincetten kan även mäta mycket små krafter och interaktioner (< 10-12 N) samt applicera både statiska och dynamiska krafter i biologiska system med utmärkt precision. Optisk pincett är därför en utmärkt teknik för mätning av intermolekylära krafter och för bestämning av biomekaniska strukturer och dess funktioner.

Vissa typer av bakterier har specifika vidhäftningsorganeller som kallas för pili. Dessa förmedlar vidhäftningen till värdceller och är därför viktiga vid bakteriens första kontakt. En djupare förståelse av pilis uppbyggnad och biomekanik kan därmed ge information, som kan vara vital i framtagandet av nya mediciner som förhindrar bakteriella infektioner. Detta är av stor vikt i skenet av den ökande antibiotikaresistensen i vårt samhälle.

I denna avhandling presenteras konstruktionen av en experimentell uppställning av kraftmätande optiskt pincett tillsammans med resultat från biomekaniska undersökningar av vidhäftande bakteriella organeller. Kraftmätande optisk pincett har använts för att karakterisera tre olika typer av pili, P, typ 1, och S pili, vilka kan uttryckas av uropatogena Escherichia coli. En kvantitativ biofysikalisk modell som beskriver deras förlängningsegenskaper under pålagd kraft har konstruerats. Modellen bygger på pilis strukturella uppbyggnad samt på dess respons som uppmäts med den kraftmätande optiska pincetten. Modellen beskriver de biomekaniska egenskaperna väl för alla tre pili. Dessutom kan ett antal specifika bindnings- och subenhetsparametrar bestämmas, t.ex. interaktionsenergier och bindningslängder. Skillnaden mellan dessa parametrar hos de tre pilis samt deras olika kraftrespons har jämförts. Detta arbete har dels resulterat i en förbättrad förståelse av pilis biomekaniska funktion och dels i en metod som, med hög noggrannhet, tillåter oss att bestämma ett antal biomekaniska egenskaper hos olika organeller på ett effektivt sätt.

Place, publisher, year, edition, pages
Umeå: Fysik, 2007. 79 p.
Keyword
optical tweezers, biological physics, unfolding, Escherichia coli, force measurements, energy landscape, dynamic force spectroscopy, manipulation, polymers, pili
National Category
Physical Sciences
Identifiers
urn:nbn:se:umu:diva-1425 (URN)978-91-7264-435-9 (ISBN)
Public defence
2007-11-30, N450, Naturvetarhuset, Umeå Universitet Campus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2009-08-19Bibliographically approved

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Fällman, ErikSchedin, StaffanAndersson, MagnusUhlin, Bernt EricAxner, Ove

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