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Detecting Bacterial Surface Organelles on Single Cells using Optical Tweezers
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.
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2016 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 32, no 18, p. 4521-4529Article in journal (Refereed) Published
Abstract [en]

Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as: intercellular communication, motility and adhesion leading to biofilm formation, infections and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nm wide and µm long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol, can in real time and within seconds assist single cell studies by distinguishing between piliated and non-piliated bacteria.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016. Vol. 32, no 18, p. 4521-4529
National Category
Physical Chemistry Materials Engineering
Identifiers
URN: urn:nbn:se:umu:diva-119441DOI: 10.1021/acs.langmuir.5b03845ISI: 000375809100015PubMedID: 27088225OAI: oai:DiVA.org:umu-119441DiVA, id: diva2:920853
Funder
Swedish Research Council, 2013-5379Available from: 2016-04-19 Created: 2016-04-19 Last updated: 2018-08-15Bibliographically approved
In thesis
1. Digital holography and image processing methods for applications in biophysics
Open this publication in new window or tab >>Digital holography and image processing methods for applications in biophysics
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Understanding dynamic mechanisms, morphology and behavior of bacteria are important to develop new therapeutics to cure diseases. For example, bacterial adhesion mechanisms are prerequisites for initiation of infections and for several bacterial strains this adhesion process is mediated by adhesive surface organelles, also known as fimbriae. Escherichia coli (E. coli) is a bacterium expressing fimbriae of which pathogenic strains can cause severe diseases in fluidic environments such as the urinary tract and intestine. To better understand how E. coli cells attach and remain attached to surfaces when exposed to a fluid flow using their fimbriae, experiments using microfluidic channels are important; and to assess quantitative information of the adhesion process and cellular information of morphology, location and orientation, the imaging capability of the experimental technique is vital.

In-line digital holographic microscopy (DHM) is a powerful imaging technique that can be realized around a conventional light microscope. It is a non-invasive technique without the need of staining or sectioning of the sample to be observed in vitro. DHM provides holograms containing three-dimensional (3D) intensity and phase information of cells under study with high temporal and spatial resolution. By applying image processing algorithms to the holograms, quantitative measurements can provide information of position, shape, orientation, optical thickness of the cell, as well as dynamic cell properties such as speed, growing rate, etc.

In this thesis, we aim to improve the DHM technique and develop image processing methods to track and assess cellular properties in microfluidic channels to shed light on bacterial adhesion and cell morphology. To achieve this, we implemented a DHM technique and developed image processing algorithms to provide for a robust and quantitative analysis of holograms. We improved the cell detection accuracy and efficiency in DHM holograms by developing an algorithm for detection of cell diffraction patterns. To improve the 3D detection accuracy using in-line digital holography, we developed a novel iterative algorithm that use multiple-wavelengths. We verified our algorithms using synthetic, colloidal and cell data and applied the algorithms for detecting, tracking and analysis. We demonstrated the performance when tracking bacteria with sub-micrometer accuracy and kHz temporal resolution, as well as how DHM can be used to profile a microfluidic flow using a large number of colloidal particles. We also demonstrated how the results of cell shape analysis based on image segmentation can be used to estimate the hydrodynamic force on tethered capsule-shaped cells in micro-fluidic flows near a surface.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 59
Keywords
Digital holographic microscopy, image processing, image reconstruction, bacterial adhesion, cell morphology, algorithm development, software design, quantitative measurement, microfluidics, multidisciplinary research
National Category
Biophysics Computer Vision and Robotics (Autonomous Systems)
Research subject
Signal Processing; Technical Physics
Identifiers
urn:nbn:se:umu:diva-150687 (URN)978-91-7601-915-3 (ISBN)
Public defence
2018-09-07, Naturvetarhuset, N430, Umeå, 13:15 (English)
Opponent
Supervisors
Available from: 2018-08-17 Created: 2018-08-15 Last updated: 2018-08-16Bibliographically approved

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Zakrisson, JohanSingh, BhupenderSvenmarker, PontusWiklund, KristerZhang, HanqingHakobyan, ShoghikRamstedt, MadeleineAndersson, Magnus

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