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Step-by-step guide to reduce spatial coherence of laser light using a rotating ground glass diffuser
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.ORCID iD: 0000-0003-1746-5157
Umeå University, Faculty of Science and Technology, Department of Physics.
Umeå University, Faculty of Science and Technology, Department of Physics.
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2017 (English)In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 56, no 19, p. 5427-5435Article in journal (Refereed) Published
Abstract [en]

Wide field-of-view imaging of fast processes in a microscope requires high light intensities motivating the use of lasers as light sources. However, due to their long spatial coherence length, lasers are inappropriate for such applications, as they produce coherent noise and parasitic reflections, such as speckle, degrading image quality. Therefore, we provide a step-by-step guide for constructing a speckle-free and high-contrast laser illumination setup using a rotating ground glass diffuser driven by a stepper motor. The setup is easy to build, cheap, and allows a significant light throughput of 48%, which is 40% higher in comparison to a single lens collector commonly used in reported setups. This is achieved by using only one objective to collect the scattered light from the ground glass diffuser. We validate our setup in terms of image quality, speckle contrast, motor-induced vibrations, and light throughput. To highlight the latter, we record Brownian motion of micro-particles using a 100x oil immersion objective and a high-speed camera operating at 2000 Hz with a laser output power of only 22 mW. Moreover, by reducing the objective magnification to 50x, sampling rates up to 10,000 Hz are realized. To help readers with basic or advanced optics knowledge realize this setup, we provide a full component list, 3D-printing CAD files, setup protocol, and the code for running the stepper motor.

Place, publisher, year, edition, pages
Optical Society of America, 2017. Vol. 56, no 19, p. 5427-5435
National Category
Atom and Molecular Physics and Optics
Identifiers
URN: urn:nbn:se:umu:diva-135625DOI: 10.1364/AO.56.005427ISI: 000404745800041OAI: oai:DiVA.org:umu-135625DiVA, id: diva2:1104338
Available from: 2017-06-01 Created: 2017-06-01 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; engineering science with specialization in microsystems technology
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)
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Supervisors
Available from: 2018-08-17 Created: 2018-08-15 Last updated: 2018-08-16Bibliographically approved

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Stangner, TimHanqing, ZhangTobias, DahlbergKrister, WiklundAndersson, Magnus

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