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Proceedings Paper

Holographic microscopy for 3D tracking of bacteria
Author(s): Jay Nadeau; Yong Bin Cho; Marwan El-Kholy; Manuel Bedrossian; Stephanie Rider; Christian Lindensmith; J. Kent Wallace
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Paper Abstract

Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

Paper Details

Date Published: 15 March 2016
PDF: 9 pages
Proc. SPIE 9718, Quantitative Phase Imaging II, 97182B (15 March 2016); doi: 10.1117/12.2213021
Show Author Affiliations
Jay Nadeau, California Institute of Technology (United States)
McGill Univ. (Canada)
Yong Bin Cho, California Institute of Technology (United States)
Marwan El-Kholy, McGill Univ. (Canada)
Manuel Bedrossian, California Institute of Technology (United States)
Stephanie Rider, California Institute of Technology (United States)
Christian Lindensmith, Jet Propulsion Lab. (United States)
J. Kent Wallace, Jet Propulsion Lab. (United States)

Published in SPIE Proceedings Vol. 9718:
Quantitative Phase Imaging II
Gabriel Popescu; YongKeun Park, Editor(s)

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