15 March 2016 Holographic microscopy for 3D tracking of bacteria
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Proceedings Volume 9718, Quantitative Phase Imaging II; 97182B (2016) https://doi.org/10.1117/12.2213021
Event: SPIE BiOS, 2016, San Francisco, California, United States
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.
Conference Presentation
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Jay Nadeau, Jay Nadeau, Yong Bin Cho, Yong Bin Cho, Marwan El-Kholy, Marwan El-Kholy, Manuel Bedrossian, Manuel Bedrossian, Stephanie Rider, Stephanie Rider, Christian Lindensmith, Christian Lindensmith, J. Kent Wallace, J. Kent Wallace, "Holographic microscopy for 3D tracking of bacteria", Proc. SPIE 9718, Quantitative Phase Imaging II, 97182B (15 March 2016); doi: 10.1117/12.2213021; https://doi.org/10.1117/12.2213021
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