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Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

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Department of Chemistry, University of Michigan, Ann Arbor, MI 48019, USA
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Department of Medical Microbiology and Immunology, University of Toledo, Toledo, OH 43606, USA
3
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48019, USA
*
Author to whom correspondence should be addressed.
Molecules 2014, 19(8), 12116-12149; https://doi.org/10.3390/molecules190812116
Received: 24 June 2014 / Revised: 1 August 2014 / Accepted: 1 August 2014 / Published: 13 August 2014
(This article belongs to the Special Issue Single Molecule Techniques)
Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. View Full-Text
Keywords: single-molecule microscopy; super-resolution imaging; single-particle tracking; fluorescence; microbiology; live-cell imaging single-molecule microscopy; super-resolution imaging; single-particle tracking; fluorescence; microbiology; live-cell imaging
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Haas, B.L.; Matson, J.S.; DiRita, V.J.; Biteen, J.S. Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology. Molecules 2014, 19, 12116-12149.

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