Abstract
This article reports a new approach to track (x, y, z, t) coordinates of multiple fluorescent particles (diameter range 1–10 μm) simultaneously using a quantitative defocusing method. We find that the defocused image of a 1-μm diameter fluorescent particle formed by the objective lens of a conventional microscope has a bright outer ring due to the spherical aberration of the lens system. The ring radius increases as the particle is moved away from its reference plane and closer to the lens. The reference plane refers to locations of the particle at which the projected image is in focus. The (x, y, z) coordinates of the particle are then inferred from the center location of the image ring as well as the ring radius. The described technique is implemented successfully for obtaining 3D trajectories of swimming Escherichia coli cells.
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Acknowledgements
We would like to thank Professor DeLisa from the Chemical and Biomolecular Engineering Department at Cornell University for providing us with bacteria samples. This work is supported by the National Science Foundation (CTS-0121340), and a startup fund from the Physics Department at Cornell University.
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Wu, M., Roberts, J.W. & Buckley, M. Three-dimensional fluorescent particle tracking at micron-scale using a single camera. Exp Fluids 38, 461–465 (2005). https://doi.org/10.1007/s00348-004-0925-9
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DOI: https://doi.org/10.1007/s00348-004-0925-9