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Polarization of excitation light influences molecule counting in single-molecule localization microscopy

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Abstract

Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from −40 up to 100 % when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins, and the background noise in the system, namely the illumination scheme. This difference can be further exaggerated or mitigated by various fixation methods, magnification, and camera settings We conclude that the final choice between circularly or linearly polarized excitation light should be made experimentally, based on the signal to noise ratio of the system.

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Acknowledgments

This work was supported by project grants and a fellowship from the National Health and Medical Research Foundation of Australia to SMR, and Australian Research Council Laureate Fellowship to MG and Future Fellowship to SMR. We thank Katharina Gaus for technical assistance and the pLifeAct-mEos2 construct.

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Authors and Affiliations

  1. Immune Signalling Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Australia

    Ye Chen, Mandy J. Ludford-Menting & Sarah M. Russell

  2. Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Australia

    Ye Chen, Han Lin, Andrew H. Clayton, Min Gu & Sarah M. Russell

  3. Department of Pathology, The University of Melbourne, Melbourne, Australia

    Mandy J. Ludford-Menting & Sarah M. Russell

  4. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia

    Sarah M. Russell

Authors
  1. Ye Chen
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  2. Han Lin
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  3. Mandy J. Ludford-Menting
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  4. Andrew H. Clayton
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  5. Min Gu
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  6. Sarah M. Russell
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Correspondence to Sarah M. Russell.

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Chen, Y., Lin, H., Ludford-Menting, M.J. et al. Polarization of excitation light influences molecule counting in single-molecule localization microscopy. Histochem Cell Biol 143, 11–19 (2015). https://doi.org/10.1007/s00418-014-1267-1

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  • DOI: https://doi.org/10.1007/s00418-014-1267-1

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