Precisely and accurately localizing single emitters in fluorescence microscopy

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Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

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Figure 1: Localization precision and accuracy.
Figure 2: Theories describing the localization precision or accuracy in SPT and localization microscopy.
Figure 3: Simulated images of an isotropic emitter at different axial positions Zp and cross-sections through the emitter position along the x direction.
Figure 4: Simplified illustrations of the detection beam path in several microscope setups that allow for 3D localization of single emitters.
Figure 5: Influence of dipole photon emission on localization precision and accuracy.
Figure 6: Influence of translational movement during camera exposure on localization precision and accuracy.
Figure 7: Experimental images illustrating the effect of sample background on the localization precision.
Figure 8: Simplified illustrations of the illumination path in several microscope setups that allow for sample background reduction.
Figure 9: Influence of localization precision, label density and label displacement on the resolution in a localization microscopy image.
Figure 10: Experimental images illustrating the influence of localization precision and label density on the resolution in localization microscopy.
Figure 11: Influence of localization precision on the analysis of single-particle trajectories in the case of Brownian motion.


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H.D. acknowledges the financial support of the Agency for Innovation by Science and Technology (IWT, Belgium). Financial support from the Ghent University Special Research Fund and the Fund for Scientific Research Flanders (FWO, Belgium) is acknowledged by K.B. with gratitude. S.T.H. is funded by R15-GM094713 from the US National Institutes of Health and MTAF 1106 and 2061 from the Maine Technology Institute. A.D. is partially funded by PF7-EU 280804-2 LANIR CP-TP and by the Italian Programmi di Ricerca di Rilevante Interesse Nazionale 2010JFYFY2-002 grant. Appreciation goes to E. Kromann for the simulation of the images of fluorescent particles with a fixed dipole orientation.

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Correspondence to Alberto Diaspro or Kevin Braeckmans.

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J.B. is a cofounder and consultant of Vutara, Inc., a company that develops super-resolution microscopes, and has personal financial interest in it.

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Deschout, H., Zanacchi, F., Mlodzianoski, M. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat Methods 11, 253–266 (2014) doi:10.1038/nmeth.2843

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