Measuring the positions and movements of single molecules enables new insights into the functioning of life at the cellular level. In some applications, the measurement range is very close to a water / glass interface, where near-field coupling (super-critical angle fluorescence or SAF) can be exploited to strongly improve the localization precision.
Specific detection systems have been developed in the past to exploit SAF in modified two-channel imaging configurations. Recently, we have shown that imaging at a slight intentional defocus allows one to effectively benefit from this effect as well using a standard research grade microscope [1, 2]. This observation raises the question if and by how much other 3D localization techniques may profit from SAF.
In my talk I will provide a quantitative comparison of the performances of popular 3D localization techniques in the near field regime. The comparison will include off-focus, cylindrical lens and biplane imaging as well as a dedicated SAF system. Important properties such as robustness to aberrations, fixed dipole emission and multi-emitter events will be discussed.
1. Zelger, P., et al., Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy. Biomedical Optics Express, 2020. 11(2): p. 775-790.
2. Zelger, P., et al., Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence. Biomedical Optics Express, 2021. 12(2): p. 802-822.