Accurate 4Pi single-molecule localization using an experimental PSF model

Yiming Li

doi:10.1117/12.2575241

10 October 2020 Accurate 4Pi single-molecule localization using an experimental PSF model

Yiming Li

Proceedings Volume 11553, Optics in Health Care and Biomedical Optics X; 1155303 (2020) https://doi.org/10.1117/12.2575241
Event: SPIE/COS Photonics Asia, 2020, Online Only

Abstract

Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) uses multiphase interferometry to localize single molecules and currently achieves the highest axial resolution of all 3D superresolution approaches. In theory, 3D sub-10 nm resolution can be achieved with only 250 photons collected in each objective for an individual molecule. However, the resolution achievable with the current image analysis workflow is substantially worse than the theoretical limit. Here, we developed an experimental PSF fitting method for the interference 4Pi-PSF. As the interference phase is not fixed with respect to the shape of the PSF, we developed a new 4Pi-PSF model, which decouples the phase term from the shape of the PSF. Using a spline-interpolated experimental PSF model and by fitting all 3 or 4 phase images globally, we showed on simulated data that we can achieve the theoretical limit of 3D resolution, the Cramér-Rao lower bound (CRLB), also for 4Pi microscope.

Conference Presentation

Citation Download Citation

Yiming Li "Accurate 4Pi single-molecule localization using an experimental PSF model", Proc. SPIE 11553, Optics in Health Care and Biomedical Optics X, 1155303 (10 October 2020); https://doi.org/10.1117/12.2575241

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