Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models.

Autor: Bates M; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. mark.bates@mpibpc.mpg.de.; Department of Optical Nanoscopy, Institute for NanoPhotonics, Göttingen, Germany. mark.bates@mpibpc.mpg.de., Keller-Findeisen J; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany., Przybylski A; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany., Hüper A; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany., Stephan T; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.; Clinic of Neurology, University Medical Center Göttingen, Göttingen, Germany., Ilgen P; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.; Clinic of Neurology, University Medical Center Göttingen, Göttingen, Germany., Cereceda Delgado AR; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.; Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany., D'Este E; Optical Microscopy Facility, Max Planck Institute for Medical Research, Heidelberg, Germany., Egner A; Department of Optical Nanoscopy, Institute for NanoPhotonics, Göttingen, Germany., Jakobs S; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.; Clinic of Neurology, University Medical Center Göttingen, Göttingen, Germany., Sahl SJ; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany., Hell SW; Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. stefan.hell@mpibpc.mpg.de.; Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg, Germany. stefan.hell@mpibpc.mpg.de.
Jazyk: angličtina
Zdroj: Nature methods [Nat Methods] 2022 May; Vol. 19 (5), pp. 603-612. Date of Electronic Publication: 2022 May 16.
DOI: 10.1038/s41592-022-01465-8
Abstrakt: Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2-3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.
(© 2022. The Author(s).)
Databáze: MEDLINE
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