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Fluorescence imaging; Single-molecule biophysics; Super-resolution microscopy
Abstract:
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 capa-bilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.
