Abstract
Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.
Highlights
Optical aberrations compromise the performance of fluorescence microscopes, which can degrade image quality
Aberrations become more serious in point-localization superresolution imaging (SR), that is Stochastic Optical Reconstruction Microscopy (STORM)[1] and Photoactivated Localization Microscopy (PALM)[2], where even nanometer-scale distortions can significantly degrade the accuracy of measurements
We demonstrate that correcting “wobble” in astigmatic 3D SR microscopy reduces distortion both for test and biological samples
Summary
Optical aberrations compromise the performance of fluorescence microscopes, which can degrade image quality. Aberrations become more serious in point-localization superresolution imaging (SR), that is Stochastic Optical Reconstruction Microscopy (STORM)[1] and Photoactivated Localization Microscopy (PALM)[2], where even nanometer-scale distortions can significantly degrade the accuracy of measurements. In SR, a structure of interest is labeled with fluorescent molecules which are imaged and localized with precisions on the order of 10–50 nm in directions lateral to the optical axis and precisions that are typically 3–4 times worse axially [3]. To minimize the deleterious effects of aberrations in SR, the localizations themselves require correction. SR techniques were first extended to three-dimensions (3D) by introducing astigmatism[4] into the imaging system with a cylindrical lens, effectively encoding molecules’ axial positions onto the microscope’s point spread function (PSF) [5]. Astigmatic 3D SR requires correction of PLOS ONE | DOI:10.1371/journal.pone.0142949 November 23, 2015
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