Abstract
Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy and (fluorescence) photoactivated localization microscopy, has enabled superresolution microscopy beyond the diffraction limit. However, the temporal resolution of SMLM is limited by the time needed to acquire sufficient sparse single-molecule activation events to successfully construct a superresolution image. Here, a novel fast SMLM technique is developed to achieve superresolution imaging within a much shortened duration. This technique does not require a faster switching rate or a higher activation density, which may cause signal degradation or photodamage/bleaching, but relies on computational algorithms to reconstruct a high-density superresolution image from a low-density one using the concept of blind image inpainting. Our results demonstrate that the technique reduces the acquisition time by up to two orders of magnitude compared to the conventional method while achieving the same high resolution. We anticipate our technique to enable future real-time live cell imaging with even higher resolution.
Highlights
Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy (STORM) [1] and photoactivated localization microscopy [(F)PALM] [2,3], is able to break the diffraction limit in optical fluorescence microscopy
Reconstruction Results—The fluorophore list of fixed microtubules obtained by STORM was converted to a subdiffraction image with a pixel size of 53 nm
The above results have proven that blind inpainting is able to recapture the microtubule structures using up to one hundredth of the frames typically needed in STORM, suggesting a potential one to two orders of magnitude of reduction in acquisition time
Summary
Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy (STORM) [1] and (fluorescence) photoactivated localization microscopy [(F)PALM] [2,3], is able to break the diffraction limit in optical fluorescence microscopy. Behind various SMLM methods, there lies a common principle: the localization precision of a single fluorophore is much lower than the width of the point spread function (PSF), given enough photons. To exploit this property, the specimen is labeled with photoswitchable (or photoactivatable) fluorophores, of which only a random sparse subset is fluorescent (on) at a time. In order to ensure that the localization density satisfies the Nyquist criterion, a large number of sequential frames are necessary to acquire, suggesting long acquisition time and low imaging speed This restriction limits the time resolution of SMLM but can result in light-induced perturbations in biological samples. There exists a strong demand for fast SMLM techniques
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
