Because conventional localization methods fail to resolve such partially overlapping fluorescent spots, several advanced algorithms have been proposed to address this issue. Another popular approach is to increase the activation density at each camera frame so that more molecules can be localized and the required number of frames is thus reduced. achieved video-rate SMLM using scientific complementary metal-oxide semiconductor (sCMOS) cameras and a new localization algorithm accounting for the pixel-dependent sCMOS noises. reduced the image acquisition time down to 1–2 s while still maintaining the 3D spatial resolution of 30 nm lateral and 50 nm axial. Using organic fluorophores and high power lasers, Jones et al. One is to apply a fast switching rate, along with high excitation power. Therefore, there exists a strong demand for fast SMLM techniques.Ĭurrently, there are two types of methods to accelerate the imaging speed without compromising the spatial resolution. This restriction not only limits the time resolution of SMLM but can also result in light-induced perturbations in biological samples. 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 thus low imaging speed. According to the Nyquist criterion, the minimum required molecular density of localized fluorescent probes in order to achieve a certain spatial resolution needs to be (2∕spatial resolution) D, where D = 1, 2, or 3 for one-dimensional (1D), two-dimensional (2D), or three-dimensional (3D) imaging, respectively. In this context, both localization precision and localization density contribute to the spatial resolution of the final subdiffractive image, but the latter is always the limiting factor. After detecting and localizing the activated fluorophores, an artificial subdiffraction image is synthesized from a fluorophore localization list accumulated from different camera frames. 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.
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. Single-molecule localization microscopy (SMLM), such as stochastic optical reconstruction microscopy (STORM) and (fluorescence) photoactivated localization microscopy, is able to break the diffraction limit in optical fluorescence microscopy. We anticipate our technique to enable future real-time live cell imaging with even higher resolution. 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. 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. Here, a novel fast SMLM technique is developed to achieve superresolution imaging within a much shortened duration. 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.
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