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Accelerated FRET-PAINT microscopy
摘要： Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.
关键词： FRET-PAINT,Super-resolution fluorescence microscopy,FRET,Single-molecule localization microscopy
更新于2025-09-23 15:22:29
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Reference Module in Materials Science and Materials Engineering || Super Resolution Fluorescence Microscopy
摘要： Superresolution in microscopy has intrigued researchers for many years. There is always a challenge in overcoming what seems to be a fundamental limit. Even since the award of the Nobel prize in Chemistry in 2014 for stimulated emission depletion (STED) microscopy and localization microscopy, there has been continuing activity to improve these techniques, by reducing specimen radiation exposure for example. The term superresolution refers to overcoming the classical limit to resolution, usually defined in terms of either the Abbe resolution limit, or the Rayleigh two-point resolution criterion. These two measures are robust, the Abbe limit in particular providing a hard limit that can only be beaten by a fundamentally new approach, almost like a ‘trick’. Rayleigh recognized that his criterion was arbitrary, but considered it robust enough. Originally defined for incoherent imaging of self-luminous objects, its applicability has been extended to coherent and partially coherent systems, and later to confocal microscopy and other developments. This is usually achieved by the so-called ‘generalized Rayleigh resolution criterion’, according to which two points are taken as being just resolved when the intensity of the image midway between the points is 0.735 times that at the points themselves. It is found that around this value the image contrast changes quickly for a small change in the separation of the points.
关键词： Abbe resolution limit,Rayleigh criterion,Localization microscopy,STED,Superresolution,Microscopy
更新于2025-09-23 15:21:01
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Super-resolution imaging of self-assembled nanocarriers using quantitative spectroscopic analysis for cluster extraction
摘要： Self-assembled nanocarriers have inspired a range of applications for bioimaging, diagnostics, and drug delivery. Non-invasive visualization and characterization of nanocarriers are important for understanding their structure to function relationship. However, quantitative visualization of nanocarriers in the sample’s native environment remains challenging using existing technologies. Single-molecule localization microscopy (SMLM) has the potential to provide both high-resolution visualization and quantitative analysis of nanocarriers in their native environment. However, non-specific binding of fluorescent probes used in SMLM can introduce artifacts, which impose challenges in quantitative analysis of SMLM images. We showed the feasibility of using spectroscopic point accumulation for imaging in nanoscale topography (sPAINT) to visualize self-assembled polymersomes (PS) with molecular specificity. Furthermore, we analyzed the unique spectral signatures of Nile Red (NR) molecules bound to the PS to reject artifacts from non-specific NR bindings. We further developed quantitative spectroscopic analysis for cluster extraction (qSPACE) to increase the localization density by 4-fold compared to sPAINT; thus, reducing variations in PS size measurements to less than 5%. Finally, using qSPACE we quantitatively imaged PS at various concentrations in aqueous solutions with ~20-nm localization precision and 97% reduction in sample misidentification relative to conventional SMLM.
关键词： nanocarriers,Nile Red,super-resolution imaging,single-molecule localization microscopy,spectroscopic analysis,polymersomes
更新于2025-09-23 15:19:57
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Photoswitchable single-walled carbon nanotubes for super-resolution microscopy in the near-infrared
摘要： The design of single-molecule photoswitchable emitters was the first milestone toward the advent of single-molecule localization microscopy, setting a new paradigm in the field of optical imaging. Several photoswitchable emitters have been developed, but they all fluoresce in the visible or far-red ranges, missing the desirable near-infrared window where biological tissues are most transparent. Moreover, photocontrol of individual emitters in the near-infrared would be highly desirable for elementary optical molecular switches or information storage elements since most communication data transfer protocols are established in this spectral range. Here, we introduce a type of hybrid nanomaterials consisting of single-wall carbon nanotubes covalently functionalized with photoswitching molecules that are used to control the intrinsic luminescence of the single nanotubes in the near-infrared (beyond 1 mm). Through the control of photoswitching, we demonstrate super-localization imaging of nanotubes unresolved by diffraction-limited microscopy.
关键词： single-molecule localization microscopy,near-infrared,super-resolution microscopy,carbon nanotubes,photoswitchable emitters
更新于2025-09-12 10:27:22
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Localization microscopy at doubled precision with patterned illumination
摘要： MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.
关键词： DNA-origami,DNA-PAINT,patterned illumination,tubulin,STORM,Localization microscopy
更新于2025-09-12 10:27:22
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Super Blinking and Biocompatible Nanoprobes Based on Dye Doped BSA Nanoparticles for Super Resolution Imaging
摘要： As one of the super-resolved optical imaging technique, single molecule localization microscopy (SMLM) imaging techniques, SMLM has one particular request for the fluorophores, that is, continuous “on” and “off” behaviors of their signals (referred to as “blinking”). Hence, we present here a kind of Super Blinking and biocompatible Nanoprobes (denoted as SBNs) for SMLM. The SBNs have two main advantages, first, they possess an outstanding fluorescence blinking. Second, they are biocompatible since they are based on bovine serum albumin (BSA). The SBNs are fabricated by doping organic dyes into BSA nanoparticles (NPs). We fabricated two kinds of SBNs, one was doped with Alexa Fluor 647 (A647) and the other was doped with Alexa Fluor 594 (A594). Especially for A594 doped SBNs, the improved blinking of A594 doped SBNs induced a better localization precision as compared with A594 alone. Moreover, SMLM imaging of breast cancer cells and exosomes using the SBNs was successfully realized with high spatial resolutions. The work demonstrated here provides a new strategy to prepare novel kinds of super blinking fluorescent agents for SMLM, which broadens the selection of suitable fluorophores for SMLM.
关键词： BSA nanoparticles,single molecule localization microscopy,super blinking,dyes,localization precision
更新于2025-09-10 09:29:36
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Sharpening emitter localization in front of a tuned mirror
摘要： Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
关键词： localization precision,metal-dielectric nanocoatings,dual-color SMLM,Single-molecule localization microscopy
更新于2025-09-09 09:28:46
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Shining New Light on the Structural Determinants of Cardiac Couplon Function: Insights From Ten Years of Nanoscale Microscopy
摘要： Remodelling of the membranes and protein clustering patterns during the pathogenesis of cardiomyopathies has renewed the interest in spatial visualisation of these structures in cardiomyocytes. Coincidental emergence of single molecule (super-resolution) imaging and tomographic electron microscopy tools in the last decade have led to a number of new observations on the structural features of the couplons, the primary sites of excitation-contraction coupling in the heart. In particular, super-resolution and tomographic electron micrographs have revised and refined the classical views of the nanoscale geometries of couplons, t-tubules and the organisation of the principal calcium handling proteins in both healthy and failing hearts. These methods have also allowed the visualisation of some features which were too small to be detected with conventional microscopy tools. With new analytical capabilities such as single-protein mapping, in situ protein quantification, correlative and live cell imaging we are now observing an unprecedented interest in adapting these research tools across the cardiac biophysical research discipline. In this article, we review the depth of the new insights that have been enabled by these tools toward understanding the structure and function of the cardiac couplon. We outline the major challenges that remain in these experiments and emerging avenues of research which will be enabled by these technologies.
关键词： couplons,localization microscopy,super-resolution,ryanodine receptors,cardiac muscle
更新于2025-09-09 09:28:46