- 标题
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- 实验方案
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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
摘要： DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.
关键词： DNA-PAINT,microfluidics,super-resolution microscopy,fluorescent proteins,molecular localization,multi-color imaging,multiplexing,single domain antibodies (sdAb),linkage error,nanobodies
更新于2025-09-19 17:15:36
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Protein‐specific, multi‐color and 3D STED imaging in cells with DNA‐labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,DNA-PAINT,STED microscopy,fluorescence,fluorescent probes
更新于2025-09-19 17:13:59
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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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Protein-specific, multi-color and 3D STED imaging in cells with DNA-labeled antibodies
摘要： Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed with exchangeable labels, which transiently bind to and off a target and thereby replenish destroyed labels by intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.
关键词： multicolor imaging,STED microscopy,fluorescent probes,DNA-PAINT,fluorescence
更新于2025-09-11 14:15:04
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Ortsspezifische Funktionalisierung von Affimeren für die DNA-PAINT-Mikroskopie
摘要： Superaufl?sende Mikroskopie erm?glicht Bildgebung weit unterhalb des klassischen Beugungslimits von Licht. Technisch gesehen erreichen aktuelle Methoden sogar eine r-umliche Aufl?sung auf Einzelmolekglebene. Dieser beeindruckende Erfolg kann jedoch nicht einfach auf die Bildgebung von zellul-ren Komponenten gbertragen werden, da Sonden, die zur Markierung von Molekglen verwendet werden, entweder durch deren Gr??e (Antik?rper) oder wegen begrenzter Verfggbarkeit limitiert sind (Nanok?rper, Aptamere, genetisch-kodierende Marker). In dieser Arbeit kombinieren wir kgrzlich entwickelte Affimer-Reagenzien mit einer ortsspezifischen DNA-Funktionalisierung, um eine F-rbung mit anschlie?ender Bildgebung durch DNA-PAINT zu erreichen. Hierfgr benutzen wir ein Aktin-bindendes Affimer. Diese kleinen, mit DNA konjugierten Affinit-tsmarker k?nnten eine zukgnftige L?sung fgr superaufl?sende Bildgebung mit hoher Multiplexkapazit-t darstellen.
关键词： Kleine Affinit-tsmarker,DNA-PAINT,Quantitative Mikroskopie,Superaufl?sende Mikroskopie
更新于2025-09-10 09:29:36
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Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy
摘要： The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.
关键词： photo-induced DNA damage,surface-integrated fluorescence correlation spectroscopy (SI-FCS),reactive oxygen species,super-resolution microscopy,DNA-PAINT
更新于2025-09-09 09:28:46
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Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT
摘要： Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.
关键词： STORM,frequency multiplexing,multicolor imaging,DNA-PAINT,superresolution microscopy
更新于2025-09-09 09:28:46
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DNA-Based Super-Resolution Microscopy: DNA-PAINT
摘要： Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.
关键词： DNA PAINT,SMLM,DNA origami,DNA,fluorescence microscopy,super-resolution microscopy
更新于2025-09-04 15:30:14