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Self-healing dyes for super-resolution fluorescence microscopy
摘要： In recent years, optical microscopy techniques have emerged that allow optical imaging at unprecedented resolution beyond the diffraction limit. These techniques exploit photostabilizing buffers to enable photoswitching and/or the enhancement of fluorophore brightness and stability. A major drawback with the use of photostabilizing buffers, however, is that they cannot be used in live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores, which undergo intramolecular photostabilization, in super-resolution microscopy examining both targeted (stimulated emission depletion (STED) microscopy) and stochastic readout (stochastic optical reconstruction microscopy (STORM)). The overall goal of the study was to identify dyes and conditions that lead to improved spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. As a result of previously shown superior performance, we identified an ATTO647N-photostabilizer conjugate as a potential candidate for STED microscopy. We have here characterized the photostability and resulting performance of this nitrophenylalanine (NPA) conjugate of ATTO647N on oligonucleotides in STED microscopy. We found that the superior photophysical performance resulted in optimal STED imaging and demonstrated that single-molecule fluorescent transients of individual fluorophores can be obtained with both the excitation- and STED-laser. In similar experiments, we also tested a nitrophenylacetic acid conjugate of STAR635P, another frequently used dye in STED microscopy, and present a characterization of its photophysical properties. Finally, we performed an analysis of the photoswitching kinetics of self-healing Cy5 dyes (containing trolox, cyclooctatetraene and NPA-based stabilizers) in the presence of Tris(2-carboxyethyl) phosphine and cysteamine, which are typically used in STORM microscopy. In line with previous work, we found that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.
关键词： STORM,super-resolution microscopy,fluorescent dyes,STED,fluorescence microscopy
更新于2025-09-09 09:28:46
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Super-resolution enhancement by quantum image scanning microscopy
摘要： The principles of quantum optics have yielded a plethora of ideas to surpass the classical limitations of sensitivity and resolution in optical microscopy. While some ideas have been applied in proof-of-principle experiments, imaging a biological sample has remained challenging, mainly due to the inherently weak signal measured and the fragility of quantum states of light. In principle, however, these quantum protocols can add new information without sacrificing the classical information and can therefore enhance the capabilities of existing super-resolution techniques. Image scanning microscopy, a recent addition to the family of super-resolution methods, generates a robust resolution enhancement without reducing the signal level. Here, we introduce quantum image scanning microscopy: combining image scanning microscopy with the measurement of quantum photon correlation allows increasing the resolution of image scanning microscopy up to twofold, four times beyond the diffraction limit. We introduce the Q-ISM principle and obtain super-resolved optical images of a biological sample stained with fluorescent quantum dots using photon antibunching, a quantum effect, as a resolution-enhancing contrast mechanism.
关键词： image scanning microscopy,quantum dots,quantum optics,super-resolution microscopy,photon antibunching
更新于2025-09-09 09:28:46
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Characterization of functionalized glass and indium tin oxide surfaces as substrates for super-resolution microscopy
摘要： Modern high-throughput biosensors with sensitivity down to a single analyte molecule may be possible with single-molecule localization microscopy (SMLM). Functionalized surfaces can be fabricated with self-assembly monolayer chemistry on indium tin oxide (ITO) substrates but not glass. However, characterizations of SMLM-compatible fluorophores are primarily performed on glass substrates. Here we collect single-molecule kinetics data of isolated Alexa Fluor 647 molecules on bare and functionalized glass and ITO surfaces. Extracting the photophysical dynamics of the fluorophores allows direct comparison of behavior of this dye on these substrates and fitting data to a model that accounts for multiple reversible dark states. All surfaces had sensitivity sufficient to image single fluorophore molecules. Photophysical kinetics observed are similar between the two substrates. The photon yield from individual fluorophores was greatest on bare glass, but functionalized ITO surfaces showed superior yield to functionalized glass surfaces and nearly matched the yield of bare glass. Together these results indicate functionalized ITO as a promising substrate for modern single-molecule biosensors.
关键词： indium tin oxide,super-resolution microscopy,biosensor,single-molecule imaging
更新于2025-09-09 09:28:46
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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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Photochemically Active Dyes for Super-Resolution Microscopy
摘要： The development of super-resolved optical microscopies has revolutionized the way we visualize cell biology. These techniques strongly rely on the use of photochemically active fluorophores that display changes in their photophysical properties upon irradiation with light. Many reversible and irreversible photochemical transformations have been explored for this purpose, and different imaging techniques require specific mechanisms of photoconversion. In this review, we provide an overview of the most common strategies used for the development of fluorophores for super-resolution microscopy and give specific examples of state-of-the-art fluorogenic probes. Furthermore, we discuss their main field of application and possible directions for future developments.
关键词： Live-cell imaging,Photoswitchable,Super-resolution microscopy,Fluorescent probes,Photoactivatable
更新于2025-09-09 09:28:46
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Super-resolution microscopy and empirically validated autocorrelation image analysis discriminates microstructures of dairy derived gels
摘要： The food industry must capitalise on advancing technologies in order to optimise the potential from emerging ingredient technologies. These can aid in product optimisation and provide quantitative empirical data to which there is a fundamental physical understanding. Super-resolution microscopy provides a tool to characterise the microstructure of complex colloidal materials under near native conditions. Coherent Anti-Stokes Raman Scattering (CARS) microscopy was used to show the presence of fluorescent dye required for imaging does not affect gel microstructure and super-resolution Stimulated Emission Depletion (STED) microscopy is used to image four dairy derived gels. Image analysis has been developed based on 2D spatial autocorrelation, and a model that extracts parameters corresponding to a typical length of the protein domains and the inter pore distance. The model has been empirically validated through the use of generated images to show the fitting parameters relate to precise physical features. The fractal dimension is extracted from Fourier space analysis. The combination of STED microscopy and image analysis is sensitive enough to significantly differentiate samples based on whether gels were made from fresh or reconstituted milk, and whether gelation was induced through acidification or rennet addition. Rheometry shows that the samples exhibit different macroscopic behaviours, and these differences become increasingly significant with time. Samples can be differentiated earlier in the gelation process with imaging as compared to rheometry. This highlights the potential of STED imaging and image analysis to characterise the size of protein domains, pore spacing and the fractal dimensions of microstructures to aid product optimisation.
关键词： Stimulated Emission Depletion (STED) microscopy,Super-resolution microscopy,Fractal dimension,Coherent Anti-stokes Raman Scattering (CARS) microscopy,2D spatial autocorrelation analysis
更新于2025-09-04 15:30:14
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Deep learning in imaging
摘要： Machine learning approaches that include deep learning are moving beyond image classification to change the way images are made. Computers are powerful tools for carrying out tasks such as image classification or identification as well as or better than human experts. Conventional machine learning approaches are widely used for segmentation and phenotyping in fluorescence microscopy. These tools are now being largely outperformed by their deep-learning-based counterparts, some of which are available as user-friendly tools. But a perhaps more astonishing wave of developments has recently come about through the use of deep learning not for image analysis but for image transformation. In these cases, deep convolutional networks are trained to transform one type of image into another. For example, two studies have shown the power of deep learning for the creation of fluorescence micrographs of cells directly from bright-field or phase images, to facilitate multiplexed and longitudinal imaging. Researchers have also used deep learning to go from low signal-to-noise images to high-quality images, which opens the door to extended imaging of even very light-sensitive living organisms. Deep learning can similarly overcome obstacles associated with super-resolution microscopy. Two approaches, ANNA-PALM and DeepSTORM, were developed to improve the speed of localization microscopy, which is one of the major hurdles of the technique. Deep learning can also enable cross-modality imaging, where applications such as a shift from confocal images to stimulated-emission-depletion-microscopy-resolution images could democratize super-resolution imaging. As with any method, the caveats associated with deep learning in such applications, such as the potential for artifacts, must be carefully considered and analyzed. Nevertheless, we think we have seen only the tip of the iceberg, and that deep learning stands to improve all aspects of imaging, from acquisition to analysis.
关键词： image transformation,machine learning,fluorescence microscopy,deep learning,super-resolution microscopy,imaging
更新于2025-09-04 15:30:14
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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
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Amorphous Quantum Nanomaterials
摘要： In quantum materials, macroscopic behavior is governed in nontrivial ways by quantum phenomena. This is usually achieved by exquisite control over atomic positions in crystalline solids. Here, it is demonstrated that the use of disordered glassy materials provides unique opportunities to tailor quantum material properties. By borrowing ideas from single-molecule spectroscopy, single delocalized π-electron dye systems are isolated in relatively rigid ultra-small (<10 nm diameter) amorphous silica nanoparticles. It is demonstrated that chemically tuning the local amorphous silica environment around the dye over a range of compositions enables exquisite control over dye quantum behavior, leading to efficient probes for photodynamic therapy (PDT) and stochastic optical reconstruction microscopy (STORM). The results suggest that efficient fine-tuning of light-induced quantum behavior mediated via effects like spin-orbit coupling can be effectively achieved by systematically varying averaged local environments in glassy amorphous materials as opposed to tailoring well-defined neighboring atomic lattice positions in crystalline solids. The resulting nanoprobes exhibit features proven to enable clinical translation.
关键词： photodynamic therapy (PDT),optical super-resolution microscopy,organic dyes,amorphous silica nanoparticles
更新于2025-09-04 15:30:14
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Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes
摘要： Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins (PA-FPs). An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.
关键词： Halo tag,single-molecule tracking,photoactivatable fluorescent protein,Escherichia coli,fluorophores,super-resolution microscopy,DNA-binding proteins
更新于2025-09-04 15:30:14