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A Review on Optical Imaging of DNA Nanostructures and Dynamic Processes
摘要： This article reviews recent advances in optical imaging methods for characterizing self-assembled DNA nanosystems, with particular emphasis on super-resolved fluorescence microscopy. Several advanced strategies are developed to obtain accurate and detailed images of intricate DNA nanogeometries and to perform precise tracking of molecular motions in dynamic processes. We present state-of-the-art instruments and imaging strategies including localization microscopy and spectral imaging. We discuss how they are used in biological studies and biomedical applications, and also provide current challenges and future outlook. Overall, this review serves as a practical guide in optical microscopy for the field of DNA nanotechnology.
关键词： super-resolution microscopy,fluorescence microscopy,optical imaging,DNA nanostructures,DNA nanotechnology
更新于2025-09-23 15:21:21
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Fabrication of Two-Dimensional Arrays of Fluorescent Centers in Single-Crystalline Diamond Using Particle Beam Writing
摘要： Micrometer-scale patterning was performed using the particle beam writing technique with a focused heavy-ion microbeam, allowing the creation of a unique two-dimensional distribution of fluorescent centers in single-crystalline diamond. The focused nitrogen microbeam was scanned over the target of single-crystalline diamond prepared by chemical vapor deposition to create nitrogen-vacancy (NV) centers at defined positions. Imaging using a custom-built confocal fluorescence microscopy system revealed that the desired NV distribution was generated in the target crystal with a spatial resolution similar to the beam resolution. A two-dimensional matrix barcode test pattern was successfully generated in a diamond substrate to demonstrate the encryption of information inside a solid-state target.
关键词： diamond,particle beam writing,two-dimensional,chemical vapor deposition,nitrogen vacancy centers,confocal fluorescence microscopy
更新于2025-09-23 15:21:21
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Increasing fluorescence lifetime for resolution improvement in STED nanoscopy
摘要： Super-resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction-unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.
关键词： confocal microscopy,super-resolution,fluorescence lifetime,fluorescence microscopy
更新于2025-09-23 15:21:01
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Restoration of Light Sheet Multi-View Data with the Huygens Fusion and Deconvolution Wizard
摘要： Light sheet fluorescence microscopy (LSFM) allows for high-resolution three-dimensional imaging with minimal photo-damage. By viewing the sample from different directions, different regions of large specimens can be imaged optimally. Moreover, owing to their good spatial resolution and high signal-to-noise ratio, LSFM data are well suited for image deconvolution. Here we present the Huygens Fusion and Deconvolution Wizard, a unique integrated solution for restoring LSFM images, and show that improvements in signal and resolution of 1.5 times and higher are feasible.
关键词： selective plane illumination microscopy (SPIM),Light sheet fluorescence microscopy (LSFM),deconvolution,Huygens,fusion
更新于2025-09-23 15:21:01
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Fluorescence Microscopy of Single Liposomes with Incorporated Pigment–Proteins
摘要： Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here we present a novel strategy employing methods of proteoliposome preparation, fluorescent labelling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment–protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of non-reconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled native-like environment.
关键词： Fluorescence Microscopy,Proteoliposomes,Pigment–Proteins,LHCII,Single Liposomes
更新于2025-09-23 15:21:01
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Visualizing pore architecture and molecular transport boundaries in catalyst bodies with fluorescent nanoprobes
摘要： The performances of porous materials are closely related to the accessibility and interconnectivity of their porous domains. Visualizing pore architecture and its role on functionality—for example, mass transport—has been a challenge so far, and traditional bulk and often non-visual pore measurements have to suffice in most cases. Here, we present an integrated, facile fluorescence microscopy approach to visualize the pore accessibility and interconnectivity of industrial-grade catalyst bodies, and link it unequivocally with their catalytic performance. Fluorescent nanoprobes of various sizes were imaged and correlated with the molecular transport of fluorescent molecules formed during a separate catalytic reaction. A direct visual relationship between the pore architecture—which depends on the pore sizes and interconnectivity of the material selected—and molecular transport was established. This approach can be applied to other porous materials, and the insight gained may prove useful in the design of more efficient heterogeneous catalysts.
关键词： porous materials,molecular transport,fluorescence microscopy,catalyst bodies,heterogeneous catalysts
更新于2025-09-23 15:21:01
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Highly Efficient Energy Transfer from Water Soluble Zinc Silver Indium Sulphide Quantum Dot to Organic J-Aggregates
摘要： The present work has been carried out with an aim to design and develop an efficient light harvesting inorganic-organic hybrid nanoscale material by employing less toxic, environment friendly inorganic substance and also to understand the mechanism of inter-particle electronic interaction between the inorganic and organic components of the nanomaterial. Specifically, the inorganic-organic hybrid associate has been made by integrating water soluble semiconductor QDs (Zinc-silver-indium-sulfide (ZAIS)) and organic J-aggregates of a cyanine dye (S2165). The fabrication of present nano-hybrid system has been achieved via electrostatically driven self-assembly of organic dyes over ZAIS QDs. The interaction between QD and J-aggregates have been investigated by steady state and time resolved fluorescence measurements. Zeta potential measurements have also been performed to understand the role of electrostatic interaction and thermodynamic feasibility of the association process. The investigations have revealed that energy transfer (ET) process between QD and J-aggregates mediated through dipole-dipole mechanism. Interestingly, data analysis based on F?rster theory has further revealed that the ET from QD to J-aggregates is very high indicating efficient electronic coupling between the inorganic QD and the organic J-aggregates. Zeta potential measurements and thermodynamic calculations have demonstrated that the interaction between QD and organic dye is electrostatically driven and the association of organic dyes over QD is thermodynamically feasible. The outcome of the present study is expected to be helpful in designing efficient nanoscale light harvesting devices. Additionally, fluorescence microscopy and toxicity studies on the QDs have also shown its suitability for biological applications.
关键词： energy transfer,fluorescence microscopy,electrostatic interaction,J-aggregates,inorganic-organic hybrid,toxicity studies,quantum dots
更新于2025-09-23 15:21:01
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Pulsed interleaved excitation-based line-scanning spatial correlation spectroscopy (PIE-lsSCS)
摘要： We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIE-lsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells.
关键词： lipid bilayer membranes,dual-color excitation,live cells,line-scanning spatial correlation spectroscopy,fluorescence microscopy,tissues,GUVs,confocal microscope,photobleaching,Pulsed interleaved excitation
更新于2025-09-23 15:21:01
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Editorial: Advances in Label Free Tissue Imaging With Laser Scanning Microscopy Techniques
摘要： Significant efforts are being spent at the time being for transferring various laser scanning microscopy (LSM) techniques to the realm of tissue characterization, because of their potential to circumvent some of the most important disadvantages of traditional histopathology approaches based on excisional biopsy and tissue staining. Although conventional histopathology is currently regarded as a golden standard for the diagnosing pathologies that reflect in tissular modification (e.g., cancers), limitations such as long diagnosis time, invasiveness, artifacts, sampling error, time consumption, high costs, and interpretive variability make such approaches to be impractical in many scenarios, while also placing considerable pressure on the sustainability of healthcare systems around the world. The potential of LSM techniques to contribute to overcoming these aspects derives from their “non-invasive” character. They can exploit various endogenous optical signals generated by tissues upon interaction with a laser beam and are able to provide optical sections (virtual biopsies) that reflect the tissular architecture at controlled depths. Many studies reported to date showed that LSM techniques can provide label-free information of similar pathologic relevance to the information collected for characterization/confirmation purposes with traditional histopathology approaches. These techniques are thus capable of probing optical properties of tissues with deep implications for resolving important anatomical and physiological aspects which represent hallmarks for disease predisposition and progression. To date techniques such as Confocal Laser Scanning Microscopy (CLSM) [1], Fluorescence Lifetime Imaging (FLIM) [2], Two-Photon Excited Fluorescence Microscopy (TPEF) [2–6], Second Harmonic Generation Microscopy (SHG) [5, 6], Third Harmonic Generation Microscopy (THG) [4], Coherent Anti-Stokes Raman Scattering Microscopy (CARS) [3, 7], as well as other LSM variants such as the Brillouin Microscopy [8] have already been demonstrated to be powerful tools for investigating tissue morphology, functionality, and biochemical composition with high spatial and temporal resolution. In the opinion of many, these techniques, together with investigations approaches based on their combined use, will soon become the central element of the default tissue characterization frameworks for both ex vivo and in vivo assays. Furthermore, emerging LSM techniques exploiting various ingenious strategies to achieve superresolved images in a label-free manner [9–12] are also likely to be transferred soon toward applications addressing tissue imaging.
关键词： tissue imaging,two photon excitation fluorescence microscopy,Brillouin microspectroscopy,laser scanning microscopy,second harmonic generation microscopy
更新于2025-09-23 15:19:57
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Digital scanned laser lighta??sheet fluorescence lifetime microscopy with widea??field timea??gated imaging
摘要： We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a sub-resolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimeters and can be utilized as a powerful and robust method for biomedical research, for example as a readout of protein-protein interactions via F?rster resonance energy transfer.
关键词： Fluorescence lifetime imaging,light-sheet fluorescence microscopy,time-resolved fluorescence imaging
更新于2025-09-23 15:19:57