- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Super-resolution Mapping of Enhanced Emission by Collective Plasmonic Resonances
摘要： Plasmonic particle arrays have remarkable optical properties originating from their collective behavior, which results in resonances with narrow line widths and enhanced electric fields extending far into the surrounding medium. Such resonances can be exploited for applications in strong light?matter coupling, sensing, lasing, and light harvesting, nonlinear nanophotonics, solid-state lighting. However, as the lattice constants associated with plasmonic particle arrays are on the order of their resonance wavelengths, mapping the interaction between point dipoles and plasmonic particle arrays cannot be done with diffraction-limited methods. Here, we map the enhanced emission of single fluorescent molecules coupled to a plasmonic particle array with ～20 nm in-plane resolution by using stochastic super-resolution microscopy. We find that extended lattice resonances have minimal influence on the spontaneous decay rate of an emitter but instead can be exploited to enhance the outcoupling and directivity of the emission. Our results can guide the rational design of future optical devices based on plasmonic particle arrays.
关键词： light?matter interaction,nanophotonics,single molecule localization,collective resonances,plasmonics,super-resolution microscopy
更新于2025-11-25 10:30:42
-
Deep learning enables cross-modality super-resolution in fluorescence microscopy
摘要： We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.
关键词： GAN,cross-modality,super-resolution,fluorescence microscopy,deep learning
更新于2025-11-21 11:24:58
-
Accelerating single molecule localization microscopy through parallel processing on a high-performance computing cluster
摘要： Super-resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high-performance computing (HPC) cluster and quantify the speed advantage for a four-node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD-enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software.
关键词： super-resolved microscopy,high-performance computing,Automated image analysis
更新于2025-11-21 11:24:58
-
Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H
摘要： Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens.
关键词： Fluorescence Lifetime Imaging Microscopy (FLIM),single-cell analysis,NADPH/NADH ratio,NAD(P)H,redox,FAD,fluorescence lifetime redox ratio (FLIRR),NAD(P)H-a2%
更新于2025-11-21 11:24:58
-
A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray
摘要： A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
关键词： gene expression,enzyme-free,fluorescence microscopy,mRNA detection,microarray
更新于2025-11-21 11:24:58
-
Fluorescence microscope light source stability
摘要： The process of fluorescence starts with the efficient generation of light that is required for the excitation of fluorophores. As such, light sources are a crucial component of a fluorescence microscope. Choosing the right illumination tool can not only improve the quality of experimental results, but also the microscope’s economic and environmental footprint. While arc lamps have historically proven to be a reliable light source for widefield fluorescence microscopy, solid-state light-emitting diodes (LEDs) have become the light source of choice for new fluorescence microscopy systems. In this paper, we demonstrate that LEDs have superior light stability on all timescales tested and use less electrical power than traditional light sources when used at lower power outputs. They can be readily switched on and off electronically, have a longer lifetime and they do not contain mercury, and thus are better for the environment. We demonstrate that it is important to measure light source power output during warm-up and switching, as a light source’s responsiveness (in terms of power) can be quite variable. Several general protocols for testing light source stability are presented. A detailed life cycle analysis shows that an LED light source can have a fourfold lower environmental impact when compared to a metal halide source.
关键词： Light source,Fluorescence,Solid state,LED,Stability,Microscopy
更新于2025-11-21 11:24:58
-
ImmunoSERS microscopy for the detection of smooth muscle cells in atherosclerotic plaques
摘要： We investigated the suitability of immuno-SERS (iSERS) microscopy for imaging of smooth muscle cells (SMCs) in atherosclerotic plaques. Localization of SMCs is achieved by using SERS-labelled antibodies direct against alpha-smooth muscle actin (SMA). The staining quality of the false-colour iSERS images obtained by confocal Raman microscopy with point mapping is compared with wide-field immunofluorescence images. Both direct (labelled primary antibody) and indirect iSERS staining (unlabelled primary and labelled secondary antibody) techniques were employed. Direct iSERS staining yields results comparable to indirect IF staining, demonstrating the suitability of iSERS in research on atherosclerosis and paving the way for future multiplexed imaging experiments.
关键词： ImmunoSERS microscopy,Smooth muscle cells (SMCs),Atherosclerosis,Direct/indirect staining
更新于2025-11-21 11:24:58
-
Immobilization of Detonation Nanodiamonds on Macroscopic Surfaces
摘要： Detonation nanodiamonds (NDs) are a novel class of carbon-based nanomaterials, and have received a great deal of attention in biomedical applications, due to their high biocompatibility, facile surface functionalization, and commercialized synthetic fabrication. We were able to transfer the NDs from large-size agglomerate suspensions to homogenous coatings. ND suspensions have been used in various techniques to coat on commercially available substrates of pure Ti and Si. Scanning electron microscopy (SEM) imaging and nanoindentation show that the densest and strongest coating of NDs was generated when using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC/NHS)-mediated coupling to macroscopic silanized surfaces. In the next step, the feasibility of DNA-mediated coupling of NDs on macroscopic surfaces is discussed using fluorescent microscopy and additional particle size distribution, as well as zeta potential measurements. This work compares different ND coating strategies and describes the straightforward technique of grafting single-stranded DNA onto carboxylated NDs via thioester bridges.
关键词： detonation nanodiamonds,bio-conjugation,fluorescent microscopy,de-agglomeration,nanoindentation
更新于2025-11-21 11:24:58
-
Surface Coatings Modulate the Differences in the Adhesion Forces of Eukaryotic and Prokaryotic Cells as Detected by Single Cell Force Microscopy
摘要： Single cell force microscopy was used to investigate the maximum detachment force (MDF) of primary neuronal mouse cells (PNCs), osteoblastic cells (MC3T3), and prokaryotic cells (Staphylococcus capitis subsp. capitis) from different surfaces after contact times of 1 to 5 seconds. Positively charged silicon nitride surfaces were coated with positively charged polyethyleneimine (PEI) or poly-D-lysine. Laminin was used as the second coating. PEI induced MDFs of the order of 5 to 20 nN, slightly higher than silicon nitride did. Lower MDFs (1 to 5 nN) were detected on PEI/laminin with the lowest on PDL/laminin. To abstract from the individual cell properties, such as size, and to obtain cell type-specific MDFs, the MDFs of each cell on the different coatings were normalized to the silicon nitride reference for the longest contact time. The differences in MDF between prokaryotic and eukaryotic cells were generally of similar dimensions, except on PDL/laminin, which discriminated against the prokaryotic cells. We explain the lower MDFs on laminin by the spatial prevention of the electrostatic cell adhesion to the underlying polymers. However, PEI can form long flexible loops protruding from the surface-bound layer that may span the laminin layer and easily bind to cellular surfaces and the small prokaryotic cells. This was reflected in increased MDFs after two-second contact times on silicon nitride, whereas the two-second values were already observed after one second on PEI or PEI/laminin. We assume that the electrostatic charge interaction with the PEI loops is more important for the initial adhesion of the smaller prokaryotic cells than for eukaryotic cells.
关键词： prokaryotic cells,poly-D-lysine,silicon nitride,laminin,cell adhesion,single cell force microscopy,surface coatings,polyethyleneimine,eukaryotic cells,maximum detachment force
更新于2025-11-21 11:24:58
-
The Role of Secondary Electron Emission in the Charging of Thin-Film Phase Plates
摘要： In the past few years, physical phase plates (PP) have become a viable tool to enhance the contrast of weak-phase objects in transmission electron microscopy (TEM). Thin-film PPs, such as the Zernike and Hilbert PP, are based on the mean inner potential of microstructured thin films [1,2]. Typically, a thin amorphous carbon (aC)-film is applied, whose thickness is adjusted to induce a well-defined phase shift between unscattered and scattered electrons. However, the illumination with high-energy electrons initiates an irreversible degeneration of the aC-film, which causes electrostatic charging and affects the phase-shifting properties. Taking even advantage of charging, hole-free PPs were recently developed [3,4]. Electrostatic charging plays a central role in the application of thin-film PPs. However, the mechanisms of charging are not well-understood. This work shows that charging is dominated by secondary electron emission. For this purpose, Hilbert PPs were fabricated from different materials to study their charging behavior under electron beam illumination. Besides aC-films, thin films of the metallic glass alloy Pd77.5Cu6.0Si16.5 (PCS) were used for PP fabrication. The PCS-alloy is characterized by an amorphous structure and a high electrical conductivity, which is three orders of magnitude higher than that of aC [5,6]. Moreover, the PCS-alloy exhibits a strong resistance towards oxidation, which suggests less charging of PCS-films.
关键词： transmission electron microscopy,thin-film,secondary electron emission,charging,phase plates
更新于2025-11-21 11:20:48