首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 42 毫秒
1.
We propose a near-infrared super resolution near field imaging system with an array of metallic nanoshell particle chain. The imaging array can plasmonically transfer the near field components of dipole sources and the super resolution images can be reconstructed in the output plane. By decreasing the metallic nanoshell’s thickness of the fixed size nanoparticle, the plasmon resonance wavelength of the isolate nanoshell particle is red-shifted to the near-infrared region. The operation wavelength of the imaging array is correspondingly red-shifted to the near-infrared region. In this paper, we study the incoherent and coherent super resolution imaging. The field intensity distributions at the different planes of imaging process are calculated using the finite element method. The simulation results demonstrate that the array has super resolution imaging capability at near-infrared wavelengths in the incoherent and coherent manners. The results also show that the image formation highly depends on the source coherence. In the same structural parameters, the reconstructed images under the illumination of incoherent light source reach to the higher image quality and spatial resolution than the images under the illumination of coherent light source of in phase. By reasonably designing parameters of the imaging array, the approximate spatial resolutions of λ/13 in incoherent case and λ/10 in coherent case are obtained at the near-infrared wavelength of 764 nm. Furthermore, the image–array distance and the chains’ spacing also affect the image reconstruction.  相似文献   

2.
We propose a wide-field super-resolved optical microscopic imaging technique based on subwavelength slit arrays embedded in a thin silver film to generate surface plasmon (SP) standing wave interference patterns. These fringes carrying high spatial frequency information serve as excitation profiles to excite the nanoscale fluorescence objects. The super-resolved fluorescence density distribution is reconstructed from a weight sum of a series of fluorescence images with differently phase-shifted SP standing wave illumination. Simulation and experimental results show that the lateral resolution of the reconstructed fluorescence density image is enhanced by 0.28?λ SP in two dimensions, which is twofold better than that of conventional high numerical aperture fluorescence microscopy. This technique benefits from a grating coupler to offer a simple way for the generation and phase shift of SP standing wave excitation profiles in two dimensions. The flat configuration, wide field, and noninvasive nature make this approach suitable for real-time analyzing the fine details of bio-samples in biochip applications.  相似文献   

3.
Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.  相似文献   

4.
Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.  相似文献   

5.
A plasmonic resonant cavity-based hyperlens is theoretically proposed and demonstrated to achieve far-field phase contrast images of nano-transparent objects. The phase contrast super-resolution imaging is mainly contributed to the excited surface plasmons inside hyperlens and cavity structure surrounding nano-objects, which help to greatly enhance evanescent waves generated by nano-transparent objects and convert weak phase information to light intensity distribution with high contrast at the zoomed imaging plane of hyperlens. As examples, nano-dielectric object imaging is numerically demonstrated with half-pitch resolution about λ/10 and a minimum distinguishable refractive index difference down to 0.15.  相似文献   

6.
Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)–based single-molecule tracking technique to analyze F-actin movements with ∼30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of ∼138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.  相似文献   

7.
In this study, we propose a plasmonic free-space filter with dual resonance wavelength by using an asymmetric T-shaped array. The structure under the T-shaped structure forms two metal/insulator/metal cavities with different cavity length. Each cavity supports a specific resonance wavelength. A notch filter for second harmonic generation Nd:YAG laser is also proposed. The filter offers two resonance dips and low sideband. In addition, the filter properties are based on the localized surface plasmon. Therefore, the angle tolerance is extremely high. This makes the proposed structure easy to align. The proposed structure can be used in dual wavelength biosensing detection and dual wavelength thermal emission applications.  相似文献   

8.
We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction (“super-resolution”) images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specialized pore-PD that connect sieve elements (SEs) with their companion cells (CCs) along the tubular sieve element reticulum (SER). The SER was shown to interconnect individual pore-PD at the SE-CC interface. 3D-SIM resolved fine (less than 100 nm) endoplasmic reticulum threads running into individual pore-PD as well as strands that crossed sieve plate pores, structurally linking SEs within a file. Our data reveal that MP entering the SE from the CC may remain associated with the SER. Fluorescence recovery after photobleaching experiments revealed that this MP pool is relatively immobile compared with the membrane probe 3,3’-dihexyloxacarbocyanine iodide, suggesting that MP may become sequestered by the SER once it has entered the SE. The advent of 3D-SIM offers considerable potential in the subdiffraction imaging of plant cells, bridging an important gap between confocal and electron microscopy.Fluorescence-based imaging has revolutionized cell biology, allowing the localization of proteins to specific cells and organelles (Shaner et al., 2007; Frigault et al., 2009). However, conventional fluorescence microscopy is limited by the diffraction of light to approximately 200 nm in the lateral (x-y) plane and to about 500 nm in the axial (z) plane (Fernandez-Suarez and Ting, 2008; Huang et al., 2009). This is because light traveling through a lens cannot be focused to a point, only to an airy disc with a diameter of about half the wavelength of the visible emitted light (Huang et al., 2009). Confocal laser scanning microscopy has produced improvements in axial resolution due to the removal of out-of-focus flare, but it is also limited by diffraction (Huang et al., 2009). Thus, objects closer than about 200 nm cannot be resolved but appear merged into one. Many subcellular structures of interest to cell biologists lie below this resolution limit and have remained below the diffraction barrier. Such structures can be seen but not resolved.Recently, major innovations in biological imaging have broken the diffraction barrier. These include photoactivation localization microscopy (PALM) and stimulated emission and depletion (STED; for review, see Fernandez-Suarez and Ting, 2008; Huang et al., 2009). Most subdiffraction or “super-resolution” approaches have improved resolution in either the lateral (x-y) plane or the axial (z) plane, but usually not both (Schermelleh et al., 2008). Many of the structures of interest within plant cells lie some distance from the cell wall, posing problems for some super-resolution approaches (e.g. PALM) where the subject of interest must lie close to the coverslip (Huang et al., 2009). Recently, Schermelleh et al. (2008) described a subdiffraction multicolor imaging protocol using three-dimensional structured illumination microscopy (3D-SIM). In this method, objects beyond the diffraction limit are illuminated with multiple interfering beams of light transmitted through a series of diffraction gratings, producing a resolution of 100 nm in x-y and 200 nm in z (Schermelleh et al., 2008; Huang et al., 2009). These substantial increases in resolution are significant for plant cell imaging. The thickness of the plant cell wall is typically in the region of about 700 nm, allowing limited optical sectioning capacity with a confocal microscope (about 500 nm in z). A further advantage of 3D-SIM is that it permits the imaging of conventional fluorescent reporters and dyes that are compatible with confocal imaging, allowing a direct correlation of 3D-SIM and confocal images (Schermelleh et al., 2008).The phloem of higher plants is a major conduit for the long-distance transport of solutes (Oparka and Turgeon, 1999) and also functions as a “superhighway” for macromolecular trafficking (Lucas and Lee, 2004; Kehr and Buhtz, 2008; Lee and Cui, 2009). However, the phloem is difficult to image with conventional optical microscopy (Knoblauch and van Bel, 1998; Oparka and Turgeon, 1999; van Bel et al., 2002). Sieve elements (SEs), the conducting cells of the phloem, are enucleate yet contain a plethora of proteins and RNAs associated with long-distance signaling and defense (van Bel and Gaupels, 2004; Lee and Cui, 2009). Many of these macromolecules are synthesized in the companion cell (CC) and passed into the SE via the specialized pore-plasmodesmata (PD) that connect the two cell types (Oparka and Turgeon, 1999; van Bel et al., 2002). Pore-PD have been suggested to be a major “lifeline” from CC to SE (van Bel et al., 2002), but the exact nature of this pathway remains unresolved.Our current understanding of PD substructure is derived largely from electron microscope studies (Roberts, 2005). Such methods are time-consuming and do not permit facile protein localization within PD. Recent proteomics approaches have been successful in identifying new proteins associated with PD (Maule, 2008). Localization of these proteins with confocal microscopy results in the appearance of discrete punctae at the cell wall, consistent with the location of pit fields (Faulkner et al., 2008), but does not pinpoint specific protein locations within PD. In general, there is a growing gap between proteomics studies of plant organelles, including PD, and the ability to ascribe accurate addresses to these proteins (Millar et al., 2009; Moore and Murphy, 2009). The advent of 3D-SIM prompted us to explore the potential of subdiffraction imaging in plant cells, with a view to obtaining improved florescence resolution of PD. We used 3D-SIM to examine PD in a transgenic tobacco (Nicotiana tabacum) line expressing the viral movement protein (MP) of Tobacco mosaic virus (TMV) fused to GFP. Using a specific antibody to callose, a wall constituent located at the PD collar, we were able to resolve clearly the structure of single, simple PD in epidermal cells at 100-nm resolution, discriminating between the neck region of the pore and the central cavity to which it connects (Roberts and Oparka, 2003; Faulkner et al., 2008). 3D-SIM also revealed details of the central cavities of complex PD seen previously only with the electron microscope (Ding et al., 1992; Ehlers and Kollmann, 2001; Faulkner et al., 2008).Using 3D-SIM, we were able to image PD sequentially from the epidermis to the phloem within vascular bundles, producing unparalleled images of sieve plate pores and the specialized pore-PD that connect SEs with their CCs. In the SEs, MP was no longer restricted to the central cavities of PD but became distributed along the SE parietal layer, connecting all the pore-PD along the SE-CC interface. We were able to detect fine threads of MP-GFP that extended for up to 40 μm along the SE and also crossed individual sieve plate pores. Fluorescence recovery after photobleaching (FRAP) experiments revealed that this MP-GFP pool was relatively immobile within the SE parietal layer, suggesting that the SE may sequester TMV MP on or within the sieve element reticulum (SER).Our data reveal that 3D-SIM is especially suited to the subdiffraction imaging of plant cells and yields spatial information not previously possible with conventional fluorescence-based imaging. The unique optical sectioning capacity of 3D-SIM and the ability to produce multicolor imaging with conventional fluorophores offer enormous potential in plant cell biology.  相似文献   

9.
The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein.  相似文献   

10.
11.
Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.  相似文献   

12.
We design two kinds of plasmonic broadband polarization splitters based on dual-core photonic crystal fiber (DC-PCF) with elliptical Au or Ag nanowire in this paper. It is analyzed for the polarization independent characterestics of the designed DC-PCF by the finite element method (FEM). In order to excite the surface plasmon resonance (SPR), the metal Au and Ag are filled into elliptical central air hole. The resonance coupling between the fourth- or fifth-order surface plasmon modes (SPMs) and core-guided modes (CGMs) are founded by this numerical simulation. The device lengths of the designed splitters with Au nanowire are 2937 and 827 μm at the wavelength of 1.31 and 1.55 μm, respectively. As the extinction ratios are better than ?20 dB, its bandwidths are better than 94 and 103 nm. For the designed Ag nanowire splitter, the device lengths are 3066 or 809 μm at 1.31 or 1.55 μm, respectively. The bandwidths with the extinction ratio better than ?20 dB are 66 and 104 nm, respectively.  相似文献   

13.
Super-resolution imaging using microspheres has attracted tremendous scientific attention recently because it has managed to overcome the diffraction limit and allowed direct optical imaging of structures below 100 nm without the aid of fluorescent microscopy. To allow imaging of specific areas on the surface of samples, the migration of the microspheres to specific locations on two-dimensional planes should be controlled to be as precise as possible. The common approach involves the attachment of microspheres on the tip of a probe. However, this technology requires additional space for the probe and could not work in an enclosed environment, e.g., in a microfluidic enclosure, thereby reducing the range of potential applications for microlens-based super-resolution imaging. Herein, we explore the use of laser trapping to manipulate microspheres to achieve super-resolution imaging in an enclosed microfluidic environment. We have demonstrated that polystyrene microsphere lenses could be manipulated to move along designated routes to image features that are smaller than the optical diffraction limit. For example, a silver nanowire with a diameter of 90 nm could be identified and imaged. In addition, a mosaic image could be constructed by fusing a sequence of images of a sample in an enclosed environment. Moreover, we have shown that it is possible to image Escherichia coli bacteria attached on the surface of an enclosed microfluidic device with this method. This technology is expected to provide additional super-resolution imaging opportunities in enclosed environments, including microfluidic, lab-on-a-chip, and organ-on-a-chip devices.  相似文献   

14.
Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes: (1) patterned illumination- based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures; (2) single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluo- rophore at subdiffraction precision; (3) bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.  相似文献   

15.
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-''light-sheet'', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-''light-sheet'' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.  相似文献   

16.
Three-dimensional (3D) localization-based super-resolution microscopy (SR) requires correction of aberrations to accurately represent 3D structure. Here we show how a depth-dependent lateral shift in the apparent position of a fluorescent point source, which we term `wobble`, results in warped 3D SR images and provide a software tool to correct this distortion. This system-specific, lateral shift is typically > 80 nm across an axial range of ~ 1 μm. A theoretical analysis based on phase retrieval data from our microscope suggests that the wobble is caused by non-rotationally symmetric phase and amplitude aberrations in the microscope’s pupil function. We then apply our correction to the bacterial cytoskeletal protein FtsZ in live bacteria and demonstrate that the corrected data more accurately represent the true shape of this vertically-oriented ring-like structure. We also include this correction method in a registration procedure for dual-color, 3D SR data and show that it improves target registration error (TRE) at the axial limits over an imaging depth of 1 μm, yielding TRE values of < 20 nm. This work highlights the importance of correcting aberrations in 3D SR to achieve high fidelity between the measurements and the sample.  相似文献   

17.
In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging.  相似文献   

18.
Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.  相似文献   

19.
20.
We propose a 3D metasurface structure with unsymmetrical metallic slices array. The tunable plasmon-induced transparency (PIT) effects and different electric field mode distributions could be realized by modulating the structure parameters and angle of incidence. The radiative and dark elements of the asymmetric metallic slices unit cell structure are analyzed. The transmission spectra and the electric fields distributions are studied by the finite element method (FEM). We demonstrate that PIT phenomena based on those metasurface array structures may have applications as tunable sensors and filters in nanophotonics and integrated optics.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号