共查询到20条相似文献,搜索用时 15 毫秒
1.
Changyong Song Masatoshi Takagi Jaehyun Park Rui Xu Marcus Gallagher-Jones Naoko Imamoto Tetsuya Ishikawa 《Biophysical journal》2014
Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens. 相似文献
2.
Murphy GE Narayan K Lowekamp BC Hartnell LM Heymann JA Fu J Subramaniam S 《Journal of structural biology》2011,(3):268-278
We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues. 相似文献
3.
Joshua Yoon Colin J. Comerci Lucien E. Weiss Ljiljana Milenkovic Tim Stearns W.E. Moerner 《Biophysical journal》2019,116(2):319-329
Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ~250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (~2–6 μm long and 200–300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10–25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest. 相似文献
4.
We report attainment of subdiffraction resolution using stimulated emission depletion (STED) microscopy with GFP-labeled samples. The approximately 70 nm lateral resolution attained in this study is demonstrated by imaging GFP-labeled viruses and the endoplasmic reticulum (ER) of a mammalian cell. Our results mark the advent of nanoscale biological microscopy with genetically encoded markers. 相似文献
5.
Daniel Haddad Stefan Huggenberger Michaela Haas-RiothLars S. Kossatz Helmut H.A. OelschlägerAxel Haase 《Zoologischer Anzeiger》2012,251(2):115-130
The structure of two preserved prenatal dolphins were visualized by 3D MR microscopy (isotropic nominal resolution up to 78.1 μm), which is a high-resolution 3D magnetic resonance imaging (MRI) technique. To determine the benefits and limitations of this method, the acquired 3D datasets were segmented manually and compared to histological sections of different specimens in corresponding developmental stages. The MR images visualize the external and internal morphology of both prenatal dolphins in detail. Various organ systems with their main components are clearly documented in the images, allowing a complete segmentation of the specimens and the calculation of volumes and surface areas of different organ systems. Due to its non-invasive character and the detailed imaging within its resolution range, MR microscopy proves to be a valuable tool in developmental biology for the visualization of the inner architecture of rare and delicate museum specimens, such as the small dolphin embryo and fetus examined. In these two prenatal dolphins, the profound structural modifications at the transition from the embryonic to the fetal stage reflect the adaptations of the mammalian bauplan to the requirements of a holaquatic cetacean life-style. However, the developmental pattern and sequence of the emerging tissues and organs in prenatal life do not resemble the hypothesized evolution of the structural and functional adaptations found in the fossil record. 相似文献
6.
Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists. 相似文献
7.
Hertz HM von Hofsten O Bertilson M Vogt U Holmberg A Reinspach J Martz D Selin M Christakou AE Jerlström-Hultqvist J Svärd S 《Journal of structural biology》2012,177(2):267-272
Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a λ=2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined. 相似文献
8.
Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells. 相似文献
9.
Cella Zanacchi F Lavagnino Z Perrone Donnorso M Del Bue A Furia L Faretta M Diaspro A 《Nature methods》2011,8(12):1047-1049
We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids. 相似文献
10.
Mokso R Quaroni L Marone F Irvine S Vila-Comamala J Blanke A Stampanoni M 《Journal of structural biology》2012,177(2):233-238
Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state. 相似文献
11.
Johnny Tam Guillaume Alan Cordier ?tefan Bálint ángel Sandoval álvarez Joseph Steven Borbely Melike Lakadamyali 《PloS one》2014,9(12)
Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. 相似文献
12.
Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. 总被引:14,自引:0,他引:14
A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos. 相似文献
13.
Heymann JA Hayles M Gestmann I Giannuzzi LA Lich B Subramaniam S 《Journal of structural biology》2006,155(1):63-73
Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications. 相似文献
14.
Fluorescence microscopy, especially confocal microscopy, has revolutionized the field of biological imaging. Breaking the optical diffraction barrier of conventional light microscopy, through the advent of super-resolution microscopy, has ushered in the potential for a second revolution through unprecedented insight into nanoscale structure and dynamics in biological systems. Stimulated emission depletion (STED) microscopy is one such super-resolution microscopy technique which provides real-time enhanced-resolution imaging capabilities. In addition, it can be easily integrated with well-established fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) in order to capture the structure of cellular membranes at the nanoscale with high temporal resolution. In this review, we discuss the theory of STED and different modalities of operation in order to achieve the best resolution. Various applications of this technique in cell imaging, especially that of neuronal cell imaging, are discussed as well as examples of application of STED imaging in unravelling structure formation on biological membranes. Finally, we have discussed examples from some of our recent studies on nanoscale structure and dynamics of lipids in model membranes, due to interaction with proteins, as revealed by combination of STED and FCS techniques. 相似文献
15.
Feng Xu Lukas Helfen Heikki Suhonen Dan Elgrabli Sam Bayat Péter Reischig Tilo Baumbach Peter Cloetens 《PloS one》2012,7(11)
Structure and composition at the nanoscale determine the behavior of biological systems and engineered materials. The drive to understand and control this behavior has placed strong demands on developing methods for high resolution imaging. In general, the improvement of three-dimensional (3D) resolution is accomplished by tightening constraints: reduced manageable specimen sizes, decreasing analyzable volumes, degrading contrasts, and increasing sample preparation efforts. Aiming to overcome these limitations, we present a non-destructive and multiple-contrast imaging technique, using principles of X-ray laminography, thus generalizing tomography towards laterally extended objects. We retain advantages that are usually restricted to 2D microscopic imaging, such as scanning of large areas and subsequent zooming-in towards a region of interest at the highest possible resolution. Our technique permits correlating the 3D structure and the elemental distribution yielding a high sensitivity to variations of the electron density via coherent imaging and to local trace element quantification through X-ray fluorescence. We demonstrate the method by imaging a lithographic nanostructure and an aluminum alloy. Analyzing a biological system, we visualize in lung tissue the subcellular response to toxic stress after exposure to nanotubes. We show that most of the nanotubes are trapped inside alveolar macrophages, while a small portion of the nanotubes has crossed the barrier to the cellular space of the alveolar wall. In general, our method is non-destructive and can be combined with different sample environmental or loading conditions. We therefore anticipate that correlative X-ray nano-laminography will enable a variety of in situ and in operando 3D studies. 相似文献
16.
Quantitative Superresolution Microscopy Reveals Differences in Nuclear DNA Organization of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance
下载免费PDF全文
![点击此处可从《Journal of cellular biochemistry》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Chirawadee Sathitruangsak Christiaan H. Righolt Ludger Klewes Pille Tammur Tiiu Ilus Anu Tamm Mari Punab Adebayo Olujohungbe Sabine Mai 《Journal of cellular biochemistry》2015,116(5):704-710
The mammalian nucleus has a distinct substructure that cannot be visualized directly by conventional microscopy. In this study, the organization of the DNA within the nucleus of multiple myeloma (MM) cells, their precursor cells (monoclonal gammopathy of undetermined significance; MGUS) and control lymphocytes of the representative patients is visualized and quantified by superresolution microscopy. Three‐dimensional structured illumination microscopy (3D‐SIM) increases the spatial resolution beyond the limits of conventional widefield fluorescence microscopy. 3D‐SIM reveals new insights into the nuclear architecture of cancer as we show for the first time that it resolves organizational differences in intranuclear DNA organization of myeloma cells in MGUS and in MM patients. In addition, we report a significant increase in nuclear submicron DNA structure and structure of the DNA‐free space in myeloma nuclei compared to normal lymphocyte nuclei. Our study provides previously unknown details of the nanoscopic DNA architecture of interphase nuclei of the normal lymphocytes, MGUS and MM cells. This study opens new avenues to understanding the disease progression from MGUS to MM. J. Cell. Biochem. 116: 704–710, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. 相似文献
17.
Schneider G Guttmann P Rehbein S Werner S Follath R 《Journal of structural biology》2012,177(2):212-223
X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed. 相似文献
18.
Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast. 相似文献
19.
Three-dimensional imaging by deconvolution microscopy 总被引:26,自引:0,他引:26
Deconvolution is a computational method used to reduce out-of-focus fluorescence in three-dimensional (3D) microscope images. It can be applied in principle to any type of microscope image but has most often been used to improve images from conventional fluorescence microscopes. Compared to other forms of 3D light microscopy, like confocal microscopy, the advantage of deconvolution microscopy is that it can be accomplished at very low light levels, thus enabling multiple focal-plane imaging of light-sensitive living specimens over long time periods. Here we discuss the principles of deconvolution microscopy, describe different computational approaches for deconvolution, and discuss interpretation of deconvolved images with a particular emphasis on what artifacts may arise. 相似文献
20.
Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of actin. Measurements done on nonmoving cellular protrusion tips showed no pair-correlation at two orbital positions indicating a lack of flow of F-actin bundles. However, in some protrusions, the pair-correlation approach revealed directional flow of F-actin bundles near the protrusion surface with flow rates in the range of ∼1 μm/min, comparable to results in two dimensions using fluorescent speckle microscopy. Furthermore, we found that the actin flow rate is related to the distance to the protrusion tip. We also observed collagen deformation by concomitantly detecting collagen fibers with reflectance detection during these actin motions. The implementation of the nanoscale precise imaging by rapid beam oscillation method with a cloverleaf-shaped trajectory in conjunction with the pair cross-correlation function method provides a quantitative way of capturing dynamic flows and organization of proteins during cell migration in 3D in conditions of poor contrast. 相似文献