共查询到20条相似文献,搜索用时 15 毫秒
1.
Sven Truckenbrodt Manuel Maidorn Dagmar Crzan Hanna Wildhagen Selda Kabatas Silvio O Rizzoli 《EMBO reports》2018,19(9)
Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge. 相似文献
2.
Plecitá-Hlavatá L Lessard M Santorová J Bewersdorf J Jezek P 《Biochimica et biophysica acta》2008,1777(7-8):834-846
Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262+/-26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311+/-36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284+/-38 nm and 417+/-110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to approximately 10%, disintegration into several reticula and numerous approximately 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4+/-0.4 microm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard. 相似文献
3.
Nikki M. Curthoys Michael J. Mlodzianoski Dahan Kim Samuel T. Hess 《Journal of visualized experiments : JoVE》2013,(82)
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described. 相似文献
4.
Sylvia Glaschick Carlheinz Röcker Karen Deuschle Jörg Wiedenmann Franz Oswald Volker Mailänder G. Ulrich Nienhaus 《Journal of biological physics》2007,33(5-6):433-443
Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200–300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to ~100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells. 相似文献
5.
A novel, multifunctional optical imaging system was developed by integrating four-dimensional (4D) real-time confocal microscopy (RT-CM), multicolor total internal reflection microscopy (TIRFM), and Nomarski differential interference contrast (DIC) microscopy based on an epifluorescence microscope platform. A microcell incubator was combined with the imaging system for extended, real-time monitoring of living cells. The 4D images were generated by a combination of 3D images and multiple spatial or time images of a specimen, obtained at 10 nm intervals. Optical sectioning was accomplished with a z-motor, which obtained 4D information with sequential layered sections. The integrated imaging system showed excellent detection sensitivity at the single-molecule level and 3D-spatial resolution (20 nm x-y and 10 nm z-axis) without moving the cell sample. This could be a tool for obtaining crucial information needed to develop approaches for characterizing and understanding the dynamics of biomolecules and nanoparticles in individual living cells and molecular interactions at the single-molecule level. 相似文献
6.
Baoju Wang Longfang Yao Yueyue Jing Yiyan Fei Qianming Bai Lan Mi Jiong Ma 《Journal of biophotonics》2020,13(5)
Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm. 相似文献
7.
Widefield fluorescence microscopy with extended resolution 总被引:1,自引:1,他引:0
Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging. 相似文献
8.
We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse fibroblast cell. Comparison with two-photon confocal resolution reveals a fourfold better axial resolution in the 4Pi-confocal case. By combining 4Pi-confocal microscopy with Richardson–Lucy image restoration a further resolution increase is achieved. Featuring a three-dimensional resolution in the range 100–150 nm, the 4Pi-confocal (restored) images are intrinsically more detailed than their confocal counterparts. Our images constitute what to our knowledge are the best-resolved three-dimensional images of entangled cellular microtubules obtained with light to date. 相似文献
9.
Jialin Wang Jia Zhang Luwei Wang Xinwei Gao Yonghong Shao Liwei Liu Zhigang Yang Wei Yan Junle Qu 《Journal of biophotonics》2020,13(8)
STED (stimulated emission depletion) microscopy is one of the most promising super‐resolution fluorescence microscopies,due to its fast imaging and ultra‐high resolution. In this paper, we present a dual‐color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual‐color STED system to achieve a spatial resolution of 75 nm in cell samples. 相似文献
10.
In plant storage tissue, lipid bodies are composed of triacylglycerides and surrounded by a phospholipid monolayer which is stabilized by oleosins. At the onset of lipid body mobilization, cells express phospholipase A2, which partially degrades the monolayer and thus provides access for the subsequently acting triacylglyceride degrading enzymes. Analyzing the lipid body surface by atomic force microscopy we show that, at the stage of maximal phospholipase A2 expression, the monolayer contains holes of approximately 80 nm in width and 2.45 +/- 0.46 nm in depth. Non-contact mode imaging was performed with a lateral resolution of approximately 10 nm and a vertical resolution of less than 0.1 nm. The depth of the holes corresponds to the width of the monolayer, while the width of the channels is sufficiently large to provide access to 100 kDa enzymes, such as lipoxygenase and lipases. 相似文献
11.
The three-dimensional structure of the regular surface layer of Bacillus sphaericus P-1 (T-layer) was determined to a resolution of ca. 2.5 nm by electron microscopy and image analysis. The T-layer has P4 symmetry, a lattice constant of 13 +/- 0.2 nm, and a thickness of ca. 8 nm. The reconstruction revealed three distinct domains: a major, a minor, and an arm domain. In the z-direction, the domains are arranged in two planes creating two different surface reliefs. 相似文献
12.
13.
Measuring the size of biological nanostructures with spatially modulated illumination microscopy
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Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes. 相似文献
14.
15.
Unusual ultrastructure of complement-component-C4b-binding protein of human complement by synchrotron X-ray scattering and hydrodynamic analysis. 总被引:2,自引:0,他引:2
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Solution X-ray-scattering experiments with the use of synchrotron radiation on the human complement-component-C4b-binding protein showed that its RG is 13 nm and that its Mr is 550,000. From the known primary amino acid sequence and estimated carbohydrate content, C4b-binding protein is inferred to have a total of 7.4 +/- 1 subunits. Heptameric computer models for C4b-binding protein were based on the X-ray-scattering curve to a resolution of 6.4 nm, and literature values for sedimentation coefficients and electron-microscopy images. The macromolecule was represented by a bundle of seven arms held together at the C-terminal end and spaced out by a base containing 23% of C4b-binding protein by volume. If the overall length of each arm is assumed to be 33 nm as seen in electron microscopy, the solution data indicate an average arm-axis angle of 5-10 degrees. The seven arms of C4b-binding protein are found to be close together, in distinction to the splayed-out images seen in electron micrographs. 相似文献
16.
Iancu CV Wright ER Benjamin J Tivol WF Dias DP Murphy GE Morrison RC Heymann JB Jensen GJ 《Journal of structural biology》2005,151(3):288-297
Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (approximately 4-8 nm) resolution in a near-native state, but restrictions in the range of accessible views are a major limitation. Here we report on the design, characterization, and demonstration of a new "flip-flop" rotation stage that allows facile and routine collection of two orthogonal tilt-series of cryosamples. Single- and dual-axis tomograms of a variety of samples are compared to illustrate qualitatively the improvement produced by inclusion of the second tilt-series. Exact quantitative expressions are derived for the volume of the remaining "missing pyramid" in reciprocal space. When orthogonal tilt-series are recorded to +/-65 degrees in each direction, as this new cryostage permits, only 11% of reciprocal space is left unmeasured. The tomograms suggest that further improvement could be realized, however, through better software to align and merge dual-axis tilt-series of cryosamples. 相似文献
17.
Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs. 相似文献
18.
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
19.
Cremer C Kaufmann R Gunkel M Pres S Weiland Y Müller P Ruckelshausen T Lemmer P Geiger F Degenhard S Wege C Lemmermann NA Holtappels R Strickfaden H Hausmann M 《Biotechnology journal》2011,6(9):1037-1051
For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (~1/100 of the exciting wavelength). 相似文献
20.
Toshihiko Ogura 《Biochemical and biophysical research communications》2009,380(2):254-1671
The indirect secondary electron contrast (ISEC) condition of the scanning electron microscopy (SEM) produces high contrast detection with minimal damage of unstained biological samples mounted under a thin carbon film. The high contrast image is created by a secondary electron signal produced under the carbon film by a low acceleration voltage. Here, we show that ISEC condition is clearly able to detect unstained bacteriophage T4 under a thin carbon film (10-15 nm) by using high-resolution field emission (FE) SEM. The results show that FE-SEM provides higher resolution than thermionic emission SEM. Furthermore, we investigated the scattered electron area within the carbon film under ISEC conditions using Monte Carlo simulation. The simulations indicated that the image resolution difference is related to the scattering width in the carbon film and the electron beam spot size. Using ISEC conditions on unstained virus samples would produce low electronic damage, because the electron beam does not directly irradiate the sample. In addition to the routine analysis, this method can be utilized for structural analysis of various biological samples like viruses, bacteria, and protein complexes. 相似文献