X-ray tomography generates 3-D reconstructions of the yeast, saccharomyces cerevisiae, at 60-nm resolution

We examined the yeast, Saccharomyces cerevisiae, using X-ray tomography and demonstrate unique views of the internal structural organization of these cells at 60-nm resolution. Cryo X-ray tomography is a new imaging technique that generates three-dimensional (3-D) information of whole cells. In the energy range of X-rays used to examine cells, organic material absorbs approximately an order of magnitude more strongly than water. This produces a quantifiable natural contrast in fully hydrated cells and eliminates the need for chemical fixatives or contrast enhancement reagents to visualize cellular structures. Because proteins can be localized in the X-ray microscope using immunogold labeling protocols (Meyer-Ilse et al., 2001. J. Microsc. 201, 395-403), tomography enables 3-D molecular localization. The time required to collect the data for each cell shown here was <15 min and has recently been reduced to 3 min, making it possible to examine numerous yeast and to collect statistically significant high-resolution data. In this video essay, we show examples of 3-D tomographic reconstructions of whole yeast and demonstrate the power of this technology to obtain quantifiable information from whole, hydrated cells.     

Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX

Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the 'water-window' wavelength region (2.34-4.37nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach - the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only.     

Hard X-ray fluorescence tomography--an emerging tool for structural visualization

Hard X-ray fluorescence microscopy is well-suited to in-situ investigations of trace metal distributions within whole, unstained, biological tissue, with sub-parts-per-million detection achievable in whole cells. The high penetration of X-rays indicates the use of X-ray fluorescence tomography for structural visualization, and recent measurements have realised sub-500-nm tomography on a 10-μm cell. Limitations of present approaches impact the duration of an experiment and imaging fidelity. Developments in X-ray resolution, detector speed, cryogenic environments, and the incorporation of auxiliary signals are being pursued within the synchrotron community. Several complementary approaches to X-ray fluorescence tomography will be routinely available to the biologist in the near future. We discuss these approaches and review applications of biological relevance.     

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of > 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.     

X-ray fluorescence microprobe imaging in biology and medicine

Characteristic X-ray fluorescence is a technique that can be used to establish elemental concentrations for a large number of different chemical elements simultaneously in different locations in cell and tissue samples. Exposing the samples to an X-ray beam is the basis of X-ray fluorescence microscopy (XFM). This technique provides the excellent trace element sensitivity; and, due to the large penetration depth of hard X-rays, an opportunity to image whole cells and quantify elements on a per cell basis. Moreover, because specimens prepared for XFM do not require sectioning, they can be investigated close to their natural, hydrated state with cryogenic approaches. Until several years ago, XFM was not widely available to bio-medical communities, and rarely offered resolution better then several microns. This has changed drastically with the development of third-generation synchrotrons. Recent examples of elemental imaging of cells and tissues show the maturation of XFM imaging technique into an elegant and informative way to gain insight into cellular processes. Future developments of XFM-building of new XFM facilities with higher resolution, higher sensitivity or higher throughput will further advance studies of native elemental makeup of cells and provide the biological community including the budding area of bionanotechnology with a tool perfectly suited to monitor the distribution of metals including nanovectors and measure the results of interactions between the nanovectors and living cells and tissues.     

X-ray mosaic nanotomography of large microorganisms

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.     

Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10 nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si₃N₄) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si₃N₄ film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.     

Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography

Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper, we describe the use of soft X-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft X-ray tomography has the advantage of not requiring the use of any staining or fixation protocols—cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft X-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.     

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

X-ray fluorescence analysis using synchrotron radiation at the photon factory

X-ray fluorescence experiments at the Photon Factory in Japan are described. An energy-dispersive X-ray fluorescence system was combined with various excitation modes, i.e., a continuum and a monochromatic excitation, which consist of a crystal monochromator or a wide band pass monochromator. These excitation modes provide a wide range of band width and photon flux of excitation beams. Minimum detection limits obtained for a thin sample were less than 0.1 ppm and 0.1 pg when there was no line interference. Advantages of using monochromatic excitation are discussed, with emphasis on the possibility of chemical state analysis. Grazing incidence X-ray fluorescence is a technique very appropriate to synchrotron radiation characteristics. Near-surface analysis and trace analysis of solution samples placed on a total reflection support were made. Future plans are discussed, including microbeam analysis, tomography, X-ray excited optical fluorescence, and applications of insertion devices (undulator and wiggler).     

光学相干层析成像技术在内窥镜中的应用

光学相干层析成像(optical coherence tomography,OCT)技术是继X射线成像、核磁共振成像、超声成像等之后的一种新型的成像技术,其可光纤化的特点使得它易于医用电子内窥镜相结合。OCT内窥镜技术可实现对人体内部器官的高速率、高分辨率、无损伤、实时成像。主要介绍了OCT技术的种类、基本原理以及包括探测深度和纵向分辨率等的参数;简述了OCT内窥镜的发展历史以及最新成果,重点分析了光源对OCT内窥镜的影响。总结了OCT内窥镜的主要应用。     

High-resolution 3-D imaging of living cells in suspension using confocal axial tomography

Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.     

Multiphoton microscopy for the in-situ investigation of cellular processes and integrity in cryopreservation

In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast.     

Quadrature demodulation in line‐scan focal modulation microscopy for imaging three‐dimensional zebrafish neural structure

Visualizing biological processes in neuroscience requires in vivo functional imaging at single‐neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide‐field fluorescence microscopes are unable to resolve cells in the presence of a strong out‐of‐focus background. Line‐scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line‐scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.      

Comparison of X-ray fluorescence and optical fluorescence measured behind scattering layers as a basis for X-ray fluorescence endoscopy.

Recent developments in the technology of capillary-fiber optics suitable for X-rays in the range of approximately 4-10keV point to the possible realization of endoscopes applicable in X-ray fluorescence analysis. A general problem is the determination of scattering and absorption processes with consideration to tissue optics, X-ray scattering and X-ray absorption in a diagnostic partial volume. Therefore comparative investigations were performed in order to answer these questions. Zinc-oxide nanoparticles configured as single particles and ZnO clusters provided the fluorescence source in cell layers. An artificial scattering material was employed, which closely approximated the tissue optical conditions and the X-ray optical application conditions in possible diagnostic situations. As a result imaging of spatially resolved X-ray contrasts was better than adequate optical fluorescence imaging by approximately one magnitude. Hence a very important precondition for realizing X-ray fluorescence endoscopy is fulfilled.     

Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits

Many biological functions depend critically upon fine details of tissue molecular architecture that have resisted exploration by existing imaging techniques. This is particularly true for nervous system tissues, where information processing function depends on intricate circuit and synaptic architectures. Here, we describe a new imaging method, called array tomography, which combines and extends superlative features of modern optical fluorescence and electron microscopy methods. Based on methods for constructing and repeatedly staining and imaging ordered arrays of ultrathin (50-200 nm), resin-embedded serial sections on glass microscope slides, array tomography allows for quantitative, high-resolution, large-field volumetric imaging of large numbers of antigens, fluorescent proteins, and ultrastructure in individual tissue specimens. Compared to confocal microscopy, array tomography offers the advantage of better spatial resolution, in particular along the z axis, as well as depth-independent immunofluorescent staining. The application of array tomography can reveal important but previously unseen features of brain molecular architecture.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Laboratory cryo soft X-ray microscopy

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.     

Use of UV excitation in confocal laser scanning fluorescence microscopy

By making only minor modifications, we adapted a conventional confocal beam-scanning laser microscope for the recording of UV-excited fluorescence. The major, and most expensive, change is that we coupled an external UV argon ion laser, providing the wavelengths 334, 351 and 364 nm, to the microscope scanner. We also replaced some optical components to obtain improved transmission and reflection properties in the UV. Only easily obtainable and inexpensive off-the-shelf components were used. The most serious problem encountered was the chromatic aberration of the microscope objective when using both UV and visible wavelengths. This is of no consequence in conventional microscopy where good imaging properties are important only in the visible region. In confocal microscopy on the other hand, good imaging properties are necessary for both the exciting and fluorescent light. Rather than having new optics designed, we tried with simple means to reduce the effects of the chromatic aberration to a tolerable level. This was done by mechanical adjustments in the ray-path. In addition we also tested two mirror objectives, which are inherently free from chromatic aberrations. However, such objectives have rather limited numerical apertures and are not of the immersion type. Their value in biomedical applications is therefore limited.The objective most frequently used in our experiments was a 63/1.25 oil-immersion fluorite. Without any compensation this objective had a depth resolution in UV-excited confocal fluorescence that was an order of magnitude worse than when using visible-light excitation. The useful field of view was also very small due to lateral chromatic aberration. By simple means we managed to improve the depth resolution by a factor of 4.4, and at the same time increase the useful field of view substantially. Still, the depth resolution was worse than what is obtained using visible light excitation. We think this is due to the fact that after compensation the objective is working with an incorrect tube length.Using the modified instrument, we recorded specimens labelled with AMCA and Fluoro-Gold, obtaining 1.5 μm thick optical sections.