Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography

Using a cryo scanning transmission X-ray microscope ( Maser, et al . (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc . 197, 68–79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

X-ray microscopy and imaging of Escherichia coli, LPS and DNA

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of speci-mens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 μm wavelength, 5 × 10¹² W cm⁻² intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20 J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E. coli , darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 Å level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

A new cryo‐STEM method for imaging food colloids in the scanning electron microscope

A new cryo‐scanning transmission electron microscopy (cryo‐STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at ?135°C showing casein micelles in their frozen hydrated state and in the size range 30–500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo‐transmission electron microscopy, suggesting that cryo‐STEM is a useful alternative technique for visualizing food colloids close to their native state. SCANNING 32: 150–154, 2010. © 2010 Wiley Periodicals, Inc.     

Computed tomography of cryogenic biological specimens based on X-ray microscopic images

Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment, rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state.     

Soft X-ray spectroscopy from image sequences with sub-100 nm spatial resolution

A method is described whereby a sequence of X-ray images at closely spaced photon energies is acquired using a scanning transmission X-ray microscope, and aligned. Near-edge absorption spectra can then be obtained both from large, irregular regions, and from regions as small as the spatial resolution of the microscope (about 40 nm in the examples shown here). The use of the technique is illustrated in examination of a layered polymer film, a micrometeorite section, and an interplanetary dust particle section.     

软X射线显微术和光谱显微术

X射线显微术可直接在水环境中对胶体颗粒尺寸范围内的颗粒进行高分辨率成像,将该项技术与高分辨率光谱相结合,还可用于光谱显微研究。其中,常用的两种X射线显微镜是透射显微镜和扫描透射显微镜,文中示出了它们的装置图。由于X射线显微镜能迅速拍下一物体的高分辨率图像,所以,作为一种分析仪器,扫描X射线显微镜更适合作光谱显微研究。作为形态学目视化的一个示例,本文用一台透射X射线显微镜拍摄了粘土和土壤样品的图像。根据X射线图像进行的低温层析实验所获得的图像得到了有关细菌构成的显微生存环境以及其它土壤胶状体的3D结构信息。对扫描透射X射线显微镜拍摄的一系列图像进行分析,得到了土壤样品的形貌特性和化学特性。     

Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples

The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30‐keV Ga⁺ beam, and cryo‐transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200–150 nm, changes characteristic of heat‐induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that ‘cryo‐FIB thinning’ of bulk frozen‐hydratred material will be capable of producing specimens for TEM cryo‐tomography with much greater efficiency than cryo‐ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.     

An environmental sample chamber for X-ray microscopy

The manufacture, construction and performance of a special specimen holder for biological samples suspended in aqueous solution is described. The holder was designed for use in a scanning transmission X-ray microscope. Using it, we have successfully obtained high-resolution images of fresh cellular and subcellular specimens in suspension and have been able to vary the sample environment during viewing in the microscope.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

The preparation,examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and X-ray microanalysis

A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X-ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen-hydrated. Criteria for assessing whether or not the tissue remains frozen-hydrated are reported. After quench freezing, 1–2 μm thick sections of mouse liver were cut at 193°K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143°K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X-ray analyser. Concentration of intra-nuclear and intracytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.     

Scanning luminescence X-ray microscopy: Imaging fluorescence dyes at suboptical resolution

Scanning luminescence X-ray microscopy is based on the use of the very small focused probe of a scanning X-ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X-ray source and a Fresnel zone plate to produce a focused X-ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X-ray probe (the microscope has imaged features at 36-nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye-tagged sites of biochemical activity at the resolution of the X-ray microscope in wet, unsectioned, and unfixed cells, especially with soft X-ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye-tagged features should be 2–22 times lower than it is in transmission X-ray microscopy. A possible extension of the technique for three-dimensional imaging at the transverse resolution of the X-ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.     

The effect of large solid angles of collection on quantitative X-ray microanalysis in the AEM

Increasing the solid angle of X-ray collection is a major factor in improving the analytical sensitivity of X-ray energy-dispersive spectrometry (XEDS) in the analytical electron microscope (AEM). A new scanning transmission electron microscope, the VG HB 603, is equipped with two XEDS detectors with the largest collection angles (0.30 and 0.17 sr) available in commercial AEMs. However, large collection angles result in a large range of take-off angles, from ~ 4° to 36°, and the low angles can cause strong X-ray absorption. In order to investigate possible detrimental effects of the low (and of the range of) take-off angles on quantitative microanalysis of specimens exhibiting significant absorption, a stoichiometric Ni₃Al thin-film, in which the Al Kα line is significantly absorbed, was analysed. Furthermore, the effect of different values of the collection angle on X-ray intensities was theoretically evaluated by numerical calculations and spectral simulation. These theoretical approaches permitted correlation of changes in the X-ray take-off angle (and hence X-ray absorption) with changes in the collection angle. It is demonstrated that ~ 0.30 sr detectors, with minimum take-off angles as small as 4°, only result in maximum errors of 4% in the quantification of Al in Ni₃Al and, therefore, further increases in collection angle can be pursued while maintaining current levels of accuracy of quantification.     

Compact water-window transmission X-ray microscopy

We demonstrate sub-100 nm resolution water-window soft X-ray full-field transmission microscopy with a compact system. The microscope operates at λ = 3.37 nm and is based on a 100 Hz table-top regenerative debris-free droplet-target laser-plasma X-ray source in combination with normal-incidence multilayer condenser optics for sample illumination. High-spatial-resolution imaging is performed with a 7.3% efficiency nickel zone plate and a 1024 × 1024 pixel CCD detector. Images of dry test samples are recorded with exposure times of a few minutes and show features smaller than 60 nm.     

Coaxial arrangement of a scanning probe and an X-ray microscope as a novel tool for nanoscience

We report on the design and first tests of a novel instrument aimed at combining the benefits of scanning force microscopy with those of X-ray spectroscopy. For this we built an instrument combining a scanning transmission X-ray microscope with a beam-deflection atomic force microscope in a coaxial geometry. This allows to combine X-ray absorption spectroscopy and high resolution topography in-situ. When replacing the conventional scanning probe tip by a coaxially shielded tip the instrument will allow detection of the photoelectrons produced by resonant X-ray absorption. This could yield spectroscopic information with a spatial resolution approaching the values achievable with atomic force microscopy.     

Quantitative amplitude and phase contrast imaging in a scanning transmission X-ray microscope

Phase contrast in X-ray imaging provides lower radiation dose, and dramatically higher contrast at multi-keV photon energies when compared with absorption contrast. We describe here the use of a segmented detector in a scanning transmission X-ray microscope to collect partially coherent bright field images. We have adapted a Fourier filter reconstruction technique developed by McCallum, Landauer and Rodenburg to retrieve separate, quantitative maps of specimen phase shift and absorption. This is demonstrated in the imaging of a germanium test pattern using 525eV soft X-rays.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.