Mark LeGros

Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.

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.

COMPUTED TOMOGRAPHY OF CRYOGENIC CELLS

COMPUTED TOMOGRAPHY OF CRYOGENIC CELLS G. SCHNEIDER, and E. ANDERSON Center for X-ray Optics, Lawrence Berkeley National Laboratory, One Cyclotron Road MS 2-400, Berkeley, CA 94720, USA S. VOGT, C. KNOCHEL, and D. WEISS Institut fur Rontgenphysik, Universitat Gottingen, Geiststrase 11 D-37073 Gottingen, Germany M. LEGROS, and C. LARABELL Life Sciences, Lawrence Berkeley National Laboratory, One Cyclotron Road MS 6-2100, Berkeley, CA 94720, USA Received (to be inserted Revised by publisher) Soft X-ray microcopy has resolved 30 nm structures in biological cells. To protect the cells from radiation damage caused by X-rays, imaging of the samples has to be performed at cryogenic temperatures, which makes it possible to take multiple images of a single cell. Due to the small numerical aperture of zone plates, X-ray objectives have a depth of focus on the order of several microns. By treating the X-ray microscopic images as projections of the sample absorption, computed tomography (CT) can be performed. Since cryogenic biological samples are resistant to radiation damage, it is possible to reconstruct frozen-hydrated cells imaged with a full-field X-ray microscope. This approach is used to obtain three-dimensional information about the location of specific proteins in cells. To localize proteins in cells, immunolabelling with strongly X-ray absorbing nanoparticles was performed. With the new tomography setup developed for the X-ray microscope XM-1 installed at the ALS, we have performed tomography of immunolabelled frozen-hydrated cells to detect protein distributions inside of cells. As a first example, the distribution of the nuclear protein, male specific lethal 1 (MSL-1) in the Drosophila melanogaster cell was studied. 1. Introduction The structure of proteins can be studied by X-ray crystallography with atomic resolution, but their location in cells remains unknown. With immunolabelling it is possible to localize these proteins in cells. Up to now light microscopy has mainly been used to study their distribution in cells by tagging the investigated protein with fluorophore-conjugated antibodies. While light microscopes allows routine investigation of whole, unsectioned cells, the obtainable resolution is diffraction limited to about 200 nm. In addition, this technique reveals mainly the distribution of the fluorophore-conjugated antibodies whereas most unlabelled cell structure is not clearly visualized. Electron microscopy can reveal cell structures at much higher resolution level, but is limited by the thickness of the sample, i.e. only less than 1 µm thick objects can be imaged. Therefore, no conventional imaging technique exists which can visualize the three- dimensional distribution of proteins inside whole hydrated cells, e.g. in the cell nucleus, with higher than light microscopical resolution. Due to the shorter wavelengths of X-rays than visible light, X-ray microscopy provides higher resolving power than light microscopes. By utilizing the natural absorption contrast between protein and water at photon energies of about 0.5 keV, smallest cell structures of about 30 nm in size embedded in vitreous ice can be detected in X-ray microscope images 1-3 . The aim of this work is to apply computed tomography, which has already been demonstrated using artificial samples 4 , mineralized sheats of bacteria 5 and frozen-hydrated algae 6 , in order to localize specific proteins and organelles in unsectioned, frozen-hydrated cells. 2. Lateral Resolution and Depth of Focus The computed-tomography experiments presented in this work are all based on tilt series of images acquired using the amplitude contrast mode of the TXM. In the amplitude contrast mode, the microscope forms enlarged images of the intrinsic photoelectric absorption contrast of the object. However, the obtained image contrast is influenced both by the condenser illuminating the object and by the imaging X-ray objective. The e-beam written condenser zone plate used for these experiments has an outermost zone width of dr N = 54 nm 7 . At 2.4 nm wavelength, the numerical aperture is given by NA cond = λ / (2 dr N ) = 0.0222.

Soft x-ray microscopy to 25 nm with applications to biology and magnetic materials

We report both technical advances in soft X-ray microscopy (XRM) and applications furthered by these advances. With new zone plate lenses we record test pattern features with good modulation to 25 nm and smaller. In combination with fast cryofixation, sub-cellular images show very fine detail previously seen only in electron microscopy, but seen here in thick, hydrated, and unstained samples. The magnetic domain structure is studied at high spatial resolution with X-ray magnetic circular dichroism (X-MCD) as a huge element-specific magnetic contrast mechanism, occurring e.g. at the L2,3 edges of transition metals. It can be used to distinguish between in-plane and out-of-plane contributions by tilting the sample. As XRM is a photon based technique, the magnetic images can be obtained in unlimited varying external magnetic fields. The images discussed have been obtained at the XM-1 soft X-ray microscope on beamline 6.1 at the Advanced Light Source in Berkeley. # 2001 Elsevier Science B.V. All rights reserved.

The first high resolution, broad band X-ray spectroscopy of ion-surface interactions using a microcalorimeter

Abstract A high resolution, broad band X-ray microcalorimeter has been used for the first time to investigate the radiative deexcitation of highly charged 7 keV/q Ar 17+ ions as they interact with a beryllium surface at normal incidence. The 20 eV energy resolution of this instrument made it possible to clearly distinguish the argon K α and K β, γ complex simultaneously. The intensity ratio of the K β, γ to K α emission is 31%, compared to the 8% found in the neutral argon atom. There is strong evidence that the relative intensities of the KL n ( n = 1–8) transitions do not agree with those obtained with crystal spectrometers.

First Use of NTD Germanium‐Based Microcalorimeters For High‐Resolution, Broadband X‐Ray Microanalysis

Broadband, high-resolution x-ray spectra from samples excited by the electron beam of a scanning electron microscope were obtained with an NTD germanium-based microcalorimeter. An energy resolution of 8 eV was used to resolve completely the silicon Kα from the tungsten Mα x-rays. This performance will make it possible to analyze efficiently the composition of thin films and surface contaminants by using low electron excitation energies.

Soft x-ray microscopy

Summary form only given. Soft X-ray microscopy is at the forefront of research with spatial resolution approaching 10 nm, and wide ranging applications to the physical and life sciences, including the dynamics of magnetic nanostructures, three-dimensional biotomography at the sub-cellular level, elemental and chemically specific environmental studies. Examples of recent work are shown below in figures 1-3. Figure 1 shows a general layout of the soft X-ray microscope XM-1 at the Advanced Light Source (ALS) synchrotron facility at Lawrence Berkeley National Laboratory. Figure 2 shows an image, at 15 nm spatial resolution, of nanomagnetic structures in a CoCrPt alloy as revealed by X-ray magnetic circular dichroism (XMCD) using synchrotron radiation tuned to the cobalt L3-edge at 778 eV (1.59 nm wavelength). Figure 3 shows a natural contrast tomographic reconstruction of a whole yeast cell imaged in the water window at 500 eV (2.4 nm wavelength).

High-resolution soft x-ray microscopy

The XM-1 is a soft x-ray full-field microscope that uses zone plates for the condenser and objective lenses. One of the main features of XM-1 is the high spatial resolution, which is made possible by the fine features of the objective zone plate. At present, the microscope uses a zone plate with an outer zone width of 25 nm. Several test patterns containing periodic lines and spaces were fabricated to measure the resolution of the microscope. Experimental data shows that the microscope can resolved 25 nm features. As simulations indicate that good contrast can be observed with even smaller features, test patterns with finer features are being fabricated to actually determine the resolution limit of the microscope.

Quantitative Analyzing the Spatial Organization of the Organelles in Cancer Cell Using Soft X-Ray Tomography

Using soft x-ray microscopy (SXM), we are able to image and quantitatively analyze the spatial organization of the organelles within cancer cells [1]. Crucial cellular activities, such as cancer invasion and metastasis, mostly take place within a three-dimensional extracellular matrix (ECM) [2]. Thus, the information of three-dimensional organization of organelles in cancer cells is required to tackle questions regarding those highly remodeling activities.

The National Center for X-Ray Tomography: Status Update

The National Center for X-ray Tomography (NCXT) develops new technologies for bio-imaging. In particular, the NCXT pioneered the development of soft x-ray tomography (SXT) as a method for imaging whole, hydrated cells, including eukaryotic cells. This presentation will describe the current status of this work, together our progress incorporating ‘super-resolution’ cryogenic SIM as a correlative partner for SXT.