J. Maser

Elemental Analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the Host Cell’s Endosomal System

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with 59Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-γ or TNF-α before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

NANOMETER FOCUSING OF HARD X RAYS BY PHASE ZONE PLATES

Focusing of 8 keV x rays to a spot size of 150 and 90 nm full width at half maximum have been demonstrated at the first- and third-order foci, respectively, of a phase zone plate (PZP). The PZP has a numerical aperture of 1.5 mrad and focusing efficiency of 13% for 8 keV x rays. A flux density gain of 121 000 was obtained at the first-order focus. In this article, the fabrication of the PZP and its experimental characterization are presented and some special applications are discussed.

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. J. Cell. Biochem. 99: 1489–1502, 2006.

Focusing of hard x-rays to 16 nanometers with a multilayer Laue lens

We report improved results for hard x-ray focusing using a multilayer Laue lens MLL. We have measured a line focus of 16 nm width with an efficiency of 31% at a wavelength =0.064 nm 19.5 keV using a partial MLL structure with an outermost zone width of 5 nm. The results are in good agreement with the theoretically predicted performance.

X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis

Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) toin vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80–90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.

Nanoparticles for applications in cellular imaging.

In the following review we discuss several types of nanoparticles (such as TiO2, quantum dots, and gold nanoparticles) and their impact on the ability to image biological components in fixed cells. The review also discusses factors influencing nanoparticle imaging and uptake in live cells in vitro. Due to their unique size-dependent properties nanoparticles offer numerous advantages over traditional dyes and proteins. For example, the photostability, narrow emission peak, and ability to rationally modify both the size and surface chemistry of Quantum Dots allow for simultaneous analyses of multiple targets within the same cell. On the other hand, the surface characteristics of nanometer sized TiO2allow efficient conjugation to nucleic acids which enables their retention in specific subcellular compartments. We discuss cellular uptake mechanisms for the internalization of nanoparticles and studies showing the influence of nanoparticle size and charge and the cell type targeted on nanoparticle uptake. The predominant nanoparticle uptake mechanisms include clathrin-dependent mechanisms, macropinocytosis, and phagocytosis.

A hard X-ray nanoprobe beamline for nanoscale microscopy

The Hard X-ray Nanoprobe Beamline is a precision platform for scanning probe and full-field microscopy with 3–30 keV X-rays. A combination of high-stability X-ray optics and precision motion sensing and control enables detailed studies of the internal features of samples with resolutions approaching 30 nm.

Topological defect dynamics in operando battery nanoparticles

Watching defects during battery cycling Dislocations affect the mechanical properties of a material. Ulvestad et al. studied the influence of dislocations on a nanoparticle undergoing charge and discharge cycles in a lithium ion battery. The defects influenced the way the material expanded and contracted during cycling. In the future, it may be possible to tune the properties of a material through controlled defect engineering. Science, this issue p. 1344 Coherent x-rays image structural transformations in battery nanoparticles during electrochemical operation. Topological defects can markedly alter nanomaterial properties. This presents opportunities for “defect engineering,” where desired functionalities are generated through defect manipulation. However, imaging defects in working devices with nanoscale resolution remains elusive. We report three-dimensional imaging of dislocation dynamics in individual battery cathode nanoparticles under operando conditions using Bragg coherent diffractive imaging. Dislocations are static at room temperature and mobile during charge transport. During the structural phase transformation, the lithium-rich phase nucleates near the dislocation and spreads inhomogeneously. The dislocation field is a local probe of elastic properties, and we find that a region of the material exhibits a negative Poisson’s ratio at high voltage. Operando dislocation imaging thus opens a powerful avenue for facilitating improvement and rational design of nanostructured materials.

Takagi-Taupin description of x-ray dynamical diffraction from diffractive optics with large numerical aperture

We present a formalism of x-ray dynamical diffraction from volume diffractive optics with large numerical aperture and high aspect ratio, in an analogy to the Takagi-Taupin equations [Acta Crystallogr. 15, 1311 (1962); Bull. Soc. Fr. Mineral. Crystallogr. 87, 469 (1964)] for strained single crystals. We derive a set of basic equations for dynamical diffraction from volume diffractive optics, which enable us to study the focusing property of these optics with various grating profiles. We study volume diffractive optics that satisfy the Bragg condition to various degrees, namely, flat, tilted, and wedged geometries, and derive the curved geometries required for ultimate focusing. We show that the curved geometries satisfy both the Bragg condition everywhere and phase requirement for point focusing and effectively focus hard x rays to a scale close to the wavelength. Our calculations were made for an x-ray wavelength of 0.064 nm (19.5 keV).

The Bionanoprobe: Hard X-ray Fluorescence Nanoprobe with Cryogenic Capabilities

The Bionanoprobe has been developed to study trace elements in frozen-hydrated biological systems with sub-100 nm spatial resolution. Here its performance is demonstrated and first results reported.