Optical projection tomography as a tool for 3D microscopy and gene expression studies

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.     

Nanoscale imaging of buried structures via scanning near-field ultrasound holography

A nondestructive imaging method, scanning near-field ultrasound holography (SNFUH), has been developed that provides depth information as well as spatial resolution at the 10- to 100-nanometer scale. In SNFUH, the phase and amplitude of the scattered specimen ultrasound wave, reflected in perturbation to the surface acoustic standing wave, are mapped with a scanning probe microscopy platform to provide nanoscale-resolution images of the internal substructure of diverse materials. We have used SNFUH to image buried nanostructures, to perform subsurface metrology in microelectronic structures, and to image malaria parasites in red blood cells.     

Plastic deformation with reversible peak broadening in nanocrystalline nickel

Plastic deformation in coarse-grained metals is governed by dislocation-mediated processes. These processes lead to the accumulation of a residual dislocation network, producing inhomogeneous strain and an irreversible broadening of the Bragg peaks in x-ray diffraction. We show that during plastic deformation of electrodeposited nanocrystalline nickel, the peak broadening is reversible upon unloading; hence, the deformation process does not build up a residual dislocation network. The results were obtained during in situ peak profile analysis using the Swiss Light Source. This in situ technique, based on well-known peak profile analysis methods, can be used to address the relationship between microstructure and mechanical properties in nanostructured materials.     

High-resolution three-dimensional imaging of dislocations

Dislocations and their interactions govern the properties of many materials, ranging from work hardening in metals to device pathology in semiconductor laser diodes. However, conventional electron micrographs are simply two-dimensional projections of three-dimensional (3D) structures, and even stereo microscopy cannot reveal the true 3D complexity of defect structures. Here, we describe an electron tomographic method that yields 3D reconstructions of dislocation networks with a spatial resolution three orders of magnitude better than previous work. We illustrate the method's success with a study of dislocations in a GaN epilayer, where dislocation densities of 1010 per square centimeter are common.     

Flash X-ray Microscopy

Soft x-ray contact microscopy, utilizing single-shot exposures of approximately 60 nanoseconds duration in polymethyl methacrylate, has been realized with a resolution of 300 angstroms. The radiation spectrum is intense in the "window" between 23 and 44 angstroms where water is transparent compared to biological materials, and therefore permits viewing of wet samples.     

Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.     

Scanning tunneling microscopy of freeze-fracture replicas of biomembranes

The high resolution of the scanning tunneling microscope (STM) makes it a potentially important tool for the study of biomaterials. Biological materials can be imaged with the STM by a procedure in which fluid, nonconductive biomaterials are replaced by rigid and highly conductive freeze-fracture replicas. The three-dimensional contours of the ripple phase of dimyristoylphosphatidylcholine bilayers were imaged with unprecedented resolution with commercial STMs and standard freeze-fracture techniques. Details of the ripple amplitude, asymmetry, and configuration unobtainable by electron microscopy or x-ray diffraction can be observed relatively easily with the STM.     

Three-dimensional readout of flash x-ray images of living sperm in water by atomic-force microscopy

The imaging of living specimens in water by x-ray microscopy can be greatly enhanced with the use of an intense flash x-ray source and sophisticated technologies for reading x-ray images. A subnanosecond [corrected] x-ray pulse from a laser-produced plasma was used to record the x-ray image of living sea urchin sperm in an x-ray resist. The resist relief was visualized at high resolution by atomic-force microscopy. Internal structure of the sperm head was evident, and the carbon density in a flagellum was estimated from the relief height.     

X-ray Linear Dichroism Microscopy

Chemical-specific x-ray linear dichroism was observed in an x-ray microscope as evidenced by changes in relative contrast upon azimuthal rotation of the sample. As a demonstration, thin sections of a partially ordered polymer fiber were examined with a transmission x-ray microscope near the carbon K-shell absorption edge to provide chemical-specific imaging at 50-nanometer spatial resolution. The observed dichroism and change in contrast upon rotation arise from the polarization dependence of the near-edge x-ray absorption cross section and can be used to image the orientation of specific chemical bonds.     

X-ray holograms at improved resolution: a study of zymogen granules

X-ray holography offers the possibility of three-dimensional microscopy with resolution higher than that of the light microscope and with contrast based on x-ray edges. In principle, the method is especially advantageous for biological samples if x-rays in the wavelength region between the carbon and oxygen K edges are used. However, until now the achieved resolution has not exceeded that of the light microscope because of the poor coherence properties of the x-ray sources and the low resolution of the detectors that were available. With a recently developed x-ray source based on an undulator on an electron storage ring, and high resolution x-ray resist, a hologram has been recorded at about 400-angstrom resolution. The experiment utilized x-rays with wavelengths of 24.7 angstroms and required a 1-hour exposure of the pancreatic zymogen granules under study.     

Electric field x-ray scattering measurements on tobacco mosaic virus

The feasibility of electric field x-ray solution scattering with biological macromolecules was investigated. Electric field pulses (1.25 to 5.5 kilovolts per centimeter) were used to orient tobacco mosaic virus in solution (4.5 milligrams per milliliter). The x-ray scattering is characteristic of isolated oriented particles. The molecular orientation and its field-free decay were monitored with a time resolution of 2 milliseconds by means of synchrotron radiation and a multiwire proportional area detector. The method should also be applicable to synthetic polymers and inorganic colloids.     

High-resolution scanning x-ray diffraction microscopy

Coherent diffractive imaging (CDI) and scanning transmission x-ray microscopy (STXM) are two popular microscopy techniques that have evolved quite independently. CDI promises to reach resolutions below 10 nanometers, but the reconstruction procedures put stringent requirements on data quality and sample preparation. In contrast, STXM features straightforward data analysis, but its resolution is limited by the spot size on the specimen. We demonstrate a ptychographic imaging method that bridges the gap between CDI and STXM by measuring complete diffraction patterns at each point of a STXM scan. The high penetration power of x-rays in combination with the high spatial resolution will allow investigation of a wide range of complex mesoscopic life and material science specimens, such as embedded semiconductor devices or cellular networks.     

X-ray diffraction from magnetically oriented solutions of macromolecular assemblies

A simple system was developed for obtaining x-ray diffraction patterns from magnetically oriented solutions of macromolecular assemblies. A small permanent magnet was designed that produces a magnetic field of 16 kilogauss in a volume of 1 cubic millimeter and is mountable on most x-ray cameras. Many subcellular structures have sufficient diamagnetic anisotropy that they exhibit orientation in dilute solution when placed between the poles of the magnet. Diffraction from solutions oriented in this magnet can provide substantially more structural information than small-angle scattering from isotropic solutions. In favorable cases, such as dilute solutions of filamentous bacteriophages, it is possible to produce oriented fiber diffraction patterns from which intensities along layer lines can be measured to 7-angstrom resolution. The magnetically induced birefringence observed in solutions of other macromolecular assemblies suggests that this technique may have broad applicability to subcellular structures.     

Age Determination by X-ray Fluorescence Rubidium-Strontium Ratio Measurement in Lepidolite

X-ray fluorescence analysis of several lepidolites whose rubidium and strontium concentrations had already been determined by neutron activation and stable isotope dilution, or both, indicates that this technique can be used for rapid nondestructive reconnaissance rubidiumstrontium studies, and that an x-ray analysis method comparable in accuracy to isotope dilution can probably be developed for dating Precambrian lepidolites, as the simple technique presently used has many obvious possibilities for improvement.     

Atomic-resolution microscopy in water

The scanning tunneling microscope is revolutionizing the study of surfaces. In ultra-high vacuum it is capable not only of imaging individual atoms but also of determining energy states on an atom-by-atom basis. It is now possible to operate this instrument in water. Aqueous optical microscopy is confined to a lateral resolution limit of about 2000 angstroms, and aqueous x-ray microscopy has yielded a lateral resolution of 75 angstroms. With a scanning tunneling microscope, an image of a graphite surface immersed in deionized water was obtained with features less than 3 angstroms apart clearly resolved. Further, an image measured in saline solution demonstrated that the instrument can be operated under conditions useful for many biological samples.     

Ex situ NMR in highly homogeneous fields: 1H spectroscopy

Portable single-sided nuclear magnetic resonance (NMR) magnets used for nondestructive studies of large samples are believed to generate inherently inhomogeneous magnetic fields. We demonstrated experimentally that the field of an open magnet can be shimmed to high homogeneity in a large volume external to the sensor. This technique allowed us to measure localized high-resolution proton spectra outside a portable open magnet with a spectral resolution of 0.25 part per million. The generation of these experimental conditions also simplifies the implementation of such powerful methodologies as multidimensional NMR spectroscopy and imaging.     

Element-selective single atom imaging

Electron energy-loss spectroscopy (EELS) is widely used to identify elemental compositions of materials studied by microscopy. We demonstrate that the sensitivity and spatial resolution of EELS can be extended to the single-atom limit. A chemical map for gadolinium (Gd) clearly reveals the distribution of Gd atoms inside a single chain of metallofullerene molecules (Gd@C82) generated within a single-wall carbon nanotube. This characterization technique thus provides the "eyes" to see and identify individual atoms in nanostructures. It is likely to find broad application in nanoscale science and technology research.     

Deformation twinning in nanocrystalline aluminum

We report transmission electron microscope observations that provide evidence of deformation twinning in plastically deformed nanocrystalline aluminum. The presence of these twins is directly related to the nanocrystalline structure, because they are not observed in coarse-grained pure aluminum. We propose a dislocation-based model to explain the preference for deformation twins and stacking faults in nanocrystalline materials. These results underscore a transition from deformation mechanisms controlled by normal slip to those controlled by partial dislocation activity when grain size decreases to tens of nanometers, and they have implications for interpreting the unusual mechanical behavior of nanocrystalline materials.     

Echinoderm calcite: single crystal or polycrystalline aggregate

Electron microscopy of natural and broken surfaces of echinoid skeletal plates reveals that the interior portions have the morphology of a single crystal, whereas the exterior is a polycrystalline aggregate with preferred orientation. These data help to resolve earlier contradictory x-ray and optical evidence.     

Post-perovskite phase transition in MgSiO3

In situ x-ray diffraction measurements of MgSiO3 were performed at high pressure and temperature similar to the conditions at Earth's core-mantle boundary. Results demonstrate that MgSiO3 perovskite transforms to a new high-pressure form with stacked SiO6-octahedral sheet structure above 125 gigapascals and 2500 kelvin (2700-kilometer depth near the base of the mantle) with an increase in density of 1.0 to 1.2%. The origin of the D" seismic discontinuity may be attributed to this post-perovskite phase transition. The new phase may have large elastic anisotropy and develop preferred orientation with platy crystal shape in the shear flow that can cause strong seismic anisotropy below the D" discontinuity.