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Abstract— We show that the calcium fiuorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nra, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17μ concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Tksapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.     

Structural and Chemical Analysis of Materials with High Spatial Resolution

 An understanding of the correlation between microstructures and properties of materials require the characterization of the material on many different length scales. Often the properties depend primarily on the atomistics of defects, such as dislocations and interfaces. The different techniques of transmission electron microscopy allow the characterization of the structure and of the chemical composition of materials with high spatial resolution to the atomic level: high resolution transmission electron microscopy allows the determination of the position of the columns of atoms (ions) with high accuracy. The accuracy which can be achieved in these measurements depends not only on the instrumentation but also on the quality of the transmitted specimen and on the scattering power of the atoms (ions) present in the analyzed column. The chemical composition can be revealed from investigations by analytical microscopy which includes energy dispersive X-ray spectroscopy, mainly quantitatively applied for heavy elements, and electron energy-loss spectroscopy. Furthermore, the energy-loss near-edge structure of EELS data results in information on the local band structure of unoccupied states of the excited atoms and, therefore, on bonding. A quantitative evaluation of convergent beam electron diffraction results in information on the electron charge density distribution of the bulk (defect-free) material. The different techniques are described and applied to different problems in materials science. It will be shown that nearly atomic resolution can be achieved in high resolution electron microscopy and in analytical electron microscopy. Recent developments in electron microscopy instrumentation will result in atomic resolution in the foreseeable future.     

3D nanoscale imaging of the yeast, Schizosaccharomyces pombe, by full-field transmission X-ray microscopy at 5.4 keV

Three-dimensional (3D) nanoscale structures of the fission yeast, Schizosaccharomyces pombe, can be obtained by full-field transmission hard X-ray microscopy with 30 nm resolution using synchrotron radiation sources. Sample preparation is relatively simple and the samples are portable across various imaging environments, allowing for high-throughput sample screening. The yeast cells were fixed and double-stained with Reynold's lead citrate and uranyl acetate. We performed both absorption contrast and Zernike phase contrast imaging on these cells in order to test this method. The membranes, nucleus, and subcellular organelles of the cells were clearly visualized using absorption contrast mode. The X-ray images of the cells could be used to study the spatial distributions of the organelles in the cells. These results show unique structural information, demonstrating that hard X-ray microscopy is a complementary method for imaging and analyzing biological samples.     

Investigation of the thermal stability of Ni/C multilayers by X-ray methods

Microstructural properties of Ni/C multilayers prepared by PLD (pulsed laser deposition) have been investigated after heat treatment in vacuum at temperatures in the range of 50 degrees C to 500 degrees C. X-ray diffractometry, X-ray reflectometry, fluorescence EXAFS (extended X-ray absorption fine structure) and HREM (high resolution transmission electron microscopy) have been applied to characterize samples in the initial state and after annealing. The multilayer reflectivity remained unchanged or increased at temperatures below 400 degrees C due to sharpening of the interfaces caused by the formation of alpha-nickel and nickel carbide. The reflectivity decreased at temperatures above 400 degrees C because of the fragmentation of the nickel layers. It can be shown, that both chemical and mechanical driving forces are responsible for the observed modifications of the initial specimen state.     

Applications of synchrotron-based X-ray techniques in environmental science

Synchrotron-based X-ray techniques have been widely applied to the fields of environmental science due to their element-specific and nondestructive properties and unique spectral and spatial resolution advantages. The techniques are capable of in situ investigating chemical speciation, microstructure and mapping of elements in question at the molecular or nanometer scale, and thus provide direct evidence for reaction mechanisms for various environmental processes. In this contribution, the applications of three types of the techniques commonly used in the fields of environmental research are reviewed, namely X-ray absorption spectroscopy (XAS), X-ray fluorescence (XRF) spectroscopy and scanning transmission X-ray microscopy (STXM). In particular, the recent advances of the techniques in China are elaborated, and a selection of the applied examples are provided in the field of environmental science. Finally, the perspectives of synchrotron-based X-ray techniques are discussed. With their great progress and wide application, the techniques have revolutionized our understanding of significant geo- and bio-chemical processes. It is anticipatable that synchrotron-based X-ray techniques will continue to play a significant role in the fields and significant advances will be obtained in decades ahead.     

A new sample substrate for imaging and correlating organic and trace metal composition in biological cells and tissues

Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and X-ray fluorescence (XRF) microscopes are becoming increasingly popular tools for imaging the organic and trace metal compositions of biological materials, respectively, without the need for extrinsic labels or stains. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2–10 μm in the mid-infrared region. The synchrotron X-ray fluorescence (SXRF) microprobe is a complementary technique used to probe trace element content in the same systems with a similar spatial resolution. However to be most beneficial, it is important to combine the results from both imaging techniques on a single sample, which requires precise overlap of the IR and X-ray images. In this work, we have developed a sample substrate containing a gold grid pattern on its surface, which can be imaged with both the IR and X-ray microscopes. The substrate consists of a low trace element glass slide that has a gold grid patterned on its surface, where the major and minor parts of the grid contain 25 and 12 nm gold, respectively. This grid pattern can be imaged with the IR microscope because the reflectivity of gold differs as a function of thickness. The pattern can also be imaged with the SXRF microprobe because the Au fluorescence intensity changes with gold thickness. The tissue sample is placed on top of the patterned substrate. The grid pattern’s IR reflectivity image and the gold SXRF image are used as fiducial markers for spatially overlapping the IR and SXRF images from the tissue. Results show that IR and X-ray images can be correlated precisely, with a spatial resolution of less than one pixel (i.e., 2–3 microns). The development of this new tool will be presented along with applications to paraffin-embedded metalloprotein crystals, Alzheimer’s disease, and hair composition.     

Investigation of the thermal stability of Ni/C multilayers by X-ray methods

Microstructural properties of Ni/C multilayers prepared by PLD (pulsed laser deposition) have been investigated after heat treatment in vacuum at temperatures in the range of 50°C to 500°C. X-ray diffractometry, X-ray reflectometry, fluorescence EXAFS (extended X-ray absorption fine structure) and HREM (high resolution transmission electron microscopy) have been applied to characterize samples in the initial state and after annealing. The multilayer reflectivity remained unchanged or increased at temperatures below 400°C due to sharpening of the interfaces caused by the formation of -nickel and nickel carbide. The reflectivity decreased at temperatures above 400°C because of the fragmentation of the nickel layers. It can be shown, that both chemical and mechanical driving forces are responsible for the observed modifications of the initial specimen state.     

NMR microscopy of heavy metal absorption in calcium alginate beads

In recent years, heavy metal uptake by biopolymer gels, such as Cal-alginate or chitosan, has been studied by various methods. This is of interest because such materials might be an alternative to synthetical ion-exchange resins in the treatment of industrial waste waters. Most of the work done in this field consisted of studies of equilibirum absorption of different heavy metal ions with dependence on various experimental parameters. In some publications, the kinetics of absorption were studied, too. However, no experiments on the spatial distribution of heavy metals during the absorption process are known to us. Using Cu as an example, it is demonstrated in this article that NMR microscopy is an appropriate tool for such studies. By the method presented here, it is possible to monitor the spatial distribution of heavy metal ions with a time resolution of about 5 min and a spatial resolution of 100 μm or even better.     

Micro-beam scanning PIXE analysis system at the National Institute of Radiological Sciences (NIRS)

In 2000, micro-beam scanning particle induced X-ray emission (PIXE) analysis system was installed in NIRS. This system provides the ability of multi-elemental mapping on maximum 2.5 mm×2.5 mm area in a spatial resolution of about 1 μm with quadrupole triplet magnets and a scanning coil. The estimated beam size on good tuning was 0.40×0.65 μm², that is one of the best capacity of micro-beam scanning PIXE system in the world. The performance was tested using small biological samples such as fish scale, pollen and small fish eye. Fine elemental maps were obtained in the samples of about 30 μm to a few mm size in the special resolution of about 1 μm. This revised version was published online in August 2006 with corrections to the Cover Date.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

 Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers.     

Novel bulk synthesis of magnesium oxide nanobelts networks by microwave hydrothermal route

Novel magnesium oxide (MgO) nanobelt structures were successfully synthesized on a large scale, low cost and catalyst free by a microwave hydrothermal route using the magnesium metal and potassium hydroxide precursors at 200 °C for 10 min. The synthesized MgO nanobelts were systemically characterized by power X-ray diffraction (XRD), scanning electron microscopy, energy dispersion spectroscopy and transmission-high resolution electron microscopy. The XRD results indicate that the MgO nanobelts have the well-crystalline cubic phase. The detailed morphological studies revealed that the synthesized products were nanobelts and were grown in large quantity. The optical energy gap of MgO nanobelts was found to be 3.1 eV. The photoluminescence spectra of the MgO nanobelts show a strong and broad green emission band, a weak ultraviolet emission band, and a weak red emission band. This novel method will open new dimensions for synthesized 1-D metal oxide and can be easily extended to the synthesis and assembly of other inorganic nanocrystals.     

Effects of optical constants on time-gated transillumination of tissue and tissue-like media.

Light transillumination was used to study structures inside turbid media. Time-gated viewing was performed to suppress multiply-scattered light and thus improve spatial resolution. We demonstrate that, for the case of scattering-dominated attenuation (scattering coefficient much greater than the absorption coefficient), the detection of early transmitted light will be practically insensitive to variations in the absorption coefficient. This is an important observation for the development of time-gated optical mammography, since optical mammography using continuous-wave light is based on increased light absorption in the tumour region caused by the neovascularization surrounding a tumour. In order to detect tumours in time-gated viewing it is the scattering coefficient of the tumour that must be characteristic. The scattering coefficient is measured to be lower in the tumour region than in the surrounding breast tissue for one resected breast specimen.     

Analytical evidence of amorphous microdomains within nitridosilicate and nitridoaluminosilicate single crystals

Single crystals of new nitridosilicates and nitridoaluminosilicates with excellent R values in X-ray investigations were analysed quantitatively using 30 to 60 μm single-spot LA-ICP-MS. Significant discrepancies between expected and measured chemical composition could not be explained by the crystallographic data. High spatial resolution analysis using electron probe microanalysis (EPMA, 10 μm) leads to the discovery of inhomogeneities in the crystalline material. The application of standard single-spot LA-ICP-MS with a spatial resolution of 30 to 60 μm is not suitable for the analysis of these crystals as the existing inhomogeneities dominate and alter the determined concentrations. However, owing to the better detection capabilities, a scanning LA-ICP-MS procedure enables a more representative analysis of single crystals of Ca₅Si₂Al₂N₈ than single-spot LA-ICP-MS as a result of a larger sampling volume. It is highly likely that these impurities consist of amorphous, vitreous phases as powder diffraction X-ray data indicates the existence of a significant fraction of an X-ray amorphous material besides crystalline silicates. These microdomains contain less aluminium, silicon and calcium or are nearly free of aluminium, which explains the detected discrepancies in the chemical composition.     

Resolving the Unpaired‐Electron Orbital Distribution in a Stable Organic Radical by Kondo Resonance Mapping

The adsorption geometry and the electronic structure of a Blatter radical derivative on a gold surface were investigated by a combination of high‐resolution noncontact atomic force microscopy and scanning tunneling microscopy. While the hybridization with the substrate hinders direct access to the molecular states, we show that the unpaired‐electron orbital can be probed with Ångström resolution by mapping the spatial distribution of the Kondo resonance. The Blatter derivative features a peculiar delocalization of the unpaired‐electron orbital over some but not all moieties of the molecule, such that the Kondo signature can be related to the spatial fingerprint of the orbital. We observe a direct correspondence between these two quantities, including a pronounced nodal plane structure. Finally, we demonstrate that the spatial signature of the Kondo resonance also persists upon noncovalent dimerization of molecules.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

Summary.  Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers. Received June 23, 2000. Accepted (revised) July 31, 2000     

PHOTOACTIVABLE FLUOROPHORES FOR THE MEASUREMENT OF FLUENCE IN TURBID MEDIA

Knowledge of the fluence distribution in biological tissue is essential for applications of lasers and light in medicine. A method using a photoactivable fluorophore as a chemical actinometer is presented to investigate the fluence (J/cm²) distribution in tissue-simulating phantoms. Such a chemical actinometer provides high spatial resolution (≤20 μm) while minimizing the disturbance of the fluence distribution. The actinometer substance, nonfluorescent in its native state, is incorporated into an acrylamide gel. Upon absorption of 351 nm radiation (λ_act), the actinometer substance becomes a Ruorophorc, which is excited at λ_ex≤ 485 nm. Thus the spatial distribution of the emitted fluorescence (λ_em≤ 515 nm) in the actinometer represents the fluence distribution of the activating radiation. Using histological techniques, 20 μm sections are cut from gel-like optical phantoms containing the actinometric substance. The fluorescence intensity in the section is recorded under a standard fluorescence microscope equipped with a sensitive video camera. To simulate different biological tissues, the scattering and absorption properties of the gel phantoms arc varicd over a wide range. The experimentally obtained fluence distributions are compared with theoretical models of light distribution in turbid media.     

Synthesis and optical properties of self-assembled flower-like CdS architectures by mixed solvothermal process

Self-assembled CdS architectures with flower-like structures have been synthesized by a mixed solvothermal method using ethylene glycol and oleic acid as the mixed solvent at 160°C for 12 h. The results of X-ray diffraction (XRD) patterns, field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) images indicate that the product exists as the hexagonal wurtzite phase and conatins of larger numbers of flower-like CdS architectures with diameters of 1.8–3 μm. The selected-area electron diffraction (SAED) pattern and the high resolution transmission electron microscope (HRTEM) image reveal that the grain has better crystallinity. The optical properties of flower-like CdS architectures were also investigated by ultraviolet-visable (UV-vis) and photoluminescence spectroscopy at room temperature. A strong peak at 490 nm is shown in the UV-vis absorption, while an emission at 486 nm and another strong emission at 712 nm are shown in the PL spectrum.      

Thinner,Smaller, Faster: IR Techniques To Probe the Functionality of Biological and Biomimetic Systems

New techniques in vibrational spectroscopy are promising for the study of biological samples as they provide exquisite spatial and/or temporal resolution with the benefit of minimal perturbation of the system during observation. In this Minireview we showcase the power of modern infrared techniques when applied to biological and biomimetic systems. Examples will be presented on how conformational changes in peptides can be traced with femtosecond resolution and nanometer sensitivity by 2D IR spectroscopy, and how surface‐enhanced infrared difference absorption spectroscopy can be used to monitor the effect of the membrane potential on a single proton‐transfer step in an integral membrane protein. Vibrational spectra of monolayers of molecules at basically any interface can be recorded with sum‐frequency generation, which is strictly surface‐sensitive. Chemical images are recorded by applying scanning near‐field infrared microscopy at lateral resolutions better than 50 nm.     

Imaging Magnetic Domain Structures with Soft X-Ray Microscopy

The current achievements in magnetic transmission soft X-ray microscopy will be reviewed. The magnetic contrast is given by X-ray magnetic circular dichroism (X-MCD), i.e., the dependence of the absorption coefficient of circularly polarized X rays on the projection of the magnetization in a ferromagnetic system onto the photon propagation direction. X-MCD contrast can reach, e.g., at L_2,3 edges in transition metals, large values up to 50%. Combined with a soft X-ray microscope where Fresnel zone plates acting as optical elements provide a lateral resolution down at 25 nm, it allows for imaging magnetic microstructures. Specific features of this photon-based technique are the recording of images in varying external magnetic fields, an inherent chemical specificity, a high sensitivity to thin magnetic layers, due to the large contrast, and the possibility to distinguish between in-plane and out-of plane contributions. In this report, recent results obtained with the XM-1 microscope at the ALS (Berkeley/CA) demonstrate the broad applicability of this novel experimental technique to both fundamental and technological relevant issues in nanomagnetism. The future potential will be briefly outlined.