Proceedings Volume 1741

Soft X-Ray Microscopy

Chris J. Jacobsen, James E. Trebes
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Proceedings Volume 1741

Soft X-Ray Microscopy

Chris J. Jacobsen, James E. Trebes
View the digital version of this volume at SPIE Digital Libarary.

Volume Details

Date Published: 13 January 1993
Contents: 11 Sessions, 45 Papers, 0 Presentations
Conference: San Diego '92 1992
Volume Number: 1741

Table of Contents

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Table of Contents

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  • X-Ray Sources for X-Ray Imaging
  • X-Ray Optics
  • Synchrotron-Based Imaging Systems
  • Pulsed-Source-Based Imaging Systems
  • New Modalities and Related Techniques
  • Image Analysis
  • Detectors
  • Pulsed-Source-Based Imaging Systems
  • Materials Applications
  • X-Ray Damage to Biological Specimens
  • Biological Applications I
  • Biological Applications II
  • Pulsed-Source-Based Imaging Systems
  • X-Ray Optics
  • Synchrotron-Based Imaging Systems
  • Biological Applications II
X-Ray Sources for X-Ray Imaging
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X-ray lasers as sources for x-ray microscopy
Brian J. MacGowan
Laboratory x-ray lasers are currently being developed by researchers worldwide as potential sources for x-ray imaging and other applications. Laser action has been demonstrated at wavelengths as short as 35.6 angstroms and there is a large effort underway to enhance the coherent power output of the Ni-like Ta x-ray laser at 44.83 angstroms as a source for x-ray imaging of live cells. Part of this effort is dedicated to producing a more affordable, compact laser to pump the x-ray laser in order to increase the accessibility of x-ray laser sources.
X-ray diode using a silicon field-emission photocathode
Wael I. Karain, Larry V. Knight, David D. Allred, et al.
We have produced arrays of 10,000 sharp p-type silicon points using an etch plus oxidation method. These points were used as electron emitters. No high vacuum caseation or high temperature cleaning was needed to observe the electron emission. These are seen to be photosensitive sources of electrons at 200 K and 300 K. They were also used to produce AlK(alpha ) x rays. This constitutes the first use of etched, point arrays for generating electrons for x-ray sources.
X-Ray Optics
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Design and analysis of a fast two-mirror soft x-ray microscope
David L. Shealy, Cheng Wang, Wu Jiang, et al.
During the past several years, a number of investigators have addressed the design, analysis, fabrication, and testing of spherical Schwarzschild microscopes for soft-x-ray applications using multilayer coatings. Some of these systems have demonstrated diffraction limited resolution for small numerical apertures. Rigorously aplanatic, two-aspherical mirror Head microscopes can provide near diffraction limited resolution for very large numerical apertures. This paper summarizes the relationships between the numerical aperture, mirror radii and diameters, magnifications, and total system length for Schwarzschild microscope configurations. Also, an analysis of the characteristics of the Head-Schwarzschild surfaces is reported. The numerical surface data predicted by the Head equations have been fit by a variety of functions and analyzed by conventional optical design codes. Efforts have been made to determine whether current optical substrate and multilayer coating technologies will permit construction of a very fast Head microscope which can provide resolution approaching that of the wavelength of the incident radiation.
Performance limitations of imaging microscopes for soft x-ray applications
Kristin L. Lewotsky, Anita Kotha Thompson, James E. Harvey
Recent advances in the fabrication of nanometer-scale multilayer structures have yielded high- reflectance mirrors operating at near-normal incidence for soft x-ray wavelengths. These developments have stimulated renewed interest in high-resolution soft x-ray microscopy. The design of a Schwarzschild imaging microscope for soft x-ray applications has been reported by Hoover and Shealy. Based upon a geometrical ray-trace analysis of the residual design errors, diffraction-limited performance at a wavelength of 100 angstrom was predicted over an object size (diameter) of 0.4 mm. In this paper we expand upon the previous analysis of the Schwarzschild x-ray microscope design by determining the total image degradation due to diffraction, geometrical aberrations, alignment errors, and realistic assumptions concerning optical fabrication errors. NASA's optical surface analysis code (OSAC) is used to model the image degradation effects of residual surface irregularities over the entire range of relevant spatial frequencies. This includes small angle scattering effects due to mid spatial frequency surface errors falling between the traditional `figure' and `finish' specifications. Performance predictions are presented parametrically to provide some insight into the optical fabrication and alignment tolerances necessary to meet a particular image quality requirement.
Synchrotron-Based Imaging Systems
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X-ray microscopy studies with the Goettingen x-ray microscopes
Peter Guttmann, Gerd Schneider, Juergen Thieme, et al.
Zone plates with improved resolution have been constructed by electron beam lithography. With these zone plates features as small as 30 nm can be imaged without astigmatism. By using the phase shifting property of the zone plate material and by an improved etching technique, groove efficiency values up to 10% have been measured. A new type of foils for the support of the zone plates as well as for the x-ray windows are more stable under synchrotron radiation than the polyimide foils used before and have a higher transmission for x-rays in the `water-window' region. The x-ray microscope installed at BESSY, Berlin, Germany was modified in such a way that the environmental chamber is now placed in air with better access to the specimens under investigation. In addition, a light microscope was incorporated in the x-ray microscope providing a better practicability to adjust object details to the object field of the x-ray microscope, selecting another object or object detail to be investigated, and prefocussing the object for x-ray imaging. With the improved x-ray microscope different wet biological specimens and test structures were investigated, showing features as small as 30 nm. The x-ray microscope using a pulsed plasma x-ray source, installed in Gottingen, Germany was modified in such a way that instead of a zone plate an ellipsoidal mirror is used as the condenser. The pulsed plasma x-ray source was improved too, i.e., the number of pulses needed for an image is reduced.
X-ray holographic microscopy at LURE and Institut d'Optique
Gabor holographic recording is an efficient way to collect information for soft x-ray imaging with undulator synchrotron radiation sources. In addition to being well adapted to the radiative properties of these sources, the high simplicity and large field of the recording operation makes it akin to a particular kind of sample preparation. This is comparable to the conditions of contact microscopy. But, to take advantage of these properties, several difficult problems concerning hologram reconstruction have to be solved. We have investigated the theoretical performances and the practical implementation of Gabor holographic imaging with optical reconstruction, with the realization of a dedicated optical instrument in mind. Such an instrument is presently under construction; it is expected to provide low noise images, with a resolution better than 0.1 micrometers .
Experimental demonstration of high-resolution three-dimensional x-ray holography
Ian McNulty, James E. Trebes, James M. Brase, et al.
Tomographic x-ray holography may make possible the imaging of biological objects at high resolution in three dimensions. We performed a demonstration experiment with soft x-rays to explore the feasibility of this technique. Coherent 3.2 nm undulator radiation was used to record Fourier transform holograms of a microfabricated test object from various illumination angles. The holograms were numerically reconstructed according to the principles of diffraction tomography, yielding images of the object that are well resolved in three dimensions.
Soft x-ray microscope with zone plates at UVSOR
Norio Watanabe, Yoshio Shimanuki, Mieko Taniguchi, et al.
A soft x-ray microscope with zone plates was set up at UVSOR (Okazaki, Japan). A 0.41 micrometers line and space pattern was clearly distinguished using an objective zone plate with the outermost zone width of 0.41 micrometers . Modulation transfer functions were measured at wavelengths of 3.1 nm and 5.4 nm, and compared with theoretical calculations. Considering the resolution of a microchannel plate used as a detector, the agreement is fairly good. With this microscope, some biological specimens such as diatoms, spicule of trepang, crab and rabbit muscles, human blood cells, human chromosomes, and magnetotactic bacterium were observed at 3.1 nm and 5.4 nm. With an environmental chamber (wet cell) using polypropylene foils as windows, wet specimens were observed at a wavelength of 4.6 nm. Images of spicule of trepang, human blood cell, and cultured protoplast of plant cell stained by methyl mercury were observed with good contrast.
Evaluation of the Schwarzschild x-ray microscope using the compact SR-ring 'AURORA'
Shin Masui, Shouichirou Mochimaru, Norio Watanabe, et al.
Using the compact SR-ring `AURORA,' imaging performance of the soft x-ray microscope with a Schwarzschild objective were evaluated. The multilayer mirrors of the objective were designed at a wavelength of 135 angstroms with 41 layers of Mo/Si. X rays from the source were filtered with a Be membrane (1.5 micrometers thickness), and then focused on a sample with a paraboloidal condenser. Visible and UV light was removed with the Be filter, and hard x ray was removed with the condenser. X rays transmitted through the sample were imaged on a detector (micro-channel plate) with the Schwarzschild objective. The image was converted to visible light by a fluorescent screen, and monitored with a CCD camera. Magnified ratio was 200. Spatial resolution was evaluated as 0.3 - 0.45 micrometers from an edge response.
Scanning x-ray microscope project at Hefei
Xinya Lei, Yong Feifei Zhao, Ming Wang, et al.
The test operation of Hefei synchrotron radiation source was started in Oct. 1991. We test the first scanning transmission x-ray microscope, which is installed in beamline U12A, designed primarily for soft x-ray microscopy in China. We also perform the studies of contact x-ray microscopy with synchrotron radiation to some biological and medical specimens. In this paper we describe the instrumentation of the scanning x-ray microscope and show some experimental results.
Pulsed-Source-Based Imaging Systems
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Flash contact x-ray microscopy of biological specimen in water
Toshihisa Tomie, Hazime Shimizu, Toshikazu Majima, et al.
From the shot noise consideration in x-ray microscopy it is shown that, for 50 nm resolution, a few tens mJ/cm2 energy of 3 nm x ray is required on the specimen, and required x ray energy density increases with the fourth power of the resolving power. X-ray images of biological specimens in water taken by flash contact microscopy with a laser-plasma source are presented to discuss the prospects of x-ray microscopy for biological applications. Forty nanometer diameter biological components are observed in an x-ray image. The attempt of observing thick specimens by short wavelength x rays produced by a 10 ps duration laser pulse is also reported. Technical issues for further improving contact microscopy, including the feasibility of stereo-microscopy, are discussed.
Zone plate x-ray microscope using a laser plasma source
Hiroaki Aritome, Shigeru Nakayama, Katsumi Mizunoe, et al.
An imaging x-ray microscope using an incoherent laser plasma source and zone plates is reported. The He-like line of 4.03 nm (1 X 1014 photons/sr/pulse) from a carbon plasma produced by a compact glass laser is used as a source. The x-ray source is monochromatized by a condenser zone plate and a pinhole. As an x-ray image detector, a Kodak 101-07 film and an MCP or a cooled backside illuminated CCD are used. An image of the #1000 copper mesh is obtained by a single x-ray pulse at a magnification of 10 by the CCD. In the case of a singe pulse, the magnification is limited so that enough photons are incident on a pixel of the CCD. Thus, the spatial resolution is limited. Further improvement such as an ellipsoidal mirror with multilayer coating is considered to improve the resolution.
Pulsed x-ray microscopy of biological specimens with laser plasma sources
Martin C. Richardson, Kunio Shinohara, Kazuo A. Tanaka, et al.
The development of a compact, affordable, high-resolution x-ray microscope will have a strong impact on the biological and medical sciences. We discuss the potential that pulsed, laser-plasma x-ray sources have to this development. Several approaches to the high-resolution analysis of dried and in-vitro biological specimens with laser-plasma sources are described. We discuss the details of the laser and plasma conditions required for optimum x-ray generation, and the various x-ray optical and x-ray electro-optical imaging systems which could be incorporated into a compact, laser-plasma x-ray microscope.
Soft x-ray contact microscopy using laser-generated plasma sources
Julian H. Fletcher, Robin A. Cotton, Colin E. Webb
Of the three main approaches to soft x-ray microscopy currently being investigated (contact, scanning, and direct imaging), we believe that, at present, contact microscopy provides the only solution to the problems of user access to working x-ray microscope systems and radiation damage to biological specimens. A small, transportable, discharge pumped, UV pre- ionized KrF laser system has been developed for the exclusive use of contact microscopy. By employing an injection seeded unstable confocal resonator, a beam of 2.5 times the diffraction limited divergence and energy of the order of magnitude 2 J is generated. By focusing this beam onto the surface of a suitable target material with an intensity of the order of magnitude 1012 W/cm2, a plasma is created which emits largely in the water window region of the spectrum with an efficiency of up to 20%. These x-rays are used to illuminate a biological specimen which is placed in close contact with a photosensitive resist such as polymethyl methacrylate. The specimen may be hydrated and requires no prior preparation such as staining or fixation. A transmission map of the specimen is recorded in the resist within 25 ns, before radiation damage can effect the specimen's structure. After chemical development, during which exposed areas of the resist are preferentially dissolved resulting in a relief map of the x-ray transmission of the specimen, the resist is viewed in an SEM or alternatively an atomic force microscope (AFM). Images of various biological specimens are presented.
New Modalities and Related Techniques
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Hard x-ray microimaging techniques based on phase zone plates
Barry P. Lai, Wenbing Yun, Dan G. Legnini, et al.
Phase zone plates of high focusing efficiency and submicron resolution have been demonstrated in the hard x-ray region. A scanning microscope based on these focusing optics will create many new applications. Preliminary results in the applications of the microscope are reported here. In the area of imaging, we have utilized absorption contrast to clearly identify the locations of Au and Ni constituents in a sample of two interleaved grids. Micro- EXAFS spectra has also been obtained on a Ni foil. Fluorescence from a nuclear fuel sample, as an example of microanalysis, has revealed the elemental distribution at the interfaces. Lastly, microdiffraction from AgBr crystallites has been studied.
Phase contrast and darkfield imaging in x-ray microscopy
Graeme R. Morrison
The transmission x-ray microscope has so far been used almost exclusively to form images with absorption contrast. Methods of forming phase contrast and darkfield images are considered, particularly in the scanning transmission x-ray microscope, and the advantages and disadvantages of these methods are reviewed, particularly in the context of the new generation of synchrotron x-ray sources that will be able to provide high brightness over a much wider range of energies than is available at present.
Determination by x-ray microscopy of the phases of the x-ray diffraction by macromolecular structures
Ronald E. Burge, Christopher J. Buckley, Guy F. Foster, et al.
The use of soft x-ray imaging is considered for the determination of the repeating macromolecular structure of biological fibers (e.g., collagen and muscle), within the available image resolution and subject to the effects of radiation damage. A comparison is made between the structure in sarcomere (2 (mu) to 3 (mu) long repeating unit) of striated muscle as seen directly by x-ray microscopy and as derived from published interpretations of x-ray diffraction data from whole muscle. The comparison shows that the loss by radiation damage of the ability of a muscle myofibril to contract is related to the loss of fine structure. Ways to minimize the effects of beam damage are discussed, including the use of images taken in phase, rather than amplitude contrast, and with photon energies above the `water window.'
Atomic force microscopy employed as the final imaging stage for soft x-ray contact microscopy
Robin A. Cotton, Mike D. Dooley, Julian H. Fletcher, et al.
Soft x-ray contact microscopy (SXCM) enables a high resolution image of a living biological specimen to be recorded in an x-ray sensitive photoresist at unity magnification. Until recently scanning electron microscopes (SEM) have been employed to obtain the final magnified image. Although this has been successful in producing many high resolution images, this method of viewing the resist has several disadvantages. Firstly, a metallic coating has to be applied to the resist surface to provide electrical conductivity, rendering further development of the resist impossible. Also, electron beam damage to the resist surface can occur, in addition to poor resolution and image quality. Atomic force microscopy (AFM) allows uncoated resists to be imaged at a superior resolution, without damage to the surface. The use of AFM is seen as a major advancement in SXCM. The advantages and disadvantages of the two technologies are discussed, with illustrations from recent studies of a wide variety of hydrated biological specimens imaged using SXCM.
Development of a linear scanning-force microscope for x-ray Gabor hologram readout
Steve A. Lindaas, Chris J. Jacobsen, Malcolm R. Howells, et al.
Recent work in Gabor x-ray holography has had a resolution limit imposed by the method used to extract the hologram information form the photoresist recording medium. In our case, we believe spiral distortions in the transmission electron microscope used for hologram readout limit resolution to 56 nm. To overcome this limitation we are building a scanning force microscope with a linear scanning stage offering < 20 nm resolution over a (70 micrometers X 70 micrometers ) field and a field linearity of 1 part in 10,000. A field linearity yielding one half or less pixel registration error across the scan is desirable so that the resulting hologram reconstruction is not significantly degraded. This desire for a large and linear scanning field necessitated designing our own stage since these conditions could not be met commercially. It is our goal to use this microscope to achieve higher resolution in reconstructed holograms. In addition, it should offer a means to explore at a macromolecular level the resolution limit of resists, such as PMMA. In this report we describe the technical strategy employed to meet these specifications.
Experiments in scanning luminescence x-ray microscopy
Chris J. Jacobsen, Steve A. Lindaas, Vivian Oehler, et al.
Scanning luminescence x-ray microscopy is based on collecting visible light emission from a sample region illuminated by an x-ray microprobe. We have tested the resolution of the method using P31 phosphor grains, and have obtained luminescence images of dye-labelled polystyrene spheres and sodium salicylate crystals. However, we have observed no light emission from the dyes DAPI, ethidium bromide, Hoecst 33258, and rhodamine phalloidon. Present efforts are aimed at improving our understanding of the luminescence process so as to find appropriate dyes for imaging dye-labelled biological specimens.
Image Analysis
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Image reconstruction for x-ray holographic microscopy
James M. Brase, Thomas J. Yorkey, James E. Trebes, et al.
Soft x-ray microscopy offers the potential of extending imaging system resolutions below 100 nm with less destructive specimen preparation than electron microscopy. Imaging in the wavelength regime between 10 and 100 angstroms has been demonstrated with several techniques including scanning microscopy, imaging zone-plate microscopy, and Gabor or Fourier holography. Good transverse resolution has been demonstrated in these systems ((iota ) 100 nm) but the longitudinal or depth resolution has been very limited. Our approach to improving depth resolution is to combine multiple views of the object topographically. These systems present unique problems for computational image formation and enhancement.
Wavefront reconstruction from three-dimensional intensity measurements
The reconstruction of a partially coherent wavefield from its three-dimensional intensity distribution is discussed. A computer simulation of the reconstruction of a Gaussian beam is also given.
Image enhancement through deconvolution
Xiaodong Zhang, Chris J. Jacobsen, Shawn P. Williams
Several groups have been developing x-ray microscopes for studies of biological and materials specimens at suboptical resolution. The X1A scanning transmission x-ray microscope at Brookhaven National Laboratory has achieved 55 nm Rayleigh resolution, and is limited by the 45 nm finest zone width of the zone plate used to focus the x rays. In principle, features as small as half the outermost zone width, or 23 nm, can be observed in the microscope, though with reduced contrast in the image. One approach to recover the object from the image is to deconvolve the image with the point spread function (PSF) of the optic system. Toward this end, the magnitude of the Fourier transform of the PSF, the modulation transfer function, has been experimentally determined and agrees reasonably well with the calculations using the known parameters of the microscope. To minimize artifacts in the deconvolved images, large signal to noise ratios are required in the original image, and high frequency filters can be used to reduce the noise at the expense of resolution. In this way we are able to recover the original contrast of high resolution features in our images.
Digital twin-image elimination in soft x-ray in-line holography
In-line holography is attractive for x-ray microscopy due to its recording simplicity. A drawback of this method is the superposition of the virtual and real images, in which structures and details can be modified or lost. This superposition effectively limits the application of in-line holography to x-ray microscopy. We present in this work an iterative constrained algorithm for twin image elimination from Gabor holograms of finite support objects. It is based in the different spatial extent of both images, together with a finite support constraint. The conditions under which the algorithm is applicable are presented, together with an alternative Monte Carlo method for holograms of complex objects recorded in the shadow regions.
Detectors
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Cooled backside-illuminated CCD for x-ray microscopy application
Hiroaki Aritome, Shigeru Nakayama, Guan-ming Zeng, et al.
In x-ray microscopy applications of CCDs in the water window region, radiation damage in the MOS structure due to x rays is a problem. The backside illuminated CCD is one of the possibilities to solve the problem. This paper reports about a CCD imaging system for x-ray microscopy applications using a backside illuminated CCD (EEV Ltd., CCD 02-06). The system is used for a zone plate x-ray microscope using a laser plasma source. It is effective to take an image with a single x-ray pulse. The dark noise is 1 electron/s/pixel (r.m.s.) at a temperature of -53 degree(s)C. The quantum efficiency is measured between the wavelength of 2.25 - 8 nm.
Microchannel plates as detectors and amplifiers of x-ray images
Jay Douglas Wiedwald
Two decades of development driven largely by military night vision applications has led to the availability of a wide selection of microchannel plates for use by the scientific community. Microchannel plates (MCPs) are electron multipliers which retain a high degree of spatial resolution making it possible to amplify electron images by factors of 1000 or more. Plates having 40 mm diameter and intrinsic spatial resolution of 8 micrometers are readily available. By coating the front surface of a microchannel plate with an x-ray sensitive photocathode material, x-ray images can be detected and amplified. While the detective quantum efficiency is relatively low, the low noise of the MCP (including the ability to construct images by single photon detection) and its high dynamic range make it suitable for some x-ray microscopy applications. The principles of MCP operation and typical performance are discussed. Examples of related applications and commercial capabilities also are presented.
Electronic zooming TV readout system for an x-ray microscope
Katsuyuki Kinoshita, T. Matsumura, Yoshinori Inagaki, et al.
The electronic zooming TV readout system using the x-ray zooming tube has been developed for purposes of real time readout of very high resolution x-ray image, e.g., the output image from an x-ray microscope. The system limiting resolution is 0.2 to approximately 0.3 micrometers and it is easy to operate in practical applications.
Pulsed-Source-Based Imaging Systems
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Development and first applications of an imaging microscope using the Vulcan x-ray laser
Ronald E. Burge, Michael T. Browne, Pambos S. Charalambous, et al.
An imaging microscope, comprising a Schwarzschild condenser and zone plate optical arrangement, has been established on the Vulcan Nd-glass laser system at the Rutherford Appleton Laboratory (RAL). Images of simple test structures have been taken in x-ray transmission using doublet x-ray laser radiation at 23.2 nm and 23.6 nm from collisionally pumped Ne-like germanium. Image resolutions of about 0.15 micrometers have been measured. The results are intended as a proof of principle and demonstrate both that images can be taken successfully using the Vulcan x-ray laser, and of specimen regions which are destroyed on passage of the x-ray beam.
Materials Applications
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New results from MAXIMUM: an x-ray scanning photoemission microscope
W. Ng, Avijit K. Ray-Chaudhuri, S. H. Liang, et al.
The scanning soft x-ray photoemission microscope (MAXIMUM) operating at the Synchrotron Radiation Center at the University of Wisconsin-Madison has been substantially upgraded. The major upgrades are: installation of a new beam line that is optimized for the microscope; new optical mount and alignment system for the Schwarzschild objective; a new scanning stage; installation of a cylindrical mirror analyzer; implementation of an ultrahigh vacuum sample preparation chamber and transfer system; a new window-driven data acquisition program that is more flexible and user-friendly. The new system had demonstrated better than 0.1 micrometers spatial resolution, and photoemission data with 350 meV energy resolution has been obtained.
Design of the second-generation scanning photoemission microscope at the National Synchrotron Light Source
Cheng-Hao Ko, Harald Ade, Janos Kirz, et al.
During the past few years, we have built and commissioned a scanning photoemission microscope (X1-SPEM). It was the first photoemission microscope to achieve submicron resolution. To improve the performance, we are designing a second generation instrument. The major changes in the instrument are the replacement of the home-made single pass cylindrical mirror analyzer (CMA) with a hemispherical sector analyzer (HSA) and the construction of a new chamber with a scheme to manipulate the zone plate and the order sorting aperture (OSA) with more flexibility. The design concepts and expected performance of the instrument are discussed.
X-ray imaging of aggregation in silica and zeolitic precursors
Graeme R. Morrison, Michael T. Browne, Theo P. M. Beelen, et al.
The resolution available in the King's College London scanning transmission x-ray microscope (STXM) can be exploited to study aggregate structures over a length scale from 100 nm to 10 micrometers that overlaps with and complements that available from small-angle x-ray scattering (SAXS) data. It is then possible to use these combined sets of data to test between different growth models for the aggregates, using the fractal dimension of the structures as a way of distinguishing the different models. In this paper we show some of the first transmission x-ray images taken of silica gels and zeolite precursors, materials that are of great practical and economic importance for certain selective catalytic processes in the chemical industry, and yet for which there is still only limited understanding of the complicated processes involved in their preparation. These images reveal clearly the fractal aggregates that are formed by the specimens.
X-Ray Damage to Biological Specimens
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Radiation damage to chromosomes in the scanning transmission x-ray microscope
Shawn P. Williams, Chris J. Jacobsen, Janos Kirz, et al.
Imaging with soft x rays having energies between the carbon and oxygen K edge (284 - 531 eV) yields large absorption contrast for wet organic specimens, but these soft x rays are known to be very effective in damaging biological specimens. The commonly used criterion of mass loss was employed for assessing radiation damage in the scanning transmission x-ray microscope. Multiple images of freeze-dried V. faba chromosomes show no significant mass loss after 150 Mrad. Experiments performed on fixed hydrated chromosomes revealed them to be radiation sensitive. The greater total mass loss observed in multiple low dose images compared to that incurred during a single high dose image suggests that the effects of radiation damage occur slower than the acquisition time for neighboring pixels. The radiation sensitivity of chromosomes depends critically on the fixative used, with damage minimized in glutaraldehyde fixed samples. Radiation damage to chromosomes is independent of ionic strength above 65 mM, but increases for ionic strengths below 65 mM. Using free radical scavengers in the buffer, and changing the design of the sample cell reduced the amount of damage incurred as a function of dose.
Effects of soft x-ray irradiation on cell ultrastructure
Thomas W. Ford, Anton M. Page, Guy F. Foster, et al.
The future of x-ray microscopy lies mainly in its potential for imaging fresh, hydrated biological material at a resolution superior to that of light microscopy. For the image to be accepted as representing the cellular organization of the living cell, it is essential that artefacts are not introduced as a result of the image collection system. One possible source of artefacts is cellular damage resulting form the irradiation of the material with soft x rays. Cells of the unicellular alga Chlorella have been examined by transmission electron microscopy (TEM) following exposure to different doses of monochromatic (380 eV) soft x rays. Extreme ultrastructural damage has been detected following doses of 103 - 104 Gy, in particular loss of cellular membranes such as the internal thylakoid membranes of the chloroplast. This is discussed in relation to dosage commonly used for imaging by soft x-ray microscopy.
Role of x-ray-induced damage in biological micro-imaging
Interactions of x rays with a sample are studied to determine the optimal wavelength, source energy, and exposure time for microcopy and holography. The optimal wavelength is influenced by two criteria: minimizing the required source energy and minimizing the absorbed dose and subsequent damage to the sample. The use of heavy element labels, such as colloidal gold, can significantly reduce the energy and dose. Limits to the exposure time due to natural motions, x-ray induced chemical damage, heat build-up, and hydrodynamic expansion are discussed.
Biological Applications I
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Measuring changes in the mass of single subcellular organelles using x-ray microscopy
Kaarin K. Goncz, Mario M. Moronne, W. Lin, et al.
Using quantitative scanning transmission x-ray microscopy, zymogen granules isolated from pancreatic acinar cells were observed suspended in aqueous medium at 50 nm resolution. From 3.64 nm x-ray absorption data, the protein content and rate of protein efflux from individual granules were determined. This was accomplished with a specially designed silicon nitride based wet-cell that allowed continuous perfusion and monitoring of individual granules in a variety of different aqueous environments. Granules suspended in 300 mM sucrose, 5 mM phosphate buffer (pH 6.0) were observed to continuously decrease in size and protein content over a period of several hours. Sudden lysis of the granules was not observed. From the flux data, the apparent protein permeability coefficients for individual granules were determined to range from 1 - 10 X 10-10 cm/sec with an average of 4.78 +/- 3.0 X 10-10 cm/sec. We believe this is the first quantitative population profile determined for a subcellular organelle developed from measurements of individual members of the population.
Visualization of the effects of electron microscopy fixatives on the structure of hydrated epidermal hairs of tomato (lycopersicum peruvianum) as revealed by soft x-ray contact microscopy
Anthony D. Stead, Robin A. Cotton, Anton M. Page, et al.
In order to examine the ultrastructure of biological specimens by electron microscopy it is necessary to stabilize the highly labile cellular contents before embedding and sectioning the specimens. This is commonly achieved by treating the tissues with various chemical fixatives. The assessment of the efficacy of these fixative is usually based upon the appearance of the specimen under the microscope although this is somewhat intuitive as the ultrastructure of the living cell cannot be studied. Previous studies, in which the structure of epidermal hairs has been followed under the light microscope as the commonly used fixatives are perfused into the tissue, have shown that the cellular contents are drastically rearranged by these fixatives. This paper describes the effects of some of the commonly used fixatives on cell ultrastructure using firstly electron microscopy and secondly soft x-ray contact microscopy. The latter technique allows not only direct comparisons of the effects of the fixatives but also allows living, hydrated specimens to be imaged so that the true ultrastructural effects of the fixatives can be seen and compared to the ultrastructure of the living material.
X-ray imaging of calcium deposits in human cartilage
Christopher J. Buckley, Ronald E. Burge, Guy F. Foster, et al.
X rays have been used extensively in the study of hard tissue such as bone. The x rays used are typically of energy 50 keV, which have an absorption depth of approximately 1.5 cm in hard tissue. These x rays are used for creating x-ray shadowgraphs (or radiographs) of bones where the finest details recorded are of the order of a few tenths of a millimeter. However, due to the advent of x-ray sources which are energy tunable, and the availability of high resolution x-ray optics, an entirely new range of contrast is now possible along with resolution down to a few tens of nanometers. These new x-ray sources and optics have been combined to create a variety of x-ray microscopes which are now being used in a range of unique applications.
Biological Applications II
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Application of x rays to the analysis of DNA packaging in mammalian sperm
Rod Balhorn, Michele Corzett, Michael J. Allen, et al.
X-ray microscopy has been performed on unlabeled and gold labeled rat sperm nuclei using a tantalum x-ray laser (44.83 angstrom) and an x-ray zone plate lense. Transmission images of nuclei labeled with gold using an antibody to protamine 1 show large clusters of gold as well as individual 400 angstrom gold particles coating the surface of the chromatin. Images of the same nuclei obtained with a scanning x-ray microscope demonstrate that the initial exposure of the sperm nucleus to a photon intensity of 3.0 X 1014 W/cm2 for a duration of 500 ps did not destroy or grossly distort their structure. Other nuclei labeled with an antibody to protamine 2 were partially decondensed during the labeling process and contained large vacuole-like regions. Images were also obtained of unlabeled sperm nuclei. Data obtained from one unlabeled nucleus imaged with both the atomic force and x-ray laser microscopes was used to determine the thickness of the chromatin and estimate the amount of water that must be associated with the DNA-protamine complex in air-dried nuclei. These results suggest that even air-dried sperm nuclei are extensively hydrated and that tightly bound water may comprise as much as one-third of the volume of the dried sperm nucleus.
Observation of human chromosome fibers in a water layer by laser-plasma x-ray contact microscopy
Kunio Shinohara, Yasuhito Kinjo, Martin C. Richardson, et al.
Soft x-ray contact microscopy was applied to hydrated human chromosomes. Chromosomes of human lymphocytes were spread on a clean surface of distilled water, attached on an x-ray resist, polymethylmethacrylate (PMMA), immediately covered with a silicon nitride window, and mounted in a simple hydrated chamber. The specimens were exposed to a single shot of laser-produced gold plasma x rays (600 ps) in a vacuum chamber. The developed images were observed with transmission electron microscope using the replica method with a plasma polymerization-film in a glow discharge. The results show that we have imaged the complicated entanglement of chromosome fibers in a hydrated condition. The thickness was estimated as 10 nm in an average of four narrow parts of these fibers. Particle like structures were observed in many places. The present results prove that a hydrated biological specimen is observable with the contrast produced by its components themselves using soft x-ray microscopy at the resolution of 10 nm. During this imaging exposure, however, the silicon nitride (SiN) window was broken. We have studied the reason for this evidence and found that the energy absorbed by the SiN window or water layer was very high. The estimated temperature increase was 870 -1470 degree(s)C for SiN and 43 degree(s)C for the water layer. These results suggest that the temperature increase may be responsible for the breakage of SiN window.
High-resolution x-ray stereomicroscopy: true three-dimensional imaging of biological samples
Billy W. Loo Jr., Shawn P. Williams, W. Lin, et al.
X-ray microscopy has the potential to become a powerful tool for the study of biological samples, allowing the imaging of intact cells and subcellular organelles in an aqueous environment at resolutions previously achievable only by electron microscopy. The ability to examine a relatively thick sample raises the issue of superposition of objects from multiple planes within the sample, making difficult the interpretation of conventional, orthogonally projected images. This paper describes our early attempts at developing three-dimensional methods for x-ray microimaging: the first to use x-ray optics, and to our knowledge, the first demonstrating sub-visible resolutions and natural contrast. These studies were performed using the scanning transmission x-ray microscope (STXM) at the National Synchrotron Light Source, Brookhaven National Laboratory.
Pulsed-Source-Based Imaging Systems
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Imaging microscopy with x-ray lasers at LLNL
Luiz Barroca Da Silva, James E. Trebes, Stanley Mrowka, et al.
Imaging microscopy with short pulse (200 ps) x-ray lasers offers the opportunity of high resolution three-dimensional imaging of specimens in an aqueous environment without the blurring effects associated with natural motion. As a first step toward this goal we have performed imaging experiments which clearly resolved 500 angstrom features on a gold test pattern. In addition, we have taken images of dried biological specimens as a basis for comparison in future wet specimen imaging. The results of these experiments are described. We also discuss some of the alignment problems involved in doing x-ray microscopy with low repetition rate systems where in situ focusing of the imaging optics is not practical as single shot exposures can alter the specimen.
Soft x-ray microscopy with a 182-angstrom soft x-ray laser
Darrell S. DiCicco, Dong-Su Kim, Leonid Polonskiy, et al.
The high brightness and short pulse duration of soft x-ray lasers provide unique advantages for x-ray microscopy. We briefly review soft x-ray laser development at Princeton University and present results from the development of novel soft x-ray microscopes. The Princeton soft x- ray laser at 18.2 nm has been used to record high resolution contact images of biological specimens. More recently we have demonstrated proof-of-principle of reflection imaging in the soft x-ray wavelength range with the first results from a soft x-ray reflection imaging microscope. The microscope used a Schwarzschild objective with Mo/Si multilayer mirrors (normal incidence reflectivity of approximately 20% per surface) to form an image in reflected 18.2 nm soft x rays. In a separate experiment a novel `diffraction plate,' designed as an alternative to conventional condenser optics, has been tested.
X-Ray Optics
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Capillary x-ray optics
X-ray optical devices based on arrays of capillaries and single tapered capillaries can focus and concentrate x rays by the reflection of near-grazing-incidence rays at the interior walls of the channels. Capillary arrays are true imaging devices and can be used as focusing, condensing,and collimating optics, suitable for x-ray microcopy and astronomy. Rays which enter channels and are reflected once or twice, via total external reflection, may be redirected towards the image. Theoretical calculations of the focusing performance of arrays consisting of capillaries of square and circular cross-section are given. Experimental investigations have been made using microchannel plate (MCP) detector blanks and various x-ray sources.
Synchrotron-Based Imaging Systems
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X-ray microscopy resource center at the advanced light source
Werner Meyer-Ilse, Masato Koike, H. Raul Beguiristain, et al.
An x-ray microscopy resource center for biological x-ray imaging will be built at the Advanced Light Source (ALS) in Berkeley, California. The unique high brightness of the ALS allows short exposure times and high image quality. Two microscopes, an x-ray microscope (XM) and a scanning x-ray microscope (SXM) are planned. These microscopes serve complementary needs. The XM gives images in parallel at comparable short exposure times, and the SXM is optimized for low radiation doses applied to the sample. The microscopes extend visible light microcopy toward significantly higher resolution and permit images of objects in an aqueous medium. High resolution is accomplished by the use of Fresnel zone plates. Design considerations to serve the needs of biological x-ray microscopy are given. Also the preliminary design of the microscopes is presented. Multiple wavelength and multiple view images will provide elemental contrast and some degree of 3-D information.
Biological Applications II
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Imaging and etching: soft x-ray microscopy on whole wet cells
John R. Gilbert, Jerry Pine
We have produced images of whole wet tissue culture cells with the Stony Brook/BNL scanning transmission x-ray microscope (STXM). For fixed cells we have taken images at theoretical resolutions of approximately 50 - 75 nm, and in practice have measured FWHM of features down to near 100 nm, without any exotic image processing. For un-fixed (i.e., initially live) cells we have imaged with 100 nm pixels and measured features down to 250 nm. In order to do this we have developed, tested, and used a wet cell for maintaining fixed or live cells on the STXM stage during imaging. Our design of the wet cell and the culture substrates that go with it make the STXM compatible with almost all standard systems for surface adherent tissue culture. We show some new images of whole wet fixed and unfixed cells, with visible sub-micron features. We also report data that helps to characterize the tissue damage due to x-ray absorption during STXM imaging.