Potential Operating Region for Ultrasoft X-Ray Microscopy of Biological Materials

Sayre, D.; Kirz, Janos; Feder, R.; Kim, DM; Spiller, Eberhard

doi:10.1126/science.867033

Cited by 107 publications

(35 citation statements)

References 7 publications

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“…Correlative microscopy provides strong This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: Johanna.Nelson@stonybrook.edu.…”

Section: Resultsmentioning

confidence: 99%

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

Nelson

Huang

Steinbrener

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.coherent imaging | immunogold labeling X -ray microscopes can be used for the imaging of unsectioned eukaryotic cells too thick to be viewed in their entirety using electron microscopy (1-3), with the potential for higher spatial resolution than even the most advanced optical microscopy methods (see, e.g., ref. 4). These advantages are being realized in a number of ways, such as the imaging of trace metals and metalloproteins with improved detection limits (5, 6) and tomographic imaging of frozen hydrated cells at 40 to 60-nm resolution (7-11). Such efforts are leading to the availability of x-ray microscopes at most synchrotron radiation research centers, and laboratorybased systems are also beginning to appear (12, 13).Although most lens-based x-ray imaging of biological specimens has been done at 40 to 60-nm resolution range, the spatial resolution of x-ray microscopes has been steadily improving (14), with demonstrations in specific test cases of optics with resolutions around 15 nm (15-17). Even so, practical challenges remain in lens-based x-ray imaging. Soft x-ray Fresnel zone plates with outermost zone widths smaller than 20 nm have had submillimeter short focal lengths as well as focusing efficiencies well below 10%. The former creates geometric complications for tomographic imaging, whereas the latter translates to an increase in radiation damage to the specimen when zone plates are used in transmission x-ray microscope systems. To reduce the damage from radiation, biological materials have been successfully imaged with x-rays in the frozen hydrated state with no artifactcausing pretreatment (11,18,19). For hydrated specimens, both phase and amplitude contrast are maximized when working in the "water window," the spectral region between the carbon and oxygen K-shell energies (18,20). However at a water window energy of 540 eVand a spatial resolution of 20 nm, the depth of focus of a standard monochromatic zone plate imaging system approaches the half-micrometer thickness at which cryoelectron tomography at 5 to 6-nm resolution becomes possible (2, 21). As a result, while progress is ongoing in lens-based x-ray imaging, it is also valuable to consider alternative approaches to high-resolution x-ray i...

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“…Correlative microscopy provides strong This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: Johanna.Nelson@stonybrook.edu.…”

Section: Resultsmentioning

confidence: 99%

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

Nelson

Huang

Steinbrener

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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“…Higher multiples of λ/4 can be used to better match the strength of the diffracted and undiffracted radiation so as to optimize the phase contrast. Since the exposure required to render a feature visible is inversely proportional to the square of the contrast [5], maximizing the contrast is an important consideration. Images of mouse liver (Fig.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

A High Resolution, Hard X-ray Bio-imaging Facility at SSRL

Andrews

Brennan

Patty

et al. 2008

Synchrotron Radiation News

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The old saying that seeing is believing has particular resonance for studying biological cells and tissues. Since 1677, when Anton van Leeuwenhoek used a simple light microscope to discover single cell organisms, scientists have relied on structural information obtained from microscopes with improving capabilities to advance the understanding of how biological systems work. Optical and electron microscopes are essential for many of these important discoveries and have been widely employed in biomedical research laboratories. However, various limitations exist in these microscopy techniques. We describe below how the new xray imaging facility at the Stanford Synchrotron Radiation Laboratory (SSRL), based on an Xradia nano-XCT full-field transmission x-ray microscope (TXM), can provide complementary and unique capabilities to the current microscopy methods for studying complex biological systems.The TXM, developed at the 54 pole wiggler beam line 6-2 (BL6-2) at SSRL, is based on zone plate optics using absorption contrast over a wide energy range from 5-14 keV and Zernike phase contrast at 8 keV [1]. The instrument offers complementary capabilities to many imaging tools that are widely deployed in biomedical research. For example, the capability of high NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript resolution imaging of thick biological specimens is of importance to the study of complex biological systems because the biology of higher living organisms relies on the cooperation of cells. This is evident in the study of development and differentiation and of intra-cellular communication, not only through synapses, but also via the interplay of cells organized into organs and the development of cancer. Even bacterial plaques, so resistant to medical intervention, involve cooperation on the multi-cell level. While these systems are routinely studied by visible light and electron microscopy, visible light microscopy is limited in resolution, and electron microscopy inevitably requires sectioning, thereby losing a great deal of information on the three dimensional architecture. The high-resolution "virtual sectioning" capability of this instrument enables imaging at 40 nm resolution of any number of pre-selected regions within a visible light microscope specimen or even a cylindrical specimen such as obtained with a needle biopsy.Overall, the instrument offers several unique and complementary capabilities that are expected to have a significant impact for biologists studying complex biological structures. These include:• High resolution of 40 nm (or better with newer zone plates)• Imaging of tissues without cross sectioning• High penetration (~1-20 μm) for nondestructive imaging of relatively thick biological specimens• High quality images using single exposure times of 0.5 seconds• Rich contrast mechanisms (Zernike phase contrast at 8 keV, and soon at 5 keV) for imaging biological and marker materials• Absorption contrast in the 5-14 keV range, and X-ray absorption near ed...

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“…where it is desirable to image biological samples due to favorable levels of absorption [15]. Although harder x-rays (and thus shorter wavelengths) would create higher resolution images, these energies damage biological images.…”

Section: Motivationmentioning

confidence: 99%

“…The wavelength is expressed to include order number, following the convention of equation (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). From equation (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), we see that for an increased harmonic, n, there is a matching such that the reflected angle of the grating remains a constant. This enables higher orders for a given K to pass through the exit slit of the monochroma wavefront division interferometers and amplitude division interferometers.…”

Section: Beamline Challengesmentioning

confidence: 99%

Tunable coherent radiation at soft X-ray wavelengths: Generation and interferometric applications

Rosfjord

2004

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The availability of high power, spectrally and spatially coherent soft x-rays (SXR) would facilitate a wide variety of experiments as this energy region covers the primary resonances of many magnetic and biological materials. Specifically, there are the carbon and oxygen K-edges that are critical for biological imaging in the water window and the L-edges of iron, nickel, and cobalt for which imaging and scattering studies can be performed.A new coherent soft X-ray branchline at the Advanced Light Source has begun operation (beamline 12.0.2). Using the third harmonic from an 8 cm period undulator, this branch delivers coherent soft x-rays with photon energies ranging from 200eV to 1keV. This branchline is composed of two sub-branches one at 14X demagnification and the other 8X demagnification. The former is optimized for use at 500eV and the latter at 800eV. Here the expected power from the third harmonic of this undulator and the beamline design and characterization is presented. The characterization includes measurements on available photon flux as well as a series of double pinhole experiments to determine the coherence factor with respect to transverse distance. The first high quality Airy patterns at SXR wavelengths are created with this new beamline.The operation of this new beamline allows for interferometry to be performed in the SXR region. Here an interferometric experiment designed to directly determine the index of refraction of a material under test is performed.Measurements are first made in the EUV region using an established beamline (beamline12.0.1) to measure silicon, ruthenium and tantalum silicon nitride. This work is then extended to the SXR region using beamline 12.0.2 to test chromium and vanadium.

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Potential Operating Region for Ultrasoft X-Ray Microscopy of Biological Materials

Cited by 107 publications

References 7 publications

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

A High Resolution, Hard X-ray Bio-imaging Facility at SSRL

Tunable coherent radiation at soft X-ray wavelengths: Generation and interferometric applications

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