Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Mäser, J.; Osanna, A.; Wang, Y; Jacobsen, Chris; Kirz, Janos; Spector, S.; Winn, Barry; Tennant, D. M.

doi:10.1046/j.1365-2818.2000.00630.x

Cited by 138 publications

(109 citation statements)

References 39 publications

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“…Although we show here 2D images, both ptychography (39) and fluorescence (57) are compatible with tomographic approaches to nanoscale 3D imaging of biological materials. Compared with "water window" soft X-ray tomography (30,58,59), tomography with multi-keV X-rays offers the capability for increased sample thickness, and increased depth of focus for a given spatial resolution, which allows this combined method to image larger cells and tissue sections in 3D. This method will aid the interpretation of studies of the localization of nanoparticles attached to therapeutic agents (1,60), or the role of metals in cell development (61), and in diseases where trace metal misregulation is implicated as a cause (62).…”

Section: Discussionmentioning

confidence: 99%

“…However, radiation damage limits the achievable resolution in X-ray microscopy of hydrated biological specimens (28). A good approach to reduce beam-induced degradation of the sample is to work with frozen-hydrated biological specimens under cryogenic conditions (29,30). Cryogenic samples can provide highfidelity structural (31) and ionic elemental (32-34) preservation, and mitigate the effects of radiation damage (35).…”

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confidence: 99%

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Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Deng

Vine

Chen

et al. 2015

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

160

131

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Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolution beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ∼90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context. ptychography | X-ray fluorescence microscopy | cryogenic biological samples X -ray fluorescence microscopy (XFM) offers unparalleled sensitivity for quantitative mapping of elements, especially trace metals which play a critical role in many biological processes (1-3). It is complementary to light microscopy, which can study some elemental content in live cells (with superresolution techniques possible) but which is more difficult to quantitate because it depends on the binding affinities of fluorophores. However, XFM does not usually show much cellular ultrastructure, because the light elements (such as H, C, N, and O, which are the main constituents of biological materials) have low fluorescence yield (4). At the multi-keV X-ray energies needed to excite most X-ray fluorescence lines of interest, these light elements show little absorption contrast, but phase contrast can be used to image cellular structure (5, 6) and this can be combined with scanned-beam XFM (7-11).One can also acquire phase-contrast X-ray images with a resolution beyond X-ray lens limits by recording the diffraction pattern from a coherently illuminated, noncrystalline sample in an approach called coherent diffraction imaging (CDI) (12). This approach has been used to image isolated dried cells (13-15), and 3-nm resolution has been achieved when imaging silver nanocubes (16). The traditional CDI approach requires that samples meet a so-called "finite support" (17) requirement with no observable scattering outside of a defined region; although some limited success has been obtained (18,19), this finite support condition has proven difficult to achieve with single cells surrounded by ice layers. Ptychography (20-22) is a recently realized CDI method [with an older history (23)] that circumvents this isolated cell requirement by instead scanning a limitedsize coherent illumination spot across the sample. Ptychography has been used to image freeze-dried diatoms at 30-nm resolution (24) and bacter...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Deng

Vine

Chen

et al. 2015

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

160

131

View full text Add to dashboard Cite

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“…Synchrotron radiation at k = 2.4 nm was used for imaging frozenhydrated samples at atmospheric pressure, where details inside cells of algae as small as 35 nm were visible [32], or to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution, using transmission SXR microscope [33]. Synchrotron-based microscope in the ''water-window'' spectral range was developed to image frozen hydrated specimens with a thickness of up to 10 lm at temperatures of around 100 K [34].…”

Section: Introductionmentioning

confidence: 99%

Water-window microscopy using a compact, laser-plasma SXR source based on a double-stream gas-puff target

et al. 2013

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We report on a compact, desktop size, laboratory type microscopy setup, based on a short wavelength gas puff target soft X-ray source, which emits incoherent radiation in ''water-window'' spectral range. The microscope employs a Wolter type I reflective objective and allows capturing magnified images of objects with *1-lm spatial resolution and exposure time as low as 5 s. A detailed characterization and optimization of both the source and the microscope setups are presented and discussed.

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“…Because no x-ray lenses are needed, the resolution of singleparticle x-ray diffraction is limited only by radiation damage to the samples. By freezing the biological samples to liquid nitrogen temperatures, previous x-ray experiments indicated that radiation damage can be greatly alleviated (15)(16)(17), and better resolution should be achievable. In the long run, with the prospects of x-ray free electron lasers providing ultrashort and extremely intense pulses, the radiation damage problem could be circumvented by recording the diffraction pattern from a single protein molecule before the molecule gets destroyed (8,18).…”

Section: Resultsmentioning

confidence: 99%

Imaging Whole Escherichia Coli Bacteria by Using Single-Particle X-Ray Diffraction

Miao¹

2004

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We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining singleparticle diffraction with x-ray free electron lasers. Single-particle diffraction is a methodology of extending crystallography to determine the 2D and 3D structures of nano crystals and noncrystalline samples by using coherent x-rays and electrons (1-6). In this approach, coherent diffraction patterns are recorded and then converted directly to highresolution images by using the oversampling phasing method (7). Due to the lack of multiple copies of the object, which constructively reinforce due to Bragg diffraction, the diffraction patterns are usually weak and continuous. This illustrates why the experiments with high Z scatters, such as those made of Au and Ni, are simpler to carry out (2-4). However, some of the most important potential applications of single-particle diffraction lie in biological materials (8, 9). Here we report the first successful experiment, to our knowledge, of imaging Escherichia coli bacteria at 30-nm resolution by using single-particle x-ray diffraction. The images show the distribution of manganese-tagged proteins, which is consistent with results from fluorescence microscopy. MethodsExperimental Setup. The coherent diffraction experiment was conducted on an undulator beamline at SPring-8 (10). Because single-particle x-ray diffraction requires good spatial (i.e., beam divergence) and moderate temporal coherence (i.e., energy spread) (4), we achieved the beam divergence of Ϸ6 ϫ 10 Ϫ6 rad by setting a 150-m pinhole at a distance of 27 m upstream of the experimental instrument, and the energy spread of 0.7 eV (1 eV ϭ 1.602 ϫ 10 Ϫ19 J) by using a Si (1,1,1) double crystal (10). To make a small clean beam, a 20-m pinhole and a corner were placed at a distance of 25.4 and 12.7 mm upstream of the sample. The pinhole and corner combination created three very clean quadrants on a charge-coupled device (CCD) detector for recording weak diffraction patterns. The intensity lost in the noisy fourth quadrant was recover...

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Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Cited by 138 publications

References 39 publications

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Water-window microscopy using a compact, laser-plasma SXR source based on a double-stream gas-puff target

Imaging Whole Escherichia Coli Bacteria by Using Single-Particle X-Ray Diffraction

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