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

Abstract

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...

A study on new approaches in coherent x-ray microscopy of biological specimens

“…In 1999 the first experimental demonstration of coherent x-ray diffractive imaging was achieved in the (soft) x-ray energy range [99], again enabled by the high degree of coherence provided by a synchrotron source. Largely driven by the potential to overcome technical restrictions in the fabrication of x-ray lenses, the technique has been applied successfully by now to single freeze-dried [103,132] and frozen-hydrated [73,86] cells, with a potential to exceed the resolution of conventional x-ray microscopy which is limited by the lenses. As a result of the Shannon theorem, for plane wave illumination sufficient sampling of the diffraction pattern and therefore reconstruction is only possible, if the object has a finite extension in real space.…”

When holography meets coherent diffraction imaging

“…The envisioned ultimate application of CDI is to obtain an image of an individual biological molecule at atomic resolution. Some biological specimens have already been imaged by CDI employing coherent X-rays [6][7][8][9][10][11][12][13] . The first results from the Linac Coherent Light Source X-FEL facility were reported and demonstrated imaging an individual unstained mimivirus (800 nm in diameter) with 6.9 Å wavelength X-rays at 32 nm resolution 14 .…”

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

“…This approach has been used to image isolated dried cells (13)(14)(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.…”