X-ray diffraction microscopy (XDM) is a new form of x-ray imaging that is being practiced at several third-generation synchrotron-radiation x-ray facilities. Nine years have elapsed since the technique was first introduced and it has made rapid progress in demonstrating high-resolution threedimensional imaging and promises few-nm resolution with much larger samples than can be imaged in the transmission electron microscope. Both life-and materials-science applications of XDM are intended, and it is expected that the principal limitation to resolution will be radiation damage for life science and the coherent power of available x-ray sources for material science. In this paper we address the question of the role of radiation damage. We use a statistical analysis based on the socalled "dose fractionation theorem" of Hegerl and Hoppe to calculate the dose needed to make an image of a single life-science sample by XDM with a given resolution. We find that for simplyshaped objects the needed dose scales with the inverse fourth power of the resolution and present experimental evidence to support this finding. To determine the maximum tolerable dose we have assembled a number of data taken from the literature plus some measurements of our own which cover ranges of resolution that are not well covered otherwise. The conclusion of this study is that, based on the natural contrast between protein and water and "Rose-criterion" image quality, one should be able to image a frozen-hydrated biological sample using XDM at a resolution of about 10 nm.6Corresponding author: mrhowells@lbl.gov, phone 510 486 4949, fax 510 486 7696.

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions. coherent x-ray diffraction imaging ͉ x-ray microscopy X -ray diffraction microscopy is a recently developed method in which only the coherent diffraction pattern of the sample is measured. It provides a path to high resolution without the limitations imposed by an x-ray optical system. The idea to image a noncrystalline object by phasing and inverting its diffraction pattern goes back to a suggestion by Sayre (1, 2) and was first demonstrated with x-rays by Miao et al. (3). In this article, we report the imaging of the complex-valued exit wavefront (both phase and magnitude) of a whole freeze-dried and unstained yeast cell. The images, at 30-nm resolution from multiple angular orientations of the cell, required an exposure of approximately one minute each using 750-eV x-rays (1 eV ϭ 1.602 ϫ 10 Ϫ19 J). This demonstration paves the way for the application of 3D x-ray diffraction microscopy (XDM) (4, 5) to frozen-hydrated samples in the future.High-resolution 3D images of biological samples are currently made by at least three methods: zone-plate x-ray microscopy (6-9), transmission electron microscopy (10, 11), and x-ray crystallography. All three have particular strengths and limitations. Both water-window (7-9) and multi-keV (12) zone-plate microscopes are currently limited to Ϸ60-nm 3D resolution by details of zone-plate resolution, depth of field, and operation. On the other hand, high-resolution transmission electron microscopes, although capable of extraordinary resolution, are limited by multiple electron scattering to specimens thinner than 0.5-1 m (10, 13). The third method, x-ray crystallography, traditionally yields the highest resolution structures and is the structural technique of choice, but it is limited to specimens that can be crystallized. In summary, the traditional structural techniques do not provide a capability for 3D imaging of an intact eukaryotic cell with resolution around 10 nm, and it is toward this end that our present efforts are directed.Since its introduction, XDM has been demonstrated with metal test objects in two dimensions (3, 14) and three dimensions (4) and with stained biological specimens (15) an...

We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below −170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.X-ray microscopes allow high resolution microscopy of intact, hydrated biological specimens with thicknesses of many micrometers, beyond the limit of biological electron microscopy [1][2][3]. Radiation damage precludes repeated imaging of live specimens [4], but this can be mitigated by working at liquid nitrogen temperature [5,6]. In addition, single view flash imaging of cells using ultrabright sources has been proposed [7,8] as a way of capturing the image before radiolytical and thermal damage become evident.In recent years, there has been much progress in developing zone plate microscopy for 3D imaging of frozen hydrated cells [9][10][11][12][13]. While there are demonstrations of x-ray optics with higher resolution [14][15][16], scientific applications using x-ray microscopes have mainly used Fresnel zone plate optics with 25-40 nm spatial resolution. These optics typically have a focusing efficiency in the 10% range [17] and the modulation transfer function for incoherent bright field imaging decreases the efficiency of utilization of higher spatial frequency information. As a result, while the practical advantages of lens-based microscopes will be the deciding factor for most studies, it is also worthwhile to consider alternative methods for high resolution x-ray imaging.X-ray diffraction microscopy (XDM), also called coherent x-ray diffraction imaging, was proposed by Sayre as an imaging method that dispenses with the technological limits of lens efficiency and resolution [18]. Instead, the far-field diffraction pattern of an isolated object illuminated by a coherent x-ray beam is recorded. If the object is finite, and the diffraction pattern is sampled finely enough, the object can be reconstructed from the measured diffraction intensities alone [19,20]. In this manner one is able to eliminate limitations due to the efficiency and finite numerical aperture of x-ray optics [21]. Following a first demonstration by Miao et *Chris.Jacobsen@stonybrook.edu. An important limitation applies to the demonstrations of x-ray diffraction microscopy of cells, chromosomes, and virions cited above: they have all involved dehydrated specimens at room temperature. Though Nishino et al. [27] have obtained a very exciting 3D XDM image of a dehydrated chromosome, they note significant resolution degradation due to accumulated radiation dose. In electron microscopy, stability a...

Using a signal-to-noise ratio estimation based on correlations between multiple simulated images, we compare the dose efficiency of two soft x-ray imaging systems: incoherent brightfield imaging using zone plate optics in a transmission x-ray microscope (TXM), and x-ray diffraction microscopy (XDM) where an image is reconstructed from the far-field coherent diffraction pattern. In XDM one must computationally phase weak diffraction signals; in TXM one suffers signal losses due to the finite numerical aperture and efficiency of the optics. In simulations with objects representing isolated cells such as yeast, we find that XDM has the potential for delivering equivalent resolution images using fewer photons. This can be an important advantage for studying radiation-sensitive biological and soft matter specimens.