Springer Handbook of Microscopy pp 2-2 | Cite as

# Diffractive Imaging of Single Particles

## Abstract

The computational methods and applications of diffractive (lensless) imaging are reviewed. Far-field scattering (e. g., by neutrons, electrons, light, or x-rays) by a nonperiodic localized potential is detected and the phase problem solved using iterative optimization methods, which allows a three-dimensional image of the object potential to be reconstructed without the need for a lens. The history of the subject and its relationship to the crystallographic phase problem are reviewed, together with a summary of theory, algorithms, uniqueness issues, resolution limits, the constraint ratio concept, and coherence requirements. Applications, from various forms of microscopy (electron, optical, and x-ray) to snapshot x-ray laser single-particle imaging, are reviewed. The method of ptychography, which achieves a similar aim, is reviewed elsewhere in these volumes.

Diffractive (or lensless) imaging refers to the use of theoretical methods and computer algorithms to solve the phase problem for scattering by a nonperiodic object. The topic is also covered in the chapter *Ptychography* (Chap. 17). Here we first review the history of the noncrystallographic phase problem by other methods, describe early experimental results with electron and x-ray beams, and the iterative phasing algorithms used, and then specialize to their application to imaging nonperiodic biological samples using coherent x-ray lasers ( ), which are not suited to ptychography when using a destructive-readout mode. We do not cover the rapidly-growing related field of phase-contrast x-ray imaging in medicine [20.1].

The name coherent x-ray diffractive imaging ( ) is used in the x-ray community, which we could generalize to coherent diffractive imaging ( ). Additional information about the object, such as the sign of the scattering potential and the approximate boundary of the object, may be combined with the measured scattered intensity to solve for the phases of the scattered amplitudes. In this way, under conditions of single scattering (and other approximations which often apply in optics, electron, x-ray, and neutron diffraction) one aims to reconstruct a real-space image of an object by Fourier transform of the complex scattering distribution, or Fraunhofer far-field diffraction pattern. This noncrystallographic phase problem has been described as *interferometry without an interferometer*. Applications to Fresnel near-field imaging are also possible, where, however, resolution may be limited by detector pixel size, since the magnification is unity, unless a lens or a diverging beam are used. The powerful transport of intensity method has been used for this purpose in the near field [20.2].

By avoiding the need for a lens, CDI avoids the aberrations and resolution limits introduced by lenses. Within the past two decades this method has been applied to experimental neutron, x-ray, light, and electron scattering data. The electron work has produced atomic-resolution experimental images in projection [20.3], while experiments with soft x-rays have produced three-dimensional () (tomographic) reconstructions at \({\mathrm{10}}\,{\mathrm{nm}}\) resolution [20.4]. It now offers the real possibility of diffraction-limited imaging with any radiation for which lenses do not exist. Since each radiation interacts differently with matter, the method can be expected to provide us with new information on matter in fields as diverse as biology, materials science, and astronomy. Much current interest focuses on tomographic imaging of whole cells, nanoparticles, chemical mapping [20.5], and mesoporous materials, and on the imaging of proteins and viruses at near-atomic resolution using x-ray lasers [20.6]. While it can be shown rigorously that some of the iterative algorithms described here do not diverge, because of the nonconvex nature of the constraints involved, no formal proof of convergence or of the uniqueness of solutions exists. Yet computational trials for simulated data with little noise practically always converge rapidly to the known solution, while the inevitable stagnation occurs in the presence of excessive noise. (The ability of these algorithms to *climb out* of the local minimum and find a global minimum in a data set with perhaps a million unknown parameters (phases) is one of their most remarkable features.) In summary, over the last two decades many algorithms for lensless imaging have been published, which work well on simulated noisy data, while increasing success has been achieved with experimental data.

## 20.1 History

This review section presents the history of ideas which led to the solution of the noncrystallographic phase problem in qualitative terms. The phase problem in optics has early origins: in a letter to Michelson, *Rayleigh* comments that ‘‘the phase problem in interferometry is insoluble without a priori information on the symmetry of the data'' [20.7]. Several fields have contributed to the development of our current working solution to this problem, including signal processing, wave-front sensing, astronomy, electron microscopy, image processing, and x-ray crystallography, in addition to applied mathematics. Thus, a wonderfully rich set of ideas has contributed, from communications theory to diffraction physics and nonconvex optimization by iterated projections.

The origins of the solution to the noncrystallographic phase problem lie in crystallography, however this was not recognized until recently [20.8]. The most successful iterative phasing methods for single particles are akin to the solvent flattening methods of crystallography. The review by *Millane* and *Lo* [20.9] describes iterative phasing in crystallography, where questions of uniqueness, and the powerful constraint ratio \(\Omega\) are discussed. (A unique solution, with the number of Fourier equations equal to the number of unknown phases, requires \(\Omega> {\mathrm{1}}\), which holds only for a protein crystal whose unit cell contains mostly solvent, as is further discussed below.) In retrospect, we can now see that the key to a successful solution was given in the crystallography literature as early as 1952. *D. Sayre*, in *Some implications of a theorem due to Shannon* [20.10], appears to have been the first to consider the relationship between Shannon's sampling theorem and Bragg's law. He pointed out that, for centrosymmetric crystals, where only the sign of structure factors \(F_{g}\) is unknown, these could be determined if intensities \(|F_{g}|^{2}\) were observable at half-integral values of \(\boldsymbol{g}\) (a reciprocal lattice vector), since a change of sign requires a zero-crossing of the molecular scattering factor there. By Shannon's theorem , these fractional and integral order intensities are needed and sufficient to completely define the autocorrelation function of the molecule. Sayre's fractional orders could thus only be generated by a molecule which filled half (in one dimension) of the cell, leaving the remaining half empty. If the density in the *empty* half of the cell is assumed known, it will be found that the number of Fourier equations relating real and reciprocal space are equal to the number of unknown phases, and the problem reduces to solving these (nonlinear) equations. This is best done by iterative search methods. (A modern application of exactly this method to a protein crystal in which the unit cell is occupied mostly by solvent can be found in *He* and *Su* [20.11].) Sayre concludes that a solution to the phase problem for centric crystals could as well be obtained from measurements of the *intensity* of half-order reflections as from study of the (complex) Bragg reflections. The paper was prompted by the observation of nonintegral reflections (following hydration) in experimental patterns from haemoglobin by Perutz and others and a talk by Gay in 1949. (Similarly, the phase problem may be solved if one has measured structure factors from the same molecule crystallized in a second space group, to provide additional sampled intensities between Bragg reflections of the first space group.) Note that the Patterson or autocorrelation function of a molecular crystal may differ from that of the isolated molecule, since the autocorrelation of a molecule which fills the cell will be twice as large as the unit cell in each direction and so produce overlap. Since diffraction from an isolated nonperiodic object is a continuous function of the scattering angle, it provides immediate access to these *fractional orders*. Thus, a solution in principle to the phase problem for a real, centrosymmetric single particle has existed in the literature since 1952.

Sayre's paper had no impact in the image processing or signal processing communities. For imaging in two dimensions (in the projection approximation), the next important development was work by *Gerchberg* and *Saxton* (G–S ) [20.12], which posed the following question: if the intensity of a general complex two-dimensional () image and its corresponding diffraction pattern intensity is known, can the complex image (and diffraction pattern) be reconstructed? (By a complex image we refer here to the sample exit-face wavefunction, as defined in the next section. The reconstruction of the sample structure from this may involve further approximations.) This data is available in a modern electron microscope. By solving the resulting quadratic equations (equal in number, as they comment, to the number of unknown phases) using an iterative algorithm, they showed successful inversion for one-dimensional ( ) data. It has often been noted that these Fourier equations, one for each pixel in the detector, may not be independent, are nonlinear if a sign constraint is used, and are corrupted by noise. Dependence in the equations may be introduced by the choice of support shape. Other approaches have been explored for solving these nonlinear Fourier equations, but the G–S paper established the use of iterations between real and reciprocal space, with known information imposed repeatedly in each domain. G–S do not establish uniqueness or convergence in general. The connection with Sayre's paper was not made, however, in retrospect we see that the sampling interval used by G–S for their analysis of a nonperiodic object necessarily corresponded to the Shannon half-integral orders of Sayre's paper.

Modern work is focused on 3-D data in which only the diffraction pattern intensity from a real or complex object is measured. (A complex object is one whose refractive index at each voxel may be complex, doubling the amount of information required.) In that case, the curvature of the Ewald sphere cannot be ignored, the projection approximation fails, and a simple Fourier transform of the phased 2-D diffraction pattern does not give a projection of the object density. However, for a pure phase object in transmission electron microscopy ( ), the modulus of the exit face wavefunction is known a priori to be unity everywhere, greatly reducing the number of unknowns in the G–S analysis. (This unit modulus constraint is, unfortunately, nonconvex.) For a real object (or, equivalently, a weak phase object), the diffraction pattern has Hermitian symmetry (Friedel's law) , resulting in a reduction in unknowns. Following the G–S paper (and a second paper [20.13]), it remained to determine the minimum information needed about a real object to solve the phase problem if the diffracted intensity was given. The role of the object boundary (defining a *support*) and the need to sample the continuous scattering finely enough to satisfy Shannon's theorem, (treating the autocorrelation function as the *bandlimit*) were all recognized at this time. The autocorrelation of a 2-D object is twice as large as the object in any direction, so Shannon's theorem requires sampling at half the Bragg angle for the periodically continued object. Bates referred to this as *oversampling*, a term still in use. (Since it is, in fact, *optimum* sampling of the diffracted *intensity* (for reconstruction of the autocorrelation function), we avoid that term here.) The problem was vigorously attacked by authors such as Bates, Fiddy, Fienup, Gonslaves, and Papoulis in the early 1980s, and the use of lensless imaging based on these ideas using x-ray scattering was advocated [20.14]. The early series of papers by Bates co-workers are especially significant and can be traced through *Bates* and *McDonnell* [20.15], while *Fiddy* provided a new approach based on the analysis of zeros in the diffraction pattern ([20.16] and earlier work). It was shown, for example, that the set of all bandlimited functions having a given set of real zero crossings is convex, and that, in the absence of noise, the data might be factorizable. By 1982, a useful working solution had been obtained [20.17] for real 2-D images, by the addition of a feedback feature to the G–S error-reduction (ER ) algorithm. An important realization at about this time was that the *landscape* (a map of error metric in the \(N\)-dimensional search space) for the phase problem was not rugged and usually consisted of a single shallow global minimum with a few small bumps—the essential difficulty is not caused by the number of local false minima, but rather by the large number of directions in which search is possible, in a space containing one dimension for each image pixel. This Fienup algorithm has come to be known as the hybrid input–output or HIO algorithm . Feedback greatly speeds up convergence and improves the ability of the algorithm to escape from local minima, however most modern work is based on a combination of the ER and HIO algorithms, since only the ER algorithm supplies a reliable error metric. *Fienup* showed that the ER algorithm converges, in the sense that the error monotonically decreases [20.18]. Real images could then be reconstructed from the Fraunhofer diffraction pattern intensity data, provided that the sign of the scattering potential was known together with an approximate estimate of the object boundary (support). Important work on the uniqueness problem for the HIO algorithm was published by *Bruck* and *Sodin* [20.19] and *Barakat* and *Newsam* [20.20], who showed that ambiguous solutions are obtained with these constraints only on *pathologically rare* occasions. The aim of this work is to show that only one image function satisfies the known sign, support, and Fourier modulus constraints. The HIO algorithm, which we give below, is the basis for much successful modern work in CDI and has led to the development of many variants and much detailed mathematical analysis. The book by *Stark* [20.21] then provided an excellent overview of the subject at that time and can be strongly recommended to students, together with the important review articles by *Millane* [20.8, 20.9] and *Marchesini* [20.22], and the very detailed description of the application of these methods to real experimental data given in *Chapman* et al [20.4]. *Millane* [20.8] unified the crystallographic and single-particle approaches and so brought the field to the attention of a much larger audience. (The resemblance of the HIO algorithm to the independently developed solvent flattening methods of x-ray crystallography can also be traced through *Wang* [20.23].) Here, the *zero-density* region outside the support of our single particles plays the same role as the water jacket around a protein in a crystal [20.11].

The HIO algorithm and its variants have been highly successful for real objects, however rather less attention has been paid to the more difficult problem of complex image reconstruction where the sample is defined by a complex number (such as a complex refractive index) at each pixel or voxel. Except for special cases, it is then found that a very precise knowledge of the support is needed, and that, in one and two dimensions this support should be disjoint (separated into two parts) [20.24]. Iterative inversion schemes fail for 1-D real data which is not disjoint and work better in higher dimensions.

The third more recent period of work since about 1990, during which the field grew rapidly, is discussed more quantitatively in the next sections. During that time a major effort was made to apply these methods to soft x-ray transmission data at the Brookhaven synchrotron [20.25, 20.26]. Our understanding of the success of the HIO algorithm has increased considerably in that period due to the contribution of mathematicians using powerful methods based on convex set theory. Bregman projections and constraint theory were first described as early as 1982 [20.27] but not taken up until recently (an excellent review is given in [20.28]). Questions of uniqueness in dimensions greater than two have been considered, where the problem is overdetermined [20.29], and it is generally found that the iterative algorithms work better. In addition, a number of developments of the HIO algorithm have been published [20.30, 20.31], the standard symbols used by mathematicians for the theory of projections has been adopted to describe the problem, and a new algorithm, which dispenses with any need for knowledge of the object support (the *shrinkwrap* algorithm [20.32, 20.33]), has been published. The problem of lost information within the synchrotron beam stop has been addressed by reconstructing the diffuse scattering around a Bragg reflection from a nanocrystal [20.34]. In 2001, the first of a bi-annual series of conferences on the noncrystallographic phase problem was held at Lawrence Berkeley Laboratory [20.35]; they have continued since, with published proceedings.

These theoretical developments, often based on computer simulations, have been supported by far fewer demonstrations of inversions from experimental data. Only experimental results can truly give confidence in theory, and early results were somewhat disappointing. An early application of the HIO algorithm to experimental optical speckle data can be found in [20.36], and applications to images formed with laser light can be found in *Spence* et al [20.37], with emphasis on the depth of focus problem. In the x-ray imaging community, the work at Brookhaven in *Janos Kirz*'s group finally paid off in 1999, when images of lithographed characters were reconstructed from (soft) x-ray diffraction patterns for the first time [20.38]. A low-resolution optical image was used to provide the support. First results of lensless imaging applying the HIO algorithm to electron diffraction data are reported in *Weierstall* et al [20.39]. More recently, an atomic-resolution image of a single double-walled carbon nanotube was reconstructed from its electron microdiffraction pattern [20.3]. These successes have led to rapid growth of the subject in the years since.

## 20.2 The Projection Approximation, Multiple Scattering, Objects, and Images

*object*,

*image*,

*diffraction pattern*,

*exit-face wavefunction*,

*real*and

*complex object*, and

*transmission function*have sometimes been confused in the literature and are defined here for clarity. We define the

*object*by its ground state charge density \(\rho(\boldsymbol{R})\) (which diffracts x-rays) and by the corresponding electrostatic potential \(V(\boldsymbol{R})\) (which diffracts electrons); \(V(\boldsymbol{R})\) is related to the density \(\rho(\boldsymbol{R})\) by Poisson's equation. As a result of inelastic processes, both may be complex and could be referred to generally as the

*complex optical potential*, but in this chapter unless otherwise stated, we take them to be real. Here, \(\boldsymbol{R}\) is a 3-D vector, while \(\boldsymbol{r}\) will be 2-D. We define the

*exit-face wavefunction*\(\Uppsi(\boldsymbol{r})\) across the downstream face of a thin slab of sample in the transmission diffraction geometry with

*transmission function*\(\mathrm{T}(\boldsymbol{r})\) by

*image*as any magnified, resolution limited, or aberrated copy of \(\Uppsi(\boldsymbol{r})\), formed by a lens. Unfortunately, this term is now widely used in the CDI literature as a synonym for either

*object*or

*exit-face wavefunction*. This use of

*image*to refer to an exit-face wavefunction is now firmly established and will be continued here, however the more important distinction between object and exit-face wave function is likely to become important in future work and should be preserved. (Further confusion arises when x-ray diffraction patterns are referred to as

*images*, making it impossible to distinguish between real and reciprocal space.) The terms

*real object*and

*complex object*have also been widely adopted in optics but should strictly refer to the nature of the exit-face wavefunction for 2-D imaging. (For a strong phase object described by (20.1) below, the object \(\Updelta n\) may be real but the exit-face wavefunction complex. Most authors would refer to this as a

*complex object*, however the object property we wish to recover is \(\Updelta n(\boldsymbol{r})\).) It is also useful to reserve the word

*image*for real-space functions and

*diffraction*for reciprocal space, so that the term

*diffraction image*, when referring to a diffraction pattern, should be avoided. The measured diffraction pattern intensity is \(I(\boldsymbol{u})=|\Upphi(\boldsymbol{u})|^{2}\), with \(\Upphi(\boldsymbol{u})\) the Fourier transform of \(\Uppsi(\boldsymbol{r})\) and scattering vector \(|\boldsymbol{u}|=\theta/\lambda\) for small scattering angles \(\theta\). (Here \(\lambda\) is the x-ray, or relativistically corrected de-Broglie wavelength for electrons.) In this projection approximation, \(I(\boldsymbol{u})=I(-\boldsymbol{u})\) if \(\Uppsi(\boldsymbol{r})\) is real, or, for complex objects, if \(\Uppsi(\boldsymbol{r})=\psi(-\boldsymbol{r})\); \(\Upphi(\boldsymbol{u})=\Upphi^{*}(-\boldsymbol{u})\) for real \(\Uppsi(\boldsymbol{r})\), while \(\Upphi(\boldsymbol{u})\) is a signed real quantity if \(\Uppsi(\boldsymbol{r})=\psi(-\boldsymbol{r})\), and \(\Uppsi(\boldsymbol{r})\) is real. The aim of diffractive imaging is to reconstruct the

*object*from the scattered intensity \(I(\boldsymbol{u})\), however, as a first step, the exit-face wavefunction \(\Uppsi(\boldsymbol{r})\), which is simply related to \(\Upphi(\boldsymbol{u})\) by a Fourier transform, is obtained. (This reduces to determination of the sign of \(\Upphi(\boldsymbol{u})\) if \(\Uppsi(\boldsymbol{r})=\Uppsi(-\boldsymbol{r})\) and \(\Uppsi(\boldsymbol{r})\) is real.) The further recovery of object properties \(\rho(\boldsymbol{R})\) or \(V(\boldsymbol{R})\) from \(\Uppsi(\boldsymbol{r})\) may only be possible in the absence of multiple scattering or inelastic scattering. For electron diffraction, the weak-phase approximation generates a

*real object*in the language of optics and diffractive imaging, as described below. For hard x-ray diffraction (or soft x-ray scattering from thin samples), where single-scattering conditions are common, the absence of spatially-dependent absorption (due to the photoelectric effect) provides such a

*real object*. A single, spatially uniform absorptive process, however, may allow the phase contrast formulation to be used. For an experimental analysis of complex images in terms of composition variations, see

*Shapiro*et al [20.5]. The introduction of the transmission function allows a simple extension to the case of coherent convergent-beam illumination and related methods for phasing [20.40].

We now relate \(\Uppsi(\boldsymbol{r})\) to the wanted object properties \(\rho(\boldsymbol{R})\) or \(V(\boldsymbol{R})\) for the case of visible light, electron beams, and x-rays in this projection, iconal, or *flat Ewald sphere* approximation. Both refractive and dissipative (inelastic) processes may occur. In each case, one must consider whether an iconal or projection approximation may be made, and the question of whether 3-D(tomographic) information (discussed later) may be extracted.

*mask-like*) object can only be obtained if the real part of \(\Updelta n_{\mathrm{p}}\) is independent of \(\boldsymbol{r}\), and all structural information is contained in the imaginary part. These experimental conditions (pure

*absorption*contrast) can only be obtained at relatively low spatial resolution (under incoherent conditions) for both electrons and x-rays.

Since \(\delta\) is about \(\mathrm{10^{-3}}\) at \({\mathrm{6}}\,{\mathrm{kV}}\) for light materials, a thickness of about \({\mathrm{0.3}}\,{\mathrm{\upmu{}m}}\) of sapphire is needed to obtain a phase shift of \(\uppi/2\), allowing a first-order expansion of (20.1). Then, the diffracted amplitudes are simply proportional to the Fourier transform of the projected charge density of the object.

*optical*potential for high-energy electrons [20.42, 20.43], and the mean refractive index for electrons is \(n=1+\Updelta n\). The real part of this, for high beam energies, is the positive electrostatic or Coulomb potential, also obtainable from x-ray scattering factors using Poisson's equation [20.44]. The imaginary part (typically about a tenth of the real part) accounts for depletion of the elastic wavefield by inelastic scattering events such as plasmon, inner-shell, and phonon scattering. The average value \(V_{0}\) of \(\mathfrak{Re}\{V_{\mathrm{c}}\}\) is about \({\mathrm{12}}\,{\mathrm{eV}}\) for light materials, and is positive, so this may be used as a constraint. Both real and imaginary parts of the potential \(V_{\mathrm{c}}(\boldsymbol{R})\) are positive if the mean inner potential is included, since electron beams are attracted predominantly to the positive nuclei. Electron diffraction in the transmission geometry is not, however, sensitive to the mean potential \(V_{0}\), which only produces a constant phase shift. The mean inner potential can only be meaningfully defined for a finite object with zero total charge [20.45]. Although the potential due to an (unphysical) isolated negative ion may have small negative excursions, the mean value depends on the volume assumed, and, in a real crystal, any long-range ionic potential is also screened. In summary, in the absence of its mean value, the real part of the optical potential used to describe electron diffraction (when synthesized from diffraction data, with resolution limited by a temperature factor) is positive, except for possible very small negative excursions around negative ions. The positivity of the imaginary part is guaranteed by the requirement that energy gain is forbidden in inelastic processes if very small virtual processes are ignored. Fourier coefficients of the total optical potential may have either sign, and those of the real (elastic) and imaginary (inelastic) potential become mixed in objects without inversion symmetry. Hence, a sign condition on the optical potential may be used as a convex constraint unless atomic resolution reconstructions are attempted of sufficiently high accuracy to detect the very weak bonding effects.

By estimating the maximum value of \((\mathfrak{Re}\{V_{\mathrm{c}}\}-V_{0})\), the maximum thickness can be estimated for which a first-order expansion of (20.1) may be made, the weak-phase approximation, in which \(\theta(\boldsymbol{r})\ll\uppi/2\). This is also a limited case of the single-scattering approximation . For x-rays, the maximum thickness allowable in the weak-phase object approximation can be estimated using the Center for X-ray Optics (CXRO) web page (x-ray database) supported by the Lawrence Berkeley Laboratory [20.46]. For electrons, the Fourier coefficients of electrostatic potential published for many materials by *Radi* may be useful [20.42], since these also provide an estimate of the imaginary part of the optical potential for electron diffraction due to inelastic scattering.

For visible light, \(\Updelta n_{\mathrm{p}}(\boldsymbol{r})\) has a similar interpretation as for x-rays (with \(n\) given by the square root of the complex dielectric constant), however \(n> {\mathrm{1}}\). Thus, visible light and electrons (\(n> {\mathrm{1}}\)) are bent toward the normal on entering a denser medium, whereas x-rays are bent away and undergo total external reflection, with \(n<{\mathrm{1}}\).

In summary, if inelastic processes are neglected (away from absorption edges), at high energies, transmission samples of thickness \(t\) are phase objects for electrons and x-rays, for which the refractive index is proportional to the electron density for x-rays and to the total electrostatic potential, including the nuclear contribution, for electrons. Poisson's equation relates these. The magnitudes of these quantities are such that a first-order expansion of (20.1) (weak phase object approximation) is justified (\(2\,\uppi\,\mathrm{i}\,n_{\mathrm{p}}(\boldsymbol{r})/\lambda<\uppi/2\)) if \(t<{\mathrm{20}}\,{\mathrm{nm}}\) for electrons (light elements, \(V_{0}={\mathrm{200}}\,{\mathrm{kV}}\)) but \(t<{\mathrm{0.3}}\,{\mathrm{\upmu{}m}}\) for x-rays (light inorganic material, \({\mathrm{6}}\,{\mathrm{kV}}\)). Higher-order terms in the expansion of (20.1) correspond to the multiple-scattering terms of the Born series in a *flat Ewald sphere* approximation. Elastic backscattering of x-rays and polarization effects are neglected in these approximations.

*flat Ewald sphere*or projection approximation, on which (20.1) is based, depends on the ratio of the wavelength to the smallest detail \(d\) of interest (with spatial frequency \(u=1/d\)), and on the thickness of the sample. Scattering kinematics restrict elastic scattering to regions of reciprocal space near the energy and momentum-conserving Ewald sphere of radius \(1/\lambda\). The projection approximation holds if the

*excitation error*distance \(S_{u}\approx\lambda u^{2}/2\) from this sphere onto a plane in reciprocal space normal to the beam (passing through the origin) is less than either \(1/t\) or \(1/\xi_{u}\), whichever is the smallest (\(\xi_{u}\) is a multiple-scattering extinction distance for spatial frequency \(u\). Thus samples never look thicker than \(\xi_{u}\), to diffracting radiation). Hence, we require

The failure of (20.1) may require either single or multiple-scattering treatments, depending on the strength of the interaction and the sample thickness. We note that (20.1) is an exact solution which sums the Born series, including all multiple scattering effects, in the limit of the vanishing wavelength. For x-ray tomography , use of (20.1) (the projection approximation) greatly simplifies the merging of data for 3-D reconstruction, since it allows the 2-D Fourier transform of the phased diffraction pattern to be treated as a projection of the sample density. This is an important consideration in soft x-ray zone-plate microscopy and in cryoelectron microscopy ( ), where the resolution, now at best about \({\mathrm{0.2}}\,{\mathrm{nm}}\) with the new single-electron detectors, is approaching the limits set by (20.5b) [20.48], on which the merging of data from different projections depends.

The preceding discussion concerned 2-D imaging. For tomography and XFEL 3-D single-particle imaging (described below in more detail), a different analysis is required. Again, a single-scattering approximation must be used. But the introduction of a curved Ewald sphere does not prevent 3-D reconstruction, even though the transform of one phased 2-D diffraction pattern can no longer be treated as a real-space projection. The x-ray scattering theory from a general 3-D nonperiodic compact sample can be applied [20.49], and this scattering may be collected for various object orientations. For each orientation, the scattering is assigned to points on the Ewald sphere in a 3-D reciprocal space, until all of the reciprocal space volume is filled. Experimentally, in CDI this is done by rotating the sample about an axis normal to the beam and recording a diffraction pattern at each orientation. For the XFEL, where each shot destroys the sample and a goniometer is not used, a determination of the sample orientation from its scattering must be made, as is described later. Planar slices taken through the origin of the filled reciprocal space then transform to projections. For microcrystalline samples, indexing of the Bragg beams provides the orientations of the randomly-oriented samples, allowing them to be merged. Use of the Fienup algorithm with 3-D Fourier transform iterations can then take advantage of the improved convergence in three dimensions. A sign constraint may or may not be applied to both real and imaginary parts of the scattering potential, and a support, enclosing the object, must be assumed.

## 20.3 The HIO Algorithm and Its Variants, Uniqueness, and Constraint Ratio

*bandlimit*to the diffraction pattern (the object is assumed compact). Shannon's theorem then specifies either the sampling interval on the continuous diffraction pattern

*intensity*needed to fully reconstruct the autocorrelation function of the object, or the sampling of the complex scattered amplitude needed to reconstruct the object. Consider a simple 1-D complex exit-face wavefunction \(f(x)\), which is nonzero only for \(0<x<W\), so the support has width \(W\). We first treat this function as the

*bandlimit*on the complex diffraction pattern \(F(u)\), which must, therefore, be sampled at intervals \(u_{n}=n/W\) by the detector to satisfy Shannon's theorem. We then have the \(N=W/d\) equations, for resolution \(d\) (one for each pixel in the detector)

However, now consider the same function placed within a domain of width \(2W\), so that the values of \(f(x)\) in \(W<x<2W\) are known a priori to be zero. The sampling interval on \(F(u)\) is now \(1/(2W)\), and we thus have \(2N\) equations, but the number of unknown values of \(f(x)\) remains at \(2N\), so that the system of equations now becomes solvable in principle. Loosely speaking, we compensate for the missing half of the data in the diffraction domain (the phases) by requiring that half of the object values be known (they are zero outside the support of width \(W\)). Note that \(2W\) is the width of the autocorrelation function obtained from \(f(x)\), and that the Fourier transform of the diffracted intensities gives this function. Thus, the Shannon sampling of the diffraction pattern \(1/(2W)\) needed to solve the phase problem could be taken to be that which results from treating the autocorrelation function as a bandlimit on the measured quantities, which are the scattered intensities. Since the HIO algorithm assigns a random set of phases initially, the equations are also linear within the algorithm unless a sign constraint is applied. Experimentally, the effects of noise must be considered. Symmetrical support shapes should be avoided, since they may lead to dependent equations. Large systems of nonlinear equations are normally extremely difficult or impossible to solve, so it is remarkable that the HIO algorithm does this so effectively, for reasons which are not fully understood, since few methods exist for analyzing nonconvex optimization. However, although the Fourier modulus constraint is nonconvex, it is known that the error surface in hyperspace is not a rugged landscape, but consists of a single rather smooth minimum with a few small bumps. Use of a boundary estimate (support) without symmetry also drives the algorithm toward one particular enantiomorph \(\rho(\boldsymbol{r})\) rather than a spurious mixture involving \(\rho(-\boldsymbol{r})\).

This example may be extended to other cases: for complex, two-dimensional objects one has \(2N^{2}\) unknowns but \(N^{2}\) equations (if the charge-coupled device (CCD ) has linear pixel dimension \(N\)). But by placing empty space around our compact object, which increases the diffracting volume by \(2^{1/2}\) in both dimensions, we sample the diffraction pattern more finely and recover the \(2N^{2}\) equations needed to solve the phase problem. It is the knowledge of the support (the object boundary) which ensures that known object pixel values may be inserted in the algorithm correctly outside the support. Experimentally, this creates the greatest difficulty of the diffractive imaging method—one must know a priori that the diffraction pattern comes from *an isolated object whose size is approximately known*. The existence of any material outside the assumed support which unwittingly contributes to the diffraction pattern will result in inconsistent constraints being applied by the algorithm, which will then not converge. Further enlargement of the space around the sample does not contribute independent equations, since sampling finer than the Shannon interval does not add information. The introduction of Friedel symmetry for a *real* object reduces the number of unknowns by two in both domains [20.26] and makes the method much less sensitive to the initial choice of outer computational boundary.

These considerations establish that we may have as many equations as unknown phases, but not know how to solve them. The modulus operation makes them nonlinear, they may not be independent, and there may be many equivalent solutions, differing perhaps trivially by inversion or origin shifts. The two solutions made possible by the modulus operation would be reduced to one if the sign of the density \(\rho(x)\) were known. The most successful approaches to solving these coupled nonlinear equations were based on iterative optimization, and the first of these to show widespread success was the hybrid input–output method of *Fienup* [20.17], later modified to allow incorporation of known symmetries [20.50] and further developed as discussed below.

*Millane*and

*Arnal*[20.52].

For nonperiodic objects, the phase problem can now be solved if the object has a finite support i. e., the image is nonzero only within a finite region of image space, and if the image is real and nonnegative. For a weak phase object, the exit-face wavefunction is \(\psi(\boldsymbol{r})\approx 1-\mathrm{i}\uptheta(\boldsymbol{r})\), where \(\theta(\boldsymbol{r})\) may be treated as real and positive. The diffracted amplitude is \(\psi(\boldsymbol{u})=\delta(\boldsymbol{u})-i\phi(\boldsymbol{u})\), for which the scattered part differs in phase by \(90^{\circ}\) from the direct beam given by the first term. This direct *unscattered* beam is absorbed by the beamstop—difficulties arising from the finite size of this are discussed later, and we note that the average value of \(\theta(\boldsymbol{r})\) is also lost within the beam stop. Including a larger area around the object which is known a priori to have a zero or unit transmission function is equivalent to *oversampling* in Fourier space, and the spatial coherence of the illumination must span this larger area, at least equal to that of the autocorrelation function of the object. Hence, diffractive imaging requires a spatial coherence width approximately twice that of coherent imaging with a lens [20.53], increasing the exposure time by a factor of 4 for a given source and resolution limit.

- 1.
Inverse Fourier transform \(\tilde{G}_{k}(u,v)\) to obtain the image \(\tilde{g}_{k}(x,y)\)

- 2.Define \(g_{k+1}(x,y)\) asThis constitutes the hybrid input–output (HIO ) version of the algorithm; \(\beta\) is a constant chosen between \(\mathrm{0.5}\) and \(\mathrm{1}\). In the error-reduction (ER) version of the algorithm, this step is replaced by$$\begin{aligned}\displaystyle&\displaystyle g_{k+1}(x,y)\\ \displaystyle&\displaystyle=\begin{cases}\tilde{g}_{k}(x,y)\;,&\text{if }(x,y)\in S(x,y)\;,\\ g_{k}(x,y)-\beta\tilde{g}_{k}(x,y)\;,&\text{if }(x,y)\notin S(x,y)\;.\end{cases}\end{aligned}$$(20.8)$$g_{k+1}(x,y)=\begin{cases}\tilde{g}_{k}(x,y)\;,&\text{if }(x,y)\in S(x,y)\;,\\ 0\;,&\text{if }(x,y)\notin S(x,y)\end{cases}$$(20.9)
- 3.
Fourier transform \(g_{k+1}(x,y)\) to obtain \(G_{k+1}(u,v)\)

- 4.Define a new Fourier domain function \(\tilde{G}_{k+1}(u,v)\) using the known Fourier modulus \(|\mathfrak{F}\{\psi(x,y)\}|\) with the computed phase$$\tilde{G}_{k+1}(u,v)=|\mathfrak{F}\{\psi(x,y)\}|\exp[\mathrm{i}\theta_{k+1}(u,v)]$$
- 5.
Go to Step 1 with \(k\) replaced by (\(k+1\)).

When experimental data are analyzed, the physical support in the object may not be known accurately. To avoid confusion we call the support actually present in the experiment the *physical support* and the support estimate used in the data analysis the *computational support*. The term *loose support* describes the situation where the computational support is bigger than the physical support. *Tight support* means the physical support is the same as the experimental support. For 2-D simulations with real objects, it is found that a *default* triangular-shaped support may invariably be used, if it is chosen to lack any symmetry and enclose the object. (A convex centrosymmetric shape such as a circle or sphere is the worst choice, and an irregular shape may bias the reconstruction toward one of two enantiomorphs.) With experimental data, convergence may be slower (or nonexistent) due to high noise levels, the absence of data around the origin at the beamstop, and an excessively loose support. Then the simplest initial choice of support is the boundary of the autocorrelation function (obtained by Fourier transform of the diffracted intensity). This estimate is rapidly improved upon by the shrinkwrap algorithm , which uses a specified intensity threshold to find an improved smaller boundary at every iteration of the HIO algorithm [20.32, 20.33]. This has become one of the most useful practical algorithms at present. Depending on noise levels, the support estimate has been found to be sufficiently accurate to deal with complex objects in many cases. Another approach to the more difficult case of complex objects has been described by *McBride* et al [20.54], who find these objects tractable if a difference map is used during iterations, and if additional oversampling is employed.

We conclude this section with some comments on additional constraints, the beam stop problem, support determination, and recent algorithm developments. In the general case of strong multiple scattering, no simple closed-form expression relates the object to the exit-face wavefunction, and the two are related only by symmetry constraints. For a general (*strong*) phase object (20.1), a unit modulus constraint may be applied, so that reconstructed pixels are forced to lie on a unit circle on an Argand diagram, however, this constraint is nonconvex and so has not been found very useful in practice. For such an object, to which a spatially-independent absorption term is added (so that \(\rho(\boldsymbol{R})\) or \(V_{\mathrm{c}}(\boldsymbol{R})\) have a constant known imaginary part), the reconstructed image pixels may be constrained to lie on a given spiral on an Argand diagram. A summary of the many constraints which have been tried and experimental results is given in [20.39].

Over the past decades, new convex constraints have been devised, such as symmetry (useful mainly for reducing computing time), positivity, atomicity , compact support, and a known histogram of gray levels for the density (as for proteins). A knowledge of phase is a convex constraint. Nonconvex constraints include the given Fourier modulus and the sign constraint. Low-resolution imaging by a different technique (e. g., (small-angle x-ray scattering) for x-rays, or (scanning electron microscopy) for electrons) may be used to provide a support estimate. In the following section, the use of prepared objects of known shape is demonstrated to allow the method of Fourier transform holography to be used to provide a support estimate. The reference object need not be a simple point scatterer [20.55] and may have an extended complicated known shape [20.56]. The shrinkwrap algorithm described below rapidly improves iteratively on any initial support estimate and is found to converge for both real and complex objects under a wide range of conditions. For arrays of semiconductor devices, the support will often be known, so that tomographic imaging of defects within an array element might be based on a support provided by the lithography pattern used to make the array. Much of the pattern on a semiconductor chip is periodic, producing low-angle Bragg x-ray diffraction.

A *charge-flipping* algorithm has been described [20.57] for the crystallographic phase problem which may also be adapted for nonperiodic objects, in which case it reduces to Fienup's output–output algorithm with feedback parameter \(\beta={\mathrm{2}}\) and a dynamic support defined by an adjustable threshold [20.58]. This algorithm operates as follows: first, random phases are assigned to the structure factors, which must extend to atomic resolution. These are transformed to yield a real charge density. All densities values below a certain threshold have their sign reversed. The result is transformed, and Fourier magnitudes replaced with measured values. The process is continued to convergence. This algorithm (far simpler than the direct methods normally used in crystallography) has been used to solve new crystal structures from x-ray data [20.59] and is found to perform very well. It does not require a support estimate or knowledge of atomic scattering factors, but does require atomic-resolution data. The *atomicity* constraint in crystallography assumes that the solution density consists of a set of smooth peaks, and requires that diffraction data extend to atomic resolution. The support then consists of spheres around each atom—most of the density within a crystal consists of empty space between atoms, akin to the zero-density band generated by oversampling around an object in the HIO algorithm. Since all matter in the cold universe consists of atoms (whose scattering factors are known), this constraint provides an extremely powerful ab-initio assumption if one has atomic-resolution data and is the basis of the direct methods algorithms of x-ray crystallography. Other known *building blocks* (such as gold balls or lithographed dots) have been used to reduce the number of unknown parameters in image definition [20.55]. (For proteins, for example, both the sequence and the atomic structure of the 20 amino acids of which they consist are usually known a priori, together with a typical gray-level histogram for the density maps and the torsion angles between amino acids, together with interatomic bond lengths.) An algorithm which has used the atomicity idea for nonperiodic data is SPEDEN [20.60], which has been applied to the data of Fig. 20.3.

The HIO algorithm has also been used for 2-D protein crystals in electron microscopy. This technique uses Fourier transforms of conventional weak-phase-object *images* (formed with a magnetic lens) to provide the phases of low-order structure factors, and the corresponding Bragg diffraction patterns for their magnitudes. The subnanometer resolution images must be obtained over as large a range of tilts as possible, creating serious experimental difficulty. These 2-D monolayer crystals are nonperiodic in the direction normal to the plane of the crystal, so that lines of diffraction are generated in reciprocal space normal to the sample. By oversampling along these lines it is possible to phase these reciprocal lattice rods individually. The phase relation linking them can then be obtained from a few high-resolution images recorded at small tilts. In this way, the number of electron microscope images needed for 3-D imaging of 2-D organic crystals can be greatly reduced, with most of the reconstruction based on easy-to-obtain diffraction data [20.61].

We note in passing that the nonuniqueness of the crystallographic phase problem has been well studied—the so-called *homometric* crystal structures studied by Pauling, Burger, and others have the same diffraction patterns, but different structures (they are not enantiomorphs). Fortunately these are very rare.

Several approaches have been made to the problem of data lost behind a synchrotron beamstop, which is essential to protect a sensitive area detector. Any additional blooming can mean much loss of low-frequency data. In several HIO applications, these missing values have simply been treated as free adjustable parameters, and the algorithm was found to converge. Calibrated absorption filters have been placed in front of the inner portion of the detector. Another solution is to use a sample consisting of an unknown object filling a small hole in an otherwise opaque mask [20.39, 20.62]. Stray scattering of x-rays from the edges of the holes in the mask can be a difficulty, however surface roughness which is smaller than the wavelength of the x-rays produces little scattering. The use of very small silicon nitride windows (e. g., of \({\mathrm{2}}\,{\mathrm{\upmu{}m}}\) width) greatly reduces the intensity of the direct beam and blooming effects, however, the detailed shape of the partially transparent silicon wedge around the window must then be modeled and used as a support for inversion. (When making samples of small particles deposited from solution, it is found most efficient to use a focused ion beam to remove all but a favorably located and isolated particle in the center of the window.) Finally, the *diffuse* x-ray scattering around Bragg peaks (which forms the *shape transform* of the object) from a crystallite has been inverted to an image, thus avoiding the direct-beam scattering [20.34, 20.63].

## 20.4 Experimental Results

Figure 20.1 shows the first x-ray images reconstructed by this lensless method in 1999 [20.38]. The test object consists of letters formed from gold dots, \({\mathrm{100}}\,{\mathrm{nm}}\) in diameter and \({\mathrm{80}}\,{\mathrm{nm}}\) thick, on a transparent silicon nitride membrane. The transmission x-ray diffraction pattern formed with \({\mathrm{1.7}}\,{\mathrm{nm}}\) monochromatic soft x-rays is shown in Fig. 20.1a, and the reconstructed image in Fig. 20.1b. The object was illuminated through a coherently filled \({\mathrm{10}}\,{\mathrm{\upmu{}m}}\) diameter pinhole, and a \({\mathrm{25}}\,{\mathrm{cm}}\) camera length was used. Missing data from the central region within the beamstop was obtained from a lower-resolution optical image. The exposure time was \({\mathrm{15}}\,{\mathrm{min}}\) at the Brookhaven synchrotron. The Fienup algorithm was used for reconstruction, with a sign constraint applied to both real and imaginary parts of the scattering potential. A square support was used (irregular shapes usually work better), and \(\mathrm{1000}\) iterations were needed for convergence. The resolution is about \({\mathrm{75}}\,{\mathrm{nm}}\). A detailed description of an improved version of the apparatus used to obtain this and other recent results at the *Advanced Light Source* is given in *Beetz* et al [20.64].

Figure 20.2a shows a transmission diffraction pattern obtained using \({\mathrm{600}}\,{\mathrm{eV}}\) monochromatic soft x-rays from clusters of gold balls, \({\mathrm{50}}\,{\mathrm{nm}}\) in diameter, lying on a silicon nitride membrane. The silicon nitride is almost transparent to the x-rays, so the object provides a useful test object for reconstruction. The pattern resembles the Airey's disk-like pattern from one ball, crossed by *speckle* fringes due to interference between different balls. An image reconstruction series using the shrinkwrap algorithm, in which the HIO algorithm refines the support during iterations, is shown in Fig. 20.2b. The top image is the centrosymmetric autocorrelation function, with the support estimate shown as a mask below. This mask was obtained by Fourier transform of the diffraction pattern intensity (to produce the autocorrelation function shown), followed by the selection of a contour corresponding to a certain threshold of intensity. This thresholding operation is repeated after each HIO–ER iteration cycle to generate a new improved estimate of the object support ( [20.32, 20.33] for details). Intermediate iterations lead to the final converged image of the gold ball clusters (Fig. 20.2f). We note that the inversion symmetry necessarily possessed by the autocorrelation function at Fig. 20.2b is lost at Fig. 20.2d as it changes smoothly into the correctly phased image.

Figure 20.3 shows an instructive case, indicating the way in which *prepared objects* may be used to assist reconstruction. (Full experimental details for CXDI are given in *He* et al [20.65], from which Fig. 20.3 is taken.) Figure 20.3a shows an SEM image of a set of gold balls lying on a silicon nitride membrane. One ball, at A, is isolated. The autocorrelation function obtained from an experimental soft-x-ray transmission diffraction pattern (not shown) taken from this object is given in Fig. 20.3b. This may be interpreted as the self-convolution of the object with its inverse, or, for a collection of point-like objects, as the set of all inter-point vectors. Some inter-ball vectors are shown in Fig. 20.3a and indicated again in Fig. 20.3b. The convolution of the single isolated ball A with the three balls at B produces in the autocorrelation function in Fig. 20.3b a faithful image of the three balls, blurred by the image of one ball. This process is similar to the heavy-atom method of x-ray crystallography, or the method of Fourier transform holography in optics [20.66]. If such a gold ball, or strong *point* scatterer can be placed near an unknown object, the autocorrelation function will contain a useful first estimate of the desired image of the unknown, which can also be used to provide a support for further HIO iterations aimed at improving resolution. This process is demonstrated in *He* et al [20.55], where, following the original suggestion of Stroke, it is found that the resolution in the autocorrelation *image* may be considerably improved beyond the size of the reference ball by simple deconvolution. By using a larger reference object, or one consisting of a cluster of small balls, the intensity of scattering from the reference object can be increased, but resolution is commonly limited by the size of the reference scatterer.

It has been noted that a randomly placed cluster of point scatterers can provide a high resolution image in Fourier transform holography when used as a reference object [20.55, 20.62, 20.66, 20.67]. A *uniformly redundant array* has been used as a reference object for x-ray Fourier transform holography [20.68], giving a resolution of about \({\mathrm{50}}\,{\mathrm{nm}}\). The method has also proven powerful, with an array of reference scatterers, for x-ray imaging of magnetic structures [20.69].

The first successful application of CDI to the electron diffraction patterns provided by a TEM is described in *Weierstall* et al [20.39], where a complete description of the method can be found. An important asset of the TEM is its ability to provide an image of the same region which contributes to the microdiffraction pattern, so that this image can be used to supply the support. The resolution of the best TEM instruments in direct phase-contrast imaging mode using lenses is now about \({\mathrm{1}}\,{\mathrm{\AA{}}}\). Figure 20.4 shows a remarkable application of CDI to an electron diffraction pattern using a TEM [20.3]. This image is the first atomic-resolution CDI image and possibly the first atomic-resolution image of a nanotube. The image gives us the helicity of the tube, its dimensions, and the number of walls. The double-walled nanotube spans a hole in a thin amorphous carbon film, while the electron beam diameter (about \({\mathrm{50}}\,{\mathrm{nm}}\)) is smaller than the hole, so that there is no background contribution from the carbon film. A conventional TEM image was used to provide the support function for HIO iterations along the edges of the tube, and it is suggested that the boundary of the support across the tube is provided by loss of coherence at the edge of the electron probe due to rapid phase variations arising from the aberrations of the probe-forming lens. (In general, a tight support is desirable for CDI.) The image shows higher resolution detail than conventional TEM images of nanotubes. Resolution is limited perhaps only by the temperature factor or by distortions in electron lenses used to magnify the diffraction pattern. It remains to be seen whether tomographic imaging at atomic resolution is simplest by this method or by direct TEM imaging using lenses. If CDI is used, the difficult problem of supporting a nanoparticle for diffraction over a range of orientations will need to be solved. Radiation damage may be reduced in diffraction mode under some conditions. A serious problem with electrons is scattering from any supporting membrane—it is usually found that even the thinnest (e. g., \({\mathrm{10}}\,{\mathrm{nm}}\) thick) carbon film will scatter too strongly to allow inversion by the HIO algorithm in the region around a particle, so that one does not have a compact support constraint. An approach to this problem of substrate background scattering was suggested by *Wu* et al [20.70], who attempted to reconstruct an atomic-resolution image of a defect in a thin crystal from the transmission electron microdiffraction pattern, which showed both Bragg reflections and diffuse scattering from the defect. The pattern from a nearby crystalline region of the sample was used to provide an independent estimate of the crystalline region, and was subtracted off during the iterations. The defect image was later found not to be a unique reconstruction.

Three-dimensional (tomographic) CDI of inorganic samples has been demonstrated in many papers since the first results appeared [20.63, 20.71, 20.72], using soft x-rays at a resolution of about \({\mathrm{10}}\,{\mathrm{nm}}\). More recently, interest has moved to the method of ptychography, since that method does not require an isolated compact sample of approximately known size (support). This approach is described in Chap. 17. The trade-off between the two methods is as follows. Where the support is approximately known, the CDI method (e. g., using HIO for reconstruction) with approximately plane-wave illumination is insensitive to vibration or sample movement during the exposure, since the far-field diffraction pattern alone is detected, and this is independent of transverse sample motion, which, therefore, does not affect resolution. Instrumentation for ptychography is much more complex, and the scattering detected is sensitive to sample motion, as for a conventional optical microscope (but not as severely, for interesting reasons, being a hybrid real and reciprocal-space method [20.73]). This can, therefore, affect resolution. However, ptychography has the great advantage of allowing the use of an extended sample such as a slab of material much wider than the focused beam. This greatly facilitates sample preparation since an *invisible* substrate (such as graphene or a silicon nitride membrane) is not needed. Recent examples of soft x-ray ptychography (which can often be accommodated on existing scanning transmission x-ray microscopes ( )) at about \({\mathrm{5}}\,{\mathrm{nm}}\) resolution, can be found in *Shapiro* et al [20.5] and *Saliba* et al [20.67].

For one experimental CXDI experiment [20.74], the details were as follows. The x-ray source was a synchrotron and undulator, providing coherent radiation at \({\mathrm{600}}\,{\mathrm{eV}}\). A simple zone plate was used as a monochromator, followed by a beam-defining aperture of about \({\mathrm{10}}\,{\mathrm{\upmu{}m}}\) diameter, coherently filled. A nude soft-x-ray CCD camera, employing \({\mathrm{1024}}\times{\mathrm{1024}}\) \({\mathrm{24}}\,{\mathrm{\upmu{}m}}\) pixels was used. The sample was mounted in the center of a silicon-nitride window fitted to a TEM single-tilt holder, which provides automated rotation about a single axis normal to the x-ray beam. The window was rectangular, with the long axis normal to both the beam and the holder axis. Diffraction patterns were recorded at one-degree rotation increments, with a typical recording time of about \({\mathrm{15}}\,{\mathrm{min}}\) per orientation. The maximum tilt angle was then limited by the thickness of the silicon frame around the window to perhaps \(80^{\circ}\), resulting in a missing wedge of data. In addition, data may be missing around the axial beamstop. The development of software for automated tomographic diffraction data collection and merging is a large undertaking [20.75] and much can be learnt from the prior experience of tomography in biological electron microscopy, where these techniques have been perfected [20.76]. In that case, however, the registration of successive images at different tilts is greatly facilitated by direct observation of image features. The use of shadow images or x-ray zone-plate images for similar purposes has been suggested. With no direct imaging mode, much time is wasted in x-ray work locating the beam on the sample, which, with current CCD detectors will typically be smaller than \({\mathrm{2}}\,{\mathrm{\upmu{}m}}\) in diameter. The final resolution (in one dimension), allowing for an *oversampling* factor of 2, will then be \({\mathrm{4000}}/{\mathrm{1024}}={\mathrm{3.9}}\,{\mathrm{nm}}\). The camera length (sample to detector distance) of the diffraction camera must then be selected to allow half this spatial frequency to fall at the edge of the CCD camera at \(u_{\text{max}}=\theta_{\text{max}}/\lambda={\mathrm{0.5}}/{\mathrm{3.9}}\,{\mathrm{nm^{-1}}}\), so that the maximum scattering angle is \(\theta_{\text{max}}={\mathrm{0.25}}\,{\mathrm{rad}}\) for \(\lambda={\mathrm{2}}\,{\mathrm{nm}}\). Then the finest periodicity in the object \(({\mathrm{3.9}}/{\mathrm{0.5}}\,{\mathrm{nm}})\) is sampled twice in every period, according to Shannon's requirement (two points are required to define the period and amplitude of a sine wave if aliasing is avoided). For a CCD with linear pixel number \(N\), the ratio of the finest detail to the largest dimension is \(N/2\), so that developments in detector technology limit CDI. The transverse spatial coherence of the beam must exceed \({\mathrm{4}}\,{\mathrm{\upmu{}m}}\), as discussed together with monochromator requirements below.

Tomographic or 3-D imaging can provide the ability to *see inside* an object, but this requires that the intensity at a point in a projection be proportional to a line integral of some simple property of the object, such as the charge density. Then methods such as filtered backprojection can re-assemble these 2-D projections into a volume density. Contours of equal density may then be isolated and presented to show internal structure. For CXDI, a different approach is used, and some simplifications occur. It is no longer necessary to make the resolution-limiting *flat Ewald sphere* approximation, since diffraction data collected at one tilt can be assigned to points lying on the curved Ewald sphere in reciprocal space. (This is the momentum and energy-conserving sphere which describes elastic scattering in reciprocal space.) The sample is then rotated through this sphere around a single axis, until all of reciprocal space is filled, out to a given resolution. Three-dimensional interpolation of data points near the sphere is needed, and careful intensity scaling may be needed if several exposures with different times are needed to cover the full dynamic range of the data. It is often found that missing data points in the central region can be treated as adjustable parameters in the HIO iterations. Once a roughly spherical volume has been filled in reciprocal space (perhaps with missing wedge and beamstop region), the 3-D iterations of the HIO algorithm may be applied ((20.8) and (20.9) extended to three dimensions). The computing demands are severe, as outlined below. The converged data will provide a 3-D density map, proportional to the local charge density, if the single-scattering approximation of x-ray diffraction theory applies, and if the spatial variation in attenuation of the beam due to the photoelectric effect can be neglected. Figure 20.5 shows such a tomographic reconstruction, from which 3-D surfaces of constant density may be obtained. These surfaces allow us to see inside materials and may eventually permit maps to be obtained which distinguish regions of different chemical composition.

The usefulness of tomographic CXDI in biology remains to be determined—at present, the method appears to have the advantages over electron microscopy of allowing observation of thicker samples under a wider range of environments (for example, in the *water window* around \({\mathrm{580}}\,{\mathrm{eV}}\) for soft x-rays). By comparison with x-ray zone-plate *full-field* imaging, the method allows a much larger numerical aperture to be used, and, hence, makes more efficient use of scattered photons, while providing potentially higher resolution. At high resolution the depth of focus \(\lambda/\theta^{2}\) may become less than the sample thickness, which prevents tomographic reconstruction by backprojection methods based on simple projections. Then tomography may best be undertaken using optical sectioning rather than reconstruction from projections. CDI, based on 3-D diffraction data, provides a third alternative. The resolution limit imposed by radiation damage in CXDI, as expressed by the Rose equation [20.77], remains to be determined experimentally, but is unlikely to be less than \({\mathrm{1}}\,{\mathrm{nm}}\), significantly larger than achieved in 3-D single-particle cryoelectron microscopy of proteins, with their new single-electron detectors [20.73]. Here, \({\mathrm{0.2}}\,{\mathrm{nm}}\) resolution has been achieved for the much smaller (\(<{\mathrm{60}}\,{\mathrm{nm}}\)) molecular structures imaged by cyro-EM in thin vitreous ice films, thin enough to avoid multiple elastic electron scattering ([20.48] for analysis). *Howells* et al [20.78] and *Marchesini* et al [20.32, 20.33] provide a detailed discussion of this large subject beyond the scope of this review, including a plot of the dose against resolution for various microscopies in biology, as shown in Fig. 20.6 [20.79]. The dose-fractionation theorem of Hegel and Hoppe is also relevant [20.80]. Images of whole yeast cells, for example, have been imaged by CDI [20.81] using the apparatus described by *Beetz* et al [20.64].

## 20.5 Iterated Projections

*Levi*and

*Stark*[20.82] (based on earlier work by

*Youla*and

*Webb*[20.27]) showed that the algorithm could be understood as a successive Bregman projection between convex and nonconvex sets . Here, an image is represented as a single vector \(\boldsymbol{R}\) in an \(N\)-dimensional space, with one coordinate for each pixel. The addition of two such vectors adds together two images. Distance between images (vectors) in this space has the form of the familiar \(\chi^{2}\) goodness of fit index, so that similar images are represented by points in Hilbert space near each other. The set of all images subject to a given constraint (e. g., known symmetry, known Fourier modulus, known sign of density, known support) are considered to occupy a volume in this space. The operation of taking a current estimate of the image, performing a Fourier transform , replacing the magnitudes of the diffracted amplitudes with the known values, and inverse transforming, was shown to be a projection onto the set of images subject to the Fourier modulus constraint. One considers vectors \(\boldsymbol{R}\) between the boundaries of two constrained sets of images. If it is shown that all the images within the only overlap between two constrained sets are equivalent solutions, then the phase problem reduces to finding this volume, where \(\boldsymbol{R}=\chi^{2}\) and the ER error metric are a minimum. Constraints may be of two types—convex and nonconvex. For a convex set, all points on any line segment starting and terminating within the set lie inside the set. A set \(\boldsymbol{P}\) is convex if

*projectors*, which takes a given vector \(\boldsymbol{R}\) to the nearest point of a nearby constrained set (usually on its boundary). Then:

- 1.The error-reduction (ER) (Gerchberg–Saxton) algorithm may be written as$$\rho^{(n+1)}=\boldsymbol{P}_{\mathrm{s}}\boldsymbol{P}_{\mathrm{m}}\rho^{(n)}$$
- 2.The charge-flipping ( ) algorithm may be written as$$\rho^{(n+1)}=\boldsymbol{R}_{\mathrm{s}}\boldsymbol{P}_{\mathrm{m}}\rho^{(n)}$$
- 3.The hybrid input–output (HIO) algorithm may be written as$$\begin{aligned}\displaystyle\rho^{(n+1)}(\boldsymbol{r})&\displaystyle=\boldsymbol{P}_{\mathrm{m}}\rho^{(n)}(\boldsymbol{r})&\displaystyle\text{ if }\boldsymbol{r}\in\mathbf{S}&\displaystyle\\ \displaystyle\rho^{(n+1)}(\boldsymbol{r})&\displaystyle=(\boldsymbol{I}-\beta)\boldsymbol{P}_{\mathrm{m}}\rho^{(n)}(\boldsymbol{r})&\displaystyle\text{ if }\boldsymbol{r}\notin\mathbf{S}&\displaystyle\end{aligned}$$
- 4.The averaged successive reflections ( ) algorithm may be written as$$\rho^{(n+1)}=0.5(\boldsymbol{R}_{\mathrm{s}}\boldsymbol{R}_{\mathrm{m}}+\boldsymbol{I})\rho^{(n)}\;. $$

*Marchesini*[20.22].

Using this approach, it has also been shown that the HIO algorithm is equivalent to the Douglas–Rachford algorithm and related to classical convex optimization methods [20.28]. The text by *Stark* [20.21] is recommended as a tutorial introduction to this large subject.

## 20.6 Coherence Requirements for CDI Resolution

It is readily shown [20.53] that the lateral or spatial coherence requirement for diffractive imaging is, in one dimension, that the coherence width \(X_{\mathrm{c}}\approx\lambda/\theta_{\mathrm{c}}\) be at least equal to twice the largest lateral dimension \(W\) of the object. (This is similar to the requirement in crystallography that \(X_{\mathrm{c}}\) exceed the dimensions of a primitive unit cell in order to avoid overlap of Bragg beams, with beam divergence \(\theta_{\mathrm{c}}\). For phasing by the oversampling method, this cell must be about twice as big as the molecule.) This fixes the incident beam divergence and, hence, the exposure time for a given object size and source. Since at the un-apertured diffraction limit (\(\theta=90^{\circ}\)) the resolution is approximately equal to the wavelength and about two pixels are required per resolution element, a total of about (\(4X_{\mathrm{c}}/\lambda)^{2}\) image pixels would be needed for a coherence width \(X_{\mathrm{c}}\) and oversampling factor 2. Physically, this just means that the coherence patch must include the *known* region of vacuum (zero density) surrounding the object boundary (support). One must diffract coherently from an area about twice as big as the isolated object of interest.

*Spence*et al [20.53]. This determines the quality of the monochromator needed. In practice, values of \(E/\Updelta E={\mathrm{500}}\) have yielded good results in soft x-ray work using CCD detectors with \(N^{2}\) pixels, where \(N={\mathrm{1024}}\). Then the in-line arrangement of a simple zone-plate monochromator can be used [20.85]. For CDI using electron beams, the coherence requirements are easily met for the nanostructures of most interest. We note that the drive for higher resolution, for a fixed number of object pixels, reduces the demand on coherence. The number of pixels, especially in 3-D tomography, is commonly limited by computer processing power to about \(1024^{3}\) in the medium-term future, as discussed below.

It is important to devise a consistent definition of resolution in CDI . Each spatial frequency (assuming small scattering angles) \(u=d^{-1}=\theta/\lambda\) in the object diffracts energy at scattering angle \(\theta\) into the far field. Consider a square area detector used for diffractive imaging for which the largest scattering angle into the midpoint of the side of the detector (not the corner) is \(\theta_{\text{max}}\). Then, this angle defines a cutoff in the *transfer function* (see below) at \(u_{\text{max}}=\theta_{\text{max}}/\lambda\), and the full period of the corresponding finest periodicity in the object which can be reconstructed is \(d_{\text{min}}=\lambda/\theta_{\text{max}}=1/u_{\text{max}}\). This value of \(d_{\text{min}}\) has frequently been quoted as the resolution limit in CXDI. However, while it is a most important experimental parameter, it fails to consider the accuracy of the phasing process and is not equal to the corresponding Rayleigh resolution limit. There are other considerations, which we now discuss.

The Rayleigh resolution limit \(d_{\mathrm{R}}\) was intended for spectroscopy and the imaging of binary stars, which are incoherent point sources, unlike the phase contrast usually important for CXDI . If we do, nevertheless, wish to apply the Rayleigh condition to this situation, we may consider that the CXDI area detector in the far field plays the role of a square aperture in the back-focal plane of an ideal lens. The numerical aperture imposed on the reconstruction is then \(\theta_{\text{max}}=\lambda u_{\text{max}}\), and, if the reconstruction is perfect (no errors in phasing), the image will be given by the ideal object charge-density convoluted with a sinc function amplitude (the impulse response for linear imaging), whose full width at half maximum is \(d_{\mathrm{R}}=d_{\text{min}}/2=0.5/u_{\text{max}}\). (The distance between first minima is \(d_{\text{min}}\). For a circular area detector the factor \(\mathrm{0.5}\) becomes \(\mathrm{0.61}\)—the square detector does slightly better because of contributions from the corners.) Thus our adapted *Rayleigh resolution* is half the finest spatial periodicity , and is, therefore, equal to the sampling interval needed in real space for linear phase-contrast imaging, in order to avoid loss of information. Two samples are needed for every full periodicity in the object. (For an incoherent imaging model, the impulse response becomes a sinc squared function, for which a sampling interval of \(d_{\text{min}}/4\) is needed, since the autocorrelation of the transform of the sinc squared function is a triangular function, doubling the bandwidth and resolution , as pointed out in Rayleigh's original paper.) For a lens-based system with aberrations, the factor \(\mathrm{0.5}\) depends on the aberrations of the lens, but takes its minimum value for the diffraction-limited CXDI case.

There are two further considerations. For phase contrast, the ability to distinguish adjacent small objects will depend on the phase shift each introduces, and thus the resolution becomes a property of the sample, not only of the instrument. It is then not possible to define resolution in a meaningful sample-independent manner. (In fact, the resolution limits under coherent and incoherent illumination conditions are equal if neighboring points in the object scatter with a \(90^{\circ}\) phase shift. Incoherent illumination does better if they are in phase, and worse if they are in antiphase). In coherent optics, this problem is partly addressed by introducing the concept of a lens-coherent transfer function ( ). Such a function has been introduced in a way which also tests the reliability of the phasing process in a proposal by V. Elser for a resolution definition for CXDI [20.86]. The algorithm is repeatedly run to convergence, each time starting with a different set of random phases. The results for image contrast are plotted as a function of spatial frequency , showing, if noise is not too severe, a relatively smooth curve which falls to zero at some \(u_{\text{max}}\). (If the phasing process fails, the average of these many runs will be zero at each spatial frequency.) The resolution is then \(0.5/u_{\text{max}}\), if a square detector is used. This appears to be the best current definition of resolution for CXDI, however, it ignores the dependence of resolution on sample properties for phase contrast.

For a known object, the faithfulness of the reconstruction may be indicated by a cross-correlation function between the reconstructed estimate and the known object, or crystallographic \(R\)-factor, as discussed in detail elsewhere [20.61]. For an unknown object, the ER error metric \(\varepsilon\) defined in (20.10) has been shown by computational trials against known objects to vary monotonically with a cross-correlation function [20.18]. The best resolution achieved in CXDI is currently about \({\mathrm{8}}\,{\mathrm{nm}}\).

## 20.7 Single-Particle Image Reconstruction from XFEL Data

The invention of the hard x-ray (free-electron) laser ( ), the first of which began operation at the US Department of Energy SLAC laboratory near Stanford in 2009, has created many new opportunities for imaging, especially fast imaging with femtosecond time resolution. More importantly, for structural biology, it has allowed time-resolved imaging to be undertaken while avoiding most effects of radiation damage . The *diffract-then-destroy* method used outruns damage, using, for example, x-ray pulses of \({\mathrm{10}}\,{\mathrm{fs}}\) duration to produce an x-ray diffraction before the onset of damage from the later cascade of photoelectrons, which subsequently destroy the sample [20.72, 20.87]. This has allowed *molecular movies* to be made under room-temperature conditions (without the need for cooling to avoid damage) and controlled chemical conditions in the correct thermal bath [20.88]. This is possible using microcrystal samples (which provide Bragg scattering to atomic resolution) where the atomic motions within each molecule of the crystal do not destroy crystallinity. A continuous supply of *identical* samples is, therefore, needed, whose scattering patterns must be merged. A typical pulse may contain about \(\mathrm{10^{11}}\) photons (at \({\mathrm{8}}\,{\mathrm{kV}}\)), which will scatter more than a million photons from a single large virus. X-ray beam diameters can be as small as \({\mathrm{0.1}}\,{\mathrm{\upmu{}m}}\) in diameter, with a fractional energy spread in the beam of \(\mathrm{10^{-3}}\), and a repetition rate for x-ray pulses of \({\mathrm{120}}\,{\mathrm{Hz}}\). This is the rate at which diffraction patterns are read out from the area detector. The field of biology with x-ray lasers , including a simple description of the functioning of an XFEL, is reviewed in *Spence* [20.89] and *Bosted* et al [20.90], and is supported by a consortium of US universities which provides assistance to interested researchers (https://www.bioxfel.org). Whereas scattering from individual viruses has been detected at \({\mathrm{0.5}}\,{\mathrm{nm}}\), the highest-resolution 3-D reconstruction from a virus single particle is currently about \({\mathrm{9}}\,{\mathrm{nm}}\), whereas Bragg reflections are routinely detected from protein microcrystals at \({\mathrm{0.2}}\,{\mathrm{nm}}\) resolution. This section is limited to *single-particle* ( ) data analysis methods in biology (with one particle, such as a virus, per shot). For reviews of the atomic-resolution XFEL work on static and time-resolved imaging of proteins in *crystals*, see *Schlichting* [20.91] and *Spence* et al [20.92].

For SP data analysis, with one particle, such as a virus, per shot, the methods of coherent diffractive imaging (CXDI) discussed in previous sections have been adapted for XFEL data. Unlike the CXDI problem, the orientational relationship between successive diffraction patterns must first be determined from the randomly oriented particles of unknown structure. This process requires a certain minimum number of detected photons. The accuracy of the orientation determination may then limit resolution. This determination must be done prior to solution of the phase problem. Approaches to the orientation determination problem include manifold embedding [20.93, 20.94] and the expectation maximization and compression ( ) algorithm; [20.6, 20.95] and references therein also used in cryo-EM.

The most commonly studied single particles at XFELs are icosahedral viruses , containing either DNA or RNA. The first experimental requirement then is that the individual shots extend to sufficiently high resolution to distinguish such a sphere from an icosahedron (Fig. 20.7). It may seem that for the merging of thousands of diffraction patterns from similar randomly-oriented single particles (such as a virus), the same methods as used in the cryoelectron microscopy (cryo-EM) community could be used. Here, noisy low-dose projection images of many copies of a particle, lying in many random orientations, are recorded within the field-of-view of each image, and must be merged to produce a 3-D image [20.73]. However, the XFEL diffraction patterns also require solution of the phase problem, and, unlike real-space cryo-EM images, there is no requirement for correction of electron lens aberrations, while an enantiomorphous ambiguity arises from the Friedel symmetry of low-resolution patterns, not present for real-space images. In addition, diffraction patterns have an origin, unlike images, and the background due to ice in cryo-EM images must be treated differently from the background in an x-ray diffraction pattern due to diffraction from a water jacket surrounding the particle. Building on previous work on iterative phasing of continuous diffraction patterns, two main approaches have been developed for the reconstruction of a 3-D image (density map) from many randomly oriented snapshot single-particle x-ray diffraction patterns, and for dealing with the associated problems of particle inhomogeneity. We will give here only a brief outline of the general principles of these methods, focusing on key issues.

The manifold embedding approach [20.93] is illustrated in Fig. 20.8a,b, simplified for the case of a three-pixel (\(x,y,z\)) detector and single-axis rotation of a particle, in order to illustrate the principle of the method. With this simplification, a snapshot diffraction pattern can be represented as a 3-D vector, with each component representing the scattered intensity value at a pixel. Rotation of a particle traces out a loop (a 1-D manifold) in this 3-D space of intensities. Determining this manifold allows one to assign an orientation to each snapshot. In general, the detector has \(N\) pixels, and particle rotation about three axes generates a 3-D manifold in the \(N\)-dimensional Hilbert space of pixel intensities. The manifold is seen to be parameterized by a 3-D latent space defined by the three Euler angles defining the particle orientation. If the experimental diffraction patterns are recorded in random orientations and assigned to points in this space, the identification of the loop will then allow them to be sequenced for a movie. Many practical difficulties arise, including the transformation from angular increment to coordinate change in \(N\) dimensions, and the effects of noise and conformational changes. In the simplest case, a second conformation would define a second distinct loop, however the effects of noise thicken the manifolds, so that they may overlap. The key issue of distinguishing changes in particle orientation from conformational changes (essential in order to show a smooth representation of conformation change, as a kind of *movie*) is resolved using the fact that the operations associated with conformational change commute, while those associated with the rotation group do not. Conformational changes alter the internal structure of a particle, unlike rotations. An important feature of this approach is that all the data is used for all the analysis, rather than selecting subclasses (e. g., of orientation or conformation) for successive analysis. However, even in the absence of noise, a minimum number of scattered counts is needed to identify a particular orientation, proportional to the number of distinct orientations sought. The computational demands of this approach are considerable and set the limit on the size of the largest molecule, which can be analyzed. This method has been used to reconstruct 3-D images of the PR772 icosohedral virus at \({\mathrm{9}}\,{\mathrm{nm}}\) resolution, using snapshot diffraction data collected at the LCLS XFEL, as shown in Fig. 20.9 [20.94]. By using correlation methods to sequence 3-D images by similarity, obtained from an ensemble of viruses in equilibrium, it was possible to map out the conformational energy landscape and perhaps show the emergence of the viral genome from a portal vertex. It is suggested that the large amount of data which can be collected from a single particle at an XFEL (at high repetition rate) will provide access to a wider range of conformational change than is possible by cryo-EM, since, for a statistical ensemble in equilibrium, the fractions of each conformational class are related by a Boltzmann exponential factor involving their energy difference.

This approach can be generalized to the problem of orientation and merging single-particle diffraction data, where the scattering from successive similar particles in random orientations is limited to the Ewald sphere, and three Euler angles must be assigned to fix the orientation of the scattering on the sphere into the three-dimensional diffraction volume. The angles must be quantized into discrete increments to limit the computational expense. We then work with the scattered intensities rather than the image intensities above. The method then reconstructs the best set of diffracted intensities for the 3-D diffraction volume which are most consistent with all the diffraction data. An experimental demonstration of the method using low-resolution 2-D x-ray shadow images has been demonstrated using only a few photons per image [20.98].

The method has some similarity to cryo-EM methods based on cross-correlation between experimental patterns to find similar orientations but has the advantage that cross-correlations are computed between experimental patterns and a model, rather than between every possible experimental pair, so that the computation time is linear, rather than quadratic, in the number of patterns. Like the GTM method , this approach uses the entire body of data in each update of the model parameters, however, the GTM method does not constrain the data to fit a 3-D model density. At least a thousand counts per image are needed to assign patterns to a particular orientation class.

In both these methods, solution of the noncrystallographic phase problem may be integrated with the problem of orientation determination. Particle inhomogeneity (which increases with particle size) is the most important problem for single-particle XFEL imaging and may be solved in principle by the ability of the above methods to distinguish conformations, if sufficient high-quality data is available. A method for obtaining a 3-D reconstruction from a single shot is described in [20.99], using multiple incident beams from a beamsplitter. Several authors have pointed out that the curvature of the Ewald sphere provides limited 3-D information from a single shot, while *Bergh* et al [20.100] describe other possibilities for extracting 3-D information from a single shot, such as Laue diffraction using harmonics, coherent-convergent beam diffraction, and multiple-pinhole Fourier transform holography. Figure 20.7 shows the diffraction pattern obtained from a single Chlorella virus (one particle per shot). The rings extend clearly to a resolution of \({\mathrm{12}}\,{\mathrm{nm}}\). A 3-D reconstruction of the Mimivirus obtained from XFEL scattering is described in *Ekeberg* et al [20.6] at \({\mathrm{125}}\,{\mathrm{nm}}\) resolution. Here the EMC method, based on model diffraction patterns, was used for orientation determination and a modified form \(f\) the HIO algorithm used for phasing. Figure 20.10 shows the diffraction patterns (one particle per shot) obtained from Mimivirus particles and the reconstructed 3-D image of the virus obtained using the EMC algorithm [20.6].

The most important experimental problems in this field are the hit-rate of the sample-delivery devices used. These typically consist of an electrospray or gas-dynamic virtual nozzle [20.101] feeding an aerodynamic lens stack [20.6]. Since the hydrated virus particles are not synchronized with the XFEL pulses, hit rates are typically about \({\mathrm{1}}\%\), producing much lower volumes of data than work with microcrystals. *Plating-out* of salts, which concentrate as droplets evaporate, may also lead to apparent differences in particle sizes. In general, heterogeneity of the particles creates challenging problems, however this is likely to be addressed in the same way as in Cryo-EM, which particles quenched from an equilibrium ensemble are sorted in software by conformation. It has been pointed out that the much larger volume of data obtainable by XFEL SP methods should permit larger conformational changes to be imaged, and data volumes will increase with the new XFELs planned which operate at much higher repetition rates. Data aquisition is limited by detector read-out speed, which is expected to increase from \({\mathrm{120}}\,{\mathrm{Hz}}\) to about \({\mathrm{20}}\,{\mathrm{kH}}\) in the near future.

A database for SFX and SP data has been established at http://cxidb.org/index.html, where published data can be found and used to evaluate new algorithms. A special issue of the Journal of Applied Crystallography has appeared, devoted to software and algorithms for single-particle (and serial crystallography) analysis [20.102], and experimental XFEL single-particle data sets have been published for analysis by any interested scientists (*Munke* et al [20.103] for rice dwarf virus, and *Reddy* et al [20.104] for the icosoheral coliphage virus PR772, both collected at the LCLS).

## 20.8 Summary

The past two decades have been exciting times for coherent diffractive imaging ( ) and lensless imaging . For static structures, there have been many successes [20.105], as reported in the continuing series of *coherence* conferences, and there is every reason to suppose that the pursuit by tomographic CXDI imaging of these types of materials to higher resolution will be possible and of great interest to scientists. Using medium-energy x-rays, the imaging of much thicker material should be possible than that studied by tomographic electron microscopy in materials science [20.106]. However, in view of the difficulty of preparing samples with compact support for inversion by the HIO algorithm (and its variants), much recent x-ray work has been devoted to ptychography [20.107, 20.5]. For fast time-resolved imaging, however, where the focused scanned beam of ptychography cannot be used, CDI remains the method of choice for single particles, and there has been a creative explosion of ideas on how to improve the original HIO algorithm. In addition, the advent of the x-ray laser now provides biologists the possibility of minimizing radiation damage by outrunning it with femtosecond pulses.

Figure 20.6 shows a plot of dose against resolution, with the domain of applicability for a variety of microscopies indicated, indicating the niche for CXDI. Whole-cell imaging has been pursued with both the zone-plate x-ray microscope and by cryoelectron microscopy, where, using the latter technique, a resolution of about \({\mathrm{2}}\,{\mathrm{nm}}\) is possible in samples up to \({\mathrm{100}}\,{\mathrm{nm}}\) thick or more. It seems likely that radiation damage will prevent competitive performance by CXDI, however, the method may provide useful images at perhaps \({\mathrm{5}}\,{\mathrm{nm}}\) resolution in much thicker samples using medium-energy x-rays in an environment of vitreous ice , and the resolution limit when using XFEL pulses, which can out-run radiation damage , has yet to be fully explored. The publication of the first conformational movie from XFEL single-particle data is an important development, [20.94], as shown in Fig. 20.9. The optimum choice of x-ray energy involves many issues, including the variation of synchrotron undulator brightness with beam energy and the variation in phase contrast with beam energy. While the coherent flux \(B\) available for a given synchrotron source brightness varies as \(\lambda^{2}\), the required fluence (from the x-ray cross-section) \(A\) scales as \(\lambda^{-2}\), so that the recording time \(A/B\) varies as \(\lambda^{-4}\) in a most unfavorable manner as the x-ray beam energy increases. The dose in grays needed to scatter a given number of photons into a voxel varies inversely as the fourth power of the resolution in tomography. An analysis of the variation of dose against resolution for several microscopies, including CDI, can be found in *Marchesini* et al [20.32, 20.33]. Here, the statistical demands of good imaging (based on the Rose equation) are compared with the maximum tolerable dose for a given resolution for single-particle imaging.

In summary, the demand for higher-resolution 3-D noninvasive imaging with old and new radiation sources continues unabated in both materials science and biology. New convex constraints are steadily being discovered, leading to steady incremental advances. Looking back, it seems clear now that diffractive imaging has made a decisive contribution to imaging science, with exciting possibilities for further development.

## Notes

### Acknowledgements

This chapter has summarized the work of many groups and many of the author's collaborators, as indicated in the References. The author is particularly grateful for the help of Malcolm Howells, Uwe Weierstall, Rick Millane, Anton Barty, Nadia Zatsepin, and Rick Kirian during the preparation of this review. The work was supported by NSF STC award 1231306 and NSF ABI 1565180.

## References

- S. Grandl, K. Scherer, A. Sztrókay-Gaul, L. Birnbacher, K. Willer, M. Chabior, J. Herzen, D. Mayr, S. Auweter, F. Pfeiffer, F. Bamberg, K. Hellerhoff: Improved visualization of breast cancer features in multifocal carcinoma using phase-contrast and dark-field mammography: An ex vivo study, Eur. Radiol.
**25**, 3659–3668 (2015)CrossRefGoogle Scholar - D. Paganin, K. Nugent: Noninterferometric phase imaging with partially coherent light, Phys. Rev. Lett.
**80**, 2586 (1998)CrossRefGoogle Scholar - J.M. Zuo, I.A. Vartanyants, M. Gao, M. Zhang, L.A. Nagahara: Atomic resolution imaging of a carbon nanotube from diffraction intensities, Science
**300**, 1419 (2003)CrossRefGoogle Scholar - H. Chapman, A. Barty, S. Marchesini, A. Noy, S. Hau-Riege, C. Cui, M. Howells, R. Rosen, H. He, J.C.H. Spence, U. Weierstall, T. Beetz, C. Jacobsen, D. Shapiro: High-resolution ab initio three-dimensional x-ray diffraction microscopy, J. Opt. Soc. Am. A
**23**, 1179 (2006)CrossRefGoogle Scholar - D.A. Shapiro, Y.S. Yu, T. Tyliszczak, J. Cabana, R. Celestre, W. Chao, K. Kaznatcheev, A.D. Kilcoyne, F. Maia, S. Marchesini, Y.S. Meng: Chemical composition mapping with nanometre resolution by soft x-ray microscopy, Nat. Photonics
**8**, 765 (2014)CrossRefGoogle Scholar - T. Ekeberg, M. Svenda, C. Abergel, F.R.N.C. Maia, V. Seltzer, J.M. Claverie, M. Hantke, O. Jönssin, C. Nettelblad, G. van der Schot, M. Liang, D.P. DePonte, A. Barty, M.M. Seibert, B. Iwan, I. Andersson, N.D. Loh, A.V. Martin, H. Chapman, C. Bostedt, J.D. Bozek, K.R. Ferguson, J. Krzywinski, S.W. Epp, D. Rolles, A. Rudenko, R. Hartman, N. Kommel, J. Hajdu: Three-dimensional reconstruction of the giant Mimivirus particle with an x-ray free-electron laser, Phys. Rev. Lett.
**114**(9), 098102 (2015)CrossRefGoogle Scholar - Lord Rayleigh: XLVII. On the interference bands of approximately homogeneous light; in a letter to Prof. A. Michelson, Philos. Mag.
**34**, 407 (1892)CrossRefGoogle Scholar - R.P. Millane: Phase retrieval in crystallography and optics, J. Opt. Soc. Am. A
**7**, 394 (1990)CrossRefGoogle Scholar - R.P. Millane, V.L. Lo: Iterative projection algorithms in protein crystallography. I. Theory, Acta Crystallogr. A
**69**, 517 (2013)CrossRefGoogle Scholar - D. Sayre: Some implications of a theorem due to Shannon, Acta Crystallogr.
**5**, 843 (1952)CrossRefGoogle Scholar - H. He, W. Su: Direct phasing of protein crystals with high solvent content, Acta Crystallogr. A
**71**, 92–98 (2015)CrossRefGoogle Scholar - R.W. Gerchberg, W.O. Saxton: Phase determination from image and diffraction plane pictures in the electron microscope, Optik
**34**, 275 (1971)Google Scholar - R.W. Gerchberg, W.O. Saxton: A practical algorithm for the determination of the phase from image and diffraction plane pictures, Optik
**35**, 237 (1972)Google Scholar - D. Sayre: Prospects for long-wavelength x-ray microscopy and diffraction. In:
*Image Processing and Coherence in Physics*, Springer Lecture Notes in Physics, Vol. 112, ed. by M. Schlenker (Springer, New York 1980) p. 229CrossRefGoogle Scholar - R. Bates, M. McDonnell:
*Image Restoration and Reconstruction*(Oxford Univ. Press, Oxford 1989)Google Scholar - C. Liao, M. Fiddy, C. Byrne: Imaging from the zero locations of far-field-intensity data, J. Opt. Soc. Am. A
**14**, 3155 (1997)CrossRefGoogle Scholar - J.R. Fienup: Phase retrieval algorithms: A comparison, Appl. Opt.
**21**, 2758 (1982)CrossRefGoogle Scholar - J.R. Fienup: Invariant error metrics for image reconstruction, Appl. Opt.
**36**, 8352 (1997)CrossRefGoogle Scholar - Y. Bruck, L. Sodin: On the ambiguity of the image reconstruction problem, Opt. Commun.
**30**, 304 (1979)CrossRefGoogle Scholar - R. Barakat, G. Newsam: Necessary conditions for a unique solution to two-dimensional phase recovery, J. Math. Phys.
**25**, 3190 (1984)CrossRefGoogle Scholar - H. Stark:
*Image Recovery: Theory and Applications*(Academic Press, New York 1987)Google Scholar - S. Marchesini: Invited article: A unified evaluation of iterative projection algorithms for phase retrieval, Rev. Sci. Instrum.
**78**, 011301 (2007)CrossRefGoogle Scholar - B.-C. Wang: Resolution of phase ambiguity in macromolecular crystallography, Methods Enzymol.
**115**, 90 (1985)CrossRefGoogle Scholar - R. Fienup: Analytic design of optimum holographic optical elements, J. Opt. Soc. Am. A
**4**, 118 (1987)CrossRefGoogle Scholar - D. Sayre, H. Chapman, J. Miao: On the extendibility of x-ray crystallography to noncrystals, Acta Crystallogr. A
**54**, 232 (1998)CrossRefGoogle Scholar - J. Miao, D. Sayre, H. Chapman: Phase retrieval from the magnitude of the Fourier transforms of nonperiodic objects, J. Opt. Soc. Am. A
**15**, 1662 (1998)CrossRefGoogle Scholar - D.C. Youla, H. Webb: Image restoration by the method of convex projections: Part 1 Theory, IEEE Trans. Med. Imaging
**1**(2), 81–94 (1982)CrossRefGoogle Scholar - H. Bauschke, P.I. Combettes, D.L. Luke: Phase retrieval, error reduction algorithm, and Fienup variants: A view from convex optimization, J. Opt. Soc. Am. A
**19**, 1334 (2002)CrossRefGoogle Scholar - R.P. Millane: Multidimensional phase problems, J. Opt. Soc. Am. A
**13**, 725 (1995)CrossRefGoogle Scholar - H. Bauschke, P.I. Combettes, D.R. Luke: Hybrid projection–reflection method for phase retrieval, J. Opt. Soc. Am. A
**20**, 1025 (2003)CrossRefGoogle Scholar - V. Elser: Phase retrieval by iterated projections, J. Opt. Soc. Am. A
**20**, 40 (2003)CrossRefGoogle Scholar - S. Marchesini, H.N. Chapman, S.P. Hau-Riege, R.A. London, A. Szoke, H. He, M.R. Howells, H. Padmore, R. Rosen, J.C.H. Spence, U. Weierstall: Coherent x-ray diffractive imaging: Applications and limitations, Opt. Express
**11**, 2344 (2003)CrossRefGoogle Scholar - S. Marchesini, H. He, H. Chapman, S. Hau-Riege, M. Noy, M. Howells, U. Weierstall, J.C.H. Spence: X-ray image reconstruction from a diffraction pattern alone, Phys. Rev. B
**140101**(R), 68 (2003)Google Scholar - I.K. Robinson, I.A. Vartanyants, G. Williams, M. Pfeifer, J. Pitney: Reconstruction of the shapes of gold nanocrystals using coherent x-ray diffraction, Phys. Rev. Lett.
**87**, 195505 (2001)CrossRefGoogle Scholar - J.C.H. Spence, M. Howells, L.D. Marks, J. Miao: Lensless imaging: A workshop on ‘‘new approaches to the phase problem for nonperiodic objects'', Ultramicroscopy
**90**, 1 (2001)CrossRefGoogle Scholar - J.N. Cederquist, J.R. Fienup, J.C. Marron, R.G. Paxman: Phase retrieval from experimental far-field speckle data, Opt. Lett.
**13**, 619 (1988)CrossRefGoogle Scholar - J.C.H. Spence, U. Weierstall, M. Howells: Phase recovery and lensless imaging by iterative methods in optical, x-ray and electron diffraction, Philos. Trans. R. Soc. A
**360**, 875 (2002)CrossRefGoogle Scholar - J. Miao, C. Charalambous, J. Kirz, D. Sayre: Extending the methodology of x-ray crystallography to allow imaging of micrometre-sized noncrystalline specimens, Nature
**400**, 342 (1999)CrossRefGoogle Scholar - U. Weierstall, Q. Chen, J.C.H. Spence, M. Howells, M. Isaacson, R. Panepucci: Image reconstruction from electron and x-ray diffraction patterns using iterative algorithms: Experiment and simulation, Ultramicroscopy
**90**, 171 (2001)CrossRefGoogle Scholar - J.M. Zuo, J.C.H. Spence:
*Advanced Electron Microsscopy*(Springer, New York 2017)Google Scholar - J. Kirz, C. Jacobsen, M. Howells: Soft x-ray microscopes and their biological applications, Q. Rev. Biophys.
**28**, 33 (1995)CrossRefGoogle Scholar - G. Radi: Complex lattice potentials in electron diffraction calculated for a number of crystals, Acta Crystallogr. A
**26**, 41 (1970)CrossRefGoogle Scholar - A. Howie, R.M. Stern: The optical potential in electron diffraction, Z. Naturforsch. A
**27**, 382 (1972)CrossRefGoogle Scholar - J.C.H. Spence: On the accurate measurement of structure factor amplitudes and phases by electron diffraction, Acta Crystallogr. A
**49**, 231 (1993)CrossRefGoogle Scholar - M.A. O'Keeffe, J.C.H. Spence: On the average Coulomb potential (\(\Phi_{0}\)) and constraints on the electron density in crystals, Acta Crystallogr. A
**50**, 33 (1993)CrossRefGoogle Scholar - K.H. Downing, R.M. Glaeser: Estimating the effect of finite depth of field in single-particle Cryo-EM, Ultramicroscopy
**184**, 94 (2017)CrossRefGoogle Scholar - G. Subramanian, S. Basu, H. Liu, J.M. Zuo, J.C.H. Spence: Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts, Ultramicroscopy
**148**, 87 (2014)CrossRefGoogle Scholar - D. Starodub, P. Rez, G. Hembree, M. Howells, D. Shapiro, H.N. Chapman, P. Fromme, K. Schmidt, U. Weierstall, R.B. Doak, J.C.H. Spence: Dose, exposure time and resolution in serial x-ray crystallography, J. Synchrotron Radiat.
**15**, 62 (2008)CrossRefGoogle Scholar - R.P. Millane, W.J. Stroud: Reconstructing symmetric images from their undersampled Fourier intensities, J. Opt. Soc. Am. A
**14**, 568 (1997)CrossRefGoogle Scholar - V. Elser, R.P. Millane: Reconstruction of an object from its symmetry-averaged diffraction pattern, Acta Crystallogr. A
**64**(2), 273 (2008)CrossRefGoogle Scholar - R.P. Millane, R.D. Arnal: Uniqueness of the macromolecular crystallographic phase problem, Acta Crystallogr. A
**71**, 592 (2015)CrossRefGoogle Scholar - J.C.H. Spence, U. Weierstall, M. Howells: Coherence and sampling requirements for diffractive imaging, Ultramicroscopy
**101**, 149 (2004)CrossRefGoogle Scholar - W. McBride, N. O'Leary, L. Allen: Retrieval of a complex-valued object from its diffraction pattern, Phys. Rev. Lett.
**93**, 233902 (2004)CrossRefGoogle Scholar - H. He, M. Howells, S. Marchesini, H. Chapman, U. Weierstall, H. Padmore, J. Spence: Use of extended and prepared reference objects in experimental Fourier transform x-ray holography, Appl. Phys. Lett.
**85**, 2454 (2004)CrossRefGoogle Scholar - A. Szöke: Holographic microscopy with a complicated reference, J. Imaging Sci. Technol.
**41**, 332 (1997)Google Scholar - G. Oszlanyi, A. Suto: Ab initio structure solution by charge flipping, Acta Crystallogr. A
**60**, 134 (2004)CrossRefGoogle Scholar - J. Wu, U. Weierstall, J.C.H. Spence, C. Koch: Iterative phase retrieval without support, Opt. Lett.
**29**, 2737 (2004)CrossRefGoogle Scholar - J. Wu, J.C.H. Spence, M. O'Keeffe, T. Groy: Application of a modifed Oszlányi and Süto ab initio charge flipping algorithm to experimental data, Acta Crystallogr. A
**60**, 326 (2004)CrossRefGoogle Scholar - S. Hau-Riege, A. Szoke, H. Chapman, S. Marchesini, A. Noy, H. He, M. Howells, U. Weierstall, J. Spence: SPEDEN: Reconstructing single particles from their diffraction patterns, Acta Crystallogr. A
**60**, 294 (2004)CrossRefGoogle Scholar - J.C.H. Spence, U. Weierstall, T.T. Fricke, R.M. Glaeser, K.H. Downing: Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support, J. Struct. Biol.
**144**, 209 (2003)CrossRefGoogle Scholar - S. Eisebitt, M. Lorgen, W. Eberhardt: Scalable approach for lensless imaging at x-ray wavelengths, App. Phys. Lett.
**84**, 3373 (2004)CrossRefGoogle Scholar - G.J. Williams, M.A. Pfeifer, I.A. Vartanyants, I. Robinson: Three-dimensional imaging of microstructure in Au nanocrystals, Phys. Rev. Lett.
**90**, 175501 (2003)CrossRefGoogle Scholar - T. Beetz, M. Howells, C. Jacobsen, C. Koa, J. Kirz, E. Lima, T. Mentes, H. Miao, C. Sanchez-Hanke, D. Sayre, D. Shapiro: Apparatus for x-ray diffraction microscopy and tomography of cryo specimens, Nucl. Instrum. Methods Phys. Res. A
**545**, 459 (2005)CrossRefGoogle Scholar - H. He, S. Marchesini, M. Howells, U. Weierstall, G. Hembree, J.C.H. Spence: Experimental lensless soft-x-ray imaging using iterative algorithms: Phasing diffuse scattering, Acta Crystallogr. A
**59**, 143 (2003)CrossRefGoogle Scholar - R. Collier, C.B. Burkhadt, L.H. Lin:
*Optical Holography*(Academic Press, New York 1971)Google Scholar - M. Saliba, J. Bosgra, A.D. Parsons, U.H. Wagner, C. Rau, P. Thibault: Novel methods for hard x-ray holographic lensless imaging, Microsc. Microanal.
**22**(S3), 110–111 (2016)CrossRefGoogle Scholar - S. Marchesini, S. Boutet, A. Sakdinawat, M. Bogan, S. Bajt, A. Barty, J.C.H. Spence, H.N. Chapman: Massively parallel x-ray holography, Nat. Photonics
**2**, 560 (2008)CrossRefGoogle Scholar - T. Wang, D. Zhu, B. Wu, C. Graves, S. Schaffert, T. Rander, L. Müller, B. Vodungbo, C. Baumier, D.P. Bernstein, B. Bräuer, V. Cros, S. de Jong, R. Delaunay, A. Fognini, R. Kukreja, S. Lee, V. López-Flores, J. Mohanty, B. Pfau, H. Popescu, M. Sacchi, A.B. Sardinha, F. Sirotti, P. Zeitoun, M. Messerschmidt, J.J. Turner, W.F. Schlotter, O. Hellwig, R. Mattana, N. Jaouen, F. Fortuna, Y. Acremann, C. Gutt, H.A. Dürr, E. Beaurepaire, C. Boeglin, S. Eisebitt, G. Grübel, J. Lüning, J. Stöhr, A.O. Scherz: Femtosecond single-shot imaging of nanoscale ferromagnetic order in Co/Pd multilayers using resonant x-ray holography, Phys. Rev. Lett.
**108**, 267403 (2008)CrossRefGoogle Scholar - J. Wu, U. Weierstall, J.C.H. Spence: Diffractive electron imaging of nanoparticles on a substrate, Nat. Mater.
**4**, 912 (2005)CrossRefGoogle Scholar - J. Miao, T. Ishikawa, E. Johnson, B. Lai, K. Hodgson: High resolution 3-D x-ray diffraction microscopy, Phys. Rev. Lett.
**89**, 088303 (2002)CrossRefGoogle Scholar - H. Chapman, A. Barty, M. Bogan, S. Boutet, M. Frank, S. Hau-Riege, S. Marchesini, B. Woods, S. Bajt, W. Benner, R. London: Femtosecond diffractive imaging with a soft-X-ray free-electron laser, Nat. Phys.
**2**, 839 (2006)CrossRefGoogle Scholar - J.C.H. Spence:
*High Resolution Electron Microscopy*, 4th edn. (Oxford Univ. Press, New York 2017)Google Scholar - H. He, U. Weierstall, J. Spence, M. Howells, H. Padmore, S. Marchesini, H. Chapman: Use of extended and prepared reference objects in experimental Fourier transform x-ray holography, App. Phys. Lett.
**85**, 2454 (2004)CrossRefGoogle Scholar - J. Frank, M. Radermacher, P. Penczek, J. Zhu, Y. Li, M. Ladjadj, A. Leith: SPIDER and WEB: Processing and visualization of images in 3-D electron microscopy and related fields, J. Struct. Biol.
**116**, 190 (1996)CrossRefGoogle Scholar - J. Frank:
*Electron Tomography*(Plenum, New York 1992)CrossRefGoogle Scholar - A. Rose: Television pickup tubes and the problem of noise, Adv. Electron.
**1**, 131 (1948)Google Scholar - M. Howells, T. Beetz, H. Chapman, C. Cui, J. Holton, C. Jacobsen, J. Kirz, E. Lima, S. Marchesini, H. Miao, D. Sayre, D. Shapiro, J.C.H. Spence: An assessment of the resolution limitation due to radiation-damage in x-ray diffraction microscopy, J. Electron Spectrosc. Relat. Phenom.
**170**, 4 (2009)CrossRefGoogle Scholar - R. Henderson: The potential and limitations of neutrons, electrons and x-rays for atomic resolution microscopy of unstained biological molecules, Q. Rev. Biophys.
**28**, 171 (1995)CrossRefGoogle Scholar - L. Reimer:
*Transmission Electron Microscopy*(Springer, New York 1989)CrossRefGoogle Scholar - D. Shapiro, P. Thibault, T. Beetz, V. Elser, M. Howells, C. Jacobsen, J. Kirz, E. Lima, H. Miao, A.M. Neiman, D. Sayre: Biological imaging by soft x-ray diffraction microscopy, Proc. Natl. Acad. Sci. U.S.A.
**105**, 15343 (2005)CrossRefGoogle Scholar - A. Levi, H. Stark: Image restoration by the method of generalized projections with application to restoration from magnitude, J. Opt. Soc. Am. A
**1**, 932 (1984)CrossRefGoogle Scholar - K.Y.J. Zhang, P. Main: Histogram matching as a new density modification technique for phase refinement and extension of protein molecules, Acta Crystallogr. A
**46**, 41 (1990)CrossRefGoogle Scholar - R. Luke: Relaxed averaged alternating reflections for diffraction imaging, Inverse Probl.
**21**, 37–50 (2005)CrossRefGoogle Scholar - M. Howells, P. Charalambous, H. He, S. Marchesini, J.C.H. Spence: An off-axis zone-plate monochromator for high-power undulator radiation. In:
*Design and Microfabrication of Novel X-ray Optics*, Proc. SPIE, Vol. 4783, ed. by D. Mancini (SPIE, Bellingham 2002), https://doi.org/10.1117/12.453809CrossRefGoogle Scholar - S. Marchesini, H.N. Chapman, A. Barty, C. Cui, M.R. Howells, J.C.H. Spence, U. Weierstall, A.M. Minor: Phase aberrations in diffraction microscopy, arXiv:physics/0510033 [physics.optics] (2005)Google Scholar
- R. Neutze, R. Wouts, D. van der Spoel, E. Weckert, J. Hajdu: Potential for biomolecular imaging with femtosecond x-ray pulses, Nature
**406**, 752 (2000)CrossRefGoogle Scholar - K. Pande, C.D.M. Hutchison, G. Groenhof, A. Aquila, J.S. Robinson, J. Tenboer, S. Basu, S. Boutet, D.P. DePonte, M. Liang, T.A. White, N.A. Zatsepin, O. Yefanov, D. Morozov, D. Oberthuer, C. Gati, G. Subramanian, D. James, Y. Zhao, J. Koralek, J. Brayshaw, C. Kupitz, C. Conrad, S. Roy-Chowdhury, J.D. Coe, M. Metz, P.L. Xavier, T.D. Grant, J.E. Koglin, G. Ketawala, R. Fromme, V. Šrajer, R. Henning, J.C.H. Spence, A. Ourmazd, P. Schwander, U. Weierstall, M. Frank, P. Fromme, A. Barty, H.N. Chapman, K. Moffat, J.J. van Thor, M. Schmidt: Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein, Science
**352**, 725 (2016)CrossRefGoogle Scholar - J.C.H. Spence: X-ray lasers in biology: Structure and dynamics. In:
*Advances in Imaging and Electron Physics*, Vol. 200, ed. by P. Hawkes (Elsevier, New York 2017) pp. 103–152Google Scholar - C. Bostedt, S. Boutet, D.M. Fritz, Z.R. Huang, H.J. Lee, H.T. Lemke, A. Robert, W.F. Schlotter, J.J. Turner, G.J. Williams: Linac coherent light source: The first five years, Rev. Mod. Phys.
**88**(1), 015007 (2016)CrossRefGoogle Scholar - I. Schlichting: Serial femtosecond crystallography: The first five years, IUCrJ
**2**, 246 (2015)CrossRefGoogle Scholar - J.C.H. Spence: XFELs for structure and dynamics in biology, IUCrJ
**4**, 322 (2017)CrossRefGoogle Scholar - C.H. Yoon, P. Schwander, C. Abergel, I. Andersson, J. Andreasson, A. Aquila, S. Bajt, M. Barthelmess, A. Barty, M.J. Bogan, C. Bostedt, J. Bozek, H.N. Chapman, J.-M. Claverie, N. Coppola, D.P. DePonte, T. Ekeberg, S.W. Epp, B. Erk, H. Fleckenstein, L. Foucar, H. Graafsma, L. Gumprecht, J. Hajdu, C.Y. Hampton, A. Hartmann, E. Hartmann, R. Hartmann, G. Hauser, H. Hirsemann, P. Holl, S. Kassemeyer, N. Kimmel, M. Kiskinova, M. Liang, N.D. Loh, L. Lomb, F.R.N.C. Maia, A.V. Martin, K. Nass, E. Pedersoli, C. Reich, D. Rolles, B. Rudek, A. Rudenko, I. Schlichting, J. Schulz, M. Seibert, V. Seltzer, R.L. Shoeman, R.G. Sierra, H. Soltau, D. Starodub, J. Steinbrener, G. Stier, L. Strüder, M. Svenda, J. Ullrich, G. Weidenspointner, T.A. White, C. Wunderer, A. Ourmazd: Unsupervised classification of single-particle x-ray diffraction snapshots by spectral clustering, Opt. Express
**19**, 16542–16549 (2011)CrossRefGoogle Scholar - A. Hosseinizadeh, G. Mashayekhi, J. Copperman, P. Schwander, A. Dashti, R. Sepehr, R. Fung, M. Schmidt, C.H. Yoon, B.G. Hogue, G. Williams, A. Aquila, A. Ourmazd: Conformational landscape of a virus by single-particle x-ray scattering, Nat. Methods
**14**, 877 (2017)CrossRefGoogle Scholar - N.T.D. Loh, V. Elser: Reconstruction algorithm for single-particle diffraction imaging experiments, Phys. Rev. E
**80**(2), 026705 (2009)CrossRefGoogle Scholar - F.J. Sigworth: Maximum-likelihood approach to the particle alignment problem, Biophys. J.
**74**, A226 (1998)Google Scholar - K. Ayyer, T.-Y. Lan, V. Elser, N.D. Loh: Dragonfly: An implementation of the expand–maximize–compress algorithm for single-particle imaging, J. Appl. Crystallogr.
**49**(4), 1320 (2016)CrossRefGoogle Scholar - K. Ayyer, H.T. Philipp, M.W. Tate, V. Elser, S.M. Gruner: Real-space x-ray tomographic reconstruction of randomly oriented objects with sparse data frames, Opt. Express
**22**, 2403 (2014)CrossRefGoogle Scholar - K.E. Schmidt, J.C.H. Spence, U. Weierstall, R. Kirian, X. Wang, D. Starodub, H.N. Chapman, M.R. Howells, R.B. Doak: Tomographic femtosecond x-ray diffractive imaging, Phys. Rev. Lett.
**101**, 115507 (2008)CrossRefGoogle Scholar - M. Bergh, G. Huldt, N. Tîmneanu, F.R.N.C. Maia, J. Hajdu: Feasibility of imaging living cells at subnanometer resolutions by ultrafast x-ray diffraction, Q. Rev. Biophys.
**41**, 181 (2008)CrossRefGoogle Scholar - U. Weierstall, J.C.H. Spence, R.B. Doak: Injector for scattering measurements on fully solvated biospecies, Rev. Sci. Instrum.
**83**, 035108 (2012)CrossRefGoogle Scholar - F.R.N.C. Maia, T.A. White, N.D. Loh, J. Hajdu: CCP-FEL: A collection of computer programs for free-electron laser research, J. Appl. Crystallogr.
**49**(4), 1117–1120 (2016)CrossRefGoogle Scholar - A. Munke, J. Andreasson, A. Aquila, S. Awel, K. Ayyer, A. Barty, R.J. Bean, P. Berntsen, J. Bielecki, S. Boutet, M. Bucher, H. Chapman, B.J. Daurer, H. DeMirci, V. Elser, P. Fromme, J. Hajdu, M. Hantke, A. Higashiura, B.G. Hogue, A. Hosseinizadeh, Y. Kim, R.A. Kirian, H.K.N. Reddy, T.-Y. Lan, D.S.D. Larsson, H. Liu, N.D. Loh, F.R.N.C. Maia, A.P. Mancuso, K. Mühlig, A. Nakagawa, D. Nam, G. Nelson, C. Nettelblad, K. Okamoto, A. Ourmazd, M. Rose, G. van der Schot, P. Schwander, M.M. Seibert, J.A. Sellberg, R.G. Sierra, C. Song, M. Svenda, N. Timneanu, I.A. Vartanyants, D. Westphal, M.O. Wiedorn, G.J. Williams, P.L. Xavier, C.H. Yoon, J. Zook: Coherent diffraction of single Rice Dwarf virus particles using hard x-rays at the Linac Coherent Light Source, Sci. Data
**3**, 160064 (2016)CrossRefGoogle Scholar - H.K.N. Reddy, C.H. Yoon, A. Aquila, S. Awel, K. Ayyer, A. Barty, P. Berntsen, J. Bielecki, S. Bobkov, M. Bucher, G.A. Carini, S. Carron, H. Chapman, B. Daurer, H. DeMirci, T. Ekeberg, P. Fromme, J. Hajdu, M.F. Hanke, P. Hart, B.G. Hogue, A. Hosseinizadeh, Y. Kim, R.A. Kirian, R.P. Kurta, D.S.D. Larsson, N.D. Loh, F.R.N.C. Maia, A.P. Mancuso, K. Mühlig, A. Munke, D. Nam, C. Nettelblad, A. Ourmazd, M. Rose, P. Schwander, M. Seibert, J.A. Sellberg, C. Song, J.C.H. Spence, M. Svenda, G. van der Schot, I.A. Vartanyants, G.J. Williams, P.L. Xavier: Coherent soft x-ray diffraction imaging of coliphage PR772 at the Linac coherent light source, Sci. Data
**4**, 170079 (2017)CrossRefGoogle Scholar - I.A. Vartanyants, I.K. Robinson, I. McNulty, C. David, P. Wochner, T. Tschentscher: Coherent x-ray scattering and lensless imaging at the European XFEL facility, J. Synchrotron Radiat.
**14**(6), 453–470 (2007)CrossRefGoogle Scholar - M. Weyland, P. Midgley: Electron tomography. In:
*Transmission Electron Microscopy*(Springer, Cham 2016) pp. 343–376CrossRefGoogle Scholar - B. Enders, P. Thibault: A computational framework for ptychographic reconstructions, Proc. R. Soc. A
**472**(2196), 20160640 (2016)CrossRefGoogle Scholar