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Review
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Imaging of Biological Materials and Cells by

X‑ray Scattering and Diffraction
Clément Y. J. Hémonnot†,‡,§ and Sarah Köster*,†
†
Institute for X-Ray Physics, University of Goettingen, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany
‡
Northwestern Argonne Institute of Science and Engineering, Northwestern University, 9700 South Cass Avenue, Argonne, Illinois
60439, United States
§
Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, United States
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ABSTRACT: Cells and biological materials are large

objects in comparison to the size of internal components
such as organelles and proteins. An understanding of the
Downloaded via 46.243.116.135 on July 23, 2020 at 11:58:43 (UTC).

functions of these nanoscale elements is key to elucidating

cellular function. In this review, we describe the advances in
X-ray scattering and diffraction techniques for imaging
biological systems at the nanoscale. We present a number of
principal technological advances in X-ray optics and
development of sample environments. We identify radiation
damage as one of the most severe challenges in the field,
thus rendering the dose an important parameter when putting different X-ray methods in perspective. Furthermore, we
describe different successful approaches, including scanning and full-field techniques, along with prominent examples.
Finally, we present a few recent studies that combined several techniques in one experiment in order to collect highly
complementary data for a multidimensional sample characterization.
KEYWORDS: X-ray imaging, X-ray optics, biological materials, biological cells, diffraction, scattering, holography, ptychography,
tomography, coherent diffractive imaging

I maging of biological materials and cells began in the late

16th and early 17th century with the advent of the first
light microscopes. For centuries, optical resolution
remained diffraction-limited as defined by Abbe and Rayleigh.
Only recently, nanoscale imaging of biological cells with visible
signal, or be too dim to be visible. Hard X-rays, by contrast,
provide a high penetration depth (for example, photons of 10
keV have an attenuation length of 1 mm in water), a small
wavelength in the angstrom range, and thereby high resolution,
and electron density is directly probed, thus circumventing the
light became possible thanks to recent developments in super-
need for labeling or staining.
resolution techniques.1−3 Electron microscopy (EM) was
Imaging of matter with X-rays began in the 1950s8 and
developed starting in the 1930s and has been improved to
nowadays reach a resolution down to a few angstroms4 and is became more prominent about 20 years later with the advent of
widely and successfully used to image biological samples.5,6 high-quality zone plates for focusing.9,10 Thus, full-field
Both fluorescence microscopy and EM have proven to be transmission X-ray microscopes (TXM) employing soft X-
extremely valuable for studying cellular processes. Fluorescent rays and exploiting absorption contrast (see Figure 1a for a
labels allow for molecule-specific imaging in living cells, thus for typical TXM setup) were developed. At about the same time,
dynamic recordings, whereas EM reaches the best spatial scanning transmission X-ray microscopes (STXM) were
resolution available today, that is, a few angstroms in cryo-EM, introduced11 with focused beams and the additional option
and a few nanometers for whole hydrated cells.6,7 However, for energy-dispersive detectors to capture X-ray fluorescence
drawbacks remain: Electrons have a small penetration power of (XRF) (see Figure 1b for a typical setup). XRF can be readily
only about 100 nm and can only visualize the surface of larger combined with other techniques, as the signal is emitted in all
specimens like whole cells. If the interior of a cell shall be
directions, hence the detector can be placed at any angle with
studied, the sample has to be sectioned. Furthermore, the fairly
involved sample preparation procedures prohibit the study of regard to the sample. The reader is referred to comprehensive
dynamic or living samples. The advantage in fluorescence reviews such as ref 8 for more details on soft X-ray microscopy.
microscopy is the ability to visualize molecules in cells and
tissues by using optical labels that can target specific molecules. Received: May 17, 2017
However, the use of labeling can be a disadvantage as the labels Accepted: August 8, 2017
may interfere with the function of the molecule, lose their Published: August 8, 2017

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Figure 1. Setups for X-ray imaging. (a) Sketch of a full-field transmission X-ray microscope (TXM) with the sample moved out of the focal
plane. (b) Sketch of a scanning transmission X-ray microscope (STXM) with an X-ray fluorescence (XRF) detector. (c) Sketch of a scanning
nanodiffraction/scanning SAXS setup. (d) Sketch of a coherent diffractive imaging (CDI) setup as used in ptychography. (e) Sketch of an in-
line holography setup, using waveguides emitting spherical waves. Sketches are not to scale.

Table 1. Characteristics of X-ray Focusing Opticsa

category optics beam diameter (energy) efficiency ref
refracting spherical compound refractive lenses 1 μm (8 keV) several 10% 20−22
parabolic compound refractive lenses 50 nm (21 keV) >60% 23−25
Kinoform lenses 100 nm (8 keV) >50% 26, 27
reflecting Kirkpatrick−Baez mirrors 7 nm (20 keV) >50% 13, 28, 29
capillaries 50 nm (8 keV) 80% 30, 31
waveguides 10 nm (15 keV) <25% 32, 33
diffracting Fresnel zone plates 35 nm (8 keV) 10−70% 34−36
multilayer zone plates 5 nm (13.8 keV) 10−20% 14, 15, 37
multilayer Laue lenses 20 nm (14.6 keV) 10−30% 38−41
absorbing pinholes/slits 1 μm (11 keV) 42, 43
a
In addition to the minimum beam diameter currently found in the literature, we provide the energy used to achieve this value.

This review focuses on X-ray imaging techniques that rely different innovative techniques, grouped into (i) scanning
not on absorption but on scattering or diffraction and the scattering and diffraction (Figure 1c) and (ii) coherent
application of these techniques to biological materials and cells. diffractive imaging (Figure 1d), including X-ray holography
X-ray scattering is the phenomenon observed when matter is (Figure 1e). For each category, we provide examples of using
exposed to radiation and waves are elastically scattered by the X-ray imaging for both biological materials and cells. We end
electrons within the matter, leading to constructive interfer- this article by presenting the combination of several X-ray
ence.12 Diffraction is a special case of scattering, occurring techniques to enable imaging at the nanometer to micrometer
when highly ordered or even crystalline material is involved. length scales. This is important in order to probe the
The X-ray electromagnetic spectrum ranges from about 100 eV relationship between the cell’s internal components and
to about 100 keV and is typically divided into “soft” lower function.
energy and “hard” higher energy X-rays. In this review, we
included research performed with X-ray energies above 5 keV, TECHNICAL CONSIDERATIONS
which we refer to as hard X-rays. X-ray Optics. Many X-ray imaging techniques, as discussed
We begin by setting the stage of what is required in terms of below, require a focused or a coherent beam, or both. In recent
X-ray optics and sample environments and address the years, researchers have invested tremendous efforts into
challenge of radiation damage. Subsequently, we describe developing suitable focusing optics. Consequently, beams on
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Figure 2. Examples of X-ray focusing optics. (a) Compound refractive lenses; (b) Kirkpatrick−Baez mirrors; (c) toroidal mirror curvatures Rh
and Rv; (d) waveguide; (e) Fresnel zone plate; (f) multilayer Laue lenses; (g) pinhole.

the order of 100 nm are now routinely available at several aluminum. Holes are drilled or punched in the blocks with
beamlines of various synchrotron sources, and even sub-10 nm parabolic-shaped tools24 or by etching silicon.25 The radius of
X-ray beam sizes have been reached.13−15 In the following, we curvature of a few micrometers of each lens is gradually
provide a summary of the most widespread methods, with the increased in order to ensure increasing focal lengths and the
most important characteristics listed in Table 1. For a more same focal point for the whole set of lenses.
detailed overview of X-ray focusing optics, the reader is referred Reflecting Optics. Reflecting optics take advantage of the
to refs 12 and 16−19. fact that when illuminating surfaces by X-rays at a small angle to
Refracting Optics. Refractive lenses for X-rays rely on the the surface, total external reflection occurs. Using two curved
same principle as in conventional visible light optics and make Kirkpatrick−Baez (KB) mirrors in orthogonal geometry28
use of Snell’s law. When visible light interacts with condensed (Figure 2b) or waveguides (Figure 2d), X-rays can be focused
matter, the refractive index is typically around 1.5 or larger. By down to 7 nm.29 Such mirrors are coated with a high Z material
stark contrast, when X-rays interact with matter, the real part of
(e.g., silicon, rhodium, or platinum). The shape of the mirrors is
the refractive index n is lower than but very close to 1 for all
ideally elliptical or parabolic, but manufacturing ideal elliptical
materials, diminishing the refractive power considerably.
Mathematically, both the refractive index and the absorption surfaces remains difficult and is more expensive than cylindrical
can be described by the complex refractive index or toroidal mirrors48 (Figure 2c) that are used as an
approximation.18,28 However, spherical lenses and mirrors are
n = 1 − δ + iβ (1) subject to the aberration artifacts and do not reach the same
where the decrement δ corresponds to the deviation of the real small focus sizes as parabolic lenses. X-ray waveguides,49 based
part of n from unity, and β is the imaginary part of the refractive on multiple reflections and interferences of the X-rays, achieve
index. Thus, δ describes the phase shift or dispersion and β the very small spot sizes (down to 10 nm),33,50 but the efficiency is
absorption, defined as44 not as high as the KB mirrors (see Table 1). Today, it is even
possible to use waveguides to bend X-rays.51 This feature may
λ r λ2 r λ2 Z be used for simultaneous multiangle exposures of a sample. In a
β= μ and δ = 0 ρe = 0 ρ
4π 2π 2π Mu m (2) similar manner, glass capillaries can be used to focus X-rays.
−15 They provide a long focal distance of a few millimeters from the
with the classical electron radius r0 = 2.818 × 10 m, the
number of electrons Z and the atomic mass M. The M/Z ratio exit of the capillary, together with a small beam divergence and
is almost always approximately equal to 2 (except for a high efficiency providing beams with a high intensity. These
hydrogen); u = 1.661 × 10−27 kg is the atomic mass number. features are advantegeous for probing the sample surface and
The electron density ρe is given in e−/m3 and the mass density for measurements of depth profiles. At the same time, they also
ρm in kg/m3. By replacing all numerical constants in eq 2, work well for wide-band polychromatic X-rays, which is
m
δ ≃ 1.3 × 1011 kg λ 2ρm can be approximated. The attenuation essential for spectroscopic applications. Thus, they are often
used for X-ray fluorescence setups.52
coefficient μ is provided in tables or calculated with software Diffracting Optics. When electromagnetic waves encounter
such as XCOM from NIST (National Institute of Standards a grating, they are diffracted, leading to constructive and
and Technology).45,46 At 10 keV photon energy (correspond- destructive interference. The interference phenomenon can be
ing to a wavelength of λ = 1.24 × 10−10 m), a model biological exploited to achieve small focal spots of about 10 nm.15 Fresnel
specimen47 of empirical formula H50C30N9O10S and thus mass zone plates34,53 (FZP, Figure 2e) or multilayer Laue lenses38
density ρm = 1.35 g/cm3 exhibits values of δ = 3 × 10−6 and β = (MLL, Figure 2f) are composed of transparent and opaque
6.8 × 10−9.
regions and are constructed such that constructive interference
As 1 − δ < 1, lenses for X-rays are “inverse”, that is, the
accumulates in one position, defining the focal point. In the
material of the lens surrounds a cavity of air or vacuum.
Furthermore, typically many (about 30−150) lenses are stacked case of FZPs, the different zones are concentric rings with radii
in series in order to achieve a small focus. These compound rn according to the condition:8
refractive lenses20 (CRL; see Figure 2a) achieve beam
dimensions down to 50 nm.25 The lenses are produced from n 2λ 2
rn2 = nλf +
blocks of low X-ray absorbing materials, such as beryllium or 4 (3)

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Table 2. Scattering Lengths of X-rays and Neutrons for Atoms Found in Biological Mattera
atom H D C N O Na Mg P S Cl K Ca
u 1 2 12 14 16 23 24 30 32 35 39 40
Ne− 1 1 6 7 8 11 12 15 16 17 19 20
fX 0.28 0.28 1.69 1.97 2.26 3.10 3.38 4.23 4.51 4.79 5.36 5.64
fN −0.37 0.67 0.66 0.94 0.58 0.36 0.56 0.51 0.28 1.16 0.37 0.48
a
u is the atomic mass, Ne− the number of electrons, f X the scattering length for X-rays, and f N the scattering length for neutrons. Both f X and f N are
given in 10−12 cm. The values of f N are taken from ref 58.

Figure 3. Radiation damage. (a) Dose dependence of the resolution for X-ray imaging. Reproduced with permission from ref 47. Copyright
2009 Elsevier B.V. (b) Scheme showing the production of free radicals during water radiolysis. Reproduced with permission from ref 55.
Copyright 2011 MDPI AG. (c) Effect of X-rays on PC12 cells: phase contrast images of cells before and after a 6 h XRF scan. The line plot
shows the difference in size of the cells. Reproduced with permission from ref 56. Copyright 2012 AIP Publishing. Diffraction patterns
acquired on hard α-keratin at increasing exposure times. The loss of the ring after 10 s of exposure and the overall change of the signal shape
is attributed to radiation damage. Reproduced with permission from ref 57. Copyright 2010 Elsevier Inc.

with the focal length f and the zone number n. By contrast, regime, multilayer Fresnel zone plates (MZP) were intro-
MLLs use planar geometry, where the layers are deposited on duced.37 MZPs differ from standard FZPs by employing
top of each other, thus enabling higher thickness-to-width ratios alternating materials such as ZrO2/Ta2O5 or W/Si to achieve
and increasing the optics’ efficiency. Wedged, tilted, or curved focal spots of 1015 or 5 nm,14 respectively.
layers in MLLs make it possible to focus beams to nanometer Beam Definition by Absorption. Pinholes and slit apertures
spot sizes by approximating a situation where Bragg’s law would are less sophisticated than the previous focusing strategies but
be ideally fulfilled for each individual layer.38 FZPs are are frequently used to decrease the beam size by absorbing the
advantageous in the soft X-ray regime, and small beam sizes outer part of the beam and to clean the beam from parasitic
are achieved (e.g., 15 nm at 1 keV).54 For the hard X-ray scattering (Figure 2g). In principle, absorbing components are
8545 DOI: 10.1021/acsnano.7b03447
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no real focusing optics but just “cut down” the beam to a tolerable dose (in Gy) to achieve a resolution between 0.1 and
smaller spot size. Nowadays, so-called scatterless slits made of 10 nm is Dtot = 108 Gy nm−1 × resolution (in nm) (see gray
single crystals, such as silicon or germanium, are used to reach area in Figure 3a). The required dose for imaging is based on
beam sizes of a few microns.43 the experimental Rose criterion of signal-to-noise and also
Many of the cell imaging techniques described below require shown in Figure 3a (black lines). Obviously, the applied dose
coherent X-ray radiation. The coherence between two waves is needs to be larger than the required dose but smaller than the
defined as a constant phase difference between waves with the tolerable dose, offering the area between the curves to the right-
same frequency and two types of coherence are distinguished: hand side for imaging. This consideration shows that resolution
(i) temporal or longitudinal and (ii) spatial or transverse in bioimaging using X-rays is limited by radiation damage to
coherence. Longitudinal coherence is determined by the about 10 nm.
chromaticity of the X-ray source, that is, for third-generation One major effect causing radiation damage is the radiolysis
synchrotron sources typically by a monochromator. Spatial and ionization of water and thus production of free radicals by
incoherence is due to the fact that usually X-rays do not ionizing radiation.60 After the interaction of X-rays with water
propagate in a perfectly well-defined direction, and the degree molecules, two processes may occur within 100 fs: (i) excitation
of coherence is defined by the collimation of the X-ray source. and (ii) ionization, removing an electron from the water
Spatial coherence indicates how uniform the phase of the molecule (see Figure 3b).61 Over 80% of the energy deposited
wavefront is. Increased coherence can be achieved by cutting in cells results from ionization.55,60,62 On a time scale of
the beam with slits; however, the gain in coherence is typically femtoseconds to picoseconds, the water molecules are further
accompanied by a loss of photons in the beam. reduced into free radicals such as H•, HO•, or HO•2 . The
Radiation Damage. The technical advancement in X-ray extracted electrons cause a particle shower, either producing
focusing optics was accompanied by the development of new photons (by bremsstrahlung) or creating hydroxyl radicals.
different imaging techniques. However, imaging biological cells The key chemical reactions involved in this process are
with X-rays remains challenging due to the composition of the summarized in Figure 3b. The radicals move within the sample
cells themselves, which contain low atomic number constitu- and cause damage at distant locations.
ents such as hydrogen, carbon, oxygen, or nitrogen. The Several measures have been developed to reduce the effect of
electron density of biological specimens is relatively low. As X- radiation damage: (i) fast data acquisition to collect data before
rays interact with the electron clouds of the atoms, the contrast radicals have diffused to the next scanning point or to collect
increases with higher atomic numbers. The scattering power, or data before ionization has occurred, (ii) use of cryogenic
scattering length, of a material is defined as the amplitude f X of temperatures to cool down the sample and reduce the diffusion
the scattered wave ψ(q) = f Xe2πiqr. As shown in Table 2, f X is of free radicals, and (iii) use of free radical scavengers to slow
proportional to the number of electrons (f X = r0 × Ne−) and down the ionization process. When employing scanning
remains small for biological matter as compared to metals such techniques, fast scanning is key to outrun radiation damage.
as gold, where f X = 22.3 × 10−12 cm. In order to collect For example, continuous instead of stepwise motor movement
sufficient information, high flux beams are used. The intensity I0 decreases the time per scan point to exposure time and readout
is defined as the number of photons per second (photons s−1), time with no additional overhead. Modern synchrotron sources
whereas the flux is the number of photons per second per unit with high brilliance allow for short exposure time. Importantly,
area (photons s−1 m−2). subsequent scan points are recorded before the highly mobile
However, high flux dramatically increases the dose to the radicals reach the corresponding position. Furthermore, placing
sample, and as biological matter is highly radiation sensitive, deep-frozen samples, which have been vitrified in a liquid
severe radiation damage may be caused. Direct or indirect ethane/propane mixture at a temperature of 77 K, in a cryo-
radiation damage may have different consequences for the stream of N2 at about 100 K preserves the sample at low
sample: (i) in direct radiation damage, the structure of temperature while scanning.63,64 This method eliminates the
molecules are altered with the possibility of loss of crystallinity solvent and radical motility, thus reducing the damage to the
to a more amorphous system (e.g., breaking chemical sample. The use of free radical scavengers such as dimethyl
bonds);57,59 (ii) in indirect radiation damage, typically sulfoxide (DMSO, interacting with hydroxyl radicals HO•)62 or
ionization of water produces free radicals that can break 2-mercaptoethylamine HCl (cysteamine, interacting with HO•,
chemical bonds or induce oxidation. Whereas some of these HClO, e−aq and H2O2)62,65 has shown to reduce radiation
consequences might not be visible with optical microscopes, damage to cells. However, each radical scavenger targets only
severe radiation damage to cells can be observed by mass loss certain radicals.66 With the advent of X-ray free electron lasers
and change of morphology of the cell.56 Two examples are (FEL), “diffract before destroy” techniques were developed.
discussed in detail below. This issue remains the most Here, the idea is to collect information before excitation and
challenging one in modern X-ray synchrotron imaging of ionization occur, that is, faster than 100 fs.67
biological matter. Radiation dose depends on the number of In order to understand radiation damage processes in more
incident photons per unit area. For X-rays of energy hν and detail, Kosior at al.56 systematically investigated the effect of X-
matter with a mass density ρm, the dose D in units of Gray (Gy ray radiation on cell structures. They performed X-ray
= Jkg−1 = m2 s−2) is calculated as47 fluorescence microscopy on PC12 cells from rat at an energy
of 17 keV (Figure 3c). The authors reached a resolution of 50
μI0Thν nm on freeze-dried cells kept at 113 K. They reported that a
D=
ρm σ (4) dose of 9 × 105 Gy already leads to significant mass loss. In
another interesting study, radiation damage on the protein level
I0 is the primary beam intensity in photons s−1, T the exposure was investigated:57 Leccia et al. studied so-called hard α-keratins
time in seconds, and σ the exposed area per scan point or from human hair, which are also found in fingernails and are
exposure in m2. According to Howells et al.,47 the maximum stiffer than keratins found in skin cells, irradiated by a
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Figure 4. Scanning SAXS and XRD. (a) Early experiment applying scanning microdiffraction to bone. Reproduced with permission from ref
83. Copyright 1997 International Union of Crystallography. (b) Inverted gray scale fluorescence micrograph of keratin networks in human
cells (left) and corresponding X-ray dark-field image from the region of interest showing the overall scattering (color scale) and the
orientation of the keratin structures (black lines, right). Reproduced with permission from ref 100. Copyright 2012 IOP Science. (c) Visible
light phase contrast image of two hydrated human cells (left) and the corresponding X-ray dark-field image (right). Reproduced with
permission from ref 59. Copyright 2014 American Physical Society. (d) Anatomical map showing the integrated scattering cross section of a
brain slice (top), map of the concentration of myelin (center), and map of the periodicity of the lamellar structure of the myelin sheaths
(bottom). Reproduced with permission from ref 109. Copyright 2011 Elsevier Inc. (e) Computed tomogram of a human trabecular bone
sample (left), orientation map of bone ultrastructure obtained by SAXS tensor tomography (center), reconstruction of one slice along with
regions of interest showing the degree of anisotropy (represented by the color and length of cylinders, right). Reproduced with permission
from ref 110. Copyright 2015 Nature Publishing Group.

synchrotron X-ray microbeam (Figure 3d). At a flux of 4.2 × X-rays and compatible with biological samples. Besides polymer
1010 photons s−1 μm−2, they gradually increased the exposure materials such as Kapton,68,69 Mylar, and polyethylene, thin
time up to 50 s and analyzed the small- and wide-angle silicon-rich nitride membranes (also referred to as Si3N4
scattering signal. They identified three levels of structural windows) are widely used, as adapted from soft X-ray
changes: (i) after 4 s, the coiled-coil architecture of the keratin microscopy.70 These membranes are commercially available at
dimers is disrupted; (ii) after 30 s, the long-range organization different thicknesses (from 30 to 1000 nm).71−73 The
along the filament is lost; and (iii) after 50 s, the lateral attenuation length, that is, the material thickness at which
organization of the filament packing is disturbed. These intensity is reduced to e−1 of the original intensity, is about 18.7,
examples show that due to the diversity of structural alteration 140.4, and 465.2 μm for energies of 5, 10, and 15 keV,
mechanisms in radiation damage across different materials, it is respectively.45,46 Furthermore, these windows are versatile, for
advisable to monitor radiation damage by complementary example, they resist many chemicals and can be used under
techniques and to take measures to diminish sample vacuum as well as in cryogenic conditions.74 Many cell types
destruction by the energy impact. grow readily on these substrates, and contrast enhancement and
Sample Environments. Imaging biological materials and reduction of radiation damage are reached by chemical fixation,
cells with hard X-rays requires adapted sample environments, plunge-freezing, and drying or by cryopreservation.
which, ideally, should mimic the physiological conditions of the For studying hydrated samples, the windows can be arranged
studied specimen as well as possible. In general, substrates and in a “sandwich” geometry, with the sample sealed between7,75,76
window materials need to be radiation resistant, transparent for or commercial wet chambers.77 More sophisticated home-
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made75 or commercial64 microfluidic chambers increase the fiber orientation with a glancing angle instead of the more
viability of living cells by constant inflow of nutrients, outflow conventional 90° angle, leading to asymmetric angular intensity
of wastes and free radicals, as well as probably a slight cooling distributions that were analyzed to retrieve the angles of the
effect. Although to our knowledge all microfluidics studies cellulose microfibrils. Further, the orientation of these micro-
using X-rays on cells have been performed on adherent cells, in fibrils could be resolved by wide-angle microdiffraction in
principle, the devices are also suitable for suspension cells. For different layers of a single wood cell wall.93 In general, scanning
diffractive tomographic imaging, the sample has to be rotated micro-XRD is a powerful technique for ordered specimens such
and data have to be taken at multiple angles. Depending on the as biomineralized tissues. Seidel et al. developed an automatic
specimen size and beam dimension, silicon nitride windows scheme to analyze the local orientation of biological fibers.114
may be used and offer an angular opening greater than 160°. A They studied chitin in the flow sensing system of crickets and
good alternative is glass capillaries made of borosilicate or identified reflections from α-chitin corresponding to d-spacings
quartz to collect data from all angles. In order to reduce of 0.954 and 0.465 nm. Simmons et al. used micro-XRD to
absorption and background scattering, the capillaries should study the spatial and temporal progression of human dental
have thin walls, typically from 0.01 to 0.1 mm.78−81 Finally, enamel, by studying the mineral formation and mineral
solid samples, such as bone, hair, or teeth can be directly organization.115 The authors found that during enamel
mounted on the acquisition stage. maturation, the increase in mineral content proceeds fast,
whereas the crystallites become more oriented and thicker in a
SCANNING X-RAY SCATTERING AND DIFFRACTION slower fashion. In another study, myelinated, glutaraldehyde
Scanning X-ray scattering and diffraction are similar techniques fixed fibers from a mouse sciatic nerve revealed different
and use a similar setup. Diffraction is a specific case of molecular organization along the fiber.116 The authors found
scattering by well ordered and crystalline samples which diffract different d-spacings in the diffraction patterns corresponding to
the incident beam in a few specific directions, determined by different parts of the fiber, that is, 45 to 60 Å in the paranodal
Bragg’s law. Whereas in small-angle scattering (SAXS) data are region, 60 to 80 Å in the internodal region, and >80 Å in the
recorded between 0.1 and 10°, in diffraction (XRD or wide- central part of the fiber.
angle X-ray diffraction, WAXD) data are recorded up to 90°. Scanning SAXS. As compared to XRD, in case of scanning
Scanning diffraction was first introduced in 1995 by SAXS, the sample does not need to have periodicity and the
Mahendrasingam et al.82 using synchrotron radiation and signal is diffuse. The particle size can be resolved between the
applied to polymer samples. Scanning SAXS was independently smallest and the highest scattering angles, or q values,
developed in 1997 by Fratzl et al.83 on laboratory sources and corresponding to length scales from 1 to 100 nm.111 Data
used to study bone samples (see Figure 4a). By scanning the treatment follows SAXS theory, taking advantage of Guinier or
sample through a focused beam and recording a scattering or Porod analysis, and fitting procedures to obtain form and
diffraction signal in each position, spatially resolved reciprocal structure factors.111 However, it has to be taken into account
space data are obtained. Thus, heterogeneity in the sample is that beam size and the size of the scatterer are on the same
captured rather than just average values as known from solution order of magnitude.64,102,104,117 Thus, the extended theory
scattering. Biological matter displaying some degree of available for solution SAXS, where a large beam is used to
(hierarchical) order particularly benefits from scanning
ensemble average over many scatterers112,118 is not directly
diffraction. Prominent examples, some of which are discussed
applicable.
in more detail below, are bone,83−88 plant fibers and wood,89−93
Bone is a prime example of a composite material, where the
teeth,94−96 hair,97−99 cellular components,59,64,77,100−106 and
hierarchical structure is perfectly well adapted to the
tissues such as muscle107 and brain.108,109 Recently, the
mechanical requirements. Pioneering work by Fratzel et al.83
technique was used to image initially living cells,59,64 and it
has been extended to three dimensions (3D).86,96,110 XRD and could resolve the size and orientation of hydroxy apatite
SAXS111−113 are both applications of elastic scattering of X- particles embedded in the collagen matrix in a spatially resolved
rays: The electrons in the sample are caused by the X-rays to manner. The eccentricity of the scattering patterns was used as
resonate elastically. They produce secondary X-ray waves which a quantitative measure of the degree of orientation as well as
interfere and lead to a scattering signal in reciprocal or Fourier principle direction of the oriented structures. The experiment
space.111 Notably, only the amplitude of the exit wave field is was performed in the late 1990s using an X-ray tube delivering
detected, and the phase is lost. Hence, direct inversion of the a comparatively large beam (diameter 100 μm). In recent years,
scattering signal by an inverse Fourier transform is not possible. the technique has been transferred to synchrotron setups,119
In scanning techniques, a raster scanning procedure is exploited where beam sizes have been reduced to a few hundreds of nm.
to compute pseudo-real-space maps in so-called dark-field Taking advantage of these developments in X-ray optics, one
contrast: all photons collected on the detector at a certain can now image internal structures of whole, unsliced
measurement position are added, and this total intensity value mammalian cells at nanometer resolution. In an early example
is plotted on a color scale at the respective position (see of such work, we studied bundles and networks of cytoskeletal
examples in Figure 4a−c). The real-space resolution is thus keratin intermediate filament proteins (see Figure 4b).100 By
limited by the beam size and the scanning step size, whichever fitting a model to these data that takes into account the beam
of the two is larger. Additionally, however, the actual SAXS diameter and shape and organization of the bundles, we
signal or diffractograms may be analyzed in reciprocal space, quantified bundle diameters and orientations as well as their
and structural information is retrieved. inner structure including filament diameter and arrangement,104
Scanning XRD/WAXD. An early example of scanning and correlated the results to visible light fluorescence
micro-XRD was in resolving the helical architecture of cellulose micrographs of the same samples. The key characteristic of
fibrils in wood cells with a microbeam (2 μm diameter).92 In the method is that nanofocused X-ray beams provide a real-
this study, the X-ray beam was oriented almost in parallel to the space resolution on the order of 100 to 400 nm while in
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reciprocal space the resolution is on the order of few were able to directly compare chemically fixed and living
nanometers, defined by the accessible q-range. cellular samples, both immersed in aqueous environments.59
Some of the most prominent protein structures in We determined the azimuthally integrated SAXS data of living
mammalian cells consist of actin filaments (microfilaments). cells and chemically fixed cells, respectively, and calculated the
By correlating visible light fluorescence microscopy with a difference in dependence of the scattering vector q. As q is
scanning SAXS approach, Priebe et al. were able to link actin inversely proportional to the corresponding length scale d, we
fiber structure and myosin II concentration from visible light were able to identify length scales, on which structures either
micrographs with the structural data obtained from scanning disappear or emerge upon fixation, from about 15 to 60 nm
SAXS.64 The same group showed that differentiated stem cells (see Figure 4c). Formaldehyde, as we used it here, is one of the
exhibit a pronounced structural orientation and high intensity most commonly used fixative agents in biological studies and
diffraction signal, whereas naive human mesenchymal stem cells according to this study, artifacts on length scales resolvable by
do not show this particularity.106 This result supports the idea superresolution microscopy are likely to be created.
that stem cells gain ordered and oriented structures at the SAXS and Diffraction Computed Tomography. As
molecular and supramolecular level during the differentiation mentioned above, one great advantage of X-rays over visible
process. light or electrons is their large penetration depth. Large objects
Scanning SAXS studies are typically static as the extensive can be imaged without slicing or sectioning. Both diffraction
scan times, which are determined by the exposure times per and SAXS have been extended to (computed) tomography
scan point and the size of the field of view, prevent the (CT) as outlined in the following. SAXS-CT was introduced in
recording of “movies”. For example, a scan of 100 × 100 steps the 80s121 and has, with the advent of focused beams,
with 1 s exposure time takes about 3 h. Continuous scanning developed into a nondestructive (apart from radiation damage)
modes and higher brilliance sources have helped tremendously technique to study the local nanostructure of specimens in
to decrease the scan times. Furthermore, the associated 3D.122 For example, SAXS-CT has been used to study the
radiation damage (e.g., 108 Gy for exposure times of 1 s per morphology of brain tumors.108 The authors used the intensity
scan point and a 100 nm step size) remains a major draw back of the SAXS signal to reconstruct high-resolution images,
of the high-resolution method. However, biological processes making use of the extended q-range, and analyzed the slope of
may be studied in an indirect way, for example, by taking “snap the SAXS signal by applying a generalized Porod power law120
shots” at different stages. We have used this approach to follow to delineate necrotic regions of the tumor from healthy tissue.
packing and unpacking and duplication of DNA in nuclei of The same authors also derived maps of the molecular
mammalian cells during the cell division cycle. Visible light organization of myelin sheaths in formalin-fixed rat brain as
phase contrast movies (1 frame every 5 min) were recorded shown in Figure 4d.109 The results are anatomical maps similar
until chemical fixation and plunge-freezing of the samples to to transmission tomography or histology and reconstructed the
characterize the cell division stage. Subsequently, scanning 3D volumes of the samples. By contrast to conventional CT,
SAXS “snap shot” data of these samples were taken.105 With a not absorption contrast, but the differential scattering cross
real-space resolution of 250 nm and the largest q-values section at the respective position is exploited. Maps of (i) the
corresponding to only 6 nm, application of a generalized Porod myelin concentration, and (ii) periodicity of the lamellar
power law120 enabled a quantitative analysis of the power law structure were obtained by quantifying the intensity of the
exponents from the azimuthally integrated scattering signal. second-order Bragg peaks and the Bragg peak positions,
The results gave rise to information about internal structure, respectively.
packing density and surface properties of the scatterers and the Very recently, Liebi et al.110 have combined scanning SAXS
DNA packing and unpacking processes could be followed over with tensor tomography123 to reveal nanostructure organization
time in a spatially resolved manner. Surface properties and of trabecular bone on micrometer length scales using a beam
packing density were retrieved by computing surface-area-to- with dimension 25 × 25 μm2 (see Figure 4e). The technique
volume ratio maps derived from the Porod analysis. The had previously been developed on two-dimensional 20 μm
surface-area-to-volume ratio provides information about the thick sections of bone samples,86 and was extended to three-
size, compactness and density of the specimen, in this case from dimensional unsliced bone structures. The authors were able to
DNA compaction into chromatin. reconstruct the 3D organization of bone by analyzing over one
In the previous examples, only dry samples provided strong million diffraction patterns, for a total acquisition time of 22.5
enough contrast in the electron density to provide information h, corresponding to a dose of about 3 × 107 Gy on the whole
on the single filament level. In SAXS, the contrast arises from bone specimen. The reciprocal space intensities could be
the average excess scattering length density of the sample, that determined, providing access to the direction and degree of
is, the difference between the electron density of the sample orientation of each scan position in the whole sample. The
and the electron density of the solvent. As cells consist mostly authors could retrieve the size and shape of the scatterers owing
of water, the electron density contrast in aqueous environment to the SAXS signal and identify domains with a low degree of
is comparatively low. When the solvent is changed from water orientation, corresponding to random orientation of the
to air, a gain in contrast greater than a factor 800 may be collagen fibers, and domains with a high degree of orientation,
achieved. However, measurements on hydrated samples, corresponding to aligned collagen fibers. Schaff et al.96 used the
especially in combination with specially designed microfluidic same approach to study the collagen fiber orientation within a
flow chambers64,75 also reveal valuable information on the tooth. They combined real and reciprocal space data in order to
nanometer scale. Such experiments are more challenging due to obtain a six-dimension space map of the specimen. The
the application of wet or microfluidic chambers and radiation technique is based on the reconstruction of the full 3D
damage is of higher concern as free radicals move freely in scattering information (qx, qy, qz) in every voxel. While the
solution. However, an aqueous environment mimics the method remains fairly slow, it provides information on length
physiological situation more closely than dried samples. We scales from a few nanometers to a few millimeters, which is
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Figure 5. Coherent diffractive imaging. (a) Early example of CDI of whole Escherichia coli bacteria. Left: diffraction pattern. Right:
corresponding image reconstruction. Reproduced with permission from ref 130. Copyright 2003 National Academy of Sciences, U.S.A. (b)
Typical speckle pattern collected by CDI (top) and the corresponding 2D electron density reconstruction of a Staphylococcus aureus cell.
Reproduced with permission from ref 137. Copyright 2016 Nature Publishing. (c) Amplitude (left) and phase (right) reconstructions of an
insect wing measured by ptychography. Reproduced with permission from ref 136. Copyright 2009 Elsevier B.V. (d) Three-dimensional
subcellular structure of a frozen-hydrated Neospora caninum cell. Reproduced with permission from ref 138. Copyright 2015 International
Union of Crystallography. (e) Ptychographic X-ray nanotomography of frozen-hydrated Chlamydomonas cells and zoom of one cell.
Reproduced with permission from ref 81. Copyright 2015 Science Direct.

particularly attractive for composite materials with highly with the sample. Speckle patterns due to interference of the
ordered hierarchical structural features and for mesoscopic phase-modulated exit wave-fields from the specimen are
materials. This technique is now not only available at recorded on an area detector (see Figure 5a,b). The positions
synchrotron sources, but also for laboratory setups.124 and intensities of the speckles reflect the structure of the
Diffraction tomography was applied to resolve components material and the phase and amplitude of the real-space image of
in a highly biomineralized holdfast organ from Anomia simplex the specimen can be retrieved by iterative algorithms. Briefly,
mussels.125,126 The authors were able to investigate aragonite the detector reading is proportional to the square modulus of
and calcite (CaCO3 polymorphs), which were not detectable in the amplitude. The image reconstruction starts by allocating
the more conventional absorption tomography, directly by the random phases to the measured amplitudes. The result is then
respective diffraction peaks. Finally, they were able to Fourier transformed into real space as a first guess. Constraints
determine the local degree of Mg substitution in the calcite about the finite-size support of the specimen are introduced,
phase as the calcite lattice constants change in the presence of that is, the electron density outside the support and any
Mg, whereas Mg does not substitute into aragonite. negative electron density inside the support are set to
zero,129−131 and the newly determined electron density
COHERENT DIFFRACTIVE IMAGING (CDI) (amplitude and phase) is then Fourier transformed into
CDI was first suggested theoretically in 1952 by Sayre.127 reciprocal space. The phase is kept, while the new amplitudes
However, due to technical challenges such as the need for are replaced by the measured ones. This procedure is iteratively
highly coherent beams and area detectors with high dynamic repeated until the result converges.132 The phase of the
range and quantum efficiency128 experimental demonstration specimen can be retrieved by oversampling the object and by
was achieved only in 1999.129 The physical principle is valid for using iterative phase-retrieval algorithms.133−136 Here, we focus
coherent beams of (X-ray) photons or electrons, which interact on those CDI methods, which have been extensively applied to
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biological matter in the past, plane-wave CDI and ptychog- Ptychograpy. Ptychography combines CDI and scanning
raphy. and is also referred to as scanning diffraction microscopy. The
Plane-Wave CDI. Plane-wave CDI is a full-field technique method was invented by Hoppe in 1969143 and exploits a
which is particularly dose-efficient (on the order of 106 Gy). A sufficiently large overlap (>60%)144 between adjacent scanning
coherent planar X-ray beam illuminates a sample in trans- positions. The size of the imaged object is not limited in the
mission geometry and the diffraction patterns are collected on same way as it is in plane-wave CDI. Owing to the redundant
an area detector placed in the far-field. In the so-called information and by using of an iterative feedback algo-
Fraunhofer far-field, where the diffraction pattern of an object rithm135,136 as outlined previously, it is possible to retrieve
of size a is observed at a distance z far away from the sample the illumination function, that is, the incoming X-ray wave field,
z≫
a2
, plane waves are observed. By contrast, if the detector is as well as the object exit wave field. Due to the phase
λ information, real-space images can be computed. The larger
a2 beam and scan step size renders ptychography a more dose-
placed in the near-field close to the specimen, z ≪ λ , spherical
waves are observed and Fresnel theory of diffraction has to be efficient (on the order of 103−106 Gy) method compared to
used. A beam stop blocks the direct, unscattered beam. scanning SAXS (on the order of a few 108 Gy).
One of the first examples of a successful application of
Diffraction patterns are taken to obey the Nyquist frequency
ptychography to a biological sample was presented by Thibault
criterion such that the diffracted intensity is oversampled (that
et al., where the authors imaged an insect’s wing (Figure 5c).136
is, sampled finely enough) and thus the phase information is
Shortly later, even more weakly scattering Magnetospirillum
encoded in the diffracted intensity. Moreover, the area outside
gryphiswaldense bacteria were imaged by ptychography.145
the specimen has to be known or transparent, corresponding to
Furthermore, ptychography has been applied to the highly
surrounding the electron density of the specimen with an
ordered actin filament bundles in inner ear hair cell stereocilia,
“empty” region. Empirically, the linear oversampling criterion O
providing direct imaging of actin bundles at a resolution of 130
> 5, which must be fulfilled for high quality phase
nm in the reconstructed phase images.102 Giewekemeyer et al.
reconstructions, is defined by the wavelength λ, the sample
even achieved a resolution of 85 nm at 6.2 keV and a dose of
size D, the sample-to-detector distance z and the detector pixel
λz 1.3 × 105 Gy and were able to reveal unique DNA packing in
size p, O = pD .131,139 Pioneering work in the field was Deinococcus radiodurans bacteria with four subunits.69 Lima and
performed by Miao et al. by imaging Escherichia coli bacteria co-authors studied frozen-hydrated yeast cells by ptychography
at an energy of 6.2 keV and with a resolution of 30 nm, as and demonstrated the advantages of using cryogenic temper-
shown in Figure 5a.130 More recently, the same level of atures when imaging cells or tissues.146
resolution was achieved for fully hydrated yeast cells,76 Tomographic CDI. Plane-wave CDI is well suited for
providing images of the specimen morphology and internal recording 3D data sets by acquiring a series of 2D diffraction
details at high contrast. A typical example of a speckle pattern is patterns at different angles, owing to the comparatively short
presented in Figure 5b, together with the 2D electron density exposure times and thus the insensitivity to sample
projection of a Staphylococcus aureus cell (dimensions: 0.5 to 1.5 vibrations.128 Furthermore, the relatively low dose greatly
μm),137 which already provides a detailed cell density map. helps in the extension to tomographic 3D imaging. The study
With the experimental parameters the authors used, they could by Rodriguez et al.138 on Neospora caninum cells was performed
reconstruct objects up to 2.5 μm. This example will be at cryogenic temperatures and by tilting the sample the authors
discussed further in the tomographic CDI section. achieved a 3D reconstruction of the structure of the cell interior
X-ray free electron lasers attracted a lot of attention owing to (see Figure 5d). In an even earlier study, Nishino and
the possibility of collecting diffraction patterns from protein colleagues147 imaged individual human chromosomes purified
crystals, viruses, or even small whole cells using femtosecond from mitotic HeLa cells at a spatial resolution of 38 nm for each
pulses and thus faster than radiation damage is occurring 2D reconstruction and a resolution of 120 nm for the 3D
(“diffract before destroy”).140 As of now, there are only a few reconstructions. They reported the presence of axial structures
examples of hard X-ray FEL imaging of whole cells and all those confirming findings from immunoelectron microscopy or
examples are particularly small (bacteria) cells. Assuming, for fluorescence microscopy, albeit without the use of labels or
example, a wavelength of 0.1 nm, a sample-to-detector distance staining.
of 1 m and a pixel size of 20 μm, the oversampling criterion Dierlof et al. developed a tomographic version of
λz ptychography148 and applied it to the interconnective canal-
defines a maximum sample size of D = 5p = 1μm. Fan and co-
icular network of cortical bone. They attempted to resolve
authors 137 recorded single 10 fs pulse exposures of structures on the length scales of 100 nm such as the osteocyte
glutaraldehyde fixed Staphylococcus aureus cells as shown in lacunae. This example nicely shows how biological specimen,
Figure 5b. By labeling the cells with gold nanoparticles (i.e., which only show a weak absorption contrast, benefit greatly
enhancing the signal), they reached a resolution of 54 nm, from exploiting phase information. In fact, even quantitative
which improved the resolution by a factor 2.6 compared to electron density contrast maps can be obtained. Whereas the
unstained cells. Living Microbacterium lacticum cells have been solid bone sample was dehydrated and then resin-embedded,
imaged by Kimura et al. employing FEL diffraction at a Diaz et al.81 studied Chlamydomonas reinhardtii algae in solution
resolution of about 37 nm.141 They observed a nonuniform after preparation in a glass capillary, facilitating the rotation
density within the cell and attributed high density regions to during tomographic ptychography (Figure 5e). They achieved a
materials with a high electron density such as DNA. A recent resolution of 180 nm and by averaging several slices, they
study by Takayama and Yonekura using FEL radiation showed measured the cell wall to about 100 nm in thickness. By a
that CDI at cryogenic temperature leads to images of segmentation procedure, the authors were additionally able to
chloroplasts from C. melorae and minicells from Escherichia determine the mass density (see eq 2) of different organelles
coli at a resolution of 192 and 52 nm, respectively.142 within the algae to 1.4 g/cm3 for lipid droplets and 1.1 g/cm3
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Figure 6. Holography. (a) Holographic intensity and phase reconstruction from freeze-dried Dictyostelium discoideum cells. Reproduced with
permission from ref 156. Copyright 2011 American Physical Society. (b) Holograms from a cardiomyocyte recorded at five different defocus
positions and reconstructed phase (projected electron density). Reproduced with permission from ref 154. Copyright 2017 International
Union of Crystallography. (c) 3D holographic reconstruction of freeze-dried Deinococcus radiodurans: effective mass density (blue and red),
surface rendering (yellow), and combined surface and mass density. Reproduced with permission from ref 157. Copyright 2017 Springer. (d)
Holotomography of mesophyll cells and palisade cells from Arabidopsis seeds. Reproduced with permission from ref 160. Copyright 2006
National Academy of Sciences, U.S.A.

for the cytoplasm. Using a similar approach, Wilke et al.101 shown in Figure 1c. To our knowledge, there are only a few
studied DNA organization in Deinococcus radiodurans. Each examples of holographic hard X-ray imaging applied to
projection was reconstructed with a resolution of about 50 nm. biological cells. Freeze-dried Dictyostelium discoideum cells
In CDI, the electron density is encoded in the quantitative have been imaged by in-line holography by employing
phase information. From the electron density maps of the waveguides to produce the coherent beam156 at a resolution
bacteria, they estimated the mass density of the high-density of 157 nm, limited by the relatively large distance z1. The
regions to about 1.6 g/cm3 and attributed this value to DNA. authors captured a large field of view of 38 × 38 μm2 and used a
X-ray Holography. The physical principle of holography low dose of 0.8 × 103 Gy. They demonstrated the use of
was first proposed for visible light in 1948 by Gabor149 and waveguides to image cells by holography and were able to
adapted to soft X-ray photons in 1965150 and only in 1996 for resolve several subcellular features such as globular particles of
hard X-rays, due to the lack of coherent radiation sources at several hundred nanometers in size attributed to mitochondria
shorter X-ray wavelengths151,152 (for review, see ref 153). By (Figure 6a). With a similar setup and using waveguides, Wilke
recording both the exit wave of the object and an undisturbed et al. studied Bacillus thuringiensis and Bacillus subtilis bacterial
coherent reference wave field, both the intensity and the phase cells.155 The technique allowed the authors to obtain images
of the scattering object can be reconstructed from the with a large field of view (100 × 50 μm2) for studying many
interference pattern of the two waves. In holographic imaging, cells at the same time or a small field of view for imaging single
the detector is placed in the near-field, where the diffraction cells at a resolution down to 65 nm. Thanks to the high
pattern is almost a perfect projection of the specimen and we resolution and phase information, the authors were able to
observe a change in hologram structure at different sample-to- measure the mass of endospores to 110−190 fg by integrating
detector distances. In CDI, the detector is placed in the far-field the mass density in eq 2 over the number of pixels.
and the diffraction pattern does not change in structure but in Bartels et al. studied Deinococcus radiodurans bacteria in 3D,
size. as shown in Figure 6c157 as well as hydrated, living cells.158 In
In in-line holography,149 a coherent beam is produced by a the first study, holograms of freeze-dried samples were imaged
FZP or a waveguide, and the sample is placed out of focus at a in a tomographic fashion by collecting 83 2D holograms in an
distance z1 from the X-ray focus in the illumination cone angular range of 162 degrees and a spatial resolution of 125 nm
produced by the optics.149 The scattered wave and transmitted was obtained.157 Furthermore, the authors were able to retrieve
wave interfere and are collected on a 2D detector placed at a 3D density maps of Deinococcus radiodurans, with values
distance z2 ≫ z1 from the sample. The magnification is then ranging from 0.8 g/cm3 to 1.2 g/cm3 (see Figure 6c). In the
z second study, they achieved a resolution of 53 nm at a
given by M = 1 + z2 . Example values are M = 130 (z1 = 38.6
1 comparatively small dose of 5.2 × 103 Gy for freeze-dried cells.
mm and z2 = 5.12 m) or M = 2800 (z1 = 1.81 mm and z2 = 5.13 As this dose is much smaller than the lethal dose of these
m).154,155 A typical setup of an in-line holography experiment is bacteria (about 20 kGy),159 the authors concluded that the
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Figure 7. Combination of techniques. (a) Ptychography (gray scale) and scanning SAXS (color) images acquired from the same cell in a serial
fashion. Reproduced from ref 104. Copyright 2016 American Chemical Society. (b) Phase map obtained by combined X-ray ptychography
and holography of magnetotactic bacteria MO-1. Reproduced with permission from ref 172. Copyright 2016 Nature Publishing Group. (c) X-
ray fluorescence (S, K, Ca, and P maps) and ptychography phase map of a Chlamydomonas alga. Reproduced with permission from ref 171.
Copyright 2017 Nature Publishing Group.

viability of the bacteria should not be compromised and applied absorption spectroscopy (XAS),162 X-ray absorption near-
the method to living samples and reconstructed holograms at a edge spectroscopy (XANES, NEXAFS),163 or resonant soft
resolution of 100−150 nm. The decrease by a factor of 2 to 3 in X-ray scattering (RSoXS).164 These spectroscopic techniques
resolution (freeze-dried, 53 nm; living, 100−150 nm) is due to are, however, mostly applied in the soft X-ray regime.
minute movements of the samples in the buffer solution. Furthermore, correlative data from complementary methods,
Arabidopsis seeds were studied by holotomography.160 The such as visible light (fluorescence) microscopy and electron
authors have imaged single mesophyll cells and palisade cells microscopy increase the information available from an
(Figure 6d) and were able to identify previously unobserved air experiment. In the following paragraph, we highlight work
space network in the seeds. They suggested that it serves to that combines several X-ray techniques.
provide oxygen at the onset of germination. A recent study The mineralization of fin bony rays from zebrafish was
used holography for low dose imaging of mammalian cells in studied by XRF, SAXS, and WAXD.165 In order to locate the
combination with scanning SAXS, as discussed further below bone within the tissue, the authors mapped the calcium
(Figure 6b).154 distribution by XRF. Simultaneously, they acquired SAXS and
WAXD data. Owing to the XRF Ca map, they were able to
COMBINATION OF TECHNIQUES analyze the SAXS and WAXD data at specific calcium-rich
In the previous sections, we have described a number of X-ray- positions within the sample, revealing insights on the mineral
based imaging techniques that are sufficiently mature to be phase containing amorphous calcium phosphate, octacalcium
applied to cells and biological matter. Each technique has its phosphate, or carbonated hydroxyapatite, including the mineral
specific strengths and drawbacks and most importantly they particle size and shape. SAXS and XRF are easily combinable, as
probe different properties of the studied object. For example in the XRF detector can be placed at any angle from the sample.
scanning SAXS, high-resolution structural information is However, the position of the detector can be optimized to
collected in reciprocal space at the expense of a high dose, collect XRF spectra. For example, in case of thick samples a
however, no real-space images are obtained, except for the detector at 90° to the beam provides the highest signal-to-noise
pseudo-real-space dark-field representations. By contrast, in ratio, whereas for thin samples or samples with high
ptychography, high-resolution real-space images are acquired, concentration of specific elements, a larger detector at 180°
but no internal structural information is revealed. Hard X-ray (backscatter geometry) leads to the highest signal-to-noise
holography can achieve a similar resolution as ptychography at ratio.166 Consequently, there are several more recent examples,
an even lower dose. such as a study of substantia nigra neurons in the elderly with
A particular strength lies in combining the techniques we additional complementary X-ray phase contrast imaging167 and
discussed above either with each other or with spectroscopic work on Scrippsiella trochoidea microalgae that were manipu-
techniques such as X-ray fluorescence (XRF),161 X-ray lated by laser-based optical tweezers.168
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Ptychograpy and XRF are easily combinable as well. For In our view, the most challenging issue in the field of X-ray
example, they have been used to study the freshwater diatom imaging at the nanoscale remains radiation damage. When
Cyclotella meneghiniana,169 the green alga Ostreococcus sp.,170 or comparing the methods described above concerning the dose
Chlamydomonas reinhardtii alga.171 Figure 7c shows the XRF that needs to be applied, scanning SAXS imposes the highest
maps (with sub-100 nm resolution) of different elements found energy on the sample, with values up to 108 Gy.104 However, in
in Chlamydomonas reinhardtii alga (sulfur, phosphorus, turn, reciprocal space structural information is retrieved with a
potassium, and calcium). The ptychogram was recorded at a resolution down to a few nanometers. Ptychography provides
spatial resolution better than 20 nm (Figure 7c central panel). highly resolved real-space images with quantitative phase
The XRF and ptychography are acquired simultaneously and contrast at the expense of 106 Gy.101 Finally, holography only
there is perfect registry between the two modalities. It is needs on the order of 103 Gy.158 Holography can identify
possible to closely overlay the chemical maps (from XRF) with weaker features compared to CDI, but CDI provides better
the electron density maps (from ptychography). The authors resolution.173 Hagemann and Salditt have compared CDI and
identified electron-dense spherical structures in the ptychogram holography using a cell phantom.174 They found that in
that are related to polyphosphate bodies (Ph in Figure 7c, right holography the performance is better than in CDI given the
panel) because they contain polyphosphate complexed with same number of photons per pixel and the same phase
calcium. The granule labeled Ca in Figure 7c has a much lower reconstruction procedure. However, holography in the hard X-
potassium concentration as compared to the other poly- ray regime only became possible with third generation
phosphate bodies. The authors suggested that this specific synchrotrons delivering highly coherent beams and is thus a
granule might undergo degeneration or aging. relatively “young” technique. At the same time, hard X-rays
Ptychography and scanning SAXS were combined on the offer the advantage of being applicable to denser and thicker
identical cells in a serial fashion.104 First, ptychography was specimens. We thus believe that in the future hard X-ray
employed to obtain high-resolution (about 60 nm) real-space holography will play a major role in the investigation of
images of the keratin network inside the cells (Figure 7a left biological structures by X-rays.
panel). After identification of regions of interests from the Cellular systems or biological tissues are particular in the way
ptychogram (Figure 7a central panel), diffraction data were that heterogeneities within the sample typically play an
acquired by scanning nanodiffraction. Thus, dark-field contrast important role for their function. Hierarchical structures, such
images (Figure 7a right panel), where each pixel respresents the as in the cytoskeleton, the nucleus, in bone, wood or teeth
integrated intensity of a full diffraction pattern, were calculated. encode certain mechanical and biological properties and it is
By analyzing the SAXS signal and fitting it to a bundle model,64 thus of high interest to image and study the different levels of
bundle and filament diameters, filament distance and arrange- hierarchy. Ensemble averaging, as it is often applied to
ment were determined from whole unsliced and unstained cells. biological matter, such as known from solution SAXS, does
Recently, hard X-ray holography was successfully combined not capture such heterogeneities and thus the methods we
with scanning SAXS on freeze-dried neo-natal rat cardiac presented here provide many tremendous possibilities for
muscle cells.154 The full-field holographic (Figure 6b) data were future discoveries.
ideal for locating the features of interest before the scanning
SAXS acquisition and for controlling structural degradation and AUTHOR INFORMATION
beam damage after the scanning SAXS measurement. Further, Corresponding Author
holography and ptychography were combined to study the *E-mail: [email protected].
magnetotactic bacteria MO-1 (Figure 7b).172 In ptychography ORCID
(and diffraction in general) of weak-phase objects (such as soft Sarah Köster: 0000-0002-0009-1024
matter), it is difficult experimentally to obtain a large dynamic Notes
range of diffraction patterns due to detector limitations. To The authors declare no competing financial interest.
overcome this challenge, the authors combined ptychography
and X-ray in-line holography, named dark-field X-ray ACKNOWLEDGMENTS
ptychography in their work. They were able to collect
ptychography data sets with a beam-stop, and complemented The authors thank T. Salditt, M. Burghammer, B. Weinhausen,
the missing low scattering angle information by the holographic and S.-C. August for fruitful discussions. This work was
information. supported by the Helmholtz Gemeinschaft in the framework of
Virtual Institute VH-VI-403 “In-Situ Nano-Imaging of Bio-
logical and Chemical Processes”, by the German Research
CONCLUSIONS Foundation (DFG) in the framework of SFB 755 “Nanoscale
In summary, we presented recent examples for the successful Photonic Imaging” within project C10 and the Cluster of
application of innovative X-ray imaging techniques at the Excellence and DFG Research Center for Nanoscale Micros-
nanoscale to biological cells and materials. X-rays provide a copy and Molecular Physiology of the Brain (CNMPB), and by
number of advantages over visible light and electrons, such as the German Ministry of Education and Research (BMBF)
the high penetration depth, enabling the study of thick objects. under Grant No. 05K13OD4.
Due to the small wavelength of X-rays, resolution limits are in
the nm range for all presented techniques and are still being VOCABULARY
pushed further. Recent years have also clearly shown a lot of scattering, change of trajectory of a radiation due to interaction
potential for combining several X-ray techniques either in series with a media, either elastic (no change of frequency) or
or in parallel, or in complementing the X-ray measurements by, inelastic (change of frequency); diffraction, special case of
for example, (fluorescence) visible light microscopy or electron scattering by periodic samples satisfying Bragg’s law (2d sin θ =
microscopy in a correlative way. nλ); SAXS, small-angle X-ray scattering, recording of small
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