Abstract

Morphometric measurements, such as quantifying cell shape, characterizing sub-cellular organization, and probing cell–cell interactions, are fundamental in cell biology and clinical medicine. Until quite recently, the main source of morphometric data on cells has been light- and electron-based microscope images. However, many technological advances have propelled X-ray microscopy into becoming another source of high-quality morphometric information. Here, we review the status of X-ray microscopy as a quantitative biological imaging modality. We also describe the combination of X-ray microscopy data with information from other modalities to generate polychromatic views of biological systems. For example, the amalgamation of molecular localization data, from fluorescence microscopy or spectromicroscopy, with structural information from X-ray tomography. This combination of data from the same specimen generates a more complete picture of the system than that can be obtained by a single microscopy method. Such multimodal combinations greatly enhance our understanding of biology by combining physiological and morphological data to create models that more accurately reflect the complexities of life.

Introduction

Measurement of cell morphology, generally known as morphometry, is central to cell biology, medical research and clinical medicine. For example, morphometry is the basis for cell cycle analysis [1,2], a guiding factor in cell culture experiments [3] and the standard method for differentiating between benign and malignant tumor cells [4]. Cell morphology also plays a key role in staging cancer and predicting how aggressively a tumor will grow and spread [5] since cell shape regulates gene function during carcinogenesis [6].

Imaging has proved to be an efficient, accurate method of quantifying both the internal (organelles) and external (cell shape) morphology of biological specimens [7,8]. Over time, many different imaging modalities have been applied to morphometric studies, each with inherent strengths and weaknesses and capable of producing a sub-set of information about the specimen. Most commonly, light- and electron-based microscopies have been used to analyze a cell's external size and shape, together with internal characteristics such as the number, shape and relative position of organelles and labeled molecules. However, even powerful modalities such as these have limitations, regarding the type of specimens they can image and the information they can generate. As a result, much effort has gone into developing new modalities for making morphometric measurements on cells and filling gaps in information that cannot be accessed by existing methods, even when they are applied in tandem to the same specimen.

An obvious path towards meeting this goal lies in the choice of specimen illumination. Bright-field light microscopes are limited in spatial resolution by the wavelength of light. Fluorescence microscopes can image beyond the ‘diffraction limit’ imposed by the wavelength of the illumination, but they can only image labeled cellular structures or molecules, leaving the other cell contents dark. Electron microscopes can image only thin specimens; cells larger than small bacteria must be sliced into sections that are less than 500 nanometers thick. X-ray illumination has the potential to overcome these limitations and image all cell components in fully intact cells and do so with high spatial resolution. Of course, X-rays have a storied history in clinical medicine and materials science. However, for many years, X-ray microscopes failed to live up to their early promise for imaging cell-scaled biological specimens at high spatial resolution. Realizing this goal required developments in nanofabrication, detectors, cryopreservation and instrument design. Fortunately, these technological advances have now been made, and soft X-ray microscopy is now gaining influence and recognition as a tool for making morphological measurements on single cells, including large eukaryotic cells.

We should point out that this review is not meant to be a general overview of X-ray microscopy, for example, we do not discuss work carried out on biology at larger than the cellular scale or discuss the use of these instruments in research fields such as materials science and magnetism. The application of X-ray microscopy in these areas is profound and has produced enormous literature. Instead, we limit our discussion to the benefits of using X-rays to visualize and quantify the internal and external morphology of cells, in 3D and even 4D. We also describe the current combination of X-ray microscopy with other techniques, in particular, X-ray spectromicroscopy and light microscopy to add physiological information to morphological studies. Finally, we will look to the future, and the challenges of implementing higher throughput, easier handling and automated data analysis into biological X-ray microscopy.

Morphometrics: historical perspective

Light microscopy

Humans can typically see objects in the size range of 6–20 µm, depending on age and eyesight [9]. So, at best we can see a large isolated eukaryotic cell but cannot distinguish sub-cellular detail. That requires a magnifying device. The earliest example of this concept was Robert Hooke's observation in 1665 that cork consists of compartments or ‘cells'. In the 200 years that followed, light microscopy gave us first sight of organisms, such as protozoa and bacteria, and key sub-cellular structures, such as the nucleus and mitochondria [1012]. Staining procedures—developed for light microscopy by Retzius in 1881—laid the foundation for discovering the microscopic anatomy of cells and the establishment of histology [13]. Light microscopy also provided the first morphological analyses of cells when bacterial pathogens that caused human diseases were described by microscopic observation of their shape by Koch, Klebs and Pasteur [14]. Light microscopy was also the basis of many other landmark studies. Examples include the first quantitative study of the chromatin morphology during the cell cycle [15], the effect of insulin-like and epidermal growth factors on cell morphology [16], and the effect of varying osmolarities on the morphology of sickle cell [17]. These are but a few examples that highlight light microscopy for studying cell morphology. Nowadays, practically every light microscope can be equipped with analysis software for cell counting and cell morphology analysis, making these routine tasks in cell culture and histopathology. Light microscopy has the advantage of being able to image living specimens and generally requires minimal sample preparation. With the use of confocal and super-resolution light microscopy, one can partly overcome some inherent limitations of the technique, such as low spatial resolution, but obtaining a complete, unaltered 3D imaging of the internal cell morphology using light microscopy remains a challenge.

Electron microscopy

The discovery of electrons, in 1897 by Thompson, opened up the possibility of a microscope with three orders of magnitude better spatial resolution than light microscopes can achieve [18]. But, the physical properties of electrons meant new, complex sample preparation protocols had to be developed for biological specimens. In an electron microscope, biological specimens have to withstand both the high vacuum needed to prevent deflection of electrons by air and elevated temperatures caused by the absorption of high-energy electrons by the specimen. Furthermore, the strong interaction of electrons with the specimen results in scattering of the illumination. This scattering limits the maximum thickness of biological specimen that can be imaged to less than a micron. The first two conditions can be mitigated by ‘fixing’ the specimen, the latter by sectioning. Protocols for epoxy embedding [19], sectioning of tissue [20] and chemical fixation protocols [21] enabled the early application of electron microscopy to cell imaging [22], and allowed, for example, the first observation of mitochondria, the Golgi apparatus, a structure later called ‘endoplasmic reticulum’, together with the visualization of viruses and other sub-cellular structures [2325]. With the establishment of methods for 3D tomographic data acquisition, together with protocols for cryogenic fixation, electron microscopy became—and remains—the gold standard for high-resolution biological imaging [24,2628]. The importance of electron microscopy in biology was recently recognized by the Nobel committee, with the 2017 Chemistry prize awarded to Joachim Frank, Richard Henderson and Jacques Dubochet for the development of cryogenic electron microscopy (cryo-EM).

What was missing?

As highlighted above, light- and electron-based microscopes are fantastic tools that have made enormous contributions to our knowledge of cells and biological systems. Nevertheless, their respective limitations result in information gaps, either because the necessary imaging data cannot be obtained, or can only be obtained from a very small number of cells (as is the case in EM tomographic reconstructions of entire eukaryotic cells). From the perspective of cell imaging, there remained the need for methods that overcome the limitations of light and electron microscope. Specifically, an imaging technique with high spatial resolution and high penetration power for hydrated specimens, ideally with minimal sample preparation prior to imaging. In short, a technique that can measure the internal and external morphologies of large numbers of intact eukaryotic cells held in a near-native state. This and other unique information needs are met by X-ray microscopy.

X-ray microscopy

Wilhelm Röntgen discovered X-rays in 1895, an achievement that earned him the Nobel prize in physics in 1901. The penetration power of X-rays was demonstrated in the famous image of the bones in the hand of Röntgen's wife, Anna Bertha Ludwig. The medical community responded to this image with enormous enthusiasm, and almost immediately adopted X-ray radiography as a clinical technique. X-ray use is now ubiquitous in medicine, to the point most people in the developed world will have a clinical X-ray procedure at least once in their life.

Despite the promise of X-rays as the basis of new imaging methods for cell biology, the initial work was disappointing. X-rays have a very low refractive index in virtually all materials [29]. Therefore, it is impossible to use a conventional magnifying lens, such as those used in a light microscope to focus X-rays. As a result, X-ray imaging of biological specimens was limited in magnification, and not particularly informative [30].

The modern era of biological X-ray microscopy began in the 1970s with the development of X-ray lithography and high-resolution detectors. Schmahl and co-workers built the first X-ray transmission microscope at the Deutsches Elektronen-Synchrotron (DESY), Hamburg [31,32]. Unlike its predecessors, this X-ray microscope produced high resolution and excellent contrast images of cells, even when they were imaging through a relatively thick water layer [33,34]. As a result of this success, several groups around the world were inspired to design, build and apply X-ray microscopes to take morphometric measurements from cell. Many excellent reviews are available that summarize developments and applications of X-ray microscopes [30,3538].

These days, a diverse family of X-ray microscopes is available. There are now microscopes with illumination in the soft (under 1 keV) and hard (1–100 keV) X-ray energy regimes. These microscopes operate with either transmission or scanning geometries with images being generated by a variety of different mechanisms, for example absorption, phase contrast or even coherent diffraction. X-ray microscopes now offer imaging capabilities that are applicable in almost any research field. However, biological specimens present unique challenges compared with other types of specimen. Specifically, living organisms have a relatively low tolerance towards ionizing radiation as compared with materials science specimens, such as magnetic materials. Susceptibility to radiation damage held up the development of soft X-ray microscopy for biology for many years. But that hurdle was overcome by the development of cryogenic specimen stages that are capable of holding flash frozen cells at liquid nitrogen temperature during data acquisition. While this technology does not eliminate radiation damage completely, it mitigates it well enough to allow data collection to be completed long before damage becomes apparent in X-ray images. Before discussing this application in more detail, we should survey the landscape of available X-ray methods and their features.

From the cell biology perspective, X-ray microscopes fall into one of two categories, full-field transmission (visualize morphology) and scanning in 2D or 3D (to access physiology information) (Table 1). In both categories of microscope, soft and hard X-rays can be used, depending on the desired penetration depth, spatial resolution and contrast mechanism [59]. While there are multiple examples of using soft X-ray coherent diffraction imaging [42,43,6064] and hard X-ray microscopy for visualizing cells [4446], these techniques are mostly proof-of-principal studies and are not yet allowing the type of systematic studies required in cell biology. The preferred method for morphological studies of single cells is transmission soft X-ray microscopy.

Table 1
List of known and commonly used X-ray microscope methods to study morphology and physiology via trace elements of cells

Contrast, resolution, penetration depth and time required for full acquisition are average values of what has been used in the past for cell biology.

Method Contrast modality Resolution (nm) Penetration depth (µm) Time required for one tomogram (min) 
Morphology (transmission) 
 Soft X-ray microscopy [39,40‘Water window' absorption edges of C, O and N 25 10 
 Soft X-ray coherent diffraction imaging [4143Phase-contrast 25 20 50 
 Hard X-ray microscopy [4446Chemical staining
Zernike-like phase contrast 
30 60 30 
 Hard X-ray projection tomography [4749Phase contrast 50 1000 
Physiology (scanning) 
 Soft scanning fluorescence microscope [5056C, N, O, P and S 25 — 80 
 Hard scanning fluorescence microscopy [57,58Ca, Fe, Co and Zn 50 — 180 
Method Contrast modality Resolution (nm) Penetration depth (µm) Time required for one tomogram (min) 
Morphology (transmission) 
 Soft X-ray microscopy [39,40‘Water window' absorption edges of C, O and N 25 10 
 Soft X-ray coherent diffraction imaging [4143Phase-contrast 25 20 50 
 Hard X-ray microscopy [4446Chemical staining
Zernike-like phase contrast 
30 60 30 
 Hard X-ray projection tomography [4749Phase contrast 50 1000 
Physiology (scanning) 
 Soft scanning fluorescence microscope [5056C, N, O, P and S 25 — 80 
 Hard scanning fluorescence microscopy [57,58Ca, Fe, Co and Zn 50 — 180 

Soft X-ray microscopy of cells

As we mentioned above, X-rays play a major role in many disciplines, including biology. Regarding the latter, X-ray microcomputed tomography and X-ray diffraction for protein crystallography are the most common uses of X-rays in biological research [65,66]. In this review, however, we will hold focus on the class of transmission X-ray microscopes (TXM), which are similar in optical design to light microscopes and use lenses to focus illuminating light onto the specimen and an objective lens to transmit a magnified image of the specimen onto a detector. Since visible light and X-rays are electromagnetic radiation, the basic principles of bright-field light microscopy apply to X-rays in a TXM. Thus, obtaining high-quality imaging requires bright, monochromatic illumination focused to a spot approximately the same size as the specimen. In a TXM, these criteria are met by using high-quality condenser and objective lenses, ideally with closely matched numerical apertures. Also, the TXM must be fitted with a high sensitivity, low noise camera for detection and a specimen mounting system. In general, it is relatively easy to meet these criteria in light microscopy—indeed a vast amount of valuable science has been carried out using light microscopes with simple lenses and a human eye acting as the detector. However, in the context of X-rays, the situation is more complex. Developing TXMs as a biological imaging tool required the development of specialized X-ray lenses, the availability of brilliant X-ray sources, such as synchrotron radiation facilities [67] and many other technological advances as we will discuss below.

Nowadays, most TXMs are located at the synchrotron radiation facilities worldwide [68,69]. Here, we will skip discussing image formation and the basic principles of X-ray microscopy, all of which can be found in significant detail elsewhere [36,37,41]. Instead, we will concentrate our attention on the criteria required for successful imaging of cell morphology with a TXM.

There is a common misconception that spatial resolution is the most important metric when assessing an imaging modality. This assumption is not limited to the TXM; it is a commonly held belief about imaging modalities per se. And there is some logic to this assumption that the more detail you can see, the better. However, a more complete metric is to consider the information content of an image. As long as a microscope produces images that answer the questions being asked, the spatial resolution is somewhat irrelevant.

In transmission X-ray microscopy, illuminating photons are absorbed as they pass through the specimen. The spectral region that lies between the absorption edges of carbon (284 eV) and oxygen (540 eV) is termed the ‘water window’. In the water window, the absorption of X-ray photons by biological specimens adheres to Beer's Law and is therefore linear with concentration and molecular composition. Subsequently, highly solvated regions in a cell are relatively transmissive towards soft X-ray illumination. Conversely, regions such as membranes or condensed genetic material in the nucleus, which are dense with carbon-containing molecules, absorb illuminating photons much more strongly. The resulting differential absorption of the illumination produces images that contain quantitative information on every part of the specimen.

Soft X-rays have sufficient penetration depth for analysis of intact, fully hydrated cells. The relatively fast acquisition time enables relatively high-throughput imaging, essential for studies like the phenotyping of cells [36,7072]. 3D imaging of very large cells, tissues and organisms requires the greater penetration depth of hard X-rays. Until now, X-ray projection tomography has demonstrated the most promising results for subcellular morphological imaging of ‘thick specimens’ [45,4749].

The strong interaction with organic material limits the penetration of soft X-rays to ∼10–15 µm, depending on the cell type [39]. Imaging cells or tissues thicker than this requires deformation of specimens, making them extended in one direction (for example, flat or cylindrical objects that are thinner in some orientations) or trimming them to the suitable thickness as done in electron microscopy. Microscopy with hard X-rays overcomes the limitation on specimen thickness but relies on chemical staining or the use of phase contrast data acquisition protocols [7375].

As in light microscopy, the depth of field of an X-ray microscope depends on the wavelength and numerical aperture of the objective. Higher resolution requires larger numerical aperture and consequently smaller depth of field. Thus, 3D imaging of a whole cell comes at the cost of spatial resolution achievable for the desired specimen thickness. Conventionally specimen trimming for soft X-ray microscopy or imaging with hard X-rays was used to overcome this limitation. However, several approaches have been proposed [7680] to extend the depth of field in soft X-ray microscopy enabling in future to image larger cells with high resolution in 3D. These approaches are either based on the acquisition of additional through-focus projections, that is to say, similar to methods used in confocal microscopy, or rely on the computational methods to compensate for the limited depth of field.

Imaging contrast and depth of field (coupled with spatial resolution) can be pushed only to the extent of X-ray radiation dose which can be tolerated by the specimen. In most cases, the dosage required is lower than with electron microscopy [34]. However, the specimen is still estimated to be exposed to a dose as high as 107 Gy [81]. The effect of X-rays on biological specimens is severe [82], but the damage can be mitigated by imaging at cryogenic temperatures [33,81,83,84]. Cryogenic temperatures used in biological X-ray microscopy to mitigate radiation damage require only one step—vitrification. However, although straightforward, this step is a crucial determinant of image quality. Here, we would like to point out a few factors for successful sample preparation. The foremost requirement is to locate the cell or region of interest to freeze down. While this can be done later with the light microscopes integrated into X-ray microscopes, the most efficient way is to select cells of interest before vitrification, for example by techniques such as fluorescence-activated cell sorting [85]. Two (specimen) holder types are currently in use for TXM imaging of cells: thin-walled glass capillaries and grids developed for electron microscopy [69]. While the use of grids has the advantage of access to well-established, existing protocols from electron microscopy, grid mounted specimens are limited in possible rotation range by as much as ±30%, resulting in missing information in 3D data series and the generation of artifacts and reduction in the axial spatial resolution. But, capillary mounted specimens can be rotated without limitation to generate tilt series with no systematically missing data [86]. Independent of the holder type, the mounted specimen is frozen using methods developed for EM cryopreservation, such as freezing with a blast of cooled helium gas, plunge freezing or high-pressure freezing.

All aforementioned criteria are typically in the hands of experts in X-ray microscopy who developed the technique. However, there is an additional criterion, which depends on the ‘users,' most probably cell biologists, being able to recognize the specimen when they see it. Since contrast and resolution differ significantly from well-known imaging methods used in most laboratories (i.e. light and electron microscopy), one should be open minded when interpreting X-ray microscopy data. TXM data contain new, never seen before information.

In the following sections, we will survey studies that analyzed both the internal and external morphology of cells. Many of these studies are based on quantitative morphological analysis, creating a niche for X-ray microscopy in the characterization of cellular architecture in 3D.

External morphology

Cells are typically classified by their size, shape and function. Size and shape are morphometric measurements, with the function being a physiological characterization.

Morphology is routinely used to differentiate and identify simple cells, such as bacteria. For example, the curved cell shape of Caulobacter crescentus easily distinguishes it from rod-shaped bacteria such as Escherichia coli [87]. In eukaryotic cells, the geometric characteristics are more complex, but the same basic principle holds. For example, fibroblasts are elongated; neurons are dendritic [88], and so on. Determining the 3D shape of cells is one of the most fundamental tasks in cell biology. Consequently, some automated high-throughput techniques have been designed for pathology [88,89] and are indispensable for everyday clinical diagnostics. For fundamental cell biology, however, a highly detailed 3D picture of cell shape is often necessary, in addition to an averaged, ensemble representation output by high-throughput methods. This is an area where SXT excels.

An example of external cell morphometrics

Red blood cells are known for their biconcave disc shape, and their deformation of normal shape serves as an indicator of sickle cell disease. About one in 4000 individuals are diagnosed with sickle cell disease only in the U.S.A. [90], preventive approaches or effective treatment strategies do not exist. Darrow et al. [91] used soft X-ray microscopy to analyze the size and shape of over 600 sickled red blood cells throughout the progression of disease. The number of protrusions, not accessible with 2D methods, was used as an indicator of the sickling severity and could be classified into four categories as a function of disease progression: none, mild, moderate and severe (Figure 1A). Furthermore, the group analyzed the effect of the anti-sickling drug compound 5C and showed that it could decrease the severity of the sickling disease in ∼80% of cases. This work is a shining example of soft X-ray tomography (SXT) being used to both classify disease states and the efficacy of candidate pharmaceuticals.

External and internal morphology of cells.

Figure 1.
External and internal morphology of cells.

(A) Morphological study of the four stages of sickle cell anemia in red blood cells carried out by SXT [91]. (B) Diagrammatic representation of cell division in the yeast S. cerevisiae [92]; NZ, nocodazole. (C) Soft X-ray tomographic study showing the internal morphological changes that takes place during S. cerevisiae cell division. Scale bar = 2 µm [93]. (D) Example of quantitative morphological information obtained using SXT. Quantification of nuclear surface areas as a function of cell size of wild-type (WT) and mutated (cdc5-nf) cells. This mutation results in ‘nuclear flares’ that appear in cells with a volume of 140 μm3 (black triangle, left hand plot). The plot on the right quantifies nuclear volume as a function of cell size, showing there is no difference in this ratio between wild-type and mutant cells [92].

Figure 1.
External and internal morphology of cells.

(A) Morphological study of the four stages of sickle cell anemia in red blood cells carried out by SXT [91]. (B) Diagrammatic representation of cell division in the yeast S. cerevisiae [92]; NZ, nocodazole. (C) Soft X-ray tomographic study showing the internal morphological changes that takes place during S. cerevisiae cell division. Scale bar = 2 µm [93]. (D) Example of quantitative morphological information obtained using SXT. Quantification of nuclear surface areas as a function of cell size of wild-type (WT) and mutated (cdc5-nf) cells. This mutation results in ‘nuclear flares’ that appear in cells with a volume of 140 μm3 (black triangle, left hand plot). The plot on the right quantifies nuclear volume as a function of cell size, showing there is no difference in this ratio between wild-type and mutant cells [92].

Internal morphology

The real power of soft X-ray microscopy lies in the deep penetration depth of these photons and the contrast generated by using ‘water window’ illumination. In tandem, these two characteristics allow visualization and quantification of both the internal and external morphologies of intact cells. Where electron microscopy would require sectioning of the cells and light microscopy would need the use of multiple fluorescent tags, soft X-ray microscopy provides natural contrast based on X-ray linear attenuation coefficient of all organelles within a cell. The number and organization of organelles in a cell depends on its complexity and tissue type [93]. These characteristics also change quickly during the cell cycle, during development and in response to environmental factors such as cell density, temperature, oxygen concentration and the availability of nutrients [94,95].

Examples of studies quantifying the internal morphology of cells

Yeast is one of the simplest eukaryotic organisms, even so it undergoes many of the same essential processes as mammalian cells. Therefore, yeast has become a widely used model organism to study cell growth and division [96]. Yeast has been studied extensively using soft X-ray microscopy [44,97101]. Based on other methods, it was already established that ploidy (the number of complete sets of chromosomes) in yeast has a direct and linear proportionality on cell size. While cell size in both budding and fission yeast has been shown to be proportional to the growth of the nucleus, other organelles and their relation to ploidy of the fungal cells has been unknown. Uchida et al. [98] have used soft X-ray microscopy to measure volumes of the cell and the significant organelles in both haploid and diploid strains of S. cerevisiae at the four stages of the yeast cell cycle. Lipid bodies, mitochondria, vacuoles, nucleus and nucleolus were visualized and measured in 3D at G1, S, G2 and M stages of the yeast cell cycle (Figure 1C). Except for vacuoles, the growth of the main classes of organelle was found to be strictly regulated with cell size at all stages of the cell cycle and in both haploid and diploid strains. These studies have shown that yeast possesses well-defined optimal volumetric ratios that are most probably independent of strain, ploidy and phenotype and these ratios are therefore common to all yeast cells. To analyze the underlying mechanism and functional importance of these constant ratios, Walters et al. [92] used SXT to study nuclear to cell volumes in wild-type and arrested cdc5-of mutant budding yeast. Both yeast cell types were staged in G1 and then released into media containing nocodazole (Figure 1B). As cells progressed towards a mitotic arrest, nuclear volume and surface area compared with cell size were measured (Figure 1D). Despite different morphologies of mutant and wild-type cells, nuclear surface area to cell volume increased at the same rate, suggesting that flare formation allows the cells to keep this ratio without altering its nuclear volume. Future soft X-ray microscopy studies on yeast mutant cells will deepen our understanding of how cells control their size and arrangement of organelles during the cell cycle.

X-ray microscopy has been also used to analyze structurally more complex cells, like green alga Chlamydomonas reinhardtii [102,103], bacteria [104], lymphocyte T-cells [105] and olfactory neurons [104106]. A recent study demonstrated that quantitative analysis based on linear attenuation coefficient of X-rays is sensitive to biomolecular concentration, resolving substructure of organelles, particularly the nuclear organization inside stem cells [107]. Nuclear organization and chromatin conformation have been studied in embryonic and hematopoietic stem cells with varying lineage potential, particularly in embryonic stem cells, hematopoietic stem cells, multipotent progenitors, granulocyte/macrophage progenitors, B cells and granulocyte/monocyte cells. While heterochromatin volume remained similar in all cell types, the euchromatin volume decreased significantly upon differentiation, suggesting that the nuclear size is determined predominantly by euchromatin. 3D analysis of nuclear shape and interface between chromatin and euchromatin revealed high nuclear folding and extensive chromatin interphase in the stem and progenitor cells indicating greater lineage potential. This chromatin compaction effect was further investigated during neurogenesis in vivo [108]. Advanced analysis of heterochromatin in nucleus showed unappreciated interconnectivity of hetero- and euchromatin in multipotent stem cell, neuronal progenitor, mature neuron and HP1β knockout mature neuron of the olfactory epithelium cells (Figure 2A). It was shown that heterochromatin and euchromatin are highly interconnected (more than 98%) in all type of cells. This continues lattice-like structure of heterochromatin serves as an architectural platform to organize parts of euchromatin. Depletion of HP1β did not disrupt this continuity, despite reduced heterochromatin and its lost connections to the nuclear envelope. Thus, HP1β might regulate chromatin compaction, reorganization and interactions with the nuclear envelope.

Visualization of ultrastructure in complex cells.

Figure 2.
Visualization of ultrastructure in complex cells.

(A) Soft X-ray analysis of chromatin masses in the nuclei of three types of olfactory epithelium cells, namely multipotent stem cells, neuronal progenitors and terminally differentiated neurons, together with similar data from HP1β knockout (KO) cells [107]. This morphological study revealed a surprising degree of connectivity exists between chromatin and this persists throughout development and differentiation. Scale bar = 2 µm. (B) Alteration of the endoplasmic reticulum–mitochondria interface in Hepatitis C virus (HCV) replicating cells [109]. Comparative analysis of the endoplasmic reticulum–mitochondria topological relationship of vitrified control (A), HCVtcp-infected (B) or HCV replicon-bearing cells (C). Volume slices, manually segmented surface representation, and surface views of the different areas of interest are shown. Yellow arrows in (A) mark mitochondrial cristae. Two types of abnormal mitochondria were found class 1 (AbMito1) and class 2 (AbMito2). White and black arrows mark matrix condensation arrows and cristae swelling, respectively. Scale bars 0.5 μm.

Figure 2.
Visualization of ultrastructure in complex cells.

(A) Soft X-ray analysis of chromatin masses in the nuclei of three types of olfactory epithelium cells, namely multipotent stem cells, neuronal progenitors and terminally differentiated neurons, together with similar data from HP1β knockout (KO) cells [107]. This morphological study revealed a surprising degree of connectivity exists between chromatin and this persists throughout development and differentiation. Scale bar = 2 µm. (B) Alteration of the endoplasmic reticulum–mitochondria interface in Hepatitis C virus (HCV) replicating cells [109]. Comparative analysis of the endoplasmic reticulum–mitochondria topological relationship of vitrified control (A), HCVtcp-infected (B) or HCV replicon-bearing cells (C). Volume slices, manually segmented surface representation, and surface views of the different areas of interest are shown. Yellow arrows in (A) mark mitochondrial cristae. Two types of abnormal mitochondria were found class 1 (AbMito1) and class 2 (AbMito2). White and black arrows mark matrix condensation arrows and cristae swelling, respectively. Scale bars 0.5 μm.

Such access to the ultrastructural organization in whole cells placed X-ray microscopy as a valuable tool to study cell–parasite interactions. Structural development of malaria parasite, for example, was extensively studied by several groups by 2D soft X-ray microscopy [110112], soft X-ray coherent diffraction imaging [113] and X-ray fluorescence diffraction [114,115]. Spatial localization of vaccinia and herpes viral factories was likewise observed by X-ray microscopy [116118]. Lately, ultrastructural alterations were studied in hepatitis C virus-infected cells [109]. While the nuclear envelope and liposomes in hepatitis C viral cells were unaltered in comparison with the typical cell, most striking differences were observed in the endoplasmic reticulum as enlarged cisternae, tubular structures, vesicular extrusions and multiple membrane vesicles (Figure 2B). The effect of clinically relevant antiviral drugs, such as telaprevir, daclatasvir and sofosbuvir, has partially reversed alterations of endoplasmic reticulum and mitochondria, suggesting that optimal antiviral drug combination could be useful in the treatment of chronic hepatitis C viral infection.

Collectively these results demonstrate the various ways by which soft X-ray microscopy is impacting our understanding of cell biology. The high spatial resolution, penetration of an entire even eukaryotic cell, imaging in the native state and 3D quantitative analysis of cell organelles are key features of this technique. Further, we would like to explore the extent of X-ray microscopy to harder energies and hence analysis of multi-cellular structures.

Multi-cellular morphology

Biological processes rely not only on the morphology of each cell but also on the intricate balance of cell–cell or cell–environment interactions. Topics of ongoing research range from morpho- and organo-genesis to wound healing, cancer cell metastasis and immune system reaction, to control cell–cell communication. Current studies demonstrate that the understanding of cell morphology is incomplete without understanding its microenvironment [119]. This is particularly true in the case of the nanoscale materials that are increasingly used in consumer goods can modulate cell morphology at the subcellular level [120]. Nanoparticles are of particular interest as biosensors and for the targeted delivery of cancer therapeutics [120]. Here, the natural contrast of soft X-ray microscopy offers a unique tool to visualize morphological cell in response to microenvironmental changes. Nanoparticle–cell interactions have been investigated in Chlamydomonas cells by near-edge soft X-ray microscopy [121] and in human skin by scanning transmission microscopy [122,123].

Beyond qualitative visualization, Chiappi et al. demonstrated the use of SXT for quantitative analysis of cell vesicles used for iron oxide nanoparticle uptake by breast cancer cells [121]. Size and distance between vesicles containing nanoparticles were evaluated at various incubation times (Figure 3A). The cells take up iron oxide nanoparticles rapidly towards the nucleus and then continuously increase the number and size of vesicles containing nanoparticles. Such a comprehensive quantitative analysis of the cell–nanoparticle interaction is beneficial for nanoparticle design and the specific development of those best suited for clinical applications.

Imaging cells with respect to environment.

Figure 3.
Imaging cells with respect to environment.

(A) Soft X-ray tomographic analysis of endosome size in breast cancer (MCF-7) cells incubated with superparamagnetic iron oxide nanoparticles (SPION) for 0 (CTR), 3, 6, 12 and 24 h [121]. The nucleus is shown in blue. Field of view, 11.5 μm x 11.5 μm. SPIONs are color-coded according to size. (B) 3D time-lapse cell tracking during gastrulation of African frog X. laevis [124]. Virtual cut through 3D embryo rendering at stage 11.5 depicting ectoderm (blue), mesoderm (orange) and endoderm (green). Abbreviations: D, dorsal; V, ventral; AP, animal pole; VP, vegetal pole; BLC, blastocoel; BC, Brachet's cleft, BLCR, blastocoel roof. Magnitude of velocity on sagittal slice for two different times. 3D rendering of the highlighted cell pairs in the archenteron and associated trajectories over a period of 30 min. 3D Velocity field showing difference in collective and singular cell motion at two various times.

Figure 3.
Imaging cells with respect to environment.

(A) Soft X-ray tomographic analysis of endosome size in breast cancer (MCF-7) cells incubated with superparamagnetic iron oxide nanoparticles (SPION) for 0 (CTR), 3, 6, 12 and 24 h [121]. The nucleus is shown in blue. Field of view, 11.5 μm x 11.5 μm. SPIONs are color-coded according to size. (B) 3D time-lapse cell tracking during gastrulation of African frog X. laevis [124]. Virtual cut through 3D embryo rendering at stage 11.5 depicting ectoderm (blue), mesoderm (orange) and endoderm (green). Abbreviations: D, dorsal; V, ventral; AP, animal pole; VP, vegetal pole; BLC, blastocoel; BC, Brachet's cleft, BLCR, blastocoel roof. Magnitude of velocity on sagittal slice for two different times. 3D rendering of the highlighted cell pairs in the archenteron and associated trajectories over a period of 30 min. 3D Velocity field showing difference in collective and singular cell motion at two various times.

While soft X-ray microscopy is superior in terms of natural contrast for visualization of cell organelles, it is limited to studying the morphology of a single cell or cell monolayers only. To visualize cell morphology in cell cultures, tissues and organisms, X-ray microscopy is pushed towards using higher energy (i.e. harder) X-rays. At higher X-ray energies, absorption contrast is lost, and cells become transparent. For imaging of cellular morphology, hard X-ray microscopy thus relies on staining or phase contrast methods. As with electron microscopy, chemical staining for X-ray microscopy could be used to visualize the structure of cells [45]. But for imaging of morphological changes in cell–cell or cell–environment studies, chemical staining is a potential source of artifacts, making interpretation of the data unreliable. Phase-contrast microscopes overcome the problem of low absorption contrast and can image unstained cells in the native state. Hard X-ray microscopy has been already used to visualize chromatid concentration in the human chromosome [60] and the spatial organization of organelles in yeast spore cell [125]. Although the potential of hard X-ray microscopy has been demonstrated, examining cell–cell interactions within macroscopic tissue samples requires an X-ray microscope that can be ‘zoomed' to the cell of interest. Such phase-contrast zoom microscopy has been developed by the groups of Tim Saldit, Peter Cloetens and Lukas Helfen [126,127]. Recently, they successfully localized and imaged macrophages in mouse lung tissues, analyzing barium-instilled macrophages distribution in healthy and asthmatic lung tissues [126]. The 3D reconstructions showed that macrophages localize predominantly in the alveolar lumen and are even able to migrate through the epithelial layer. Use of hard X-ray microscopy for morphological cell analysis of tissues overcomes time-consuming sectioning and histology, enabling visualization of morphological changes in a single cell within macroscopically thick multicellular tissues. Imaging cell–cell interactions in vivo using hard X-rays produces even greater insights into multicellular tissues and organisms. However, the required highly damaging dose of X-rays has always been a barrier for such studies. Nevertheless, 4D X-ray microscopy studies have been carried out successfully in small organisms, i.e. fly pupae [128], weevil joints [129] and frog embryos [124,130]. While insects are more resistant to X-ray radiation [131], the 4D microscopy of the frog embryo required elaborated imaging experiments [130]. In the frog embryo study, morphology and movement of cells were captured by hard X-ray phase-contrast microscopy during the gastrulation of Xenopus laevis. Cell shape and subcellular structures, such as nuclei and yolk platelets, were used to differentiate between ectoderm, mesoderm and endoderm cell layers (Figure 3B). Furthermore, morphologically distinct cells were tracked during the gastrulation process, allowing discrimination between collective and relative cell motion. Such a global view on morphogenetic analysis helps us relate to changes in cellular shape and forces regulating cell development towards a multi-layered organism.

The studies described above demonstrate the immense potential of soft and hard X-ray microscopy for the morphological analysis of multi-cellular systems. Though in vivo 3D imaging with submicrometer resolution is still not achieved, manifold applications can be envisioned for time-lapse analysis of the cell morphology, such as the response of cells to infection, the reaction of immune cells to cancer, migration of cancer cells in healthy tissue, and the response of cells exposed to materials such as nanoparticles or biomimetic scaffolds.

Connecting morphology with physiology

The X-ray techniques discussed so far are best suited to morphometric measurements. Still, this is only part of the story. Biologists also need physiological data, ideally measured from the same cell. This can be achieved by imaging trace elements using X-rays. Since element specificity depends on the energy of the incoming X-rays, the decision between using soft or hard X-rays is based on the element of interest. Despite challenges, like absorption of the emitted photons and the complexity of reconstructing 3D spectroscopic data, there is a growing number of applications for investigating the physiology of cells [132134]. We would like to point out that this summary of methods is limited by space, and in no way approaching comprehensive coverage. For example, we do not discuss combinations or multimodal approaches, for example, X-ray ptychography and fluorescence imaging [132]. The exact resolution, penetration depth and time for 3D imaging will vary between X-ray microscopes, depending on the instrument, acquisitions geometries, available signal and type of specimen. Thus, we advise you to use the examples below as a guideline to find X-ray microscope beneficial to the question of interest and recommend contacting an expert in the corresponding field to guarantee success in applying X-ray microscopy your research.

Correlated light microscopy: molecular localization

Cell morphometric data are of limited use without knowledge of cell function. Physiological processes, like motility, ion transport, metabolism, including protein location and associations are typically visualized by fluorescence light microscopy [135]. The recent development and commercialization of super-resolution fluorescence microscopy have enabled fluorescence imaging at the nanometer scale. Fluorescence microscopy is common to virtually every cell biology laboratory. Consequently, its correlation with X-ray microscopy is a natural progression. In fact, some of the early TXM publications relied on data acquired by both X-ray and fluorescence light microscopy for interpretation of the X-ray tomographic data [96]. Typically, fluorescence microscopy confirmed what had been discovered through X-ray microscopy, or vice versa. Accurate correlative imaging became possible thanks to the development of high numerical aperture cryogenic light microscopy [136]. The cryogenic specimen environment in this microscope significantly enhances the working lifetime of fluorescent proteins and molecules, allowing the collection of through-focus or even fluorescence tomography data. Using the cryo-light microscope allows analysis of the same vitrified specimen by fluorescence and X-ray microscopy. Correlated fluorescence and X-ray microscopy is an area of active development [85,86,105,137,138]. Early works linked the 3D structure of a cell with molecular localization data, for example by identifying fluorescently tagged inactive X chromosome in female thymic lymphoma cells in the context of a soft X-ray tomographic reconstruction [85], or following the dynamics of granule and vesicles in activated mast cells [139] and analyzing ER–mitochondria contacts [140].

The latter study followed morphological changes during mitochondria fission. Mitochondria are known to be highly dynamic organelles which undergo fission and fragmentation throughout the cell lifetime. Though some proteins are known to play a role in the fragmentation of mitochondria, their interplay and their connection with other organelles were studied recently by Elgass et al. [140]. They used SXT to analyze the 3D structure of mitochondria and ER and linked it to the mitochondrial outer membrane proteins MiD49/51 by correlated cryogenic fluorescence microscopy (Figure 4A–C). 3D visualization showed that ER protrudes towards mitochondria using short fingerlike extensions, creating ER–mitochondria contact sites (Figure 4D–H). Fluorescence microscopy revealed that these contact sites contain membrane protein MiD51-GFP foci. Furthermore, quantitative analysis of the MiD51 expression level and X-ray linear attenuation coefficient allowed us to differentiate fragmented and normal cell phenotype. Loss of constriction sites and lower protein concentrations were observed for the fragmented phenotype. These results suggest that either MiD expression or mitochondria fragmentation is involved in ER–mitochondria exchange [140].

Correlated approaches in x-ray microscopy.

Figure 4.
Correlated approaches in x-ray microscopy.

(A) Two-dimensional computer-generated section (typically known as an orthoslice) from a reconstruction of a mouse lymphoblastoid cell expressing MiD51-GFP (green), generated using correlated cryo-fluorescence tomography and SXT [140]. (B) The same computer-generated slice from the SXT reconstruction as presented in (A) without fluorescence overlay. The orange rectangle outlines the area of concentrated MiD51-GFP fluorescence. (C) Magnification of the area shown in (A) and (B) containing a concentration of MiD51-GFP. White arrowheads indicate positions of ER–mitochondria contact sites. (D) Maximum intensity projection of the full 3D SXT reconstruction with the contrast reversed so that features that are low-absorbing are shaded black and features that are highly absorbent are shaded white. (E) ER (green) and mitochondria (red) segmented out and overlaid with the reconstruction. (F) Surface rendering of segmented cellular features, including the nucleus (orange), lipid droplets (blue), ER (green) and mitochondria (red). (G) Three-dimensional cutaway of the SXT-generated reconstruction reveals the same 3D location of the MiD51-GFP fluorescence of that shown in (A). (H) Detailed view of small ER extensions contacting the mitochondria at the MiD51 foci. Scale bars: 2 µm (A, B, DF); 400 nm (C); 1 µm (H). (I) Localization of elements in the different tissues of the eye, such as lens fiber and retinal pigment epithelium, of a 3-dpf (days post fertilization) zebrafish embryo [58].

Figure 4.
Correlated approaches in x-ray microscopy.

(A) Two-dimensional computer-generated section (typically known as an orthoslice) from a reconstruction of a mouse lymphoblastoid cell expressing MiD51-GFP (green), generated using correlated cryo-fluorescence tomography and SXT [140]. (B) The same computer-generated slice from the SXT reconstruction as presented in (A) without fluorescence overlay. The orange rectangle outlines the area of concentrated MiD51-GFP fluorescence. (C) Magnification of the area shown in (A) and (B) containing a concentration of MiD51-GFP. White arrowheads indicate positions of ER–mitochondria contact sites. (D) Maximum intensity projection of the full 3D SXT reconstruction with the contrast reversed so that features that are low-absorbing are shaded black and features that are highly absorbent are shaded white. (E) ER (green) and mitochondria (red) segmented out and overlaid with the reconstruction. (F) Surface rendering of segmented cellular features, including the nucleus (orange), lipid droplets (blue), ER (green) and mitochondria (red). (G) Three-dimensional cutaway of the SXT-generated reconstruction reveals the same 3D location of the MiD51-GFP fluorescence of that shown in (A). (H) Detailed view of small ER extensions contacting the mitochondria at the MiD51 foci. Scale bars: 2 µm (A, B, DF); 400 nm (C); 1 µm (H). (I) Localization of elements in the different tissues of the eye, such as lens fiber and retinal pigment epithelium, of a 3-dpf (days post fertilization) zebrafish embryo [58].

Such 3D correlation of morphology with fluorescence expression links function of the cell with its ultrastructure. Upcoming combination of X-ray microscopy with super-resolution microscopy will bring these methods to similar and isotropic spatial resolution, making this emerging modality superior for cell analysis.

Correlated x-ray fluorescence: major and trace elements

So far, we have discussed X-ray microscopy as a tool for imaging the distribution of three out of the ‘big four' molecular building blocks in a cell, i.e. carbon, oxygen, hydrogen and nitrogen (X-rays are not sensitive to bond hydrogen). The water window with soft X-rays and the phase contrast technique available for hard X-rays both allow the analysis of the morphology of cells with sufficient contrast resulting from these elements. Addressing the overall physiology of cells requires the other factors essential for life and the function of the cells to be imaged. Calcium, for example, is a messenger that helps to regulate multiple cellular functions such as secretion, contraction, cellular excitability and gene expression in all organs. Thus, calcium imaging in visible light microscopy was pioneered in 1980 by Tsien [141,142]. Since then, calcium imaging has become the method of choice for imaging intracellular communications mainly in neural tissue [143,144]. Zinc represents another primary element, which has a broad range of functions in various cellular processes, such as replication and repair, transcription and translation, metabolism and signaling, cell proliferation and apoptosis [145]. It has also been widely used in imaging, as an artificial transcription factor and even as specific modules for protein recognition [146]. Indeed, other significant elements, like phosphorus, sulfur and magnesium, are also essential for studies on DNA and protein folding and signaling. Unlike these major elements, trace elements, like iron, molybdenum and silicon, constitute just 0.5% of the cells in organisms. Still, this small fraction exerts a tremendous influence on all functions of cells [147].

Many highly sensitive microanalytical techniques and instruments have been developed for in situ analysis of major and trace elements [148]. Among them, X-ray fluorescence imaging offers the best combination of sensitivity and spatial resolution. Even though the radiation dose hinders in vivo X-ray fluorescence imaging, the ability to quantitively analyze all major and trace elements with one measurement lead to the development of X-ray spectromicroscopy as a stand-alone technique.

On one hand, X-ray spectromicroscopy allows 3D imaging of the cell morphology. On the other hand, the energy scan performed while image acquisition gives access to different elements present in the physical volume of the cell. Thus, functional elements can be localized within the cell structure. Such correlative X-ray microscopy was traditionally used to address environmental impact by imaging uptake of trace elements in plant and marine cells [50,51,133]. Sviben et al. showed that the unicellular organism E. huxleyi concentrate large amounts of calcium in a vacuole-like compartment [52]. Co-localized with calcium, high concentrations of phosphorus and minor levels of other cations may explain why E. huxleyi is thriving in waters with a low amount of phosphorus, where other phytoplankton cannot survive. Subcellular partitioning and allocation of Calcium ions within the 3D volume allowed the development of a conceptual model for the calcium pathway.

Similar to imaging unicellular organisms, X-ray spectromicroscopy can be applied to multicellular organisms and tissues to determine the intracellular distribution of metals such as iron, copper and zinc.

Kim and co-workers correlated elements in X. laevis oocytes as they mature and initiate embryonic development [53]. The total number of atoms per cell at each developmental stage has shown that copper and zinc levels both rise significantly during meiotic stages, making zinc among three other transition metals the most abundant and crucial for healthy development.

More importantly, trace elements can be linked to the pathology of various diseases. The relationship between iron biochemistry and Alzheimer's disease in intact cortex from a mouse model has been recently investigated by Telling et al. [54]. Here, X-ray spectromicroscopy helped to identify that iron biomaterial deposits in the cortical tissue as magnetite. This demonstrates that iron reduced to a pure ferrous state is associated with the amyloid complex, which is a signature characteristic of Alzheimer's disease. This observation may lead to the development of novel iron-modifying therapy strategies for Alzheimer's disease. Altered levels of iron have also been proposed to be involved in other pathological condition, i.e. cataract formation [58]. Here, the distribution of 23 elements, metals and other trace elements was visualized in the eye of a zebrafish wild-type and mutant embryos (Figure 4I). Many elements were highly enriched in the pigment epithelial layer in a wild-type embryo but were eliminated in knockdown of the zebrafish. These results suggest that by buffering trace elements, melanosomes in pigment epithelial cells protect the lens from stress during eye development.

The main advantages of X-ray spectromicroscopy are subcellular resolution and a high degree of chemical specificity in unstained or chemical stained specimens. With the ongoing research on acquisition strategies, 3D reconstruction algorithms and the development of X-ray spectromicroscopy for in vivo imaging [55,56,115], the correlation of major and trace elements at the subcellular level is a promising method for a fundamental understanding of the roles of the different components within the cell, cell toxicity studies in connection with worsened environmental factors and the etiology of diseases.

Conclusion and summary

X-ray imaging is continuing its rapid evolution as a tool for measuring cell morphology. Though constantly expanding, the technological concept of X-ray microscopy is established and well understood. By itself and in combination with fluorescence light microscopy, it has been applied to almost all cell types and tissues [68]. It is time to look broader and to challenge our current understanding of cell morphology. The exact number of organelles inside of the cell is surprisingly still unknown. With the discovery of membrane-less organelles, it seems to be a right time to draw a new 3D map of cell structure. In combination with visible light and electrons, X-ray microscopy paves a way to a comprehensive 3D phenotyping of cells. The human cell atlas within Chan Zuckerberg initiative and multi-scale model of the human pancreatic beta cell are two examples of such pioneering works [149]. With such need for X-ray microscopy, its future is predestined to be a new ‘workhorse' for 3D phenotypic analysis of cells.

Looking to the future

‘Automation of the acquisition and interpretation of data in microscopy has been a focus of biomedical research for almost a decade'. You might imagine this statement came from a recent publication or talk. However, it is the opening sentence of a paper by Prewitt and Mendelson published in 1966 [150]. The most valuable imaging techniques provide high content, fast, reproducible and reliable, ideally with moderate effort, data on the specimen of interest. X-ray microscopy is not an exception. The state-of-the art X-ray microscopes offer a quantitative analysis of the external and internal morphology of cells, cell cultures and even organism. They are indispensable to look at cells in 3D. However, three fundamental components, initially outlined by Prewitt and Mendelsohn, remain unresolved for X-ray microscopy. Primarily, most operating X-ray microscopes for biological research are located at synchrotron facilities, making them hard to access and not a complement to day-to-day imaging techniques used in most laboratories. Ideally, table-top scale X-ray microscopes should be collocated with light microscopes on and accessible at any time of the day. Current development of table-top and compact X-ray sources initiated first laboratory X-ray microscopy prototypes and proof-of-principal experiments [47,151]. The only commercialization of such X-ray microscopes, ideally at the affordable scale, can assure its benefit to cell biology. The next component is automated data processing, such as online tomographic reconstructions and automatic segmentation. To some extent, algorithms and pipelines can be adopted from the existing fast X-ray tomography routines [152,153]. However, for routine and reliable use, they have to be optimized for X-ray microscopy data [154]. The last but not the least is an automatic analysis of discrimination criteria. While looking at the beautiful 3D visualization of cell morphology is genuinely fascinating at the first time, it is not as stunning at the 100th of them, especially if it should be annotated, measured and compared with other datasets. Probably, all these developments will come soon with active users of X-ray microscopy.

Abbreviations

     
  • DESY

    Deutsches Elektronen-Synchrotron

  •  
  • SXT

    soft X-ray tomography

  •  
  • TXM

    transmission X-ray microscopes

Funding

V.W. is supported by German Research Foundation research fellowship WE 6221/1-1. The National Center for X-ray Tomography is supported by NIH (P41GM103445), DOE's Office of Biological and Environmental Research (DE-AC02-5CH11231) and the Gordon and Betty Moore Foundation.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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