Soft X-ray tomograms provide a structural basis for whole-cell modeling.

Developing in silico models that accurately reflect a whole, functional cell is an ongoing challenge in biology. Current efforts bring together mathematical models, probabilistic models, visual representations, and data to create a multi-scale description of cellular processes. A realistic whole-cell model requires imaging data since it provides spatial constraints and other critical cellular characteristics that are still impossible to obtain by calculation alone. This review introduces Soft X-ray Tomography (SXT) as a powerful imaging technique to visualize and quantify the mesoscopic (~25 nm spatial scale) organelle landscape in whole cells. SXT generates three-dimensional reconstructions of cellular ultrastructure and provides a measured structural framework for whole-cell modeling. Combining SXT with data from disparate technologies at varying spatial resolutions provides further biochemical details and constraints for modeling cellular mechanisms. We conclude, based on the results discussed here, that SXT provides a foundational dataset for a broad spectrum of whole-cell modeling experiments.

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus.

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules' diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths.

Imaging cell morphology and physiology using X-rays.

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.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization.

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

PSF correction in soft X-ray tomography.

In this article, we introduce a linear approximation of the forward model of soft X-ray tomography, such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction of data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs.

Mesoscale imaging with cryo-light and X-rays: Larger than molecular machines, smaller than a cell.

In the context of cell biology, the term mesoscale describes length scales ranging from that of an individual cell, down to the size of the molecular machines. In this spatial regime, small building blocks self-organise to form large, functional structures. A comprehensive set of rules governing mesoscale self-organisation has not been established, making the prediction of many cell behaviours difficult, if not impossible. Our knowledge of mesoscale biology comes from experimental data, in particular, imaging. Here, we explore the application of soft X-ray tomography (SXT) to imaging the mesoscale, and describe the structural insights this technology can generate. We also discuss how SXT imaging is complemented by the addition of correlative fluorescence data measured from the same cell. This combination of two discrete imaging modalities produces a 3D view of the cell that blends high-resolution structural information with precise molecular localisation data.

Imaging and characterizing cells using tomography.

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft X-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft X-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft X-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells.

Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.

Putting molecules in their place.

Each class of microscope is limited to imaging specific aspects of cell structure and/or molecular organization. However, imaging the specimen by complementary microscopes and correlating the data can overcome this limitation. Whilst not a new approach, the field of correlative imaging is currently benefitting from the emergence of new microscope techniques. Here we describe the correlation of cryogenic fluorescence tomography (CFT) with soft X-ray tomography (SXT). This amalgamation of techniques integrates 3D molecular localization data (CFT) with a high-resolution, 3D cell reconstruction of the cell (SXT). Cells are imaged in both modalities in a near-native, cryopreserved state. Here we describe the current state of the art in correlative CFT-SXT, and discuss the future outlook for this method.

Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source.

Beamline 2.1 (XM-2) is a transmission soft X-ray microscope in sector 2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. XM-2 was designed, built and is now operated by the National Center for X-ray Tomography as a National Institutes of Health Biomedical Technology Research Resource. XM-2 is equipped with a cryogenic rotation stage to enable tomographic data collection from cryo-preserved cells, including large mammalian cells. During data collection the specimen is illuminated with `water window' X-rays (284-543 eV). Illuminating photons are attenuated an order of magnitude more strongly by biomolecules than by water. Consequently, differences in molecular composition generate quantitative contrast in images of the specimen. Soft X-ray tomography is an information-rich three-dimensional imaging method that can be applied either as a standalone technique or as a component modality in correlative imaging studies.

Visualizing and quantifying cell phenotype using soft X-ray tomography.

Soft X-ray tomography (SXT) is an imaging technique capable of characterizing and quantifying the structural phenotype of cells. In particular, SXT is used to visualize the internal architecture of fully hydrated, intact eukaryotic and prokaryotic cells at high spatial resolution (50 nm or better). Image contrast in SXT is derived from the biochemical composition of the cell, and obtained without the need to use potentially damaging contrast-enhancing agents, such as heavy metals. The cells are simply cryopreserved prior to imaging, and are therefore imaged in a near-native state. As a complement to structural imaging by SXT, the same specimen can now be imaged by correlated cryo-light microscopy. By combining data from these two modalities specific molecules can be localized directly within the framework of a high-resolution, three-dimensional reconstruction of the cell. This combination of data types allows sophisticated analyses to be carried out on the impact of environmental and/or genetic factors on cell phenotypes.

Nitrogen-fixing organelle in a marine alga.

Symbiotic interactions were key to the evolution of chloroplast and mitochondria organelles, which mediate carbon and energy metabolism in eukaryotes. Biological nitrogen fixation, the reduction of abundant atmospheric nitrogen gas (N2) to biologically available ammonia, is a key metabolic process performed exclusively by prokaryotes. Candidatus Atelocyanobacterium thalassa, or UCYN-A, is a metabolically streamlined N2-fixing cyanobacterium previously reported to be an endosymbiont of a marine unicellular alga. Here we show that UCYN-A has been tightly integrated into algal cell architecture and organellar division and that it imports proteins encoded by the algal genome. These are characteristics of organelles and show that UCYN-A has evolved beyond endosymbiosis and functions as an early evolutionary stage N2-fixing organelle, or "nitroplast."

Correlative microscopy methods that maximize specimen fidelity and data completeness, and improve molecular localization capabilities.

Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.

Visualizing cell architecture and molecular location using soft x-ray tomography and correlated cryo-light microscopy.

Living cells are structured to create a range of microenvironments that support specific chemical reactions and processes. Understanding how cells function therefore requires detailed knowledge of both the subcellular architecture and the location of specific molecules within this framework. Here we review the development of two correlated cellular imaging techniques that fulfill this need. Cells are first imaged using cryogenic fluorescence microscopy to determine the location of molecules of interest that have been labeled with fluorescent tags. The same specimen is then imaged using soft X-ray tomography to generate a high-contrast, 3D reconstruction of the cells. Data from the two modalities are then combined to produce a composite, information-rich view of the cell. This correlated imaging approach can be applied across the spectrum of problems encountered in cell biology, from basic research to biotechnological and biomedical applications such as the optimization of biofuels and the development of new pharmaceuticals.

The influence of spatial variation in chromatin density determined by X-ray tomograms on the time to find DNA binding sites.

In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process.

Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear-vacuolar junctions and perinuclear ESCRT.

Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear-vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear-vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear-vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.

Automatic alignment and reconstruction of images for soft X-ray tomography.

Soft X-ray tomography (SXT) is a powerful imaging technique that generates quantitative, 3D images of the structural organization of whole cells in a near-native state. SXT is also a high-throughput imaging technique. At the National Center for X-ray Tomography (NCXT), specimen preparation and image collection for tomographic reconstruction of a whole cell require only minutes. Aligning and reconstructing the data, however, take significantly longer. Here we describe a new component of the high throughput computational pipeline used for processing data at the NCXT. We have developed a new method for automatic alignment of projection images that does not require fiducial markers or manual interaction with the software. This method has been optimized for SXT data sets, which routinely involve full rotation of the specimen. This software gives users of the NCXT SXT instrument a new capability - virtually real-time initial 3D results during an imaging experiment, which can later be further refined. The new code, Automatic Reconstruction 3D (AREC3D), is also fast, reliable, and robust. The fundamental architecture of the code is also adaptable to high performance GPU processing, which enables significant improvements in speed and fidelity.

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum.

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer-Lambert's law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.

Soft X-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans.

The opportunistic pathogen Candida albicans can undergo phenotypic switching between a benign, unicellular phenotype and an invasive, multicellular form that causes candidiasis. Increasingly, strains of Candida are becoming resistant to antifungal drugs, making the treatment of candidiasis difficult, especially in immunocompromised or critically ill patients. Consequently, there is a pressing need to develop new drugs that circumvent fungal drug-resistance mechanisms. In this work we used soft X-ray tomography to image the subcellular changes that occur as a consequence of both phenotypic switching and of treating C. albicans with antifungal peptoids, a class of candidate therapeutics unaffected by drug resistance mechanisms. Peptoid treatment suppressed formation of the pathogenic hyphal phenotype and resulted in striking changes in cell and organelle morphology, most dramatically in the nucleus and nucleolus, and in the number, size, and location of lipidic bodies. In particular, peptoid treatment was seen to cause the inclusion of lipidic bodies into the nucleus.

Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells.

Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporospatial remodeling of cells during the infection and observed changes in cellular structures, such as the presence of cytoplasmic stress granules and multivesicular structures, formation of nuclear virus-induced dense bodies, and aggregates of capsids. Our results demonstrate the power of SXT imaging for scouting virus-induced changes in infected cells and understanding the orchestration of virus-host remodeling quantitatively.