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.

Engineering yeast endosymbionts as a step toward the evolution of mitochondria.

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.

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.

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.

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.

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.

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.

A protocol for full-rotation soft X-ray tomography of single cells.

The protocol describes step-by-step sample preparation, data acquisition, and segmentation of cellular organelles with soft X-ray tomography. It is designed for microscopes built to perform full-rotation data acquisition on specimens in cylindrical sample holders, such as the XM-2 microscope at the Advanced Light Source, LBNL; however, it might be generalized for similar sample holder designs for both synchrotron and table-top microscopes. For complete details on the use and execution of this profile, please refer to Loconte et al. (2021).

Soft X-ray tomography to map and quantify organelle interactions at the mesoscale.

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic ß cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies.

Quantitative analysis of yeast internal architecture using soft X-ray tomography.

We used soft X-ray tomography (SXT)--a high-resolution, quantitative imaging technique--to measure cell size and organelle volumes in yeasts. Cell size is a key factor in initiating cell division in yeasts, whereas the number and volume of the organelles have a profound impact on the function and viability of a cell. Consequently, determining these cell parameters is fundamentally important in understanding yeast biology. SXT is well suited to this type of analysis. Specimens are imaged in a near-native state, and relatively large numbers of cells can be readily analysed. In this study, we characterized haploid and diploid strains of Saccharomyces cerevisiae at each of the key stages in the cell cycle and determined the relationships that exist cellular and organelle volumes. We then compared these results with SXT data obtained from Schizosaccharomyces pombe, the three main phenotypes displayed by the opportunistic yeast pathogen Candida albicans and from a coff1-22 mutant strain of S. cerevisiae. This comparison revealed that volumetric ratios were invariant, irrespective of yeast strain, ploidy or morphology, leading to the conclusion these volumetric ratios are common in all yeasts.

Switchable resolution in soft x-ray tomography of single cells.

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.