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.

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.

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.

Visualizing subcellular rearrangements in intact β cells using soft x-ray tomography.

Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway.

Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography.

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine.

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.

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.

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 and An ADP/ATP translocase-expressing provided ATP to a respiration-deficient yeast mutant and enabled growth of a yeast- chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic thiamin auxotroph.

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.

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.

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.

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.

Correlative cryogenic tomography of cells using light and soft x-rays.

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved.

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.

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.

Nanoimaging cells using soft X-ray tomography.

Soft X-ray microscopy is ideally suited to visualizing and quantifying biological cells. Specimens, including eukaryotic cells, are imaged intact, unstained and fully hydrated, and therefore visualized in a near-native state. The contrast in soft X-ray microscopy is generated by the differential attenuation of X-rays by the molecules in the specimen-water is relatively transmissive to this type of illumination compared to carbon and nitrogen.

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.

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.

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.

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.

Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging.

Soft X-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50nm, or better. In SXT, cells are imaged using photons from a region of the spectrum known as the 'water window'.

Conformational change of the AcrR regulator reveals a possible mechanism of induction.

The Escherichia coli AcrR multidrug-binding protein represses transcription of acrAB and is induced by many structurally unrelated cytotoxic compounds. The crystal structure of AcrR in space group P222(1) has been reported previously. This P222(1) structure has provided direct information about the multidrug-binding site and important residues for drug recognition.