Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

THE INFLUENCE OF SPECIMEN TOPOGRAPHY ON X-RAY MICROANALYSIS ELEMENT MAPPING

The effect of specimen topography on x-ray microanalysis element mapping was studied with an electron microprobe and a scanning electron microscope equipped for x-ray detection. Using the lemma and palea of rice inflorescences as models, we determined that specimen topography can physically limit the detection of x-rays and thus lead to erroneous element mapping data. Any geometrical point on a specimen interfering with a straight line from the point of excitation to the detector will cause an absorptive shadow area on the element map. Electrons impinging on a sample surface cause emissions to occur in all directions. Emissions with sufficient energy (x-rays and backscattered electrons) can strike a topographical point different from the location of the focused electron beam, causing detectable x-ray excitation. This phenomenon will also result in erroneous element map data. Methods of recognition of specimen topographical effects on x-ray microanalysis are discussed.     

ELECTRON BOMBARDMENT OF BIOLOGICAL MATERIALS : II. THE RATE OF DEATH OF FUNGUS SPORES BOMBARDED IN VACUUM WITH CATHODE RAY BEAMS FROM 4 KV.–15 KV.

A study has been undertaken of the rate of inactivation of spores of the ascomycete fungus Aspergillus niger when bombarded in vacuum, with homogeneous beams of cathode rays of energies from 4 to 15 electron kv. and current densities of 1 x 10^–7 to 3 x 10^–6 amperes per square cm. These velocities and densities are in the range of those of showers of secondary electrons produced in biological materials irradiated with moderately soft x-rays, and so may be made to serve as quantitative indicators of the mechanics of x-ray action. Four qualitative effects are described.     

Contact x-ray microscopy. A new technique for imaging cellular fine structure.

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.     

Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes

A new form of super-resolution fluorescence microscopy has emerged in recent years, based on the high accuracy localization of individual photo-switchable fluorescent labels. Image resolution as high as 20 nm in the lateral dimensions and 50 nm in the axial direction has been attained with this concept, representing an order of magnitude improvement over the diffraction limit. The demonstration of multicolor imaging with molecular specificity, three-dimensional (3D) imaging of cellular structures, and time-resolved imaging of living cells further illustrates the exciting potential of this method for biological imaging at the nanoscopic scale.     

Near Field Scanning Optical Microscopy (NSOM): Development and Biophysical Applications

A new method for high-resolution imaging, near-field scanning optical microscopy (NSOM), has been developed. The concepts governing this method are discussed, and the technical challenges encountered in constructing a working NSOM instrument are described. Two distinct methods are presented for the fabrication of well-characterized, highly reproducible, subwavelength apertures. A sample one-dimensional scan is provided and compared to the scanning electron micrograph of a test pattern. From this comparison, a resolution of > 1,500 Å (i.e., λ/3.6) is determined, which represents a significant step towards our eventual goal of 500 Å resolution. Fluorescence has been observed through apertures smaller than 600 Å and signal-to-noise calculations show that fluorescent imaging should be feasible. The application of such imaging is then discussed in reference to specific biological problems. The NSOM method employs nonionizing visible radiation and can be used in air or aqueous environments for nondestructive visualization of functioning biological systems with a resolution comparable to that of scanning electron microscopy.     

Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.     

From functional genomics to functional immunomics: new challenges, old problems, big rewards

The development of DNA microarray technology a decade ago led to the establishment of functional genomics as one of the most active and successful scientific disciplines today. With the ongoing development of immunomic microarray technology—a spatially addressable, large-scale technology for measurement of specific immunological response—the new challenge of functional immunomics is emerging, which bears similarities to but is also significantly different from functional genomics. Immunonic data has been successfully used to identify biological markers involved in autoimmune diseases, allergies, viral infections such as human immunodeficiency virus (HIV), influenza, diabetes, and responses to cancer vaccines. This review intends to provide a coherent vision of this nascent scientific field, and speculate on future research directions. We discuss at some length issues such as epitope prediction, immunomic microarray technology and its applications, and computation and statistical challenges related to functional immunomics. Based on the recent discovery of regulation mechanisms in T cell responses, we envision the use of immunomic microarrays as a tool for advances in systems biology of cellular immune responses, by means of immunomic regulatory network models.     

Biomolecular sample considerations essential for optimal performance from cryogenic probes

For compounds dissolved in non-polar solvents, nuclear magnetic resonance spectroscopic investigations have benefited greatly from the advent of cryogenically cooled probes. Unfortunately the allure of significant increases in sensitivity may not be realized for compounds such as metabolites that are dissolved in solvents with high ionic-strengths such as solutions typically utilized for metabolomic or biomolecular investigations. In some cases there is little benefit from a cryogenically cooled probe over a conventional room temperature probe. Various sample preparation methods have been developed to minimize the detrimental effects of salt; for large numbers of metabolomic samples these preparation methods tend to be onerous and impractical. An alternative to manipulating the sample, is to utilize a probe that is designed to have a higher tolerance for solutions with high ionic-strengths. In order to acquire high-quality optimal data and choose the appropriate probe configuration (especially important for comparative quantitative investigations) the effects of salts and buffers on cryogenic probe performance must be understood. Herein we detail sample considerations for two cryogenic probes, a standard 5 mm and a narrow diameter 1.7 mm, in an effort to identify via integrals, intensities and noise levels the optimal choice for biomolecular investigations.     

Correlative Nanoscale 3D Imaging of Structure and Composition in Extended Objects

Structure and composition at the nanoscale determine the behavior of biological systems and engineered materials. The drive to understand and control this behavior has placed strong demands on developing methods for high resolution imaging. In general, the improvement of three-dimensional (3D) resolution is accomplished by tightening constraints: reduced manageable specimen sizes, decreasing analyzable volumes, degrading contrasts, and increasing sample preparation efforts. Aiming to overcome these limitations, we present a non-destructive and multiple-contrast imaging technique, using principles of X-ray laminography, thus generalizing tomography towards laterally extended objects. We retain advantages that are usually restricted to 2D microscopic imaging, such as scanning of large areas and subsequent zooming-in towards a region of interest at the highest possible resolution. Our technique permits correlating the 3D structure and the elemental distribution yielding a high sensitivity to variations of the electron density via coherent imaging and to local trace element quantification through X-ray fluorescence. We demonstrate the method by imaging a lithographic nanostructure and an aluminum alloy. Analyzing a biological system, we visualize in lung tissue the subcellular response to toxic stress after exposure to nanotubes. We show that most of the nanotubes are trapped inside alveolar macrophages, while a small portion of the nanotubes has crossed the barrier to the cellular space of the alveolar wall. In general, our method is non-destructive and can be combined with different sample environmental or loading conditions. We therefore anticipate that correlative X-ray nano-laminography will enable a variety of in situ and in operando 3D studies.     

Distinct conformers of amyloid beta accumulate in the neocortex of patients with rapidly progressive Alzheimer's disease

Amyloid beta (Aβ) deposition in the neocortex is a major hallmark of Alzheimer''s disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain–like model of disease heterogeneity suggests the existence of different conformers of Aβ. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aβ and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aβ conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of Aβ with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aβ plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs—quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of Aβ deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of Aβ accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aβ conformers (strains) in the rapid progression of AD.     

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.     

Effects of Macromolecular Crowding on the Structure of a Protein Complex: A Small-Angle Scattering Study of Superoxide Dismutase

Macromolecular crowding can alter the structure and function of biological macromolecules. We used small-angle scattering to measure the effects of macromolecular crowding on the size of a protein complex, SOD (superoxide dismutase). Crowding was induced using 400 MW PEG (polyethylene glycol),TEG (triethylene glycol), α-MG (methyl-α-glucoside), and TMAO (trimethylamine n-oxide). Parallel small-angle neutron scattering and small-angle x-ray scattering allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. Considering the osmotic pressure due to PEG, this deformation corresponds to a highly compressible structure. Small-angle x-ray scattering done in the presence of TEG suggests that for further deformation—beyond a 9% decrease in volume—the resistance to deformation may increase dramatically.     

Mammalian intestinal allometry,phylogeny, trophic level and climate

An often-stated ecomorphological assumption that has the status of ‘textbook knowledge’ is that the dimensions of the digestive tract correlate with diet, where herbivores—consuming diets of lower digestibility—have longer intestinal tracts than faunivores—consuming diets of higher digestibility. However, statistical approaches have so far failed to demonstrate this link. Here, we collated data on the length of intestinal sections and body mass of 519 mammal species, and test for various relationships with trophic, climatic and other biological characteristics. All models showed a strong phylogenetic signal. Scaling relationships with body mass showed positive allometry at exponents greater than 0.33, except for the caecum, which is particularly large in smaller species. Body mass was more tightly linked to small intestine than to large intestine length. Adding a diet proxy to the relationships increased model fit for all intestinal sections, except for the small intestine when accounting for phylogeny. Thus, the diet has a main effect on the components of the large intestine, with longer measures in herbivores. Additionally, measures of habitat aridity had a positive relationship with large intestine length. The small intestine was longer in species from colder habitats at higher latitudes, possibly facilitating the processing of peak intake rates during the growing season. This study corroborates intuitive expectations on digestive tract anatomy, while the dependence of significant results on large sample sizes and inclusion of specific taxonomic groups indicates that the relationships cannot be considered fixed biological laws.     

Advances in micro-CT imaging of small animals

PurposeMicron-scale computed tomography (micro-CT) imaging is a ubiquitous, cost-effective, and non-invasive three-dimensional imaging modality. We review recent developments and applications of micro-CT for preclinical research.MethodsBased on a comprehensive review of recent micro-CT literature, we summarize features of state-of-the-art hardware and ongoing challenges and promising research directions in the field.ResultsRepresentative features of commercially available micro-CT scanners and some new applications for both in vivo and ex vivo imaging are described. New advancements include spectral scanning using dual-energy micro-CT based on energy-integrating detectors or a new generation of photon-counting x-ray detectors (PCDs). Beyond two-material discrimination, PCDs enable quantitative differentiation of intrinsic tissues from one or more extrinsic contrast agents. When these extrinsic contrast agents are incorporated into a nanoparticle platform (e.g. liposomes), novel micro-CT imaging applications are possible such as combined therapy and diagnostic imaging in the field of cancer theranostics. Another major area of research in micro-CT is in x-ray phase contrast (XPC) imaging. XPC imaging opens CT to many new imaging applications because phase changes are more sensitive to density variations in soft tissues than standard absorption imaging. We further review the impact of deep learning on micro-CT. We feature several recent works which have successfully applied deep learning to micro-CT data, and we outline several challenges specific to micro-CT.ConclusionsAll of these advancements establish micro-CT imaging at the forefront of preclinical research, able to provide anatomical, functional, and even molecular information while serving as a testbench for translational research.     

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 µm x 200 µm x 200 µm using LED illumination, to 5 mm x 5 mm x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.     

Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells

In subcellular light-sheet fluorescence microscopy (LSFM) of adherent cells, glass substrates are advantageously rotated relative to the excitation and emission light paths to avoid glass-induced optical aberrations. Because cells are spread across the sample volume, three-dimensional imaging requires a light-sheet with a long propagation length, or rapid sample scanning. However, the former degrades axial resolution and/or optical sectioning, while the latter mechanically perturbs sensitive biological specimens on pliant biomimetic substrates (e.g., collagen and basement membrane). Here, we use aberration-free remote focusing to diagonally sweep a narrow light-sheet along the sample surface, enabling multicolor imaging with high spatiotemporal resolution. Further, we implement a dithered Gaussian lattice to minimize sample-induced illumination heterogeneities, significantly improving signal uniformity. Compared with mechanical sample scanning, we drastically reduce sample oscillations, allowing us to achieve volumetric imaging at speeds of up to 3.5 Hz for thousands of Z-stacks. We demonstrate the optical performance with live-cell imaging of microtubule and actin cytoskeletal dynamics, phosphoinositide signaling, clathrin-mediated endocytosis, polarized blebbing, and endocytic vesicle sorting. We achieve three-dimensional particle tracking of clathrin-associated structures with velocities up to 4.5 μm/s in a dense intracellular environment, and show that such dynamics cannot be recovered reliably at lower volumetric image acquisition rates using experimental data, numerical simulations, and theoretical modeling.     

Uptake of iron nanoparticles by Aphanorrhegma patens (Hedw.) Lindb.

Iron nanoparticles were produced in a radiofrequency levitation furnace and their dimensions and purity determined by scanning electron microscopy and x-ray microanalysis. Confocal microscopy revealed that these particles penetrated the leaves of Aphanorrhegma when applied to the plants as mineral water suspensions. This, the first demonstration of nanoparticle uptake by a bryophyte, opens the way to nanotoxicological studies paralleling those in higher plants. The actual penetration mechanism in both groups remains unknown.     

Clonogenic survival of human keratinocytes and rodent fibroblasts after irradiation with 25 kV x-rays

Low energy x-rays (E_ph 50 keV) are widely used in diagnostic radiology and radiotherapy. However, data on their relative biological effectiveness (RBE) are scarce. Of particular importance for risk estimation are the RBE values of x-rays in the range which is commonly used in mammography (10–30 keV). We have determined clonogenic survival after low-energy x-ray irradiation for three cell lines: primary human epidermal keratinocytes (HEKn), mouse fibroblasts (NIH/3T3) and Chinese hamster fibroblasts (V79). Experiments were performed with a 25 kV x-ray tube and compared to 200 kV x-rays as a reference. Compared to the effect of 200 kV x-rays, irradiation with 25 kV x-rays resulted in a decreased survival rate in the murine fibroblasts, but not in the human epithelial cell line. The RBE value was calculated for 10% surviving fraction. For HEKn cells, RBE was 1.33±0.27, for NIH/3T3 cells 1.25±0.07 and for V79 cells 1.10±0.09, respectively. No consistently increased RBE was observed in the various cell lines. Nevertheless, a potential of increased cytogenetic changes has to be considered for risk estimation of low-energy x-rays.