Remarkable progress has been made over the past few decades in biological imaging, from the atomic (subnanometer) scale through the cellular (micron) scale, creating an opportunity of extraordinary scope and significance for scientists working to understand and harness biological systems for addressing mission-relevant challenges of the Department of Energy’s (DOE) Office of Biological and Environmental Research (BER). Many of these advances are made possible by using unique resources of DOE national user facilities available at the neutron and synchrotron light sources funded by the Office of Basic Energy Sciences. Studying biological structures across a wide range of length and time scales—from molecules to complex biological machines to cellular and tissue architecture—is becoming possible using these X-ray and neutron techniques, often together with complementary advanced optical- and electron-based methodologies and computational tools. Living matter and native cellular structures can now be imaged at or near atomic-level resolution, and molecular structures can be studied with high temporal resolution, enabling an understanding of fundamental physical and chemical processes that control dynamic biological pathways. Enabling techniques and instruments have been developed with BER support at five of DOE’s national user facilities (see the Facilities page). An overview of these techniques and the kinds of BER mission-relevant scientific information they can provide is found here.
X-Ray Macromolecular Crystallography (MC)
This is a widely used technique based on diffraction from crystalline biological materials [including proteins, large protein complexes, and nucleic acids (RNA and DNA)] to obtain high-resolution structural information (often in the ~1–2 Å range). MC requires that the biomolecules be crystallized, but can provide specific atom locations in very complex systems, enabling detailed insight into how these macromolecules carry out their functions in living cells and organisms.
Neutron Macromolecular Crystallography
Neutron MC provides complementary structural information to its X-ray based counterpart, described above. Because photons and electrons interact with the atomic electric field, hydrogen (H) is all but invisible to them. In contrast, neutrons interact with nuclei, making it possible to observe lighter elements such as H and deuterium (D) and distinguish these light elements next to heavy ones. Hence, study of H bonding networks and protonation states of catalytic residues is feasible. In addition, neutrons do not cause radiation damage as is often the case with X-rays, but significantly larger crystal volumes are required for neutron MC experiments as compared to X-rays.
Small-Angle X-Ray Scattering (SAXS)
In contrast to crystals, X-rays are scattered from non-periodic materials. The SAXS technique is used to study the size and shape of biological material including nucleic acids, proteins, protein assemblies, virus particles, and biological fibers as well as lipid membranes and membrane-protein/DNA complexes. It provides lower-resolution information (typically below 5–10 Å). Time-resolved SAXS can be used to investigate structural changes such as folding and conformational changes on a submillisecond timescale and longer, enabling studies of biomolecules close to their physiological state.
Small-Angle Neutron Scattering (SANS)
Like SAXS, SANS is used to study structures of non-periodic biological materials at lower resolution. SANS, however, can take advantage of the very different neutron scattering cross-sections of H and D making it possible to selectively highlight different components within a complex system. In combination with H2O/D2O contrast variation and D-labeling, SANS provides unique information about complexes of biomolecules and hierarchical structures (1–500 nm resolution). Ultra-SANS extends accessible length scales to several microns. Time-resolved SANS experiments are also possible, but accessible timescales are typically longer than for SAXS (seconds to minutes). Neutron reflectivity provides information about surface and interfacial structures at similar length scales accessible to SANS.
X-Ray Absorption and Emission Spectroscopy (XAS)
This suite of related techniques provides information on metal sites in biomolecules. X-ray absorption edge spectroscopy probes primarily electronic structure (oxidation state, bonding) and extended X-ray absorption fine structure (EXAFS) provides metrical information around the metal ion. Related techniques, collectively often called advanced X-ray spectroscopy (X-ray emission, resonant inelastic scattering, and X-ray Raman), can provide extensive and highly detailed electronic information on the metal. As metal ions have key roles in biological structure and function, including being active sites of many enzymes to shuttling electrons in key metabolic or signaling pathways, XAS methods provide very complementary information to that from X-ray crystallography and small-angle scattering studies. This technique also is applied to studies of biologically important ligands (carbon, nitrogen, sulfur, and chlorine) and their interactions with metals.
This is a two-dimensional (2D) technique that provides information about atomic motions in both time and space. Inelastic and quasi-elastic neutron scattering provide information about vibrational modes, molecular motions, and diffusive properties of biomolecules and their hydration water on the picosecond to nanosecond timescale. Neutron spin echo spectroscopy probes slower motions at microseconds to milliseconds such as motions associated with undulating membranes and domain motions in proteins.
There are several variants of X-ray imaging, including both 2D and 3D (tomography) and using both hard and soft X-rays. Soft X-ray tomography can be used to image biological materials such as whole, hydrated cells in 3D down to a resolution of around 50 nm. Hard X-ray tomography can provide 2D and 3D information on more strongly absorbing and less radiation sensitive biological materials with a resolution of 30 nm or less. A variant of X-ray imaging, called spectromicroscopy, provides spatially resolved information about metal distribution and chemical speciation in materials of biological and medical relevance, including tissues with resolutions typically from submicrons to millimeters.
Neutron imaging includes neutron radiography and computed tomography. Taking advantage of H/D contrast and the nondestructive nature of neutrons, it is possible to study the structure and dynamics of a wide range of hierarchical and complex materials of biological relevance at a resolution of ~50 µm. Examples include transport and interactions of fluids in porous media, plant-plant and plant-fungal interactions, pore structure and voids in soil under environmentally relevant conditions, and cavitation and gas embolism in plant-soil-groundwater systems.
Infrared (IR) Imaging
IR radiation probes vibrational modes of molecules. Hence it can be used to study chemical functional groups and their changes under reacting conditions and is nondestructive. IR can probe the chemistry in biological tissues, chemical identification and molecular conformation, and also serve as a fingerprint for molecular species. Resolution in 2D is typically 2-10 µm. 3D imaging also can be done, with a resolution of around 15 µm.
Multiscale Imaging (or Hybrid) Approaches
Structure and function in biology occurs across a wide range of distance (subnanometer to millimeter) and time (subpicosecond to seconds). No single method can access these spatial and temporal scales, so integrating methodologies to connect molecular properties to system-level functions is important. Examples include understanding the structures of macromolecules and their complexes, how interactions at the atomic level between macromolecules in these larger assemblies confer and control function (i.e., the workings of “molecular machines”), how these assemblies are organized and networked in the cellular environment, and, ultimately, how their pathways are regulated to keep the organism functional. Computational biology is essential for integrating and connecting these measurements across multiple scales of length and time. Besides the X-ray- and neutron-based techniques highlighted on this website, super resolution optical, electron microscopy, and magnetic resonance imaging also play important roles.