Summary of imaging and other selected measurement technologies discussed at the workshop. Mature technologies that will benefit from further development include X-ray and neutron crystallography and scattering, scanning probe microscopies, X-ray tomography, synchrotron spectroscopy, and con-focal microscopy. Techniques undergoing rapid development and with potential application to the mission of the U.S. Department of Energy’s Office of Biological and Environmental Research (BER) include X-ray free-electron laser ultrafast diffraction, time-resolved X-ray scattering, cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET), infrared imaging methods, super-resolution fluorescence imaging. New technologies not yet fully developed and applied to research supported by BER’s Biological Systems Science Division include dynamic EM, fluctuation scattering, ptychography, and in soil sensors. Key: Å, Angstrom; EXAFS, extended X-ray absorption fine structure; fs, femtosecond; ks, kilosecond; µm, micrometer; µs, microsecond; mm, millimeter; ms, millisecond; nm, nanometer; ns, nanosecond; ps, picosecond; s, second; SAXS, small-angle X-ray scattering; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TIRF, total internal reflection fluorescence; USANS, ultrasmall-angle neutron scattering; WAXS , wide-angle X-ray scattering; XANES, X-ray absorption near edge structure.
Remarkable progress has been made over the past few decades in biological imaging, from the atomic (sub-nm) through the cellular (micron) scale, creating an opportunity of extraordinary scope and significance for scientists working to understand and harness biological systems for addressing DOE-BER mission relevant challenges. Many of these advances are made possible by using unique properties of DOE National User Facilities available at the neutron and synchrotron light sources. Study of 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 the DOE National User Facilities (see the Facilities page). An overview of these techniques, and the kinds of information relevant to BER-mission scientific problems that they can provide, is found here.
X-ray Macromolecular Crystallography (MC)
This is a widely-used technique that is 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 detailed 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 the lighter elements such as H and deuterium (D) and distinguish these light elements next to heavy ones. Hence, study of hydrogen 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. 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, 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 sub-millisec time scale and longer, and enables 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). U-SANS extends accessible length-scales to several microns. Time-resolved SANS experiments are also possible but accessible time scales 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
This is a suite of related techniques that provide information on metal sites in biomolecules. X-ray absorption edge spectroscopy probes primarily electronic structure (oxidation state, bonding) and extended 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, 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. The technique is also applied to studies of biologically important ligands (C, N, S, Cl) and how they interact with the metals.
This is a two-dimensional 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 micro- to milliseconds such as motions associated with undulating membranes and domain motions in proteins.
There are several variants of x-ray imaging which include both 2-D and 3-D (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 2-D and 3-D 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 sub-µm’s to mm’s.
Neutron Imaging includes neutron radiography and computed tomography. Taking advantage of H/D contrast and the non-destructive nature of neutrons it is possible to study the structural and dynamics of a wide range 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, cavitation and gas embolism in plant-soil-groundwater systems
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 non-destructive. IR can probe the chemistry in biological tissues, chemical identification and molecular conformation and also serve as a fingerprint for molecular species. Resolution in 2-D is typically 2-10 µm. 3D imaging can also 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 (sub-nm to mm) and time (sub-psec to seconds). No single method can access this and hence integrating methodologies to connect molecular properties to system-level functions is important – e.g., understanding the structures of macromolecules and their complexes, how the 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 x-ray and neutron based techniques highlighted herein, super resolution optical, electron microscopy and magnetic resonance imaging also play important roles.