The imaging techniques that are currently available — including X-ray crystallography, cryo-EM and super-resolution microscopy — all started as cutting-edge research in physics. In addition to these established imaging technologies our physics program is conducting cutting-edge research in emerging fields largely driven by the recent development of X-ray Free Electron Laser (XFEL) facilities.
Our physics program is involved with the Single Particle Imaging (SPI) initiative that is being driven by LCLS to realise the long held ambition of determining the structure of biomolecules in their native state without crystallisation.
The earlier suggestions that free electron lasers could be used to image biomolecules emphasised the brightness of the pulses to increase the number of scattered photons. There are, however, many other processes that occur when a pulse interacts with matter. Photons are absorbed exciting inner shell electrons into the continuum. The internal structure of the molecules then rearranges emitting more electrons and photons the molecule then becomes highly charge recapturing some of the emitted electrons. Finally, the molecule explodes. Very little is known about the detailed descriptions of these processes in systems as complex as biomolecules.
Femtosecond nanocrystallography: The ultra fast nature of XFEL pulses offers some protection against the degradation of information caused by radiation damage. There is mounting evidence to suggest that femtosecond nanocrystallography may offer improved resolution for a given sample. And, may allow the use of crystals that are too small to be accessible to conventional crystallography using synchrotron sources.
Theoretical modelling: The interaction physics of XFEL pulses and molecules is a special case of the theory of quantum electro dynamics (QED) while the fundamental principles of this field are well understood their application to studies of biomolecules is made difficult by the complexity of the target and by the intensity of the pulse.
We are developing detailed theoretical and computational models to describe the molecular structures throughout the entire duration of the pulse from the ground state that we wish to determine to the ultimate coulomb experiment due to ionization. The approaches of the Imaging Centre utilize expertise borrowed from atomic and molecular physics, quantum chemistry and molecular dynamics to span the spatial and temporal ranges encountered in current experiments.
Image reconstruction: The determination of molecular structures from crystallographic data is a well established technology that gas been developed for protein crystallography. For systems that are not crystals, however, the methods of coherent diffractive imaging are required to determine the electron density of the target molecules from measurements of the scattered intensities of X-rays. The Imaging Centre has developed a large number of algorithms able to reconstruct images taking into account the degradation of the sample due to damage and the imperfections of the source due to partial spatial and temporal coherence.
Target orientation: When the molecular targets are presented to the XFEL pulses it is not practical to control their relative orientation. The data collected therefore correspond to any of the allowable orientations and the challenge is to assemble these data into a complete three dimensional diffraction volume. The identification of a particular orientation corresponding to a particular diffraction set is a challenge especially if we take account of the degradation of these images due to damage processes. The Imaging Centre is a participant in a number of international collaborations that work towards the classification and processing of data taken from randomly orientated samples.
Sample handling and delivery
One of the greatest challenges is coordinating the interception of the target sample by the XFEL pulse. In particular we wish to image biomolecules in a state as close to their natural environment as possible. While crystallography has been the backbone of structural biology for over 60 years the ideal scenario would deliver hydrated samples in their natural confirmations to the pulses for imaging in a non-crystalline state.
Nanofabrication: Imaging Centre scientists have developed expertise in nanofabrication techniques that enable us to produce sample holders, test samples and sample delivery systems using local and in-house resources. This facilitates our own innovation program for the determination of structures using both XFEL and synchrotron sources.
Microfluidics: The manipulation of nanoparticles using microfluidic devices will enable the Imaging Cenre to perform studies of crystallization and reaction dynamics by exploiting the time resolution of XFEL sources. These devices can be produced in-house at comparatively low cost and incorporated within our program of single particle imaging.
Laser pumping: The spectroscopy of target molecules can be controlled using laser-pumping techniques that are coordinated with the XFEL pulse delivery and sample delivery in the interaction region. Control of the electronic state of the target will enable us to study changes in structure that are driven by interactions with optical photons.
Aerosols: Careful engineering of aerosol nozzles facilitates the controlled delivery of individual particles into the XFEL beam. This produces an aerosol jet of samples that may also be hydrated and which may be focused into the interaction region. One of the target developments of the Imaging Centre is an aerosol delivery system that can be tested using local facilities.
Liquid Injection: Target molecules may be maintained in a controlled aqueous environment and injected within a collimated liquid jet into the interaction region this guarantees delivery of fully hydrated molecules but brings its own challenges with regard to the background signal that accompanies the liquid delivery system.
Sample handling and delivery: Keith Nugent and Brian Abbey
Interaction physics: Harry Quiney