Imaging using XFEL sources involves a competition between scattering and absorption of X-rays. We have investigated the effects of radiation damage and illumination variability on signal-to-noise ratio in the GroEL molecule. Gureyev et al., Acta Crystallographica A, 76, 664-676 (2020)

. . . angular intensity variations in SAXS data provide previously inaccessible information about local structures . . . and has the potential to impact diverse research areas of chemistry, biology and materials science.” A.V.Martin et al., Communications Materials, (2020)

Single-molecule imaging reveals biologically relevant heterogeneities within proteins and protein complexes. Rather than producing an average or dominant structure of a protein or complex, single-molecule techniques can capture the ‘heterogeneity landscape’ occupied by a single particle. The details of this landscape convey information about biochemical reactions and the functionality of biomolecules within living systems.



Imaging CoE scientists perform single-particle experiments that encompass X-ray free-electron laser single-particle imaging (XFEL-SPI), cryogenic electron microscopy (cryo-EM), micro-electron diffraction (micro-ED) and fluctuation microscopy. Each of these techniques faces different and significant challenges in order to achieve single-molecule imaging. It is not enough merely to achieve atomic-scale molecular imaging because this has no biological value if the molecule is not in its native state. The real challenge is to adapt and develop our techniques to better accommodate the intrinsic inhomogeneity of the systems we wish to image.

Imaging CoE researchers are advancing this field by combining XFEL-SPI or cryo-EM studies with complementary experiments using crystallography and molecular fluorescence. Such advances can come only though a collaboration spanning physics, chemistry and biology and involving both experiment and theory to determine the real behaviour of the target molecules under imaging conditions.

We have engaged with experimental work performed by Professor Ilme Schlichting, a member of the Imaging CoE International Scientific Advisory Committee, on the effects of electronic damage on pump-probe XFEL measurements of molecular dynamics. We developed closer links with the Hamburg-based CFEL group led by Imaging CoE Partner Investigator Professor Henry Chapman and recruited Dr. Andrew Morgan from Hamburg to join the Melbourne node of the Centre. He has recently taken up a DECRA Fellowship to continue this work for the next few years.


  1. Explored electronic damage processes in XFEL imaging techniques and developed new models for plasma formation in imaging experiments.
  2. Continued the development of speckle-tracking techniques and their use in the development of new lenses for XFEL experiments.
  3. Developed new methods of multiple scattering diffraction analysis and tomographic reconstruction using the Imaging CoE’s expertise across both theoretical analysis and experimental studies.
  4. Performed analysis of ion conformation effects in scattering experiments using empirical and ab initio models of aqueous dynamics.


Imaging CoE research has revealed challenges that will continue to attract attention into the future. The effects of electronic damage on the potential imaging applications of X-ray free-electron laser sources represents a challenge to theoretical models of electronic structure central to the underlying analysis of experimental scattering data used in structure determination.

Collaborating with Dr. Andrew Martin (RMIT) we have developed detailed models of these processes that include the formation of plasma during the imaging process and the effect this has on the interactions between atoms and their dynamics. Dr. Martin has developed insights into complex molecular processes using correlative approaches to the analysis of X-ray scattering data. These methods depend on the femtosecond snapshot capabilities of XFEL sources capturing structural correlations otherwise lost in synchrotron-based approaches. The multiple-scattering problem in electron scattering analysis has a long history; fortunately,

we have collaborated with a pioneer of this field, Professor Les Allen, in recent years. Dr. Tim Gureyev has led a wide collaboration of imaging experts to develop Differential Holographic Tomography algorithm that accommodates Fresnel diffraction and the effects of the curvature of the Ewald sphere in single-molecule imaging and performs well in the presence of multiple scattering. This new approach, which extends conventional applications employing image projections in tomographic reconstructions, shows early promise in improving imaging resolution in experimental cryo-EM data. It can in future gain from Bayesian or machine-learning approaches.