During its intra-erythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, outside the confines of its own plasma membrane. These structures are involved in trafficking of virulence proteins to the host cell surface however their ultrastructure is only partly defined and there is on-going debate regarding their origin, composition and organisation. We have developed a number of imaging paradigms to probe sub-cellular structure in parasitised red blood cells (RBCs).

Confocal microscopy has made a major contribution to our understanding of cell biology. It offers superb resolution and allows the selective capture of light from a particular plane within the specimen, thus allowing studies of proteins within intracellular compartments. The introduction of green fluorescent protein (GFP) as a versatile, specific and non-invasive probe of biological components has revolutionised studies of live cells. We have developed Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS) methods to probe protein dynamics in transfectanted malaria parasites expressing GFP chimeras.

Electron tomography is increasingly used as technique for obtaining 3-D information at resolutions better than 5 nm. We have employed electron tomography to image parasite-derived structures within the host RBC. We have characterised flattened structures with a complex and convoluted morphology. These structures have extensions with a more tubular profile that are modified with surface nodules, each with a circular cross-section of ~25 nm. Permeabilisation of infected RBCs with a pore-forming toxin, Equinatoxin II, allows the introduction of specific labels. We have used immunogold labelling to shows that different sub-structures have different protein components.

Non-crystallographic methods for imaging cellular architecture and, ultimately macromolecular complexes and individual membrane proteins, within a cellular environment, is a "holy grail" of cell and molecular biology. As part of the Centre of Excellence for Coherent X-ray Science we are developing methods to use X-ray Coherent Diffractive Imaging (CDI) to obtain information about the architecture of cells and biological macromolecules. CDI is a method of lensless imaging that can be applied to any individual finite object. A diffraction pattern from a single biological structure is recorded and an iterative Fourier transform between real space and reciprocal space is used to reconstruct information about the architecture and chemical composition of the sample. Highly intense and coherent X-rays are needed to obtain sufficient signal to noise. The phase and intensity profiles of malaria parasite-infected cells have been successfully reconstructed revealing the major features of the cells. The data suggest that the ultimate goal of obtaining 3D structural information from non-crystalline biological samples at a resolution of 10-40 nm is achievable.