A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Improving electrocoagulation floatation for harvesting microalgae.

Electro-coagulation floatation (ECF) is a foam-floatation dewatering method that has been shown to be a highly effective, rapid, and scalable separation methodology. In this manuscript, an in-depth analysis of the gas and flocculant levels observed during the process is provided, with microbubbles observed in the 5-80 µm size range at a concentration of 102-103 bubbles mL-1. Electrolysis of microalgae culture was then observed, demonstrating both effective separation using aluminium electrodes (nine microalgal species tested, 1-40 µm size range, motile and non-motile, marine and freshwater), and sterilisation of culture through bleaching with inert titanium electrodes. Atomic force microscopy was used to visualise floc formation in the presence and absence of algae, showing nanoscale structures on the magnitude of 40-400 nm and entrapped microalgal cells. Improvements to aid industrial biotechnology processing were investigated: protein-doping was found to improve foam stability without inducing cell lysis, and an oxalate buffer wash regime was found to dissolve the flocculant whilst producing no observable difference in the final algal lipid or pigment profiles, leaving the cells viable at the end of the process. ECF separated microalgal culture had an algal biomass loading of 13% and as such was ideal for direct down-stream processing through hydrothermal liquefaction. High bio-crude yields were achieved, though this was reduced slightly on addition of the Al(OH)3 after ECF, with carbon being distributed away to the aqueous and solid residue phases. The amenability and compatibility of ECF to integration with, or replacement of, existing centrifugation and settling processes suggests this process may be of significant interest to the biotechnology industry.

Algal Viruses: The (Atomic) Shape of Things to Come.

Visualization of algal viruses has been paramount to their study and understanding. The direct observation of the morphological dynamics of infection is a highly desired capability and the focus of instrument development across a variety of microscopy technologies. However, the high temporal (ms) and spatial resolution (nm) required, combined with the need to operate in physiologically relevant conditions presents a significant challenge. Here we present a short history of virus structure study and its relation to algal viruses and highlight current work, concentrating on electron microscopy and atomic force microscopy, towards the direct observation of individual algae⁻virus interactions. Finally, we make predictions towards future algal virus study direction with particular focus on the exciting opportunities offered by modern high-speed atomic force microscopy methods and instrumentation.