Imaging Bacterial Biofilms with Neutral Atom Microscopy

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Poster Session Two
Dr Katherine Brown (1, 3), Dr Benjamin Butler (1), Dr Matthew Bergin (2), Dr David Ward (1), Mr Nick von Jeinsen (1), Professor Paul Dastoor (2)
1. Cavendish Laboratory, University of Cambridge
2. University of Newcastle
3. The Oden Institute for Computational Engineering and Sciences, The University of Texas at Austin

Neutral Atom Microscopy; Bacteria; Pathogen; Biofilm

Abstract text

Summary: Neutral atom microscopy studies were undertaken on bacterial biofilms grown from Pseudomonas aeruginosa strain PA14. The atom microscopy images reveal unique atom beam contrast arising from distinct surface features different to those seen by electron microscopy. These results demonstrate that the SHeM can directly interrogate the biofilm surface without altering the biomaterials in any way (damage, coating, staining, or labelling). 

Introduction: Biological microscopy has evolved a long way beyond the imaging of biological organisms simply for identification and classification purposes. Currently, there is an international scientific focus on the use of biological imaging to study how bacterial systems perceive and respond to mechanical cues. In particular, driven by the mechanosensing processes, bacterial pathogens typically form biofilms, facilitating growth and offering them protection against antibiotics, complicating current treatment efforts. However, probing biofilms using conventional microscopies (light/electron/ion) represents a considerable challenge due to damage and induced movement from the incident imaging beam. Thus, there is an urgent need for the development of new non-damaging tools to shed new light on biofilm formation.

The scanning helium microscope (SHeM) is a new instrument that uses neutral helium atoms to form the image, rather than light or electrons. The imaging process is entirely non-destructive since the ultra-low energy (meV) neutral helium beam provides a chemically, electrically, and magnetically inert probe of the sample surface. The specific nature of the SHeM probe-sample interaction is well understood [1] and over the past decade the field has become well established [2–5]. Neutral helium scatters from the outermost electronic corrugation of the sample; giving SHeM its extreme surface sensitivity and non-destructive qualities. Elastically scattered helium atoms provide topographical contrast, which is the dominant mechanism in SHeM images, whereas inelastically scattered helium atoms deliver further unique imaging mechanisms; enabling chemical [6] and diffraction based contrast [7] of materials and structures. Thus, the SHeM enables a wide range of delicate surfaces to be studied for the first time.

The aim of this work was to determine the unique contrast mechanisms exhibited when neutral atom microscopy is used to probe biofilm formation. In particular, it is known that the extracellular matrix (ECM) plays a key role in the establishment and progression of bacterial biofilms, with important implications for new therapeutic approaches. 

Methods/Materials: Pseudomonas aeruginosa strain PA14 forms biofilms in a wide range of ecological niches (e.g., surfaces and solutions) and is a highly virulent strain that causes disease in organisms including humans. Biofilms were produced from a dense culture, harvested in the late-log phase of growth. For SHeM imaging the helium source was operated at 200 bar, 297 K stagnation temperature and with a 10 μm nozzle, with the resultant free-jet expansion progressively apertured by a Beam Dynamics skimmer (Model 2, 100 μm diameter) and a pinhole (5 μm diameter). During imaging, the sample chamber pressure was typically of the order 1 × 10−8 mbar.

Results and Discussion: The SHeM images reveal surface features distinctly different to those seen by the SEM. In particular, the helium atom beam appears highly sensitive to the surface topography of the ECM surrounding the bacterial cells, whereas the electron beam passes through the ECM and images the subsurface bacterial colonies This hypothesis is further confirmed by SHeM-confocal images of the Concanavalin A stain, which is a carbohydrate-binding protein) that indicates the presence of the ECM. When the SHeM and confocal images are overlaid, SHeM contrast is clearly seen in regions between bacteria that also have ECM present.

Conclusion: Neutral atom microscopy is well-suited to imaging delicate biological materials such as bacterial biofilms. The first SHeM images of PA14 bacterial biofilms highlight the instruments extreme sensitivity to the surface topography of the extra-cellular matrix surrounding the bacteria.

Funding: EOARD (RG96360A) and the University of Texas Planet Texas 2050 Bridging Barriers Initiative. Australian Research Council Linkage Program.

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