Novel imaging technologies for characterization of microbial extracellular polysaccharides

Abstract

Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects.

Keywords: AFM; SHG; high resolution AFM; superresolution optical microscopy.

FIGURE 1

Atomic force (A,C) and electron microscopic (B) images of cyanobacterial polysacaccharide sacran. The atomic force microcsopy (AFM) topograph (A) was obtained from a 1 ppm sacran aqueous aliquot dried on mica. The white arrow highlights a bundle of sacran chains. (B) Transmisssion electron micrograph of sacran dried on carbon-coated Cu-grid and schematic illustration of intertwined (helical) structure adopted by sacran chains as a possible interpretation of the TEM micrograph. (C) AFM topograph of an aliquot of 0.1 mg/ml sacran aqueous solution dried on mica. Scale bars for lateral dimensions (A–C) and height (A,C) are indicated on the topographs. Reprinted with permission from Okajima et al. (2009). Copyright (2009) American Chemical Society.

FIGURE 2

Atomic force microcsopy topographs of chitosan– xanthan polyelectrolyte complexes (PECs) as prepared at low concentrations (xanthan concentration of 2 μg/ml and chitosan with degree of acetylation 0.49 at a concentration of 10 μg/ml) at room temperature and treated at 44%#x000B0;C for 30 min (A) and following annealing for 30 min at a temperature of 90%#x000B0;C (B). Reproduced with with permission from Maurstad and Stokke (2004). Copyright (2004) Wiley.

FIGURE 3

High speed AFM imaging of Trocoderma reesei Cel7A cellulase acting on crystalline I_α cellulose. (A) AFM topographs of the same area of the cellulose crystal imaged at intervals of 0.9 s revealing different localization of individual cellulases. (B) Time dependence from the initial position of individual cellulase positions when acting on cellulose crystal as deduced by image processing of HS-AFM topographs. The colored symbols reflect data extracted from individual enzymes whereas the dotted line depict an average velocity as reported (Igarashi et al., 2009). Reprinted with permission from Igarashi et al. (2011). Copyright (2011) AAAS.

FIGURE 4

Force modulation AFM topographs of plasmid pUC18 DNA (2686 basepairs) in aqueous solution. The aqueous solution contained Ni²⁺ to stabilize the interaction between DNA and mica. (A) High resolution force modulation AFM topograph of a section of the duplex DNA with major (red arrows) and minor (blue arrows) grooves identified along the B-DNA double-helical structure. The white dotted area of the topograph in (A) are shown at higher magnification in (B). (B) Height variations along the (A–B) cross-sectional line are shown in the original publication. Height profiles along the L_exp and R_exp lines are compared with simulated data in the original publication. Reprinted with permission from Ido et al. (2013). Copyright (2013) American Chemical Society.

FIGURE 5

(A) Sectretion of Vibrio polysaccharide (VPS) stained with Cy3 attached to wheat germ agglutinin (green). (B) 3D STORM superresolution image of a single cell showing sectretion of VPS around a single cell. The color represents height in the z-direction. (C–F) Multicolor microscopy images of VPS (red) and RbmC (green). (C,D,F) are STORM superresolution images while (E) is a conventional confocal of the same region as (F), illustrating the increased understanding of the organization of VPS and RbmC in superresolution microscopy. (G) STORM image showing the individual localization points as white dots. Reprinted with permission from Berk et al. (2012). Copyright AAAS.