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The general goal of this research is to use scanning probe microscopy as a cornerstone technology for the development of a multi-technique imaging test bed that allows concurrent spatially-resolved analytical examinations of interactions between microorganisms and surfaces found in food and agriculture system. Specific to this project, the test bed will be used to advance our fundamental understanding of: (i) the attachment of pathogenic microorganisms to natural, and/or engineered surfaces; (ii) the propagation of attach micro-organisms (including pathogens) to form biofilms; and (iii) survival of the pathogens within biofilms in food and agricultural systems. <P>Objective 1: Develop a multi-technique imaging test bed that offers several types of examinations including: scanning probe (e.g., atomic force and localized force manipulations and analyses), optical, electrochemical, and/or spatially-resolved spectral imaging techniques. <P>Objective 2:Develop analysis-enclosures, sensors, probes and/or analytical methodologies for the multi-technique imaging test bed to study important processes, interaction, and structures found in agricultural and food systems. <P> Objective 3: Use the test bed to study the pathogen/substratum interactions including attachment mechanisms, formation of microbial biofilms, and responses of the microorganisms to chemical agents.
NON-TECHNICAL SUMMARY: Our nation must protect our agriculture and food systems from both unintentional and deliberate acts of chemical and biological contamination that can degrade human health and/or weaken our economic base. For biological agents, improved protection of our agricultural assets will require an increased understanding of pathogen survival and propagation, discovering new treatment and preventative strategies. The purpose of this project is the development of a multi-technique analytical test bed to allow dynamic concurrent near- and far-field imaging of substances and microorganisms at solid surfaces. The technology will provide researchers with a novel system to: (i) examine a sample at the molecular and cellular scales under conditions that mimic natural environments, (ii) rapidly locate regions of interest for nano-scale examination, (iii) measure sample heterogeneity, (iv) obtain confirmatory evidence to substantiate research hypotheses from a single region of interest, and (v) provide data for predictive modeling of macro-scale processes. Information about microbial adhesion, survival, and propagation will provide key knowledge to help minimize future foodborne illness and crop damage.
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APPROACH: Scanning probe microscopy (SPM) is a family of analysis and imaging techniques that uses a cantilevered-probe system to interact with a sample to analyze localized regions of interests and/or map surface properties at the nano- and micro-scale. Techniques in SPM are classified either by the type of surface property being probed or the signal used for analysis. For example, atomic force microscopy (AFM) is a scanning probe technology that senses and maps the atomic forces (e.g., van der Waals) occurring between the atoms of the probe-tip and the nearest atoms on a surface as the tip is raster-scanned across the sample (or the sample is raster-scanned under a stationary tip). Since atomic forces are ubiquitous at surfaces, an atomic force microscope can analyze or image many types of solid surfaces, while operating in air, vacuum, or liquid. Probes can be particles, micron-size beads, or sharp nano-structures with high aspect ratios (with a few nanometers of tip radius), which are either fabricated or adhered onto the free end of flexible cantilevers which have various stiffness and geometries that are optimized for different applications. Probes also can be chemically modified and used to measure selective nano-scale interactive forces or properties. As a probe approaches the surface and the gap between the tip and surface is decreased to a few nanometers, the cantilever is deflected by force(s). The magnitude of the deflection is generally quantified with an extremely sensitive laser diode-based detection system, where the deflection can be used to calculate applied force for cantilevers with calibrated spring constants. The cantilever can be oscillated to increase sensitivity and minimize lateral forces that damage soft biological samples. The deflection can be used for feedback-based imaging modes and to quantify the forces between the probe-tip and a particular location on the surface or to apply known forces to a particular location to determine mechanical or viscoelastic properties of localized areas. By modifying the tip with magnetic or electrochemically active material, magnetic and electrochemical scanning probe examinations are possible. Sharpened optical probes in near-field scanning optical microscopy are used to exceed the spatial resolution limits of far-field techniques. In this project, a closed-loop SPM system will be combined with an inverted microscope and other analytical components to create a unique test bed that allows concurrent analysis for increase analytical power by allowing identification and examination of regions of interest and/or multi-parametric examinations of important localized sites within food and agriculture systems. Knowledge ascertained from these studies will be used to (i) promote the positive aspect of a given interaction, (ii) optimize mitigative treatments against problematic microorganisms, (iii) develop mathematical models, and/or (iv) aid in the development of devices.