Optical coherence tomography imaging of plant root growth in soil.

Complex interactions between roots and soil provide the nutrients and physical support required for robust plant growth. Yet, visualizing the root-soil interface is challenged by soil's opaque scattering characteristics. Herein, we describe methods for using optical coherence tomography (OCT) to provide non-destructive 3D and cross-sectional root imaging not available with traditional bright-field microscopy. OCT is regularly used for bioimaging, especially in ophthalmology, where it can detect retinal abnormalities. Prior use of OCT in plant biology has focused on surface defects of above-ground tissues, predominantly in food crops. Our results show OCT is also viable for detailed, in situ study of living plant roots. Using OCT for direct observations of root growth in soil can help elucidate key interactions between root morphology and various components of the soil environment including soil structure, microbial communities, and nutrient patches. Better understanding of these interactions can guide efforts to improve plant nutrient acquisition from soil to increase agricultural efficiency as well as better understand drivers of plant growth in natural systems.

A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis.

The accumulation of bacteria in surface-attached biofilms can be detrimental to human health, dental hygiene, and many industrial processes. Natural biofilms are soft and often transparent, and they have heterogeneous biological composition and structure over micro- and macroscales. As a result, it is challenging to quantify the spatial distribution and overall intensity of biofilms. In this work, a new method was developed to enhance the visibility and quantification of bacterial biofilms. First, broad-spectrum biomolecular staining was used to enhance the visibility of the cells, nucleic acids, and proteins that make up biofilms. Then, an image analysis algorithm was developed to objectively and quantitatively measure biofilm accumulation from digital photographs and results were compared to independent measurements of cell density. This new method was used to quantify the growth intensity of Pseudomonas putida biofilms as they grew over time. This method is simple and fast, and can quantify biofilm growth over a large area with approximately the same precision as the more laborious cell counting method. Stained and processed images facilitate assessment of spatial heterogeneity of a biofilm across a surface. This new approach to biofilm analysis could be applied in studies of natural, industrial, and environmental biofilms.

Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain.

Rapid nondestructive measurement of bacterial cultures with 3D interferometric imaging.

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline and is a staple bioanalytical method for efforts ranging from research to standard clinical diagnostic tests. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we demonstrate white-light interferometry (WLI) as a practical tool for accelerated and improved measurement of bacterial cultures. High resolution WLI surface profile imaging was used for nondestructive characterization and counting of bacterial colonies on agar before they became visible to the naked eye. The three-dimensional (3D) morphology of Gram-negative (Pseudomonas fluorescens) and Gram-positive (Bacillus thuringiensis) bacterial species were monitored with WLI over time by collecting surface profiles of colonies on agar plates with high vertical resolution (3-5 nanometers) and large field of view (3-5 mm). This unique combination of sensitive vertical resolution and large field of view uniquely provided by WLI enables measurement of colony morphologies and nondestructive monitoring of hundreds of microcolonies. Individual bacteria were imaged within the first few hours after plating and colonies were accurately counted with results comparing favorably to counts made by traditional methods that require much longer wait times. Nondestructive imaging was used to track single cells multiplying into small colonies and the volume changes over time in these colonies were used to measure their growth rates. Based on the results herein, bioimaging with WLI was demonstrated as a novel rapid bacterial culture assay with several advantageous capabilities. Fast nondestructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates and colony morphology with this method may be beneficial in research and clinical applications where current methods are either too slow or are destructive.

Monitoring bacterial biofilms with a microfluidic flow chip designed for imaging with white-light interferometry.

There is a need for imaging and sensing instrumentation that can monitor transitions in a biofilm structure in order to better understand biofilm development and emergent properties such as anti-microbial resistance. Herein, we describe the design, manufacture, and use of a microfluidic flow cell to visualize the surface structure of bacterial biofilms with white-light interferometry (WLI). The novel imaging chip enabled the use of this non-disruptive imaging method for the capture of high resolution three-dimensional profile images of biofilm growth over time. The fine axial resolution (3 nm) and the wide field of view (>1 mm by 1 mm) enabled the detection of biofilm formation as early as 3 h after inoculation of the flow cell with a live bacterial culture (Pseudomonas fluorescens). WLI imaging facilitated the monitoring of the early stages of biofilm development and subtle variations in the structure of mature biofilms. Minimally-invasive imaging enabled the monitoring of biofilm structure with surface metrology metrics (e.g., surface roughness). The system was used to observe a transition in the biofilm structure that occurred in response to exposure to a common antiseptic. In the future, WLI and the biofilm imaging cell described herein may be used to test the effectiveness of biofilm-specific therapies to combat common diseases associated with biofilm formation such as cystic fibrosis and periodontitis.

In situ non-destructive measurement of biofilm thickness and topology in an interferometric optical microscope.

Biofilms are ubiquitous and impact the environment, human health, dental hygiene, and a wide range of industrial processes. Biofilms are difficult to characterize when fully hydrated, especially in a non-destructive manner, because of their soft structure and water-like bulk properties. Herein a method of measuring and monitoring the thickness and topology of live biofilms of using white light interferometry is described. Using this technique, surface morphology, surface roughness, and biofilm thickness were measured over time without while the biofilm continued to grow. The thickness and surface topology of a P. putida biofilm were monitored growing from initial colonization to a mature biofilm. Measured thickness followed expected trends for bacterial growth. Surface roughness also increased over time and was a leading indicator of biofilm growth.

Antimycobacterial efficacy of silver nanoparticles as deposited on porous membrane filters.

Environmental mycobacteria pose a significant health burden. Non-tuberculous mycobacteria infections have been traced to water treatment networks, where mycobacterial biofilms are ubiquitous. Filters that remove potential pathogens have significant medical applications. The purpose of this study is to demonstrate that an antibacterial silver nanoparticle (AgNP) coating can prevent colonization and growth of a mycobacterial biofilm on a filter material. The antibacterial efficacy of commercially available AgNPs was measured against Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium marinum after 48 h in liquid culture. Nanoparticles were deposited on micro-porous track etched polycarbonate membranes. The growth of biofilms on the membranes was observed by microscopy and counting colony forming units. M. smegmatis was most susceptible to AgNPs, with a 98.7% reduction at 100 µM AgNP concentration. M. avium was reduced by 97.3% at 539 µM AgNP after 48 h. Deposited nanoparticles inhibited colonization and growth for both M. smegmatis and M. avium on the membrane surface. Similar to the liquid culture, M. avium (84.2% survival) was more resistant than M. smegmatis (0.03% survival).