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.

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.

In Situ High Pressure Hydrogen Tribological Testing of Common Polymer Materials Used in the Hydrogen Delivery Infrastructure.

High pressure hydrogen gas is known to adversely affect metallic components of compressors, valves, hoses, and actuators. However, relatively little is known about the effects of high pressure hydrogen on the polymer sealing and barrier materials also found within these components. More study is required in order to determine the compatibility of common polymer materials found in the components of the hydrogen fuel delivery infrastructure with high pressure hydrogen. As a result, it is important to consider the changes in physical properties such as friction and wear in situ while the polymer is exposed to high pressure hydrogen. In this protocol, we present a method for testing the friction and wear properties of ethylene propylene diene monomer (EPDM) elastomer samples in a 28 MPa high pressure hydrogen environment using a custom-built in situ pin-on-flat linear reciprocating tribometer. Representative results from this testing are presented which indicate that the coefficient of friction between the EPDM sample coupon and steel counter surface is increased in high pressure hydrogen as compared to the coefficient of friction similarly measured in ambient air.

An in situ tribometer for measuring friction and wear of polymers in a high pressure hydrogen environment.

High pressure hydrogen effects on the friction and wear of polymers are of importance to myriad applications. Of special concern are those used in the infrastructure for hydrogen vehicle refueling stations, including compressor sliding seals, valves, and actuators. While much is known about potentially damaging embrittlement effects of hydrogen on metals, relatively little is known about the effects of high pressure hydrogen on polymers. However, based on the limited results that are published in the literature, polymers also apparently exhibit compatibility issues with hydrogen. An additional study is needed to elucidate these effects to avoid incompatibilities either through design or material selection. As part of this effort, we present here in situ high pressure hydrogen studies of the friction and wear on example polymers. To this end, we have built and demonstrated a custom-built pin-on-flat linear reciprocating tribometer and demonstrated its use with in situ studies of friction and wear behavior of nitrile butadiene rubber polymer samples in 28 MPa hydrogen. Tribology results indicate that friction and wear is increased in high pressure hydrogen as compared both with values measured in high pressure argon and ambient air conditions.

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.

Characterization of a swept external cavity quantum cascade laser for rapid broadband spectroscopy and sensing.

The performance of a rapidly swept external cavity quantum cascade laser (ECQCL) system combined with an open-path Herriott cell was evaluated for time-resolved measurements of chemical species with broad and narrow absorption spectra. A spectral window spanning 1278 - 1390 cm(-1) was acquired at a 200 Hz acquisition rate, corresponding to a tuning rate of 2x10(4) cm(-1)/s, with a spectral resolution of 0.2 cm(-1). The capability of the ECQCL to measure < 100 ppbv changes in nitrous oxide (N(2)O) and 1,1,1,2-tetrafluoroethane (F134A) concentrations on millisecond timescales was demonstrated in simulated plume studies with releases near the open-path Herriott cell. Absorbance spectra measured using the ECQCL system exhibited noise-equivalent absorption coefficients of 5x10(-9) cm(-1)Hz(-1/2). For a spectrum acquisition time of 5 ms, noise-equivalent concentrations (NEC) for N(2)O and F134A were measured to be 70 and 16 ppbv respectively, which improved to sub-ppbv levels with averaging to 100 s. Noise equivalent column densities of 0.64 and 0.25 ppmv × m in 1 sec are estimated for N(2)O and F134A.

Demonstration of the coupling of optofluidic ring resonator lasers with liquid waveguides.

Optofluidic lasers are of particular interest for lab-on-a-chip-type devices, with broad spectral tunability, convenient microfluidic integration, and a small footprint. Optofluidic ring resonator (OFRR) lasers are advantageous in terms of size but typically generate nondirectional emission that is of minimal practical use. We introduce two unique geometries for soft-lithography-based OFRR lasers--side-coupled rings and spiral rings--both of which can be produced in polydimethyl siloxane substrates with contact molding. These rings utilize evanescent and direct butt-coupling, respectively, to effectively couple the OFRR laser emission into microfluidic channels. A laser threshold of a few to tens of microJ/mm(2) is achieved.

Label-free DNA methylation analysis using opto-fluidic ring resonators.

The opto-fluidic ring resonator (OFRR) is a sensitive label-free optical biosensor that is uniquely well suited for photonic and fluidic integration. For the first time we have explored the utility of this novel instrument for the analysis of methylation in oligonucleotides using the MBD-2 (methyl binding) protein as the capture molecule. This application has strong relevance to cancer research and future clinical tools through the study of methylation patterns in important gene promoters. In this work we quantitatively characterized the OFRR's response to artificially methylated ssDNA and dsDNA as a function of the number of methylated cytosines and DNA concentration. The effect of hemi- versus fully methylated oligonucleotides was also investigated. Additionally, anti 5-methylcytidine antibody was also used as the capture molecule and compared with MBD-2. It is found that the antibody has stronger affinity for ssDNA, whereas MBD-2 is much better at binding dsDNA.

Label-free analysis of DNA methylation using optofluidic ring resonators.

We demonstrate the utility of the opto-fluidic ring resonator (OFRR) sensor for analyzing methylated oligonucleotides. Cytosine methylation, a regular epigenetic function in cellular growth and metabolism, may have ties to abnormal suppression of key genes involved with cellular proliferation. Such behavior is suspected to be strongly related to the occurrence of several types of cancers. The OFRR is demonstrated as a tool both for detecting DNA hybridization and methylated cytosines residues.

Overview of the optofluidic ring resonator: a versatile platform for label-free biological and chemical sensing.

Highly sensitive detection of biological and chemical analytes has significant importance within medical science, environmental monitoring, food quality, national security and defense. The opto-fluidic ring resonator (OFRR) is a relatively new solution for label-free optical sensing that is compatible with a versatile range of analytes. A capillary-based platform, the OFRR supports whispering gallery modes within its circular cross-section and conducts evanescent sensing within its hollow core. Herein, we provide an overview of the basic operation principles of the OFRR and some examples of its most important applications, including the detection of proteins, virus, DNA molecules, whole cells, vapors and pesticides.

Robust integrated optofluidic-ring-resonator dye lasers.

We demonstrate a robust optofluidic dye laser that integrates fluidics with a high Q-factor ring resonator. In this optofluidic laser the ring resonator is formed by an optical fiber fused on the inner surface of a glass capillary serving as a fluidic channel. Laser oscillation is achieved with a threshold of 7 microJ/mm2 per pulse. Furthermore, the laser emission can be directionally outcoupled through a fiber prism for easy and efficient light delivery.

Label-free detection with the liquid core optical ring resonator sensing platform.

Optical label-free detection prevents the cost and complexity of fluorescence and radio labeling while providing accurate quantitative and kinetic results. We have developed a new optical label-free sensor called the liquid core optical ring resonator (LCORR). The LCORR integrates optical ring resonator sensors into the microfluidic delivery system by using glass capillaries with a thin wall. The LCORR is capable of performing refractive index detection on liquid samples, as well as bio/chemical analyte detection down to detection limits on the scale of pg/mm2 on a sensing surface.

PDMS embedded opto-fluidic microring resonator lasers.

Opto-fluidic ring resonator (OFRR) dye lasers are embedded in low index polydimethylsiloxane (PDMS) to achieve enhanced portability, mechanical stability, and potential integration with conventional soft lithography based microfluidics for development of micro total analysis systems. The OFRR retains high Q-factors (> 10(6)) and exhibits low lasing threshold (<1 microJ/mm(2)). Fiber prisms and tapered optical fibers are used to directionally couple out the laser emission. At 2.2 microJ/mm(2) pump intensity, the laser output from the fiber prism is 80 nW, corresponding to 50% power extraction efficiency. A microarray structure of parallel OFRRs is also demonstrated, allowing simultaneous multi-color emissions.

Phage-based label-free biomolecule detection in an opto-fluidic ring resonator.

We have developed a sensitive and inexpensive opto-fluidic ring resonator (OFRR) biosensor using phage as a receptor for analyte detection. Phages have distinct advantages over antibodies as biosensor receptors. First, affinity selection from large libraries of random peptides displayed on phage provides a generic method of discovering receptors for detecting a wide range of analytes with high specificity and sensitivity. Second, phage production can be less complicated and less expensive than antibody production. Third, phages withstand harsh environments, reducing the environmental limitations and enabling regeneration of the biosensor surface. In this work, filamentous phage R5C2, displaying peptides that bind streptavidin specifically, was employed as a model receptor to demonstrate the feasibility of a phage-based OFRR biosensor. The experimental detection limit was approximately 100pM streptavidin and the K(d(apparent)) is 25pM. Specificity was verified using the RAP 5 phage, which is not specific to streptavidin, as the negative control. Sensing surface regeneration results show that the phage maintained functionality after surface regeneration, which greatly improves the sensors' reusability. The phage-based OFRR biosensor will become a promising platform for universal biomolecule detection with high sensitivity, low cost, and good reusability.

Sensitive optical biosensors for unlabeled targets: a review.

This article reviews the recent progress in optical biosensors that use the label-free detection protocol, in which biomolecules are unlabeled or unmodified, and are detected in their natural forms. In particular, it will focus on the optical biosensors that utilize the refractive index change as the sensing transduction signal. Various optical label-free biosensing platforms will be introduced, including, but not limited to, surface plasmon resonance, interferometers, waveguides, fiber gratings, ring resonators, and photonic crystals. Emphasis will be given to the description of optical structures and their respective sensing mechanisms. Examples of detecting various types of biomolecules will be presented. Wherever possible, the sensing performance of each optical structure will be evaluated and compared in terms of sensitivity and detection limit.

Opto-fluidic micro-ring resonator for sensitive label-free viral detection.

We have demonstrated sensitive label-free virus detection using the opto-fluidic ring resonator (OFRR) sensor. The OFRR is a novel sensing platform that integrates the microfluidics and photonic sensing technology with a low detection limit and small volume. In our experiment, filamentous bacteriophage M13 was used as a safe model system. Virus samples were flowed through the OFRR whose surface was coated with M13-specific antibodies. We studied the sensor performance by monitoring in real-time the virus and antibody interaction. It is shown that OFRR can detect M13 with high specificity and sensitivity. The detection limit is approximately 2.3 x 10(3) pfu mL(-1) and the detection dynamic range spanned seven orders of magnitude. Theoretical analysis was also carried out to confirm the experimental results. Our study will lead to development of novel OFRR-based, sensitive, rapid, and low-cost micro total analysis devices for virus detection.

Label-free quantitative DNA detection using the liquid core optical ring resonator.

We demonstrated quantitative real-time label-free detection of DNA sequences using the liquid core optical ring resonator (LCORR) sensor. The LCORR is a recently developed sensing platform that integrates microfluidics and photonic sensing technology with low detection limit and sub-nanoliter detection volume. We analyzed experimentally and theoretically the LCORR response to a variety of DNA samples that had different strand lengths (25-100 bases), number of base- mismatches (1-5), and concentrations (10 pM to 10 microM) to evaluate the LCORR sequence detection capability. In particular, we established the linear correlation between the LCORR sensing signal and the molecule density, which allows us to accurately calculate the molecule density on the surface. It is found that the probe surface coverage was 26-51% and the extent of hybridization was 40-50%. The titration curve for 25-base probe and 25-base target DNA yields a dissociation constant of 2.9 nM. With a 37.1 nm/RIU LCORR, detection of 10 pM bulk DNA concentration was demonstrated. The mass detection limit was estimated to be 4 pg/mm(2), corresponding to a density of 10(10) molecules/cm(2) on the surface. We also showed that the LCORR was sensitive enough to differentiate DNA with only a few base-mismatches based on the raw sensing signal and kinetic analysis. Our work will provide important insight into the light-DNA interaction at the ring resonator surface and lay a foundation for future LCORR-based DNA label-free microarray development.

Integrated refractive index optical ring resonator detector for capillary electrophoresis.

We developed a novel miniaturized and multiplexed, on-capillary, refractive index (RI) detector using liquid core optical ring resonators (LCORRs) for future development of capillary electrophoresis (CE) devices. The LCORR employs a glass capillary with a diameter of approximately 100 mum and a wall thickness of a few micrometers. The circular cross section of the capillary forms a ring resonator along which the light circulates in the form of the whispering gallery modes (WGMs). The WGM has an evanescent field extending into the capillary core and responds to the RI change due to the analyte conducted in the capillary, thus permitting label-free measurement. The resonating nature of the WGM enables repetitive light-analyte interaction, significantly enhancing the LCORR sensitivity. This LCORR architecture achieves dual use of the capillary as a sensor head and a CE fluidic channel, allowing for integrated, multiplexed, and noninvasive on-capillary detection at any location along the capillary. In this work, we used electro-osmotic flow and glycerol as a model system to demonstrate the fluid transport capability of the LCORRs. In addition, we performed flow speed measurement on the LCORR to demonstrate its flow analysis capability. Finally, using the LCORR's label-free sensing mechanism, we accurately deduced the analyte concentration in real time at a given point on the capillary. A sensitivity of 20 nm/RIU (refractive index units) was observed, leading to an RI detection limit of 10-6 RIU. The LCORR marries photonic technology with microfluidics and enables rapid on-capillary sample analysis and flow profile monitoring. The investigation in this regard will open a door to novel high-throughput CE devices and lab-on-a-chip sensors in the future.

Thermal characterization of liquid core optical ring resonator sensors.

The liquid core optical ring resonator (LCORR) has recently shown promise as a high-sensitivity label-free lab-on-a-chip biological-chemical sensor. We investigate experimentally and theoretically the temperature dependence of the LCORR to establish a noise baseline, which will enable us to implement a temperature stabilization mechanism to reduce the thermally induced noise and to improve the sensor detection limit. Our studies involve analysis of the thermo-optic and thermomechanical effects of fused silica and aluminosilicate glass as they impact LCORR performance. Both thick-walled and thin-walled LCORRs are investigated to elucidate the contribution of water in the core to the thermal response of the LCORRs. Theoretical calculations based on Mie theory are used to verify the experimental observations.