optical coherence tomography and optical coherence elastography system setup and imaging granular biofilms for elastic wave field analysis

Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

In wastewater treatment and water resource recovery, beneficial biofilms in attached growth reactors are increasingly employed to enable microbes to convert undesirable pollutants, such as organic matter, nitrogen, and phosphate, into stabilized forms that can be easily removed from the water1. In these systems, the biofilm&#39;s emergent function, namely biochemical transformations, is closely associated with the diversity of microbes residing in it and the nutrients these microbes receive2. Accordingly, ongoing biofilm growth can pose a challenge to maintaining consistent reactor functionality because the new biofilm growth may alter the biofilm&#39;s overall metabolic processes, mass transfer characteristics, and community composition. Stabilizing the biofilm environment as much as possible can protect against such changes3. This includes ensuring a consistent flow of nutrients and keeping the structure of the biofilm stable with a steady thickness4. Monitoring the biofilm&#39;s stiffness and physical structure would enable researchers to gain insight into the overall health and functioning of the biofilm.
Biofilms exhibit viscoelastic properties5,6,7. This viscoelastic nature results in a combination of an instantaneous and slow, time-dependent deformation in response to external mechanical forces. One unique aspect of biofilms is that, when they are subjected to substantial deformation, they respond like viscous liquids. Conversely, when subjected to minor deformation, their response is comparable to solids5.&#160;Moreover, within this small-deformation region, there is a deformation range under which biofilms exhibit a linear force-displacement relationship5,6,7. Deformations within this linear range are optimal for assessing biofilm mechanical characteristics because these yield reproducible measurements. Several techniques can quantify the elastic response within this range. Optical coherence elastography (OCE) is an emerging technique that is being adapted for analyzing biofilms in this linear range (strains on the order of 10-4-10-5)8,9.
OCE&#39;s most established application so far is in the biomedical field, where the technique has been applied to characterize biological tissues that only require superficial optical access.&#160;For example, Li et al. used OCE to characterize the elastic properties of skin tissue10. Other authors characterized the anisotropic elastic properties of porcine and human corneal tissues and how they are affected by intraocular pressure11,12,13,14,15,16. Some advantages of the OCE method for studying biofilms are that it is non-destructive and provides mesoscale spatial resolution, it does not require any sample preparation, and the method itself is rapid; it provides co-registered measurements of physical structure&#160;and elastic properties (e.g., porosity, surface roughness, and morphology)8,9,17,18.
The OCE method measures the local displacement of propagating elastic waves in a specimen using phase-sensitive optical coherence tomography (OCT). OCT is a low-coherence optical interferometer that transforms local changes in the sample displacement into an intensity change that is recorded with an optical spectrometer. The OCT technique has also been utilized in biofilm research for the characterization of mesoscale structure, porosity distribution in three dimensions, and biofilm deformation17,19,20,21. In addition, Picioreanu et al. estimated biofilm mechanical properties using fluid-structure interaction inverse modeling of OCT cross-sectional deformation images22.
On the other hand, OCE measurements, coupled with inverse elastodynamic wave modeling, yield the wave speed of elastic waves in the sample, which enables the characterization of the elastic and viscoelastic properties of the sample. Our group adapted the OCE technique for quantitative measurement of biofilm elastic and viscoelastic properties8,9,18 and validated the technique against shear rheometry measurements in agarose gel plate samples18. The OCE approach provides precise and reliable estimates of the biofilm properties since the measured elastic wave speed is correlated with the elastic properties of the sample. Furthermore, the spatial decay of the elastic wave amplitude can be directly correlated with the viscoelastic properties due to viscous effects in the material. We have reported OCE measurements of viscoelastic properties of mixed culture bacterial biofilms grown on coupons in a rotating annular reactor (RAR) and granular biofilms with complex geometries using elastodynamic wave models18.
The OCE technique is also a powerful alternative to traditional rheometry18which is used for viscoelastic characterization. Rheometry methods are best suited for samples with planar geometry. As such, granular biofilms, which have arbitrary shapes and surface morphologies, cannot be accurately characterized on a rheometer8,23. In addition, unlike OCE, rheometry methods may be challenging to adapt for real-time measurements, for example, during biofilm growth in flow cells24,25.
In this paper, we show that OCE measurements of the frequency-independent wave speed of surface waves can be used to characterize the biofilm elastic properties without the need for complicated models. This development will make the OCE approach more accessible to the broader biofilm community for studying the biofilm mechanical properties.
Figure 1 shows a schematic illustration of the OCT system used in this study. The system incorporates several instruments, including a commercial spectral-domain phase-sensitive OCT system, a delay generator, a function generator, and a piezoelectric transducer. The OCT system operates on the principle of interferometry by employing a broadband light source with a center wavelength of 930 nm. The collected light intensity, which is correlated with intricate structural details in the sample, is analyzed in the postprocessing unit and then converted to a cross-sectional image of the sample - commonly referred to as an OCT image. The OCT imaging depth depends on the severity of the optical scattering in the sample that stems from local variation in the refractive index and is limited to 1- 3 mm in biological tissues and biofilms. Since the optical phase in the sample and the interference intensity are modulated by motion, the OCT can be used to detect the local sample displacement. We leverage the displacement sensitivity of the OCT in the OCE method to track the steady state displacement field of elastic waves in the sample. Specifically, the function generator outputs a sinusoidal voltage to drive the piezoelectric transducer. The transducer, in turn, stretches and contracts with an oscillatory time history. The oscillatory displacement of the transducer imparts a sinusoidal force on the sample surface through a 3D-printed wedge tip at the apex of the transducer, leading to the generation of harmonic elastic waves in the sample. The wedge tip makes light contact with the sample, such that the sample remains intact after the actuator is retracted from the sample surface. To record the local displacement in the sample, adjacent depth scans separated by a fixed time delay are acquired at each pixel in the sample. The optical phase difference between consecutive scans at each pixel point is proportional to the local vertical displacement at the same point. Synchronization between the displacement of the transducer and the scanning optics in the OCT system is achieved through a trigger pulse that originates from the function generator and is delayed in the delay generator. This synchronization step facilitates the acquisition of consistent cross-sectional images of the local optical phase distribution in the sample. These images are directly proportional to the local vertical harmonic displacement in the sample and are known as the OCE image. OCE images are acquired at different transducer actuation frequencies to obtain the elastic wavelength and wave speed as a function of frequency. The wave speeds measured are analyzed with an elastodynamic model to determine the elastic properties of the sample.

Author Spotlight: Characterizing Environmental Biofilm Mechanics Using Optical Coherence Elastography and its Applications in Wastewater Treatment

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

Our research consists of characterizing the mechanical properties of environmental biofilms using the optical coherence and elastography technique. We aim to understand and predict how biofilm composition and structure correlate to mechanical properties. Ultimately, this knowledge will be leveraged to enhance or eliminate different biofilms, depending on the application.Okay, for the specific focus of characterizing the mechanical properties of biofilms, we have Rheometry, we have non-indentation, atomic force microscopy. We also have some new developmental techniques, for example, like optical tweezers, magnetic tweezers, digital image correlation, and a host of others. Most of the previously used techniques either focus on overall mechanical property measurements, overlooking those at the mesoscale, or highly localized measurements that are not relevant at the mesoscale.And the mesoscale is crucial because that is the level that is relevant to the physical attributes of biofilms. First, we've adapted the optical clearance elastography technique to characterize the mechanical properties of environmental biofilms. In this context, we've developed the methods, we've also developed inverse modeling tools to be able to deal with layered viscoelastic properties and also to analyze the complex microstructure of biofilms.Our group is interested in quantifying the mechanical properties of biofilms from wastewater treatment plants, so we're interested in learning what factors impact the stability and performance of biofilms. For example, does the stiffness of a biofilm impact the rate of sloughing?

Optical Coherence Tomography and Optical Coherence Elastography System Setup and Imaging Granular Biofilms for Elastic Wave Field Analysis

Research

JoVE Journal

Environment

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances ...