The authors thank Aqua-Aerobic Systems, Inc. (Rockford, IL, USA) for providing the granular biofilms studied in this work. The authors also acknowledge the National Science Foundation&#39;s support via Award #210047 and #193729.

1. System setup
<ol>
	<li>Gather the system components which include the commercial OCT system (base unit, stand, imaging head, and computer), waveform generator, transducer, delay/pulse generator, a switch with BNC connections, BNC cables and adapters, optical posts, and clamps.</li>
	<li>Connect the sync signal from the function generator to a switch. Connect the other port of the switch to the delay generator.</li>
	<li>Connect the output of the function generator to the transducer leads.</li>
	<li>Connect the outputs of the delay generator to the trigger channel at the back of the OCT base unit. The output signal from the delay generator is a trigger pulse to initiate the motion of the scanning optics in the OCT system.</li>
	<li>Turn on the system components (OCT base unit, computer, function generator, and delay generator) and launch the OCT software.</li>
	<li>Configure the delay generator to send a transistor-transistor logic trigger signal to the OCT base unit. Refer to the OCT system manual for the trigger signal requirements.</li>
	<li>Position the transducer beneath the OCT lens. The transducer has a 3D-printed wedge tip glued onto one of its ends that serves as a line source for elastic waves.</li>
</ol>
2. Image acquisition
<ol>
	<li>On the OCT software, select the Doppler Acquisition Mode and enable the external trigger.</li>
	<li>Place the granular biofilm under the lens in a sample holder and move it towards the tip of the transducer using a translation stage. Ensure that the transducer makes gentle contact with the sample surface, as shown in Figure 2. We used two granular biofilms (also known as granular sludge) with different nominal diameters (4.3 mm and 3.3 mm). This selection was made to investigate the impact of biofilm size on its mechanical properties. These were commercially obtained. 
	NOTE: The sample holder employed in this study consists of a 3D-printed plastic plate with multiple hemispherical indentations. This holder does not allow for measurements under native conditions. Therefore, we introduced water from the natural environment during measurements to prevent the sample from drying.</li>
	<li>Specify the scan region by clicking the Start and Endpoints of the Line of Interest (wave propagation path) in the sample monitor window. Center this line with respect to the transducer tip and ensure it is perpendicular to the edge of the tip.</li>
	<li>Specify the number of pixels along the scan region and the depth of the sample and increase the number of B-scans (2D cross-sectional images) to be recorded to improve the signal-to-noise ratio of the OCE images. The presented results were obtained using 1523 pixels along the scanning path and 1024 pixels along the depth. A total of 50 B-scans were taken.</li>
	<li>Click the Scan button and turn ON the switch. The OCT and OCE images should appear on the screen. Activate the switch within the trigger timeout time and the scan preparation time.</li>
	<li>Ensure that the reference intensity is within the optimum range and position the sample within the focal region of the OCT microscope objective. A properly focused sample should have its top edge close to the top of the image.</li>
	<li>Adjust the phase contour in the OCE image on the display toolbar by increasing the higher value of the left-hand side color bar and decreasing the lower value of the right-hand side color bar. This will increase the fringe contrast.</li>
	<li>Configure the function generator to produce a single-frequency sinusoidal voltage by pressing the Sine button in the front panel and specify the starting excitation frequency for the measurements. The measurements in this study start at 4 kHz and end at 9.6 kHz.&#160;Enable the Output connector by pressing the Output key.</li>
	<li>Set an acceptable voltage for the measurement. This value should maximize fringe visibility but also avoid phase wrapping. For the biofilms in this study and the frequency range of the measurements, a voltage between 5 and 10 V typically results in a phase map with good contrast.</li>
	<li>Acquire the OCT and OCE images by clicking the Record button.</li>
	<li>Repeat the measurements at different frequencies to obtain cross-sectional images of the elastic wave field with different wavelengths (or fringe periods).</li>
</ol>
3. Image Analysis
<ol>
	<li>Obtain the physical size of the pixels. The physical pixel size in x is obtained by dividing the field of view in the x direction by the image size in the x direction and then multiplying by a factor of two.&#160;The physical pixel size in z is obtained by dividing the field of view in the z direction by the image size in the z direction. The field of view and image size values are stored in the structure array with the image information which can be accessed with the OCTFileOpen function provided in the MATLAB SDK in the ThorImageOCT package.</li>
	<li>Obtain the OCT and OCE matrices using the OCTFileGetIntensity and OCTFileGetPhase functions, respectively, and take the average of the recorded frames. These functions are provided in the MATLAB SDK in the ThorImageOCT package.</li>
	<li>Obtain the pixel locations of the top edge of the sample by binarizing the image and detecting the white pixels from top to bottom for each column.</li>
	<li>Extract the phase distribution of the OCE image along this edge using the improfile function and compute the cumulative arc length in real dimensions. Compute the arc length by taking the cumulative sum of the norm of scaled differences between consecutive points in the x and z directions.</li>
	<li>Compute the spatial fast Fourier transform of the measured OCT phase distribution (i.e., from the OCE images) with respect to the cumulative arc length using the plomb function.</li>
	<li>Determine the location of the peak in the spectrum. This location represents the spatial frequency of the wave. Calculate the wave speed (or phase velocity) from the ratio of the transducer excitation frequency (units of Hz) and the spatial frequency (unit of inverse length).</li>
</ol>

Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

<ol>
	<li>Mahto, K. U., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilm+and+extracellular+polymeric+substances+in+the+moving+bed+biofilm+reactor+for+wastewater+treatment:+A+review.">Bacterial biofilm and extracellular polymeric substances in the moving bed biofilm reactor for wastewater treatment: A review.</a> Bioresour Technol. 345, 126476 (2022).</li><li>Pholchan, M. K., Baptista, J. d. e. C., Davenport, R. J., Curtis, T. 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The authors declare no conflicts of interest.

The attainable imaging depth in the OCT system is determined by the degree of light penetration from the light source, which depends on the wavelength of the source. Moreover, the wavelength determines the axial resolution. Longer wavelengths can penetrate more deeply into the sample but at the expense of reduced axial resolution compared to shorter wavelengths. Transverse&#160;resolution, on the other hand, is dependent on both the numerical aperture of the system and the wavelength, with shorter wavelengths delivering ...

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.

<table><tbody><tr><td>3D printed sample holder</td><td></td><td></td><td></td></tr><tr><td>3D printed wedge tip</td><td></td><td></td><td>3 mm width</td></tr><tr><td>BNC cables</td><td>Any brand</td><td></td><td></td></tr><tr><td>Delay generator</td><td>Stanford Research Systems</td><td>DG535</td><td>DG535 Digital delay/ Pulse Generator&amp;nbsp;</td></tr><tr><td>Function generator</td><td>Agilent Technologies</td><td></td><td>33250A 80 MHz Function / Arbitrary Waveform Generator</td></tr><tr><td>Granular biofilm</td><td>Aqua-Aerobic Systems</td><td></td><td>Obtained from an Aerobic Granular Sludge reactor (Aqua-Aerobic Systems, Inc.)</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td>Release 2022a (MATLAB 9.12)</td></tr><tr><td>Piezoelectric transducer</td><td>Thorlabs</td><td>PK2JUP1</td><td>Discrete Piezo Stack, 75 V, 30.0 &amp;micro;m Displacement</td></tr><tr><td>SD-OCT System</td><td>Thorlabs</td><td></td><td>Ganymede II, LSM03 scan lens</td></tr><tr><td>ThorImageOCT</td><td>Thorlabs</td><td></td><td>Version: 5.5.5</td></tr></tbody></table>

quantifying elastic properties of environmental biofilms using optical coherence elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.

In this study, we used granular biofilms (also known as granular sludge), which were commercially obtained. Granules are spherical biofilms that form through self-aggregation, meaning that they do not require a carrier or surface on which to grow26. Figure 3A shows a representative cross-sectional OCT image that arises due to the spatial variation of the local refractive index in a granular biofilm. The biofilm has a nominal diameter of 3 mm. Some of ...

Watch this Scientific Journal Video about Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography at JoVE.com

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

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

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

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Research

JoVE Journal

Environment

922 Views.  Northwestern University. This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

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

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Bioengineering

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Immunology and Infection

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

A Bead Movement Based Computational Framework for 3-Dimensional Analysis of Biofilm Material Heterogeneity

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under different growth conditions and after treatment with matrix modifying molecules. To better quantitate the material properties of 3D biofilms, an experimental and computational workflow was developed and applied to examine differences between Enterococcus faecalis, Salmonella enterica serotype Typhimurium and Escherichia coli biofilms as well as the role of the amyloid curli in confirming rigidity to Enterobacteriaceae biofilms. The spatio-temporal dynamics of 1 &#181;m carboxylate beads in biofilms were tracked in 20 &#181;m 3D biofilms over 20 minutes. The 4D image stacks were processed using the Mosaic plugin in ImageJ to produce 3D trajectory data of bead movement. This trajectory data was analyzed with a newly developed Bead Evaluator toolbox, where movement data, including trajectory lifespans, bead velocities, cell densities along trajectories, and bounding box information were computed and stored in csv-files. This paper presents the workflow from experimental setup and image recording to bead trajectory computation and analysis. The structure of curli-containing biofilms resulted in more stable bead interactions and less bead movement than in curli-mutant and Enterococcal biofilms. Bead movement did not appear strongly dependent on cell density when measuring the bead velocity and trajectory bounding box volume, supporting the hypothesis that other material properties of the biofilms control the bead dynamics. This technique is widely applicable to quantitating differences in biofilms of different matrix compositions as well as biofilms before and after matrix-modifying treatments.

We describe a method for analyzing and quantifying the movement pattern of 1 &#181;m carboxylate beads through heterogeneous bacterial biofilms. Comparison of the movement patterns can be used to quantitate differences in material properties of biofilms.

Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under ...

Biology

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination

Source: Bastounis, E. E. et al., <a href="https://app.jove.com/v/57361">A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.</a> J. Vis. Exp.&#160;(2018)
This video showcases quantitative time-lapse microscopy for evaluating extracellular matrix stiffness-dependent Listeria monocytogenes dissemination through endothelial cells. A softer hydrogel facilitates faster bacterial dissemination through endothelial cells compared to stiffer counterparts, highlighting the influence of matrix stiffness on bacterial dissemination.

1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates

	Dissolve ammonium persulfate (APS) in distilled ultrapure water to ...

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

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