This research is supported by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Programme, the Start-up Grants (M4330002.C70) from Nanyang Technological University, and AcRF Tier 2 (MOE2014-T2-2-172) from Ministry of Education, Singapore. The authors thank Joey Yam Kuok Hoong for participating in the demonstration of this protocol.

1. Biofilm Cultivation
<ol>
	<li>Preparation of Bacterial Strains
	<ol>
		<li>1 day prior to biofilm cultivation, prepare planktonic bacterial cultures by inoculating 2 ml of appropriate growth medium from frozen bacterial culture. Use Luria-Broth medium (10 g L-1 NaCl, 10 g L-1 yeast extract, and 10 g L-1 tryptone) for mucoid P. aeruginosa and its &#916;pel and &#916;psl defective mutants. Incubate overnight at 37 &#176;C and 200 rpm shaking conditions. Dilute overnight cultures to an OD600 of 0.40 using a spectrophotometer.</li>
		<li>Assemble flow cell setup, which has been described previously 14, preferably on a mobile station or trolley that can be brought into the microscope room for imaging the flow cell without disassembly.</li>
		<li>Prepare sterile growth medium in 2 L or 5 L bottles for each flow cell. Use minimal medium M9 (48 mM Na2HPO4, 22 mM KH2PO4, 19 mM NH4Cl, 9 mM NaCl, 2 mM MgSO4, and 0.1 mM CaCl2) supplemented with 0.04% glucose (wt/vol) and 0.2%(wt/vol) casamino acids) for P. aeruginosa flow cell biofilms.</li>
		<li>Aliquot fluorescent microspheres into microcentrifuge tubes and spin down in sterile H2O for 5 min in centrifuge at 9,391.2 g. Remove supernatant and resuspend in 1 ml growth medium to remove sodium azide (disinfectant) prior to adding to growth medium in 2 L or 5 L medium bottles. 
		NOTE: Different sizes of microspheres come in different concentrations and should be diluted accordingly to instructions. For example, disperse 120 &#181;l of 1.0 &#181;m diameter microspheres, 15 &#181;l of 0.5 &#181;m diameter microspheres and 0.96 &#181;l of 0.2 &#181;m diameter microspheres into 2 L of growth medium to give 2.18 &#215; 106 microspheres ml-1 for each particle size.</li>
		<li>Attach medium bottle to upstream of flow cell and allow growth medium to flow through entire setup. Stop flow and inject diluted overnight cultures into flow cell chambers. Allow bacteria to attach to the coverslip substratum for 1 hr before continuing flow of growth medium at a flow rate of approximately 5.5 x 10-3 m sec-1,or 8 rpm with a peristaltic pump. 
		NOTE: Biofilms are usually grown at room temperature (25 &#176;C) for 3-7 days.</li>
		<li>Alternatively, for static culture setup, dilute overnight cultures 100 fold in microcentrifuge tubes by adding 10 &#181;l of overnight culture to 1 ml growth medium with particles. Increase the particle concentration in the growth medium for static culture by approximately 500-fold compared to that used for flow biofilms (e.g. wash and resuspend 600 &#181;l of 1.0 &#181;m diameter spheres in 10 ml of growth medium) as flow cell biofilms are constantly replenished with particles but static cultures are not.
		<ol>
			<li>Add 200 &#181;l of diluted overnight culture with particles to the chambers of 8 well slides. Incubate at 37 &#176;C under static conditions. Biofilms are usually grown for 1 day. Replace spent growth medium daily with fresh growth medium with particles if biofilm is grown for more than 1 day.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Microscopy
<ol>
	<li>On day 3 and 5, switch off flow from peristaltic pump and bring flow cell setup into the microscopy room. Clamp tubing near the entrance and exit of the flow cell chambers to prevent drift (a systematic error cause by changes in the environment) from flow. Place the flow cell onto the microscope stage of an upright microscope. 
	NOTE: Use an inverted microscope for imaging of microwells.</li>
	<li>Use fluorescent microscopy with 40X oil objective to take videos of microsphere motion embedded in the biofilm at various locations (microcolonies and flat undifferentiated layers) and in different days (day 3 and 5) for temporal and spatial investigations. Take shorter videos with higher frame rate to investigate events occurring within short time scales (e.g. 1.5-3 min videos at frame rate of 25-50 frames per second for fast dynamics), and longer videos with lower frame rate to investigate events over longer time scales (e.g. 15-30 min videos at frame rate of 2.5-5 frames/sec for slower dynamics). 
	NOTE: A video typically contains 5-10 particles, and is typically of 1.0-2.5 GB in file size. Larger microcolonies may hold more particles. Take 5-10 videos for each category (e.g. 5-10 videos of different microcolonies and of different locations in flat undifferentiated layers). The biofilm has a heterogeneous architecture that may consist of microcolonies, channels, voids and flat undifferentiated layers.</li>
	<li>Save videos in appropriate format that can be read by Fiji/ImageJ (e.g. czi, tif).</li>
	<li>Check videos for drift by scrolling through finished video and observing that the particles do not move in the same direction simultaneously. Drift can be caused by z-stage motion, currents in the flow cell, imbalance of anti-vibration table, air pressure or temperature changes. Correct minor drifting using post-processing techniques usually provided with microscope software. Discard any videos wherein drift cannot be corrected. Record the following parameters, which are required during Particle Tracking Analysis: Resolution (px/um), Number of Frames, Duration.</li>
	<li>Remove flow cell from microscope stage and restart peristaltic pump to continue cultivating the biofilm.</li>
</ol>
3. Particle Tracking Analysis
<ol>
	<li>Obtain particle trajectories by using the plug-in TrackMate in open source software (ImageJ). Download Fiji at&#160; <a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a>. Open the video with Fiji and under the Plugins menu, select Tracking and TrackMate.</li>
	<li>Check or adjust settings in the window suggesting initial calibration settings to match the video parameters as recorded in step 2.4.</li>
	<li>Detect particles in TrackMate using ImageJ.
	<ol>
		<li>Select &#39;LoG detector&#39;, input particle diameter and threshold value (e.g. 1,000). Scroll through the video whilst clicking on &#39;Preview&#39; button to check that the purple circles follow the particles throughout the video. Adjust the diameter and threshold values as necessary: increase the threshold value if purple circles appear in the empty space. Reduce the threshold value if not enough particles are detected. Click the next button to complete detection the particles.</li>
		<li>Click the next button when presented with the option to add or remove particles detected in &#39;Initial thresholding&#39;, &#39;Select a view&#39; and &#39;Select a filter&#39; windows to continue without adjustment if initial particle detection was satisfactory.</li>
		<li>Select &#39;Simple LAP tracker&#39; in the option bar of &#39;Select a tracker method&#39; window as particles rarely move out of focus in the biofilm and are easily tracked. Use the suggested or low linking and gap-closing max distance values, and click next.</li>
		<li>Check that particle tracking is satisfactory by scrolling through the video to see that particles follow the tracks drawn by TrackMate, and that there are no unwanted or missing tracks. Skip the various filtering steps. Go to the final window &#39;Select an action&#39;. Select &#39;Export tracks to xml file&#39; from the drop down menu and click &#39;Execute&#39;. Choose a folder to save the tracks. 
		NOTE: There are various programs available in the public domain for the analysis of particle trajectories and use of microrheology. msdanalyzer and TrackArt are examples of particle tracking analysis programs that run in the Matlab environment.</li>
	</ol>
	</li>
	<li>Prepare Matlab to import the particle trajectories in the xml files by selecting in the menu of Matlab File&#62;Set Path... and add the scripts folder available with the Fiji package.</li>
	<li>To analyze the particle trajectories with msdanalyzer, download the link to the zip file or the tar.gz file at <a href="http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories">http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories</a>.
	<ol>
		<li>Extract the @msdanalyzer folder15 and drop it in a folder that belongs to the Matlab path (e.g. C:\Documents and Settings\&#60;Username&#62;\My Documents\Matlab). Start Matlab and initiate the analyzer by typing: 
		ma = msdanalyzer(2, &#39;um&#39;, &#39;sec&#39;) 
		where um and sec are the physical space and time units in the video.
		<ol>
			<li>Import the particle trajectories by typing: 
			[tracks, md] = importTrackMateTracks(&#39;FileName.xml&#39;, &#39;clipZ&#39;, &#39;scaleT&#39;) 
			ma = ma.addAll(tracks); 
			where &#39;clipZ&#39; removes the z dimension for a 2D video, and &#39;scaleT&#39;.</li>
			<li>Plot the trajectories onto graph using: 
			ma.plotTracks; 
			ma.labelPlotTracks;</li>
			<li>Compute the MSDs of the particles using: 
			ma = ma.computeMSD; 
			ma.msd</li>
			<li>Plot the MSDs of each particle: 
			figure 
			ma.plotMSD</li>
			<li>Combine particle trajectories from other videos of the same categery (e.g. particles embedded in wild-type microcolonies at day 3) by repeating 3.5.1.1 to 3.5.1.4).</li>
			<li>Plot the ensemble mean or the average over all curves: 
			ma.plotMeanMSD 
			Change the y- and x-axis from linear to logarithmic scale. At long lag times, the MSD curves become noisy due to insufficient statistics at longer timescales. The MSD curves may also rise steeply due to dynamic error. These regions of the curve can be removed by using the brush tool to select the end of the curve and selecting &#39;Brushing&#39; &#62; &#39;Remove Unbrushed&#39; in the Tools menu.</li>
		</ol>
		</li>
		<li>Download Ezyfit at <a href="http://www.fast.u-psud.fr/ezyfit/">http://www.fast.u-psud.fr/ezyfit/</a> and add Ezyfit to a folder belonging to the Matlab path (e.g. C:\Documents and Settings\&#60;Username&#62;\My Documents\Matlab). Install the Ezyfit menu by typing in Matlab workspace efmenu install. Restart MATLAB with Ezyfit menu in the figure window. Fit the MSD curves to the power law by selecting in the Ezyfit menu &#39;Show Fit&#39; &#62; &#39;Power&#39; &#62; &#39;a*x^n&#39; were n is the estimated diffusive exponent &#945;. 
		NOTE: msdanalyzer requires the Curve Fitting Toolbox in Matlab for fitting of MSD curves. Ezyfit is an alternative and free software that can perform curve fitting.</li>
		<li>Clear current particle trajectories and MSDs to calculate new particle MSDs at other locations or time: 
		clear</li>
		<li>After calculation of various mean MSD curves of particles in medium (control), and microcolonies and undifferentiated areas of various strains, copy and paste the curves into one graph for comparison. Sample stiffness and crosslinking increase with lower MSD values. Flat curves have lower &#945; and sample is more elastic than steeper curves with higher &#945;.</li>
		<li>Select the curve and go to &#34;Tools&#34; to look at the data statistics, such as the median and range. The MSD of the bead is proportional to the creep compliance, J(t) of the material in which the bead is embedded according to the relation, 
		<img alt="Equation 1" src="/files/ftp_upload/53093/53093eq1.jpg" /> 
		where J = creep compliance, d = particle diameter, kB = Boltzman constant, T = temperature and t = time.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Microrheology is a useful tool for local rheological measurements in heterogeneous systems, such as microbial biofilms. It is a non-destructive technique, enabling the real-time monitoring of rheological changes within the same biological sample over multiple time points. In this protocol, particle-tracking microrheology was applied to Pel and Psl exopolysaccharide mutants in order to investigate how they affect the elasticity and effective crosslinking of the biofilm matrix. Psl favors the development of elastic biofilm...

Most bacterial cells are able to employ both planktonic (free-living) and surface-attached (sessile) modes of growth 1. In the surface-attached mode of growth, bacterial cells secrete and encase themselves in large amounts of extracellular polymeric substances (EPS) to form biofilms. The EPS mainly consists of proteins, exopolysaccharide, extracellular DNA and is essential to biofilm formation 2. It serves as a physical scaffold by which bacteria can use to differentiate spatially and protects the bacteria from harmful environmental conditions and host responses. Different components of EPS have distinct roles in biofilm formation 3 and changes in the expression of EPS components can dramatically remodel biofilm structures 4. EPS components can also function as signaling molecules 5, and recent studies has shown certain EPS components interacting with microbial cells to guide their migration and biofilm differentiation 6-8.
Research on the EPS has greatly advanced based upon the morphological analyses of biofilms produced by mutants defective in a specific component of the EPS 9,10. In addition, the EPS is usually characterized at the macro-scale (bulk characterization) 11. Morphological analyses however can lack quantitative detail and bulk characterization, which returns average values, loses the detail that exists within the heterogeneity of the biofilm. There is now an increasing trend to progress to real-time characterization of the mechanic properties of EPS at the micro-scale. This protocol demonstrates how particle-tracking microrheology is able to determine the spatiotemporal effects of matrix components Pel and Psl exopolysaccharides on the viscoelasticity and effective crosslinking of Pseudomonas aeruginosa biofilms 4.
Passive microrheology is a simple and inexpensive rheology method that provides the highest throughput of spatial microrheological sampling of a material to date 12,13. In passive microrheology, probe spheres are placed in the sample and their Brownian motion, driven by thermal energies (kBT) is followed by video microscopy. Several particles can be tracked simultaneously, and the time-dependent coordinates of the particles follow a conventional random walk. Therefore, on average, the particles remain at the same position. However, the standard deviation of the displacements or the mean squared displacement (MSD) of the particles, is not zero. Since viscous fluids flow, the particle MSD in a viscous fluid grows linearly as time progresses. In contrast, the polymeric crosslinking found in viscoelastic or elastic substances help them to resist flow, and particles become limited in their displacement, leading to plateaus in the MSD curve (Figure 1A). This observation follows the relation MSD&#8733;t&#945; , where &#945; is the diffusive exponent that is related ratio of elastic and viscous contributions of the substance. For particles moving in viscous fluids &#945; = 1, in viscoelastic substances 0 &#60; &#945; &#60; 1, and in elastic substances &#945; = 0. The MSD may also be used to calculate the creep compliance, which is the tendency of the material to deform permanently over time and estimates how easily a material spreads.
The size, density and surface chemistry of the particle are critical to the correct application of microrheological experiment and are chosen with respect to the system studied (in this case the polymers of the biofilm matrix, see Figure 1B). Firstly, the particle measures the rheology of the substance with structures that are much smaller than the particle itself. If the substance&#39;s structures are of similar scale to the particle, the motion of the particle is perturbed by the shape and orientation of the individual structures. However, if the structures surrounding the particle are much smaller, this effect is small and averaged out, presenting a homogeneous environment to the particle (Figure 1B). Secondly, the density of the particle should be similar to the medium (1.05 g ml-1 for water based mediums) such that sedimentation is avoided and inertial forces are negligible. Most particles with polystyrene lattices meet the above criteria. Ideally, the particle does not interact with the polymers of the biofilm matrix as the rheological interpretation of particle MSD is only valid if motion is random, driven by thermal energy and collision with substance structures. This can be observed by checking whether the probe particle tends to bind or bounce off the surface of a pre-grown biofilm. However, despite the lack of attraction to the biofilm, the particles must be able to be incorporated into the matrix. In addition, the physiochemical heterogeneity of the biofilm may result in different particles being more suitable as probes in different regions of the biofilm. Thus, particles of different sizes and surface chemistry should be applied to the biofilm.
As such, the particle MSD is able to provide useful information on how different components contribute to the rheology and spreading of the biofilm. Furthermore, the use of different probes allows one to derive information on the spatial physiochemical heterogeneity of the biofilm. This method can be used to test the effect antimicrobial treatment on the mechanical properties of the biofilm, or applied to mixed species biofilms to investigate how the mechanical properties of the biofilm are changed from introduction of another species. Particle MSDs may also be useful for characterizing biofilm dispersal. Such studies would be helpful in our understanding of biofilms, potentially improving biofilm treatments and engineering of biofilms for useful activities.

<table><tbody><tr><td>Fluorspheres</td><td>Invitrogen</td><td>F-8821</td><td>1.0 &amp;#956;m red fluorescent (580/605) microspheres with carboxylate modification</td></tr><tr><td>Zeiss Axio Imager M1</td><td>Carl Zeiss</td><td></td><td>Epifluorescent Microscope</td></tr><tr><td>Masterflex L/S Digital Drive 07523-80</td><td>Cole-Parmer</td><td>EW-07523-80</td><td>Peristaltic pump</td></tr><tr><td>Flow Cell Chambers</td><td>Technical University of Denmark</td><td></td><td></td></tr><tr><td>Bubble Trap</td><td>Technical University of Denmark</td><td></td><td></td></tr><tr><td>Silicone Tubing</td><td>Dow Corning</td><td></td><td>3 mm outer diameter, 1&amp;nbsp;mm inner diameter</td></tr><tr><td>Clear polypropylene plastic connectors&amp;nbsp;</td><td>Cole Parmer</td><td>06365-83</td><td>1/16 in. (1.588 mm)</td></tr><tr><td>Binder Clips</td><td></td><td></td><td>To clamp tubing</td></tr><tr><td>Coverslips</td><td>Thermo Scientific&amp;trade; Nunc&amp;trade;</td><td>50 x 24 mm</td><td></td></tr><tr><td>Syringe 3 ml</td><td>Terumo</td><td></td><td></td></tr><tr><td>27 &amp;nbsp;G Needle</td><td>Terumo</td><td></td><td></td></tr><tr><td>2 &amp;nbsp;L Storage/Media Bottles</td><td>VWR&amp;reg;</td><td></td><td></td></tr><tr><td>Trolley</td><td></td><td></td><td>To hold biofilm setup</td></tr></tbody></table>

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.

The local viscoelastic properties of the biofilm in different regions of the biofilm, which included the voids (medium above the biofilm), plains (undifferentiated flat layer of cells) and microcolonies (see labels in Figure 2A) were investigated. The temporal changes in viscoelastic properties of the biofilm during maturation from days 3 to 5 were also determined. The MSD of the particles in the voids was used as a control and comparable to the MSD of particles in pure medium. In contrast, particles tra...

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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.

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 ...

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Research

JoVE Journal

Bioengineering

9.7K Views.  Nanyang Technological University. 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.

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

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

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 ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Chemistry

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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 ...

Environment

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Engineering

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

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.

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.

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

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

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