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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PAO1 and Desulfovibrio sp. EX265 biofilms.</a> Water Science &amp; Technology. 43, 113-120 (2001).</li><li>J&#228;ger-Z&#252;rn, I., Grubert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podostemaceae+depend+on+sticky+biofilms+with+respect+to+attachment+to+rocks+in+waterfalls.">Podostemaceae depend on sticky biofilms with respect to attachment to rocks in waterfalls.</a> International Journal of Plant Sciences. 161, 599-607 (2000).</li><li>Matysik, A., Kraut, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackArt:+the+user+friendly+interface+for+single+molecule+tracking+data+analysis+and+simulation+applied+to+complex+diffusion+in+mica+supported+lipid+bilayers.">TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.</a> BMC Research Notes. 7 (1), 274-283 (2014).</li></ol>

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 um 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 colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Bubble Trap</td>
			<td colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Silicone Tubing</td>
			<td>Dow Corning</td>
			<td></td>
			<td>3 mm outer diameter, 1 mm inner diameter</td>
		</tr>
		<tr>
			<td>Clear polypropylene plastic connectors&#160;</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 colspan="2">Thermo Scientific&#8482; Nunc&#8482;</td>
			<td>50 x 24 mm</td>
		</tr>
		<tr>
			<td>Syringe 3 mL</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2L Storage/Media Bottles</td>
			<td>VWR&#174;&#160;</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 ...

in-situ-mapping-mechanical-properties-biofilms-particle-tracking

Research

JoVE Journal

Bioengineering

9.6K 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

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

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 ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...