This work was in part supported by grants from the Agence Nationale pour la Recherche, PIRIbio program Dynabiofilm and from CNRS Interdisciplinary Risk program. We thank Philippe Thomen for his critical reading of the manuscript and Christophe Beloin for providing the E. coli strain used in this work.

1. Bacteria Culture and Suspension Preparation
<ol>
	<li>Pick a freshly grown colony from a Lysogeny Broth (LB) agar plate, inoculate it in 5 ml liquid LB medium containing 100 &#181;g/ml ampicillin and 7.5 &#181;g/ml tetracycline and incubate it for 5 to 6 hr at 37 &#176;C on a shaking platform.</li>
	<li>Then, add 100 &#181;l of the bacterial culture in 5 ml minimum medium (M63B1) supplemented with 0.4% glucose and the same antibiotic concentrations. Incubate this freshly diluted culture overnight at 37 &#176;C on a shaking platform.</li>
	<li>After 16 hr&#160;of incubation, add 100 &#956;l the overnight culture to 5 ml M63B1, 0.4% glucose. Keep the tube at 37 &#176;C to the shaking platform until a 0.5 OD is reached. The suspension is then ready for injection into the experiment channel for biofilm formation.</li>
</ol>
2. Magnetic Particle Preparation
<ol>
	<li>Take 10 &#956;l magnetic particles &#8212; 2.8 &#181;m in diameter &#8212; from the stock and wash them 3x&#160;in 190 &#956;l minimum medium with the aid of a magnetic sample rack.</li>
	<li>Adjust the particle concentration to 5 x 106 beads/ml. Typically 50 &#956;l of the washed bead solution is mixed with a further 950 &#181;l of M63B1 with 0.4% glucose.</li>
</ol>
3. Channel Preparation and Biofilm Growth
<ol>
	<li>Channel Mounting
	<ol>
		<li>Cut two square (800 &#181;m side length) borosilicate glass capillaries 10&#160;cm&#160;long to obtain two 8&#160;cm&#160;long pieces.</li>
		<li>Glue the two capillary pieces on two glass slides &#8212; cut in half first &#8212; 2 cm apart with 1 cm overhang on either end as in Figure&#160;1 using a fast-acting cyanoacrylate glue (so called super-glue).</li>
		<li>Autoclave the entire setup and the necessary tubing required for further channel connection.</li>
		<li>Gather all the sterile materials under a laminar flow hood: i) the mounted channel and trap 1, ii) the tubing and connectors, iii) the two bubble traps &#8212; the bubble filter commonly used to secure children&#39;s drip-feeding (trap 1) and the home-made bubble trap as a 4&#160;cm&#160;long tube of larger diameter (trap 2), iv) clamps, v) 30 ml syringes filled with M63B1, 0.4% glucose, and vi) the waste bottle.</li>
		<li>Connect the whole setup with Luer Lock connecters or junctions in the following order: 50 ml M63B1 syringe controlled by the syringe pump, pediatric bubble filter, homemade bubble trap, capillary (Figure 1, panel B), and the tube to the waste bottle. Then fill the setup with sterile M63B1, 0.4% glucose, turning on the syringe pump at a rate of about 10 ml/hr, higher than the experimental rates. Carefully track and eliminate all the bubbles in the circuit.</li>
		<li>Flow the medium through the system for 10-15 min; concurrently mix 1 ml of the bacteria suspension at OD 0.5 from Section 1.3 with 1 ml of the washed bead solution prepared in Section 2.2.</li>
		<li>Attach (but don&#39;t close) clamps to the tubing at two positions: before and after the capillaries. Switch the flow off.</li>
		<li>Introduce the bacterial-bead mixture into the capillary after the home made bubble trap using a 1 ml syringe, taking care to hold up the tube ends to prevent air entry. Re-attach the tubing and then close the clamps.</li>
		<li>Repeat the same procedure for the second capillary and check all the tubes for bubbles.</li>
		<li>Transfer the apparatus to the microscope and allow it to stand for 15-20 min to enable the bacteria to settle and attach to the surface of the capillary. Install the capillary onto the microscope stage with the waste container at a slightly higher level. Place the syringe pump on the counter top beside the microscope. Elevate the bubble trap before the capillary slightly higher than the capillary plane to capture bubbles.</li>
	</ol>
	</li>
	<li>Biofilm Growth
	<ol>
		<li>Adjust the flow rate on the syringe pump and start the flow, the biofilm will now develop on the capillary surface during the required period &#8212; usually 24 or 48 hr in these experiments.</li>
		<li>Focus on the capillary bottom plane and start the time-lapse recording of the sample images &#8212; usually an acquisition frequency of 2 images/min will adequately report the biofilm growth. This video monitors biofilm growth post-control (see extracts in Videos 1, 2 and 3).</li>
	</ol>
	</li>
</ol>
4. Magnetic Tweezers Installation
<ol>
	<li>Screw the magnetic tweezers on manually controlled X-Y-Z micromanipulators and screw the micromanipulators on the microscope stage to adjust the position of the tweezers relatively to the capillary. Place the tweezers as in Figure&#160;2 to ensure the appropriate magnetic field gradient is generated in the observation zone.</li>
	<li>Connect the tweezers to the function generator via the power amplifier to generate a 40 sec period of time made of 24 sec zero signal and 16 sec of 4 A direct current with a trigger sent to the bright field light shutter after 20 sec for signal synchronization providing a sequence of events as in Figure 3. 
	Note: These two operations can be achieved at any time between capillary installation and measurement beginning. See experiment overview sketch in Figure 4.</li>
</ol>
5. Creep Curve Acquisition
<ol>
	<li>Use the xy movement control of the microscope stage to bring the edge of the left-hand magnetic pole and the left-hand edge of the capillary in the same observation field. Take the origin of the analysis referential at the intersection of x- and y-axes defined by the edge of the capillary and the edge of the pole piece, respectively (see Figure 2).</li>
	<li>Adjust the vertical position of the capillary using fine focus control knob of the microscope position. Typically the first examined plane is located between 4 to 7 &#181;m above the capillary bottom. Video 1 corresponds to the xy field that has its top left-hand corner at the origin of the spatial referential.</li>
	<li>Trigger the 40 sec sequence of events described in section 4.2 and Figure 3 by switching on the current generator and at the same time, manually trigger the image acquisition sequence of Video 1.</li>
	<li>Move the capillary to the neighbor right field by a 250 &#181;m translation of the microscope stage to the left and operate as in section 5.3 to generate Video 2 of slice 2 and so on to obtain the required videos. Typically 3 to 4 fields of 250 x 250 &#181;m2 are collected along the x-axis before changing the plane and repeating same operations for the new plane.</li>
</ol>
6. Force Calibration
<ol>
	<li>Prepare a glycerol solution by mixing 39.8 g of glycerol with 190 &#181;l of bi-distilled water and 10 &#956;l of magnetic particles at a 2 x 109 particles/ml and fill up an experimental channel with this mixture and place it on the microscope stage as described for the biofilm sample.</li>
	<li>After installing the magnetic tweezers as indicated in section 4, apply the magnetic force as indicated in section 5.4 and make time-lapse images to extract each single particle velocity (v) and its position in the capillary to derive the calibration file. This file should contain the applied force as a function of its position in the zone of analysis of the capillary according to stokes law, F = 6&#960;R&#951;v (R: radius of the particle).</li>
</ol>
7. Analysis
<ol>
	<li>Use a &#34;particle tracker&#34; software to obtain text files with the particle positions in each frame for all the stacks of images acquired as indicated in section 5. Using the image stack acquisition frequency, calculate the particle displacement as a function of time (e.g. Figure 5 and Video 4).</li>
	<li>Using the force calibration file, convert the displacement curves to compliance curves (total compliance of the material &#8212; J(t)&#8212; as a function of time) according to the compliance formula: 
	<img alt="" fo:content-width="1in" src="/files/ftp_upload/50857/50857eq1.jpg" width="106" /> 
	which gives the relation between material strain and applied stress for a probe particle of radius R embedded in an incompressible, homogeneous viscoelastic medium as previously established by Schnurr and co-workers19.</li>
	<li>Adjust the creep compliance curves to the general Burgers model for viscoelastic materials and derive the viscoelastic parameters, J0, J1, &#951;0, &#951;1 for each particle (Figure 6) according to the Burgers&#39; formula: 
	<img alt="" fo:content-width="2in" src="/files/ftp_upload/50857/50857eq2.jpg" width="169" /> 
	Note: This phenomenological analysis has been previously employed for a wide range of materials including biological materials such as biofilms to interpret macroscopic rheology data20-22.</li>
</ol>

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We have nothing to disclose.

This magnetic particle seeding and pulling experiment enabled in situ 3D mapping of the viscoelastic parameters of a growing biofilm in its original state. This approach revealed the mechanical heterogeneity of the E. coli biofilm grown here and gave clues to point out the biofilm components supporting the biofilm physical properties, strongly suggesting a fundamental implication of the extracellular matrix and more precisely its degree of cross-linking.
The recognition of me...

Bacterial biofilms are communities of bacteria associated with biological or artificial surfaces1-3. They form by an adhesion-growth mechanism coupled with the production of polysaccharide-rich extracellular matrix that protects and stabilizes the edifice4,5. These biofilms are not simply passive assemblages of cells stuck to surfaces, but organized and dynamic complex biological systems. When bacteria switch from planktonic to biofilm lifestyle, changes in gene expression and cell physiology are observed as well as increased resistance to antimicrobials and host immune defenses being at the origin of many persistent and chronic infections6. However, the controlled development of these living structures also offer opportunities for industrial and environmental applications, such as bioremediation of hazardous waste sites, bio-filtration of industrial water or formation of bio-barriers to protect soil and groundwater from contamination.
While molecular features specific to biofilm way of life are increasingly described, the mechanisms driving the community development and persistence remain unclear. Using the recent advances on microscale measurements using scanning electrochemical or fluorescence microscopy, these living organizations have been shown to exhibit considerable structural, chemical and biological heterogeneity7. Yet, until now, biofilm mechanics have been mainly examined macroscopically. For instance, observation of biofilm streamers deformation due to variations in fluid flow rates8,9, &#160;uniaxial compression of biofilm pieces lift from agar medium or grown on cover slides10,11, shear of biofilm collected from the environment and then transferred to a parallel plate rheometer12,13, atomic force spectroscopy using a glass bead and coated with a bacterial biofilm attached to an AFM cantilever14 or a dedicated microcantilever method for measuring the tensile strength of detached biofilm fragments15,16 have been implemented during the ten last years, providing useful information on the viscoelastic nature of the material17. However, it seems likely that information on in situ biofilm mechanical properties is lost when the material is removed from its native environment, which was often the case in these approaches. Moreover, the treatment of the biofilm as a homogeneous material misses the information on the possible heterogeneity of the physical properties within the community. Therefore, the exact implications of the structure mechanics in the biofilm formation and biological traits such as gene expression patterning or chemical gradients can hardly be recognized. To progress towards a microscale description of the biofilm physical properties, new dedicated tools are required.
This paper details an original approach conceived to achieve measurement of local mechanical parameters in situ, without disturbing the biofilm and enabling drawing of the spatial distribution of the microscale material properties and then the mechanical heterogeneity. The principle of the experiment rests on the doping of a growing biofilm with magnetic microparticles followed by their remote loading using magnetic tweezers in the mature biofilm. Particle displacement under controlled magnetic force application imaged under the microscope enables local viscoelastic parameter derivation, each particle reporting its own local environment. From these data, the 3D mechanical profile of the biofilm can be drawn, revealing spatial and environmental condition dependences. The whole experiment will be shown here on an E. coli biofilm made by a genetically engineered strain carrying a derepressed F-like plasmid. The results detailed in a recent paper18 provide a unique vision of the interior of intact biofilm mechanics.

<table border="0" cellpadding="0" cellspacing="0" width="1258">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 1: Reagents and cells</td>
		</tr>
		<tr height="18">
			<td height="20" rowspan="2" style="height:22px;width:216px;">Magnetic particles</td>
			<td rowspan="2" style="width:323px;">Life technologies</td>
			<td rowspan="2" style="width:224px;">14307D</td>
			<td colspan="2" rowspan="2" style="width:496px;">Micrometric magnetic particle, 2.8 &#181;m diameter</td>
		</tr>
		<tr height="2">
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Ampicillin (Antibiotic)</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">A9518</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracycline (Antibiotic)</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">87128</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacterial strain MG1655gfpF</td>
			<td style="width:323px;">UGB, Institut Pasteur, France</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">produces F pili at its surface, resistant to Ampicilllin and tetracycline</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 2:&#160; Capillaries and tubing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Filters for pediatric perfusion</td>
			<td style="width:323px;">Prodimed-Plastimed</td>
			<td style="width:224px;">6932002</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Hollow Square Capillaries</td>
			<td style="width:323px;">Composite Metal Scientific</td>
			<td style="width:224px;">8280-100</td>
			<td colspan="2" style="width:496px;">Manufactured in Borosilicate glass. Square 0.8mm x0.8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing silicone peroxyde</td>
			<td style="width:323px;">VWR international</td>
			<td style="width:224px;">228-0512</td>
			<td colspan="2" style="width:496px;">Diameter 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing silicone peroxyde</td>
			<td style="width:323px;">VWR international</td>
			<td style="width:224px;">228-0700</td>
			<td colspan="2" style="width:496px;">Diameter 3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Table 3: Biofilm growth</td>
			<td style="width:323px;"></td>
			<td style="width:224px;"></td>
			<td style="width:495px;"></td>
			<td style="width:1px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysogeny Broth (LB) solution</td>
			<td style="width:323px;">Amresco-VWR</td>
			<td style="width:224px;">J106-10PK</td>
			<td colspan="2" style="width:496px;">standard medium used to grow bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">M63B1 solution</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">Standard minimum&#160; medium used to grow bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">G8270</td>
			<td colspan="2" style="width:496px;">Used to make M63B1 medium with 0.4% glucose</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 4: Electronics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera EMCCD&#160;&#160;</td>
			<td style="width:323px;">Hamamatsu</td>
			<td style="width:224px;">C9100-02</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Heater controller</td>
			<td style="width:323px;">World precision instruments</td>
			<td style="width:224px;">300354</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Function generator</td>
			<td style="width:323px;">Agilent technologies</td>
			<td style="width:224px;">33210A</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power amplifier</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">It gives a current signal with amplitudes up to 4 A.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pumps</td>
			<td style="width:323px;">Kd Scientific</td>
			<td style="width:224px;">KDS-220</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shutter</td>
			<td style="width:323px;">Vincent Associates</td>
			<td style="width:224px;">Uniblitz T132</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:216px;">Magnetic tweezers</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">Two electromagnetic poles, each made of a copper coil with 2,120 turns of 0.56 mm in diameter copper wire and soft magnetic alloy cores (Supra50-Arcelor Mittal, France) square shaped according to the blueprint shown in Fig. 10. The two cores are mounted north pole facing south pole, in order to generate a magnetic force in one direction along the length of the capillary. See coil wiring details in Figure 11.</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 5: Optics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope&#160;</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:224px;">TE-300</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">S Fluor x40 Objective (NA 0.9, WD0.3)</td>
			<td>Nikon</td>
			<td style="width:224px;"></td>
			<td>This a long working distance ojective enabling observation of the biofilm in the depth</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Epifluorescence filters: 1) for green fluorescence: Exc 480/20 nm; DM 495; Em 510/20&#160; 2) for Red fluorescence: Exc 540/25 nm; DM 565; Em 605/55</td>
			<td style="width:323px;">Chroma</td>
			<td style="width:224px;">1)#49020 2)#31002</td>
			<td>Particle displacement upon force application is recorded using the red fluoresecnce filter block.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 6: Image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td style="width:323px;">NIH - particle tracker plugin</td>
			<td style="width:224px;"></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A typical analysis will provide the spatial distribution of the viscoelastic parameters at the micron scale on a living biofilm without disturbing its original arrangement. Typical results are shown in Figure 7 where the values of J0 &#8212; the elastic compliance &#8212; are given as a function of the z-axis along the depth and of the y-axis along a lateral dimension of the biofilm. Each point corresponds to a bead which creep curve analysis has provided a J0 value. The data reveal...

Watch this Scientific Journal Video about Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties at JoVE.com

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.

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 ...

remote-magnetic-actuation-micrometric-probes-for-situ-3d-mapping

Research

JoVE Journal

Bioengineering

9.1K 조회수.  Sorbonne Universités, UPMC.  이 논문은 세균 바이오 필름과 현장에서 인터페이스에서 미생물에 의해 만들어진 복잡한 생활 소재의 로컬 기계적 특성을 측정 할 수있는 전용 자기 핀셋의 개발에 씨앗을 품고 자기 입자의 원격 작동에 따라 원래의 방법을 보여줍니다. 

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

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. ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

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 ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...