NIH grants R01 HL085369, HL067322, HL082729, EB008999

1.	Preparation
<ol>
<li>Prepare freshly made Tyrodes' solution (in mM, 128.2 NaCl, 1.3 CaCl2, 4.7 KCl, 1.19 NaH2PO4, 1.05 MgCl2, 20.0 NaHCO3, and 11.1 glucose). To expedite the daily preparation of solutions, prepare two stock solutions in advance and store them at +4&deg;C refrigerator: (1) Stock I (in g/2L, 374.6 NaCl, 9.56 CaCl2, 17.52 KCl, 8.21 NaH2PO4, 10.67 MgCl2) and (2) Stock II (in g/2L, 84.01 NaHCO3). To make 2L of Tyrodes' solution sufficient for one experiment take 1840mL of deionized water and mix in it 80mL of Stock I, 80mL of Stock II, and 4g of glucose.</li>
<li>Prepare stock solutions of dyes and uncouplers: (1) excitation-contraction uncoupler blebbistatin stock solution (Tocris Bioscience, 2mg/mL solution in DMSO); (2) voltage-sensitive dye di-4-ANEPPS stock solution (Invitrogen, 1mg/mL solution in DMSO); (3) voltage-sensitive dye RH 237 stock solution (Invitrogen, 1.25mg/ml solution in DMSO); (4) calcium indicator Rhod-2AM stock solution (Invitrogen, 1mg/ml solution in DMSO). A single rabbit experiment requires approximately 30 &mu;L of the di-4-ANEPPS stock solution, 30 &mu;L of the RH237 stock solution, and 200 &mu;L of the Rhod-2AM stock solution. To avoid repeated freezing and thawing, we store 100 &mu;L aliquots of di-4-ANEPPS at -20&deg;C. Other dyes are also stored at -20&deg;C. Store the dissolved blebbistatin in +4&deg;C refrigerator.</li>
<li>Prior to harvesting the heart, transfer Tyrodes' solution into the 2L bottle and place it into a water bath (Fisher Scientific) which maintains the solution temperature at 37 &deg;C. The solution is oxygenated with 95% O2 - 5% CO2. The pH is maintained at 7.35&plusmn;0.05 by adjusting the oxygenation level. The solution is circulated in the Langendorff perfusion system and is filtered by a nylon net filter (pore size: 11&mu;m, Millipore) placed in the perfusion line before the cannula.</li>
<li>Prepare monitoring equipment prior to harvesting the heart. A pressure transducer (WPI) is used to monitor the aortic pressure during the experiment. Baseline of the pressure transducer is adjusted to zero mmHg when the heart is not attached to the perfusion system. Pseudo-ECG electrodes are placed in the chamber to approximate Lead I, II, and III of the Einthoven triangle ECG.</li>
</ol>
2.	Harvesting and Perfusion of Rabbit Hearts
<ol>
<li>Fix the rabbit in the rabbit restrainer. Euthanize the rabbit by an intravenous injection of sodium pentobarbital (50 mg/kg) with 2000 U heparin. When the rabbit is completely euthanized, which is determined by the lack of pain reflex, the thoracic cavity is quickly opened and the heart and lungs are excised.</li>
<li>Make a cut at the upper end of ascending aorta before all the branches of aortic arch. Flush out the air from the ascending aorta and then quickly cannulate the heart to a 16-gauge cannula, which has been previously attached to a bubble trapper that is very important for keeping air out of coronaries. Once the heart is retrogradely perfused in a non-recirculating Langendorff perfusion system, make a cut to open the pericardium quickly.</li>
<li>Remove the lung, trachea, fat, and connective tissues, while the blood is flushed out by the perfusion.</li> 
<li>Very important! A silicone tubing (&tilde;3cm long, and 2mm in diameter) is inserted through a pulmonary vein and mitral valve into the left ventricle (LV) and kept there throughout the experiment. This tube releases the solution that is trapped in the LV. Without circulation for hours during the Langedorff-perfusion experiment in a mechanically immobilized heart, it is likely to cause severe ischemia in the LV cavity and to produce arrhythmia.</li>
<li>Move the heart with the cannula to the recirculating Langendorff-perfusion system with the optical mapping apparatus.</li>
</ol>
3.	Conducting Experiments using the Panoramic Optical Mapping System
<ol>
<li>Place the heart in a custom-made hexagon chamber and connect the cannula to the perfusion system. Maintain the aortic pressure at 60 &plusmn; 5 mmHg by adjusting the flow rate of the perfusion pump. Monitor lead I pseudo-ECG. Maintain the pH at around 7.35 &plusmn; 0.05.</li>
<li>Turn off the room light, because blebbistatin is photoinactivated by UV and low end (450-490 nm)17 of the visible part of the spectrum. Slowly inject blebbistatin stock solution through the injection port in the air-bubble trapper located above the cannula. Slowly inject 0.1ml blebbistatin for every bolus injection. Wait for the pressure to stabilize before the next injection.</li>
<li>Gently place the pacing electrode onto the location specific to your experimental design.</li>
<li>Focus the image of the heart in the frosted glass located at the image plane of each photo-diode array (PDA) from three evenly spaced angles surrounding the heart. Adjust the position of the cannula and the distances between each PDA and the heart in order to fit the heart in the field-of-view of all three PDAs. Take a picture of each focused image in the frosted glass.</li>
<li>Slowly inject 10&tilde;20&mu;L di-4-ANEPPS stock solution through the injection port in the air-bubble trap into the perfusion solution. Wait 1&tilde;3 minutes before taking optical recordings.</li>
<li>For the first recording, turn the green LED light (no excitation filter, LED FLOOD, LUMILEDS) on, take optical recordings simultaneously from three PDAs connected with the custom-made data acquisition system,5 and turn off the LED light. Check the quality of the signals from different pixels of all three PDAs. Add 0.1&tilde;0.2mL blebbistatin stock solution if motion artifacts in the optical action potential are noticed. Add another 5 &mu;L di-4-ANEPPS stock solution if signal-to-noise ratio is low.</li>
<li>Finish the designed experimental protocol for the functional study. Re-stain the heart with additional 5&mu;L di-4-ANEPPS stock solution if the signal deteriorates during the experiments due to photobleaching or washout.</li>
<li>Turn on the room light after the completion of the functional study. Take pictures of the heart from 36 evenly spaced angles. This is achieved by rotating the heart at a step of 10&deg; with a digital camera fixed at the location of one PDA.</li>
<li>Take the heart out of the chamber. Drain all the solutions. Wash the perfusion system in the sequence of DI water, 70% reagent alcohol, and again DI water.</li>
<li>Data analysis includes the reconstruction of the heart geometry from the 36 digital photographs, registration of the optical signal onto the surface of the reconstructed geometry, and quantification of action potential duration (APD), conduction velocity (CV), phase, etc.6</li>
</ol>
4.	Conducting Experiments using the Dual Mapping System
<ol>
<li>(Continue after part 2) Place the cannulated heart in a glass chamber (Radnoti) and connect the cannula to the perfusion system. Pin down the heart to the silicon bottom of the chamber at the ventricular apex and atria.</li>
<li>Turn off the room light. Slowly inject blebbistatin stock solution (15&tilde;20 min to reach 10&mu;M) through the injection port prior to perfusion cannula to immobilize the heart.</li>
<li>Put a plastic Petri dish, or other glass window cover, above the epicardial surface to reduce the motion of the solution surface.</li>
<li>Focus two CMOS cameras in the dual mapping system (Ultima-L, SciMedia) at the same field of view. Emitted fluorescence is separated by a dichroic mirror (635nm cutoff, Omega Optical), and filtered by a 700nm longpass filter (Thorlabs) for voltage signals and by a 590/30 nm bandpass filter (Omega Optical) for calcium signals.</li>
<li>Aim the light guides of two halogen lamps (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) towards the mapping field of view to achieve even illumination. Excitation filters (531/40 nm, SemRock) are used.</li>
<li>Stain the heart with the voltage-sensitive dye RH 237 stock solution (10&tilde;30 &mu;L) through the injection port.</li>
<li>Mix the Rhod-2AM (0.2mL) stock solution with Pluronic F-127 (Invitrogen, 1:1 mixture). Sonicate for 1min in a water-bath sonicator. Inject the mixture through the bubble trapper's port. Wait for approximately 20 minute to allow the de-esterification of the Rhod-2 AM before mapping starts.</li>
<li>For one recording, turn off the superfusion pump to avoid motion at the surface of the solution; turn on the excitation light source (the halogen lamps); take optical recordings using both cameras connected to the data acquisition system (Ultima-L, SciMedia); turn off the excitation light; and turn on the superfusion pump. Check the quality of the optical signals. Re-stain the tissue if necessary.</li>
<li>Finish the rest of the designed experimental protocol for a study.</li>
<li>Turn on the room light and take a photograph of the heart containing the field of view. Take the heart out of the chamber. Drain all the solutions. Wash the perfusion system in the sequence of DI water, 70% reagent alcohol (Fisher Scientific), and DI water.</li>
<li>Data analysis contains measurements of APD, CV, calcium transient duration (CaTD), the delay between AP upstroke and CaT rise, the rise time of the calcium transient, and the time constant of a monoexponential fit of the CaT decay.</li>
</ol>
Representative Results: 
<img src="/files/ftp_upload/3160/3160fig1.jpg" alt="Figure 1"/> Figure 1. Representative results of a Langendorff-perfused rabbit experiment using the panoramic optical mapping system. (A) The anterior view of the rabbit heart and the reconstructed rabbit heart geometry in the form of a three-dimensional mesh-grid surface. (B) The unwrapped epicardial surface color-coded by the phase (obtained from the phase plane analysis18) indicating the wavefront shown in red during an episode of tachyarrhythmia. (C) The optical action potential recordings from five locations around the phase singularity marked by 1-5 in panel B. (D) Eight snapshots of activation wavefront (red color) propagation during a cycle of a stable reentrant arrhythmia. The wavefront circles clockwise around a phase singularity, which is visible at the anterior surface of the heart. The color for repolarization (blue) is set to be partially transparent so that the posterior wavefront is visible (e.g., at 80ms, 100ms, and 120ms). A movie of this reentrant arrhythmia is provided in the supplemental video 1. The methods for geometry reconstruction, signal registration, calculation of phase map, and surface unwrapping are described in details elsewhere6. 
<img src="/files/ftp_upload/3160/3160fig2.jpg" alt="Figure 2"/> Figure 2. Representative results from a Langendorff-perfused rabbit heart experiment using the dual mapping system (simultaneous mapping of action potential and calcium transient). (A) The anterior surface of the heart with the mapping field of view covered by black dots. (B) A close-up view of recordings from one site. (C) Sample traces of action potential (blue) and calcium transient (red) from an array of evenly spaced locations marked by the black dots in panel A. Note that not all the pixel recordings are shown and the spatial resolution is 200&mu;m.

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M., Pieske, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+Ca(2+)-handling+and+sarcoplasmic+reticulum+Ca(2+)-content+in+isolated+rat+and+rabbit+myocardium.">Differences in Ca(2+)-handling and sarcoplasmic reticulum Ca(2+)-content in isolated rat and rabbit myocardium.</a> J Mol Cell Cardiol. 32, 2249-2258 (2000).</li><li>Bray, M. A., Lin, S. F., Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+surface+reconstruction+and+fluorescent+visualization+of+cardiac+activation.">Three-dimensional surface reconstruction and fluorescent visualization of cardiac activation.</a> IEEE Trans Biomed Eng. 47, 1382-1391 (2000).</li><li>Qu, F., Ripplinger, C. M., Nikolski, V. P., Grimm, C., Efimov, I. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+voltage+changes+during+unipolar+stimulation+of+rabbit+ventricle.">Transmembrane voltage changes during unipolar stimulation of rabbit ventricle.</a> Circ Res. 77, 1229-1239 (1995).</li><li>Bishop, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photon+scattering+in+optical+signal+distortion+during+arrhythmia+and+defibrillation.">The role of photon scattering in optical signal distortion during arrhythmia and defibrillation.</a> Biophys J. 93, 3714-3726 (2007).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. 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No conflicts of interest declared.

Based on our experience, the keys for a successful Langendorff-perfused rabbit heart experiment include well-prepared Tyrodes' solution, quick harvest of the heart, well-maintained perfusion pressure, and appropriate pH of the oxygenated solution in the perfusion system. In order to record the signal with highest possible signal-to-noise ratio, we need to consider factors including light source, optical filters, focusing optics, photodetectors, etc19. Details of these aspects are discussed elsewhere19. Young rabbits (age: 4-5 months; weight: 7-9 pounds) could be used to avoid the epicardial fat, which decreases the signal to noise ratio of the optical signals. 
The signal recorded by each pixel is a weighted integration of emitted light from a volume of tissue. The depth of this tissue volume depends on the excitation and emission wavelengths of the dye used. For di-4-ANEPPS, as an example, the estimated penetration depth is 300&mu;m in the rabbit heart20. Thus, interpretation of the optical signal should be done with caution when the local heterogeneity of electrical function are present in sinoatrial node, atrioventricular node, and during ventricular arrhythmia 1,21,22. 
One limitation of the optical mapping technique compared with electrode recording is that the repolarization phase of the optical action potential is often distored by motion artifact caused by cardiac contraction. Mechanical constraint could be used to reduce the artifact, but cannot completely eliminate it. In comparison, pharmacological excitation-contraction uncouplers are effective in removing the motion artifact. However, these uncouplers (e.g. 2,3-Butanedione Monoxime) could have significant electrophysiological side effects. Blebbistatin was demonstrated to have no adverse side-effects on the cardiac electrophysiology in the normal heart23, and is thus a promising uncoupler for optical mapping. It should be noted that the acceleration of edema due to the abolishment of the contraction could also affect the electrophysiology.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S271-1</td>
		</tr>
		<tr>
			<td>CaCl2 (2H2O)</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>C79-500</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S217-500</td>
		</tr>
		<tr>
			<td>MgCl2 (6H2O)</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>M33-500</td>
		</tr>
		<tr>
			<td>NaH2PO4 (H2O)</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S369-500</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S233-3</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>D16-1</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Tocris Bioscience, Ellisville, MO</td>
			<td>1760</td>
		</tr>
		<tr>
			<td>Di-4-ANEPPS</td>
			<td>Invitrogen, Carlsbad, CA</td>
			<td>D1199</td>
		</tr>
		<tr>
			<td>RH237</td>
			<td>Invitrogen, Carlsbad, CA</td>
			<td>S1109</td>
		</tr>
		<tr>
			<td>Rhod-2AM</td>
			<td>Invitrogen, Carlsbad, CA</td>
			<td>R1244</td>
		</tr>
		<tr>
			<td>Pluronic F127</td>
			<td>Invitrogen, Carlsbad, CA</td>
			<td>P3000MP</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>Sigma, St. Louis, MO</td>
			<td>D2650</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart at JoVE.com

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

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 ...

multiparametric-optical-mapping-langendorff-perfused-rabbit

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JoVE Journal

Bioengineering

22.4K Views.  Washington University in St. Louis. 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.

Video: Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

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. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

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 ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...