NIH grants R01 HL085369.

1. Advanced preparation of stock solutions
<ol>
	<li>Prepare two stock solutions of Tyrode's solution (16x) in advance in deionized water and store them at 4&deg;C:
	<ol class="lalpha">
		<li>Stock I (119.872 g/L NaCl, 3.056 g/L CaCl2 (2H2O), 5.6 g/L KCl, 2.6274 g/L NaH2PO4, 3.408 g/L MgCl2 (6H2O), (Fisher Scientific, Fair Lawn, NJ));</li>
		<li>Stock II (26.88 g/L NaHCO3, (Fisher Scientific, Fair Lawn, NJ)).</li>
	</ol>
	</li>
	<li>Prepare stock solutions of fluorescent dyes. To avoid repeated freezing and thawing, we store 30 &mu;l aliquots of both dyes at -20&deg;C, which is sufficient for one experiment:
	<ol class="lalpha">
		<li>Voltage-sensitive dye RH237 (Invitrogen, Carlsbad, CA) stock solution, 1.25 mg/mL solution in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO);</li>
		<li>Calcium indicator Rhod-2AM (Invitrogen, Carlsbad, CA) stock solution, 1 mg/mL solution in DMSO.</li>
	</ol>
	</li>
	<li>Prepare excitation-contraction uncoupler blebbistatin stock solution (Tocris Bioscience, St. Louis, MO, 2 mg/mL solution in DMSO) in advance and store the dissolved blebbistatin at 4&deg;C.</li>
</ol>
2. Prepare perfusion solutions and experimental setup7
<ol start="4">
	<li>Freshly prepare 2L Tyrode's solution (128.2mM NaCl, 1.3mM CaCl2 (2H2O), 4.7mM KCl, 1.05mM MgCl2 (6H2O), 1.19mM NaH2PO4, 20mM NaHCO3, 11.1mM D-Glucose in deionized water, pH=7.35&plusmn;0.05). If stock solution is being used to make 2L of Tyrode's solution (sufficient for one experiment) take 1750 mL of deionized water and mix in 125 mL of Stock I, 125 mL of Stock II, and 4g of glucose.</li>
	<li>Turn on the two pumps of the perfusion systems. Set the peristaltic pump (Peri-Star, WPI, Sarasota, USA) that is used for retrograde perfusion to 40 mL/min. Set the other peristaltic pump (Cole-Parmer Masterflex Peristalic Pump L/S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) that is used for superfusion and to return the perfusate back to the holding reservoir to 80 mL/min.</li>
	<li>Wash the perfusion system with 70% ethanol for 30 min and then with 2L deionized water.</li>
	<li>Once all the deionized water is evacuated from the chamber, circulate the Tyrode's solution and pass it through a 5-&mu;m filter (Millipore, Billerica, MA, USA). Warm the perfusate to +37&deg;C with a water jacket and circulator (ThermoNESLAB EX7, Newtown, USA) and oxygenate the perfusate by bubbling O2/CO2 (95% / 5%) gas into the solution. Monitor the pH of the solution with a pH meter (Oakton Instruments, Vernon Hills, IL) and adjust the rate of O2/CO2 bubbling to keep the pH at 7.35 &plusmn; .05. Continue monitoring pH and temperature during the experiment.</li>
	<li>The dual optical mapping apparatus consists of two MiCAM Ultima-L CMOS cameras (SciMedia, Costa Mesa, CA) that have high spatial (100x100 pixels, 230&plusmn;20 &mu;m per pixel) and temporal (1,000-3,000 frames/sec) resolution. Fix a band-pass filter (590&plusmn;15 nm, Thorlabs, Newton, NJ) in front of the designated calcium imaging camera; while, a long-pass filter (&gt;700 nm, Thorlabs, Newton, NJ) needs to be positioned in front of the designated voltage imaging camera. The cameras are arranged perpendicular to one another by a holder, which contains a dichroic mirror (635 nm cutoff, Omega Optical, Brattleboro, VT). Immediately below the dual camera holder there is a lens (Nikon NIKKOR 55 mm 1:1.4 235052), which focuses the emission light coming from the heart onto the dichroic mirror. The working distance is approximately 3cm. 
	The excitation light is generated by a halogen lamp (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) and is passed through a heat filter, shutter, and band-pass filter (520&plusmn;45 nm). A flexible light guide directs the band-pass filtered light onto the preparation, and a shutter is used to ensure that the preparation is exposed to light only during image acquisition to avoid photobleaching of the dyes.</li>
	<li>Prepare Ag/AgCl2 electrodes for pacing and sensing in advance and install them in the chamber prior to placing the heart. Make sure that amplifiers and filters are adjusted to appropriate levels.</li>
</ol>
3. Harvest the mouse heart, cannulate, and set up Langendorff perfusion
<ol start="10">
	<li>Anesthetize the mouse with ketamine/xylazine (ketamin, 80mg/kg bodyweight; xylazine, 10 mg/kg bodyweight) and heparin (100 units) by intraperitoneal injection. Assure appropriate level of anesthesia by the lack of pain reflex.</li>
	<li>After a mid-sternal incision, quickly remove the heart and wash it in oxygenated (95% O2, 5% CO2), constant-temperature (37&plusmn;1&deg;C) Tyrode's solution.</li>
	<li>Using a dissecting microscope, rapidly identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery. A short section of aorta is then attached to a custom made 21-gauge cannula with a flared tip. 4-0 black-braided silk suture (Surgical Specialties Corporation, Reading, PA) is used to fix the heart onto the cannula. After cannulation, the heart is retrogradely perfused and superfused with Tyrode's solution. The retrograde perfusion rate is adjusted in the range of 2-5 mL/min to keep the aortic pressure between 60 and 80 mmHg (Pressure transducer, World precision instruments Inc (WPI), Sarasota, USA; Bridge Amplifier TBM4M, WPI, Sarasota, USA).</li>
	<li>After the heart is cannulated, lung, thymus, and fat tissue are then dissected and removed.</li>
	<li>The isolated heart is pinned (Fine Science Tools) at the apex to the bottom of the perfusion chamber (Sylgard coated) to prevent stream-induced movement. The right and left atrial appendages are also stretched and pinned (Fine Science Tools, Inc, Foster City, CA) to the bottom of the chamber, which provides maximal surface area for optical measurements of the atria. 
	Very important! A small silicon tube is inserted into the left ventricle through the pulmonary veins and fixed by silk suture to nearby connective tissue. This prevents solution congestion and acidification of the perfusate trapped in the left ventricle, which is especially important after the suppression of ventricular contractions with an excitation-contraction uncoupler (see Part 4 step 19).</li>
	<li>A custom made electrode is placed on the surface of the heart to conduct the pacing stimulations, which is generated by Master-8 (A.M.P Instruments Ltd, Jerusalem, Israel) or PowerLab 26T (AD Instruments, Sydney, Australia).</li>
	<li>A small cover glass is fixed on the surface of the solution over the heart to reduce motion artifact from the vibrating solution.</li>
	<li>Focus the excitation light on the heart. In addition, adjust the distance between the dual camera apparatus and the heart so that maximum resolution will be obtained.</li>
	<li>Turn off all lights in the room and commence electrical recordings using PowerLab 26T.</li>
</ol>
4. Load voltage and calcium sensitive dyes and excitation-contraction uncoupler
<ol start="19">
	<li>Warm-up 0.6 mL of blebbistatin. Mix 0.5 mL of the blebbistatin with the perfusate in the holding reservoir. Dilute the remaining 0.1 mL of blebbistatin in 1 mL of Tyrode's solution and slowly inject it (over a 20 minute period) through a drug port located near the cannula. Observe as blebbistatin gradually reduces the motion artifact.</li>
	<li>Dilute 30 &mu;L of voltage sensitive dye RH237 stock solution in 1 mL Tyrode's solution and slowly inject over 5-7 minutes into the same injection port as blebbistatin.</li>
	<li>30 &mu;L calcium indicator Rhod-2 AM is 1:1 mixed with Pluronic F127 (Invitrogen, Carlsbad, CA, 20% solution in DMSO) and then diluted in 1 mL Tyrode's solution and slowly applied over 5-10 min through the same injection port.</li>
	<li>Wait for 5-10 minutes for blebbistatin and dyes to reach the cell membrane and cytosol. Continue with the protocol when motion is completely suppressed.</li>
	<li>Continuously monitor ECG recordings over the entire procedure to assure normal electrical function of the heart.</li>
	<li>Commence fluorescent signal recordings using the SciMedia custom software (SciMedia, Costa Mesa, CA).</li>
</ol>
5. Representative results:
<img alt="Figure 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> 
Figure 1. Experimental setup for the perfusion, electrical recordings and optical mapping. 
EM=emission; Lp=longpass
<img alt="Figure 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> 
Figure 2. Experimental preparation and signal examples recorded during ventricular pacing. Left: ECG signals are collected from Ag/AgCl2 disk electrodes (Top) and an example of S1S1 stimulation protocol is shown (Bottom). Center: Langendorff mouse heart preparation. Right: Representative optical action potentials and calcium transient signals from atria (Top) and ventricles (Bottom) are shown. The yellow arrow (Top) points to the fluorescent signal scattering coming from the ventricles, which is seen in the atrial recordings. 
LV=left ventricle; RV=Right ventricle; LA=left atrium; RA=right atrium
<img alt="Figure 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> 
Figure 3. Representative optical recordings of Vm and CaT from the ventricles of a wild type mouse heart. A. The experimental preparation with an array of evenly spaced locations marked by black dots whose optical recordings can be seen in (C). B. Example tracing of Vm and CaT from a central location on the array (see box in (C)). C. Vm (blue) and CaT (red) from the array of evenly spaced locations. Signals were binned 3x3.
<img alt="Figure 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> 
Figure 4. Activation map and conduction. A. An example activation map from a wild type mouse heart with transverse (T) and longitudinal (L) directions indicated by white arrows. B. Vm signals (Top) and dV/dt (Bottom) corresponding to the three points seen in A (T1, T2, T3; L1, L2, L3).
<img alt="Figure 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> 
Figure 5. Action potential and calcium transient duration analysis. A. Action potential duration at 80% repolarization (APD80) and calcium transient duration at 80% relaxation (CaD80) maps are shown from a heart under control conditions (Left) and after 30 nM isoproterenol application (Right). The yellow/green color in the ventricles (Right) indicates isoproterenol shortened APD80 and CaD80. B. Example tracings of APD80 and CaD80 from the wild type mouse ventricles (Top) and atria (Bottom).

<ol>
	<li>Efimov, I. R., Rendt, J. M., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+maps+of+intracellular+[Ca2+]i+transients+and+action-potentials+from+the+surface+of+perfused+guinea-pig+hearts.">Optical maps of intracellular [Ca2+]i transients and action-potentials from the surface of perfused guinea-pig hearts.</a> Circulation. 90, 1-1 (1994).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+repolarization+and+refractoriness+from+intact+hearts.">Optical mapping of repolarization and refractoriness from intact hearts.</a> Circulation. 90, 1469-1480 (1994).</li><li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> J Physiol. 529, 171-188 (2000).</li><li>Pruvot, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+calcium+cycling+versus+restitution+in+the+mechanism+of+repolarization+alternans.">Role of calcium cycling versus restitution in the mechanism of repolarization alternans.</a> Circ Res. 94, 1083-1090 (2004).</li><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95, 21-33 (2004).</li><li>Fast, V. G., Dhein, S., Delmoar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+action+potential+using+voltage+sensitive+dyes.">Recording action potential using voltage sensitive dyes.</a> Practical methods in cardiovascular research. , 233-255 (2005).</li><li>Glukhov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. , (2009).</li><li>Baker, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dispersion+of+repolarization+and+refractoriness+in+transgenic+mouse+hearts+promotes+reentrant+ventricular+tachycardia.">Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.</a> Circ Res. 86, 396-407 (2000).</li><li>Sutherland, F. J., Hearse, D. 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+isolated+blood+and+perfusion+fluid+perfused+heart.">The isolated blood and perfusion fluid perfused heart.</a> Pharmacol Res. 41, 613-627 (2000).</li><li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+electrode+polarization+in+the+far+field:+implications+for+external+defibrillation.">Virtual electrode polarization in the far field: implications for external defibrillation.</a> Am J Physiol Heart Circ Physiol. 279, 1055-1070 (2000).</li><li>Hammouda, M., Kinosita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coronary+circulation+in+the+isolated+heart.">The coronary circulation in the isolated heart.</a> J Physiol. 61, 615-628 (1926).</li><li>Salama, G., Hwang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Curr Protoc Cytom. 12, 17-17 (2009).</li><li>Lou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+panoramic+imaging+of+epicardial+electrical+activity.">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li><li>Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> J Membr Biol. 208, 125-140 (2005).</li></ol>

No conflicts of interest declared.

In this experiment we modified the Langendorff perfusion method by adding a small silicon tube, which is especially crucial after the suppression of ventricular contractions with an excitation-contraction uncoupler. The silicon tube is used to prevent solution congestion, acidification of the perfusion solution, and development of ischemia in the left ventricle. The mouse heart is very sensitive to hypothermia; thus, temperature variations across the heart will cause artificial differences in action potential durations. Consequently, a heating system was implemented in the perfusion chamber in order to maintain a constant temperature of 37&deg;C during the entirety of the experiment8. Since a Langendorff model does not retain innervation of the heart, one needs to consider adding neurotransmitters to the perfusate in order to investigate physiological sympathetic and parasympathetic tone9. Besides retrograde perfusion, addition of superfusion of the heart helps to maintain suitable environmental parameters such as pH and temperature. In this method, the Langendorff perfused heart was horizontally placed. A vertical Langendorff perfusion setup can also be used10, but may result in slightly different cardiac mechanics11. In addition to CMOS cameras, alternative detectors are also available and can be applied to map Vm and CaT simultaneously12.
Application of CMOS cameras of high spatio-temporal resolution assures the accuracy of the recordings; however, optical mapping signals are not from a single cell. Rather, each fluorescent signal comes from hundreds or thousands of cells, depending on optical magnification. The much larger ventricular fluorescence can distort atrial signals by optical scattering; therefore, careful interpretation of the optically recorded signals is required. Another limitation of the mouse preparation is the signal distortion and noise induced by the curvature of the surface due to the small size of the heart13. Conduction velocity measurements can be altered not only from the curvature of the mouse heart but also from electrode polarity and virtual electrodes. To achieve accuracy for conduction velocity, activation anisotropy, and repolarization maps, correct focusing of the camera at the surface of the heart is essential. 
In this method, real time ECG recordings can supplement the optical investigation of cardiac electrophysiology. Voltage-sensitive dye (RH237) and calcium indicator (Rhod-2AM) are used in the protocol because of their fast response, similar excitation, and distinct emission spectra3,7. There are alternative combinations of dyes that can be used to measure Vm and CaT other than RH237 and Rhod-2AM3. A novel voltage-sensitive dye, PGHI, with a large Stoke's shift (&gt;200nm) was found to allow better Vm and CaT signals because of the greater separation of the emission wavelengths between PGHI and Rhod-2AM14. Future improvements may focus on exploring novel fluorescent probes, development of new imaging detectors, and improved image processing software. Higher resolution and novel optical imaging modalities for 3D optical mapping are also important future directions of optical mapping5.

<table border="1">
	<tbody>
		<tr>
			<td>Chemical</td>
			<td>Company</td>
			<td>Catalog 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>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>
		<tr>
		 <td>Material</td>
		 <td>Company</td>
		 <td>Catalog Number</td>
	 </tr>
		<tr>
		 <td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank">PowerLab 26T </a></td>
		 <td><a href="http://www.adinstruments.com/" target="_blank">AD Instruments</a>, Sydney, Australia</td>
		 <td>&nbsp;</td>
	 </tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

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

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. ...

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

27.1K Views.  Washington University in St. Louis. 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.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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 ...

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 ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

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 ...