This study was funded by Doctoral Scholarships from The University of Auckland (awarded to JD&#160;and MC), Sir Charles Hercus Health Research Fellowships (20/011 and 21/116) from the Health Research Council of New Zealand (awarded to J-CH to KT, respectively), a Doctoral Scholarship awarded by the National Heart Foundation (awarded to AA), Marsden Fast-Start grants (UOA1504 and UOA1703) from the Royal Society of New Zealand (awarded to J-CH and KT, respectively), and a James Cook Research Fellowship from the Royal Society of New Zealand (awarded to AT). The original development of this instrument was funded by a Marsden grant (11-UOA-199) from the Royal Society of New Zealand (awarded to AT and PN).

<ol>
	<li>Han, J. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+stress+production+in+isolated+cardiac+trabeculae+from+the+rat.">Energetics of stress production in isolated cardiac trabeculae from the rat.</a> American Journal of Physiology. Heart and circulatory physiology. 299 (5), 1382-1394 (2010).</li><li>Ter Keurs, H. E. D. J., Rijnsburger, W. H., Van Heuningen, R., Nagelsmit, 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=Tension+development+and+sarcomere+length+in+rat+cardiac+trabeculae.+Evidence+of+length-dependent+activation.">Tension development and sarcomere length in rat cardiac trabeculae. Evidence of length-dependent activation.</a> Circulation Research. 46 (5), 703-714 (1980).</li><li>Shen, X., Cannell, M. B., Ward, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+SR+load+and+pH+regulatory+mechanisms+on+stretch-dependent+Ca2++entry+during+the+slow+force+response.">Effect of SR load and pH regulatory mechanisms on stretch-dependent Ca2+ entry during the slow force response.</a> Journal of Molecular and Cellular Cardiology. 63, 37-46 (2013).</li><li>Dowrick, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+slow+force+response+to+stretch:+Controversy+and+contradictions.">The slow force response to stretch: Controversy and contradictions.</a> Acta Physiologica. 226 (1), 13250 (2019).</li><li>Stuyvers, B. D. M. Y., Miura, M., Ter Keurs, H. E. 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=Diastolic+viscoelastic+properties+of+rat+cardiac+muscle;+involvement+of+Ca2+.">Diastolic viscoelastic properties of rat cardiac muscle; involvement of Ca2+.</a> Advances in Experimental Medicine and Biology. 430, 13-28 (1997).</li><li>Tang, E. J. L. P., Laven, R. C., Hajirassouliha, A., Nielsen, P. M. F., Taberner, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+displacement+in+isolated+heart+muscle+cells+using+markerless+subpixel+image+registration.">Measurement of displacement in isolated heart muscle cells using markerless subpixel image registration.</a> Conference Record - IEEE International Instrumentation and Measurement Technology Conference. , (2019).</li><li>Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+excitation-contraction+coupling.">Cardiac excitation-contraction coupling.</a> Nature. 415 (6868), 198-205 (2002).</li><li>Cheuk, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+markerless+tracking+of+the+strain+distribution+of+actively+contracting+cardiac+muscle+preparations.">A method for markerless tracking of the strain distribution of actively contracting cardiac muscle preparations.</a> Experimental Mechanics. 61 (1), 95-106 (2020).</li><li>Lippok, N., Coen, S., Nielsen, P., Vanholsbeeck, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersion+compensation+in+Fourier+domain+optical+coherence+tomography+using+the+fractional+Fourier+transform.">Dispersion compensation in Fourier domain optical coherence tomography using the fractional Fourier transform.</a> Optics Express. 20 (21), 23398 (2012).</li><li>Cheuk, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-Dimensional+imaging+of+cardiac+trabeculae+contracting+in+vitro+using+gated+OCT.">Four-Dimensional imaging of cardiac trabeculae contracting in vitro using gated OCT.</a> IEEE Transactions on Biomedical Engineering. 64 (1), 218-224 (2017).</li><li>Ritland, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+refractive+index+and+density+of+a+glass+at+constant+temperature.">Relation between refractive index and density of a glass at constant temperature.</a> Journal of the American Ceramic Society. 38 (2), 86-88 (1955).</li><li>Tuchina, D. K., Bashkatov, A. N., Genina, E. A., Tuchin, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+glucose+and+glycerol+diffusion+in+myocardium.">Quantification of glucose and glycerol diffusion in myocardium.</a> Journal of Innovative Optical Health Sciences. 8 (3), (2015).</li><li>Taberner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamometer+for+nature's+engines.">A dynamometer for nature's engines.</a> IEEE Instrumentation and Measurement Magazine. 22 (2), 10-16 (2019).</li><li>Arganda-Carreras, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trainable+Weka+Segmentation:+A+machine+learning+tool+for+microscopy+pixel+classification.">Trainable Weka Segmentation: A machine learning tool for microscopy pixel classification.</a> Bioinformatics. 33 (15), 2424-2426 (2017).</li><li>Jiang, Y., Julian, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacing+rate,+halothane,+and+BDM+affect+fura+2+reporting+of+[Ca2+](i)+in+intact+rat+trabeculae.">Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+](i) in intact rat trabeculae.</a> American Journal of Physiology - Cell Physiology. 273 (6), 2046-2056 (1997).</li><li>Miura, M., Boyden, P. A., Ter Keurs, H. E. 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=Ca2++waves+during+triggered+propagated+contractions+in+intact+trabeculae.">Ca2+ waves during triggered propagated contractions in intact trabeculae.</a> American Journal of Physiology - Heart and Circulatory Physiology. 274 (1), (1998).</li><li>Han, J. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radius-dependent+decline+of+performance+in+isolated+cardiac+muscle+does+not+reflect+inadequacy+of+diffusive+oxygen+supply.">Radius-dependent decline of performance in isolated cardiac muscle does not reflect inadequacy of diffusive oxygen supply.</a> American Journal of Physiology-Heart and Circulatory Physiology. 300 (4), 1222-1236 (2011).</li></ol>

The authors have nothing to disclose.

In this study, we present a configuration that enables the assembly of three optical systems combining brightfield, fluorescence, and OCT imaging to gather data from an actively contracting ex vivo cardiac trabecula (Figure 1 and Figure 2). Such an orchestrated integration is possible due to the design of the measurement chamber (Figure 3) to enable the orthogonal arrangement of the OCT to the brightfield-fluorescence axis. The muscle-mounting system plays an equally important role in the success of simultaneous quantifications of key indices in characterizing cardiac muscle excitation-contraction dynamics. Its novelty resides in enabling muscle-scanning procedures with no apparent disturbance to the mechanical performance of the muscle (Figure 6). With the combined imaging configuration and motorized-hook system for force measurement, this system can evaluate regional heterogeneity in the Ca2+ transient, displacement, and sarcomere length, together with macroscopic geometric information of a contracting trabecula throughout the time course of the twitch (Figures 7 and Figure 8).
Given the ubiquity of brightfield-epifluorescence imaging systems within cardiac research laboratories, the reproduction of these results can be achieved with some minor hardware considerations. Here, we present the image-processing toolkit for combining brightfield-epifluorescence and OCT, which is essential in analyzing the underlying contractile heterogeneity. The integration of the OCT requires an unobstructed optical path, while the gated imaging requires an external trigger line between the stimulus and both the OCT and brightfield imaging camera, and muscle mounting hooks that are capable of moving the sample throughout the measurement chamber. The required post-processing software and methods are freely available. In particular, the segmentation software used, WEKA14, is open-source. The technique of markerless tracking of material points8, sarcomere length, gated volumetric imaging10, and mesh generation codes are likewise accessible and can be made available on request from the corresponding author.
Muscle viability, optimal loading of Fura-2, and image focus are the three pillars that form the foundations of a successful experiment. Using a dissection solution containing BDM to prevent contracture, transportation of the muscle in a syringe, continuous oxygenation of the solution, and preparing new experimental solutions on the day of an experiment, all contribute to a high muscle viability rate. Before loading the trabecula with Fura-2AM, autofluorescence must be collected for each condition one is interested in studying as it can have a significant effect on the measured Ca2+ transient15. Oxygenation of Fura-2AM loading solution is complicated by the necessary inclusion of the surfactant pluronic-F127 to aid dye loading. To combat the resultant excess bubble formation caused by this surfactant, a small drop of anti-foam in the loading solution allows the user to increase the oxygenation rate, thereby improving the chance that the trabecula maintains functional viability throughout the loading process. Finally, the imaging focus must be uniform along muscle length to maximize the signal to noise ratio of the brightfield and fluorescence information.
There are two limitations to consider with the methods presented here. First is the spatial resolution of the fluorescence microscope. While the spatial resolutions of the OCT and brightfield imaging are high, the resolution of the fluorescence microscope is limited to the integral of the fluorescence from the volume captured within a 540 &#181;m by 540 &#181;m imaging window. There is scope to increase the spatial resolution of the fluorescence microscope by using a high-gain charge-coupled device camera, instead of a PMT, to capture the fluorescence signal at the expense of the signal to noise ratio16. Second is the diameter of the trabecula that can be studied in terms of measurable sarcomere length and geometric&#160;depth. The windowed-FFT approach for computing sarcomere length exploits the benefit of improved spatial resolution but is associated with reduced robustness (Figure 8D). In cases where turbid or large-diameter trabeculae are to be studied, the resolvability of the FFT will be greatly reduced due to the reduced contrast associated with the sarcomeric banding in larger tissue samples. Likewise, within the OCT, the back-reflections from an imaging depth of greater than 300 &#181;m will be too weak to be resolved during the segmentation stage. Hence, our technique is limited to trabeculae of a diameter less than 300&#160;&#181;m. However, it is not recommended to study large diameter samples as there may be problems with diffusive oxygenation of the muscle core during high rates of stimulation17.
Our method enables the assessment of ionic mechanical function in association with muscle geometry in healthy and diseased muscle, providing a powerful approach to understanding cardiac muscle physiology, pathophysiology, and pharmacology. The image-processing pipeline outlined here extracts data that will be pivotal for garnering a deeper understanding of contractile heterogeneity. One avenue to fully realize the potential of such a rich dataset is in the construction of mathematical models that integrate and interpret these data, and to make predictions that can be tested experimentally using our device.

Superfusion of isolated cardiac muscle tissue preparations is a standard and widely used protocol for studying cardiac ionic activation and mechanics1. In particular, the isolation of trabeculae, rod-like structures from the ventricular walls, has enabled assessment of phenomena including the length-dependent activation of contraction2, stretch-dependent response of contraction3,4, and diastolic viscoelasticity5 of cardiac tissue. Ter Keurs, the initiator of this technique of superfusing isolated trabeculae, initially used a combination of fluorescence imaging for Ca2+ measurements and laser diffraction for determining sarcomere lengths2,5. Since these early studies, it has become increasingly common to extract sarcomere length information with a greater spatial resolution using 2D fast Fourier transform (FFT)-based techniques6 on brightfield microscopy images. The two imaging systems allow a partial assessment of the underlying relationship between Ca2+ release and sarcomere length-dependent force production.
Cardiac muscle is striated, with the visible banding associated with an underlying series of contractile units consisting of thick and thick filaments. The interaction of these constituent filaments that make up sarcomeres underlies force generation, which commences as follows:&#160;a&#160;depolarizing electrical signal, or action potential, causes voltage-dependent L-type Ca2+ channels in the cell membrane to open;&#160;the ensuing cellular influx of Ca2+ induces the release of Ca2+ from the sarcoplasmic reticulum (SR), an intracellular Ca2+ store, in a process known as Ca2+-induced Ca2+ release7; this sudden increase in intracellular Ca2+ concentration from nanomolar to micromolar range enables force production to occur;&#160;Ca2+ pumps continuously extrude Ca2+ out of the cytosol back into the SR and extracellular compartment;&#160;when the intracellular Ca2+ concentration returns to the nanomolar range, force production ceases, and the muscle relaxes. During force production, the constituent thick and thin filaments slide over one another. The sarcomere length dictates the relative extent of overlap and, therefore, the potential for force production of the muscle macroscopically.
In this paper, we extend these fluorescence-brightfield imaging techniques to include optical coherence tomography (OCT). OCT utilizes the physical principle of interference and is capable of garnering the geometric deformation of tissue to understand muscle contractile heterogeneity8. Our device (Figure 1) uses a spectral-domain OCT (SD-OCT) system. In SD-OCT, a beam splitter splits the light from a broadband short coherence-length superluminescent diode into reference and measurement arms. The reference arm contains a fixed-mirror, and the measurement arm contains a 2D-galvanometer to steer the light. Light backscattered from the sample is collected and interferes with the reflected light in the reference arm to form an interference pattern. The depth information is encoded in the frequency of the spectral fringe. To extract the information, the signal is passed through a spectrometer and an inverse FFT is applied to the result. The corresponding 1D signal represents the structures at different depths, corresponding to changes in refractive index9 (A-scan). By steering the laser in a single axis, one can construct a cross-section of the sample of interest (B-scan) and, similarly, by repeating the process in a step-wise pattern in the remaining axis, a three-dimensional image can be generated (C-scan). By extension, one can collect a series of B-scans at a single slice for a repeating time-varying subject based on an external trigger and repeat to generate a three-dimensional scan, representing a time-varying planar image10.
In integrating the three imaging systems, we have considered the following two principles. First, imaging sensors should not detect light from an alternative imaging modality, and second, the physical design should contain free space for at least three simultaneous imaging planes. To address the first requirement, the brightfield microscope uses a 660 nm wavelength LED to illuminate the sample in an inverted configuration. The fluorescence microscope is in an epifluorescence configuration where the same objective lens is used for both excitation and collection of the emitted light. The excitation light has a wavelength of between 340 nm and 380 nm, and a photomultiplier tube (PMT) measures the emitted light at a wavelength of 510 nm. A pair of dichroic mirrors enable these two optical paths to share the same physical footprint without interfering with the opposite measurement (Figure 2). Finally, the OCT uses broadband (100 nm spectral width) light with a central wavelength of 840&#160;nm, distinct from the other two modalities. Due to the low-coherence nature of the light used for OCT, any scattered light from the brightfield-fluorescence sources will not contribute to the interference pattern that encodes depth information. For the second requirement, the housing design for the capillary tube has accessible optical pathways to the anterior, inferior, and superior planes of the sample. During experiments, two platinum hooks hold a trabecula within a capillary tube perfused with oxygenated Krebs-Henseleit (KH) solution. The galvanometer head of the OCT is oriented orthogonally to the brightfield-fluorescence imaging pathway to take advantage of the third orthogonal optical plane (Figure 3).
This paper outlines the design considerations for building a device capable of simultaneously imaging calcium, sarcomere length, and muscle geometry. To demonstrate these measurement capabilities, we describe the process of isolating a ventricular trabecula, the preparation of the necessary buffer solutions, along with the critical steps involved in the handling and fluorescence loading of an ex vivo trabecula. Finally, this paper outlines the processes required to translate the dataset into more useful visualizations.

<table><tbody><tr><td>2,3-Butanedione monoxime</td><td>Acros Organics</td><td>150375000</td><td></td></tr><tr><td>20&amp;times; microscope lens</td><td>Nikon</td><td>CFI Super Fluor 20&amp;times;</td><td>NA 0.75</td></tr><tr><td>2D Galvanometer</td><td>Thorlabs</td><td>GVSM002/M</td><td></td></tr><tr><td>50-50 beam splitter</td><td>Thorlabs</td><td>FC850-40-50-APC</td><td></td></tr><tr><td>90-10 beam-splitter</td><td>Thorlabs</td><td>TW850R2A2</td><td></td></tr><tr><td>Analogue input module</td><td>National Instruments</td><td>NI-9205</td><td>Records the PMT signal at 200 kHz</td></tr><tr><td>Brightfield imaging light source</td><td>CoolLED</td><td>PE-2</td><td>660&amp;nbsp;nm LAM</td></tr><tr><td>Broadband light source</td><td>Superlum</td><td>Broadlighter-840</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Cameralink card</td><td>National Instruments</td><td>NI-1429</td><td>Brightfield imaging frame grabber</td></tr><tr><td>Carbogen 5</td><td>BOC</td><td>Gas code: 181</td><td></td></tr><tr><td>Condensor lens</td><td>Nikon</td><td>LWD 0.52</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Merck</td><td>108337</td><td></td></tr><tr><td>DAQ</td><td>National Instruments</td><td>NI-6259</td><td>Triggers the galvanometer movement</td></tr><tr><td>Dichroic mirror 1</td><td>Semrock</td><td>FF409-Di03</td><td></td></tr><tr><td>Dichroic mirror 2</td><td>Semrock</td><td>FF552-Di02</td><td></td></tr><tr><td>Diffraction grating</td><td>Wasatch Photonics</td><td>1200 lines/mm @840 nm</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Direct-Q 3 UV System</td><td>Merck Millipore</td><td>ZRQSVR3WW</td><td>Distilled water machine</td></tr><tr><td>Dry bath</td><td>Corning</td><td>6875-SB</td><td>LSE digital dry bath</td></tr><tr><td>FIJI</td><td>ImageJ</td><td></td><td>Open-source image processing software</td></tr><tr><td>Fura-2AM pentapotassium salt</td><td>Thermofisher</td><td>F14186</td><td></td></tr><tr><td>Hardware FPGA card</td><td>National Instruments</td><td>NI-7813R</td><td>Also controls the triggering of the brightfield capture</td></tr><tr><td>Heparin</td><td>Pfizer</td><td>61024</td><td></td></tr><tr><td>HEPES</td><td>PanReac AppliChem</td><td>A1069</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>TI-DH illumination pillar</td><td></td></tr><tr><td>Isofluorane</td><td>MedSource</td><td>VAPDRUGISO250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>Line-scan camera</td><td>Basler</td><td>spL2048-70km</td><td>Spectrometer camera</td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>3810000</td><td>RCT basic</td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td>Data processing code</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M1880</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71376</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>71505</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>OCT FPGA card</td><td>National Instruments</td><td>NI-1483R</td><td></td></tr><tr><td>Oxygen tank</td><td>BOC</td><td>Gas code: 100D</td><td></td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>MP220</td><td></td></tr><tr><td>Photomultiplier tube</td><td>Hamamatsu</td><td>H7422-20</td><td></td></tr><tr><td>Powerload</td><td>Thermofisher</td><td>P10020</td><td></td></tr><tr><td>Superluminescent diode</td><td>Broadlighter</td><td>D-840</td><td></td></tr><tr><td>Transimpedance amplifier</td><td>Custom</td><td></td><td></td></tr><tr><td>Tris(hydroxymethyl)amino-methane</td><td>Sigma-Aldrich</td><td>252859</td><td></td></tr><tr><td>Wistar rat</td><td>Vernon Jansen Unit</td><td></td><td>8 &amp;ndash; 10 weeks</td></tr><tr><td>Xenon arc lamp</td><td>Sutter Instrument</td><td>DG-4</td><td>Lambda DG-4</td></tr></tbody></table>

simultaneous brightfield, fluorescence, and optical coherence tomographic imaging of contracting cardiac trabeculae ex vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

In order to capture the regional Ca2+ and brightfield information for the entire length of the trabecula presented here, seven muscle positions were required. Figure 6 suggests that the twitch force was undisturbed by this motion, revealing that there was no position dependence of the active force production.
B-scans collected using optical coherence tomography at a rate of 100 Hz were segmented using the ImageJ plugin WEKA14 (Figure 7A). Each cross-section appears distorted due to the difference between the lateral (10 &#181;m) and depth (1.73 &#181;m (in myocardium)) resolutions. This distortion was corrected by scaling the image&#39;s depth axis by the lateral resolution-depth resolution ratio. Figure 7B,C demonstrate that after scaling the raw C-scan of the trabecula it is approximately cylindrical in geometry. The reflection of the measurement chamber wall can sometimes overlap with the muscle data (Figure 7A,B), but the segmentation software can be trained to account for this (Figure 7D,E). Once segmented, the cross-sectional area along the length of the muscle can be calculated throughout the twitch (Figure 7F). Note that this particular trabecula has a small appendage branching from it. The motion of the branch is evident ~0.75&#160;mm along the trabecula. Finally, the segmented images can be converted into meshes to aid the construction of geometric&#160;models (Figure 7G).
Imaging data captured at each of the different trabecula positions at a rate of 100 fps were stitched together to create a single complete image of the trabecula (Figure 8A). The resolution of these images is 0.535 &#181;m/pixel. The use of linear weighting functions in the overlapping regions of neighboring windows aids visualization and minimizes the impact of the vignette present in the brightfield images. To measure the fluorescent signal, a 540 &#181;m by 540 &#181;m window of the trabecula is cyclically illuminated with 340 nm, 365 nm, and 380 nm wavelength light at a rate of 600 Hz. The ratio of the emitted fluorescence associated with the 340 nm and 380 nm excitation light correlates to the intracellular calcium after the trabecula has been loaded with Fura-2. As this measurement is a ratio, the effective measurement rate is 200 Hz. The averaged (n = 10) intracellular Ca2+ transients from each window are aligned with the region they were imaged (Figure 8B). While the peak of the transients appear reasonably consistent, the diastolic [Ca2+]&#160;is lower within the region between 900 &#181;m and 1800 &#181;m along the trabecula. Similarly, the results of the displacement tracking (Figure 8C) and sarcomere length (Figure 8D) calculations also indicate the presence of regional variability. The markerless tracking technique used is able to process the displacement of each pixel, given enough contrast. When mapping the distribution of sarcomere lengths in post, a cross-correlation area of 128 pixels by 128 pixels (~67 &#181;m by 67 &#181;m) was used to calculate regional sarcomere length. This area encapsulates approximately 29 sarcomeres when close the sample is close to optimal sarcomere length. The step-size (in both the x- and y-directions) between the centroid of each cross-correlation window was set to 50 pixels (~26 &#181;m) for processing these data. The suitability of sarcomere length estimates were tested based on the width and amplitude of the Gaussian fit to the FFT signal. These conditions were not met in the muscle region between 0 &#181;m and 500 &#181;m so no sarcomere length information could be computed there. Given the associated displacements, it is likely that the sarcomeres in this region elongated during the contractile phase. In keeping with this speculation, the average sarcomere lengths on the right-hand side of the trabecula shorten during that period. By combining the information provided by each of the panels, it appears that the region with the largest cross-sectional area does not produce the most significant force. On the assumption that the regional variation of Ca2+ transients has an approximately smooth gradient, Figure 8B indicates that the largest amplitude Ca2+ transient occurs somewhere between 1300 &#181;m and 1600 &#181;m along the trabecula. The displacement map indicates that the region that undergoes the least motion aligns well with the peak Ca2+ transient. However, this region has the smallest cross-sectional areas of the sample. With these data in mind, one could infer that this region generated the most stress.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62799/62799fig01.jpg" /> 
Figure 1: Annotated image of the cardiomyometer. Each of the main optical components is outlined. The inset contains a close-up, rear-view, of the microscope objective in situ, below the measurement chamber. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62799/62799fig02.jpg" /> 
Figure 2: Optical pathway for simultaneous brightfield and fluorescence microscopy. The illumination source for the fluorescence microscope is a Xenon arc lamp, the output of which cyclically switches between 340 nm, 365 nm, and 380&#160;nm light. The arc lamp output path contains a dichroic mirror with a cut-off wavelength of 409 nm that reflects the UV light onto a mirror that directs the light into a fluorescence microscope objective. The lens focuses the excitation light onto the sample and collects the emitted light, which has a longer wavelength of 510 nm. This emitted light passes through the first dichroic mirror, but not the second, as it has a cut-off wavelength of 552 nm. A field lens then focuses the reflected light onto the sensor of the PMT. Meanwhile, the illumination source (660 nm LED) for the brightfield microscope is located above the sample. The transmitted light is focused onto the sample by a condenser lens, and the 20&#215; fluorescence objective captures the resultant transmission image. The wavelength used for brightfield illumination exceeds the cut-off wavelength of each dichroic mirror, so it passes through both of them before the image is focused onto the sensor of a CMOS camera. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62799/62799fig03.jpg" /> 
Figure 3: Measurement chamber holder design. (A)&#160;Isometric view of the measurement chamber holder with optical paths overlaid. Brightfield illumination occurs from the superior surface (z-axis); fluorescence illumination occurs from the inferior surface (z-axis), and the measurement arm OCT signal is orthogonal to the other illumination axis (y-axis). During the experiment, two platinum hooks hold a trabecula within a glass capillary tube that functions as the measurement chamber. Voice-coil motors control each hook and their positions are measured using laser interferometry. The current position is compared with a user-defined set point and, using a PID controller encoded within an FPGA, the error is minimized. (B)&#160;Measurement chamber in situ with the brightfield illumination turned on. The back-view is shown in the Figure 1 inset. (C)&#160;Schematic of the superfusate flow through the measurement chamber holder. Superfusate enters the rear of the block and flows in the direction indicated by arrows. The upstream and downstream electrodes establish the field stimulation for eliciting the contraction of a mounted trabecula in the measurement chamber. Blue shading indicates the regions where the superfusate flows during an experiment. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62799/62799fig04.jpg" /> 
Figure 4: Front panel of the image acquisition and control software. (A) Brightfield imaging user interface. (B) OCT imaging user interface. (C)&#160;Hardware control user interface. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62799/62799fig05.jpg" /> 
Figure 5: Trabecula dissection and mounting protocol. (A)&#160;Langendorff-perfused rat heart in the dissection chamber. (B) The same heart with the atria removed. Dashed lines indicate the excision trajectory to open the ventricles. (C)&#160;An opened heart to expose the interior of both ventricles. The dashed box indicates the region where trabeculae are typically found. (D)&#160;Excised right-ventricular wall region (the same as indicated by the dashed box in C). Dashed lines highlight three trabeculae. (E)&#160;A trabecula selected from the three in D. (F)&#160;The trabecula from panel E with the wall tissue removed. (G)&#160;The isolated trabecula in the end of a 1 mL syringe. (H)&#160;The trabecula in the mounting chamber. (I)&#160;The trabecula mounted between two platinum hooks. (J)&#160;The trabecula, mounted between hooks, within the measurement chamber (Figure 3B). The green spot is an artefact from the first dichroic filter. (K) Secondary angle of the mounted trabecula within the measurement chamber. The distance between the trabecula and the microscope objective lens is approximately 1 mm. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62799/62799fig06.jpg" /> 
Figure 6: Position dependence of the force measurement. The force produced by the muscle from each of the imaging positions (n = 7) overlaid. The average active force production was 0.527 mN &#177; 0.003 mN, time to 50 % contraction 77.1 ms &#177; 0.3 ms, and time to 50 % relaxation 328.1 ms &#177; 0.9 ms (all data are presented as the mean &#177; SE). <a href="https://www.jove.com/files/ftp_upload/62799/62799fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62799/62799fig7v2.jpg" /> 
Figure 7: OCT imaging analysis. (A)&#160;Example of WEKA segmentation. The segmented cross-section of the muscle is highlighted in red, and the background is highlighted in green. (B)&#160;Superior view of the raw C-scan data of a trabecula. The bright angled line towards the top of the image is the reflection of the measurement chamber wall. (C)&#160;Lateral view of the raw C-scan data of a trabecula. (D) Superior view of the segmented OCT data. (E) Lateral view of the segmented OCT data. (F)&#160;The cross-sectional area along the length of the trabecula (x-axis) through time (y-axis). The average cross-sectional area along the muscle length was 0.0326 mm2 &#177; 0.0005 mm2 (mean &#177; S.E.) (G)&#160;A mesh of the trabecula. The mesh has been approximately aligned with the cross-sectional area plot of panel F. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig7v2lage.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62799/62799fig08.jpg" /> 
Figure 8: Brightfield and fluorescence imaging analysis. (A)&#160;Stitched image (seven imaging windows) of the trabecula. (B)&#160;Ca2+&#160;transients along the length of the trabecula. (C)&#160;Average x-displacement from each imaging window. A positive displacement represents a motion to the right and negative to the left. (D)&#160;Average sarcomere lengths from each imaging window that had the necessary image contrast. <a href="https://www.jove.com/files/ftp_upload/62799/62799fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Table of Solutions <a href="https://www.jove.com/files/ftp_upload/62799/Tables_of_solutions.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo at JoVE.com

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

simultaneous-brightfield-fluorescence-optical-coherence-tomographic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.2K Views.  University of Auckland. This protocol presents a collection of sarcomere, calcium, and macroscopic geometric data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Video: Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

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 ...

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Medicine

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Light-Sheet Microscopy and Tissue Clearing for Comprehensive Cardiac Imaging

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures in Rodent Hearts

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

Summary

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Chapters in this video

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

Optical Mapping of Langendorff-perfused Rat Hearts

3D Whole-heart Myocardial Tissue Analysis

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Mesoscopic Optical Imaging of Whole Mouse Heart