Name: simultaneous brightfield, fluorescence, and optical coherence tomographic imaging of contracting cardiac trabeculae ex vivo
Uploaded: 2021-10-02T15:00:00
Duration: 12 min 54 s
Description: Watch this Scientific Journal Video about Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo at JoVE.com

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>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Acros Organics</td>
			<td>150375000</td>
			<td></td>
		</tr>
		<tr>
			<td>20&#215; microscope lens</td>
			<td>Nikon</td>
			<td>CFI Super Fluor 20&#215;</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&#160;nm LAM</td>
		</tr>
		<tr>
			<td>Broadband light source</td>
			<td>Superlum</td>
			<td>Broadlighter-840</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</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>KH2PO4</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>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</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>NaH2PO4.2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>71505</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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 &#8211; 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 ...

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