Name: Light-sheet Fluorescence Microscopy for the Study of the Murine Heart (Video) | JoVE Journal
Uploaded: 2018-09-15T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Learn light-sheet fluorescence microscopy for cardiac imaging using dual-sided illumination and optical clearing in murine hearts. This method enables rapid 3D visualization of heart structure and gene therapy effects.

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5 minutes

The authors would like to express gratitude to Thao Nguyen and Atsushi Nakano from UCLA for providing the mice sample to image. This study was supported by grants NIH HL118650 (to Tzung K. Hsiai), HL083015 (to Tzung K. Hsiai), HD069305 (to N. C. Chi and Tzung K. Hsiai.), HL111437 (to Tzung K. Hsiai and N. C. Chi), HL129727 (to Tzung K. Hsiai), and University of Texas System STARS funding (to Juhyun Lee).

All the procedures involving the use of animals have been approved by the Institutional Review Committees (IACUC) at the University of California, Los Angeles, California.
1. Imaging System Setup
Note: See Figure 1 and Figure 2.
<ol>
	<li>Retrieve a continuous wave (CW) laser with 3 wavelengths: 405 nm, 473 nm, and 532 nm. Place 2 mirrors (M1 and M2) 150 mm apart and align them with their mirror planes at 45&#176; to the beam. 
	Note: This step is performed to redirect the laser away from the dual-sided illumination setup. The resulting beam will be in the same direction as the initial beam.</li>
	<li>Pass the beam through a 25-mm diameter iris diaphragm/pinhole (PH), a 50-mm diameter neutral density filter (NDF) with an optical density range of 0 - 4.0, a beam expander (BE), a 30 mm slit (S) with the width of ~0 - 6 mm, and a mirror (M3), all positioned 150 mm from each other. Place the mirror with its mirror plane at 45&#176; to the beam.</li>
	<li>Pass the beam through a 50:50 beam splitter placed 150 mm from M3. Place a mirror (M6) 150 mm from the beam splitter and align it so that its mirror plane is at 45&#176; to the beam that is emitted in the forward direction. Use the reflected beam to form one side of the dual-illumination light sheet.</li>
	<li>Place a mirror (M4) 150 mm from the beam splitter and align it so that its mirror plane is at 45&#176; to the beam that was emitted at a 90&#176; angle from the beam splitter. Place a second mirror (M5) 150 mm from M4 and align it so that its mirror plane is at 45&#176; to the beam reflected from M6. Use the beam emitted from M5 to form the second side of the dual-illumination light sheet.</li>
	<li>Set up the dual-sided illumination system in a symmetric fashion (see Figure 1). Place one cylindrical lens (CL1) [diameter (d) = 1 in; focal length (f) = 50 mm] 150 mm in line with the beam emitted from M5 on one side and another identical cylindrical lens (CL2) 150 mm in line with the beam emitted from M6 on the other side of the dual-illumination setup. Place 2 mirrors (M7 and M8) each in line with the cylindrical lenses at distances of 50 mm to reflect the beam at 90&#176;.</li>
	<li>Form an achromatic doublet from a pair of lenses. Place the first lens, L1 (d = 1 in; f = 100 mm), 100 mm from M7, and the second lens, L2 (d = 1 in; f = 60 mm), 160 mm from L1. Repeat this on the other side of the dual-illumination with identical lenses (L3, L4) placed the same distance from M8.</li>
	<li>Place mirrors, M9 and M10, 60 mm from lenses L2 and L4, respectively, and in line with the beam. Place the illumination objectives, OL1 and OL2 (d = 2 in; f = 150 mm), 150 mm from M9 and M10 and in line with the beam. The beam emitted from the objectives forms the light sheet for imaging the samples.</li>
	<li>Use a 3-D printer to print the sample holder from acrylonitrile butadiene styrene (ABS). Embed a piece of cover glass [refractive index (RI) = 1.4745] on each side of the chamber, perpendicular to the illumination beam, to minimize refractive index mismatching. Evenly place the chamber in between the beams emitted from the objective lenses.</li>
	<li>Install a stereo microscope with a 1X magnification objective and a scientific complementary metal oxide semiconductor (sCMOS) camera perpendicular to the illumination plane (see Figure 2).</li>
</ol>
2. Imaging System Calibration
<ol>
	<li>Retrieve fluorescent polystyrene beads that are 0.53 &#956;m in diameter.</li>
	<li>Prepare the fluorescent bead sample by diluting the bead solution in a refractive index matching solution (RIMS) with 1% low-melt pointing agarose to 1:150,000. Cut a piece of borosilicate glass tubing with an inner diameter of 12 mm and an outer diameter of 18 mm to a length of 30 mm. 
	Note: The borosilicate glass tubing is used to match the refractive index (1.47).</li>
	<li>Mix the diluted bead sample with 1% agarose and pipette the bead/agarose solution into the borosilicate tubing. Allow the agarose to solidify at room temperature (23 &#176;C).</li>
	<li>Fill the ABS chamber (from step 1.7) with a 99.5% glycerol solution. Place the borosilicate glass tubing containing the beads inside the chamber. Place the ABS chamber in the imaging system so that it is in the center of the Gaussian beam created by the dual illumination system.</li>
	<li>Attach a 3-D motorized translational stage to the borosilicate glass tubing to control the movement and orientation of the sample within the ABS chamber (see Supplemental Figure 1).</li>
	<li>Acquire images using the sCMOS camera at a rate of 30 frames per second (fps). Using the motor controller, move the sample 1 mm in the lateral direction and acquire images at each 1-mm increment using the sCMOS camera. Continue until the entire sample has been imaged.</li>
	<li>Stack the acquired images using a visualization software (see Table of Materials). Measure the point spread function (PSF) of the system using these bead images. Use this PSF for deconvolution during the image processing in later steps.</li>
</ol>
3. Sample Preparation
<ol>
	<li>Dissect hearts from a wild type P1 mouse and from a double heterozygous sarcolipin-Cre knockin mouse with the Rosa26-YFP gene (SlnCre/+; R26YFP reporter/+) at P7. 
	Note: Euthanasia was performed using pentobarbital and the technique described by Robbins et al.10. The dissection was performed according to the technique described by the National Heart, Lung, and Blood Institute (NHLBI)10,11.</li>
	<li>Perform a chemical clearing of the murine hearts as described by Kevin Sung et al.12. 
	Note: The following steps should be performed in a fume hood since the chemicals being used are toxic.
	<ol>
		<li>Rinse the hearts in 1x phosphate-buffered saline (PBS) 3x for 10 min. After rinsing, place the hearts in a 4% paraformaldehyde solution and incubate these samples at 4 &#176;C overnight.</li>
		<li>Place the samples in a 4% acrylamide solution containing 0.5% w/v of 2,2&#8217;-Azobis dihydrochloride. Allow the samples to incubate at 4 &#176;C overnight. After the overnight incubation, remove the samples and incubate them at 37 &#176;C for 2 - 3 h.</li>
		<li>Rinse the samples with PBS and then place them in a solution of 8% w/v sodium dodecyl sulfate and 1.25% w/v boric acid. Incubate the samples at 37 &#176;C until they are clear. This can take several hours depending on the thickness of the sample. After clearing, remove the samples and place them in 1x phosphate-buffered saline for 1 day.</li>
		<li>Prepare a refractive index matching solution with 40 g of nonionic density gradient medium (see Table of Materials) in 30 mL of 0.02 M phosphate buffer (PB), 0.1% Tween-20, and 0.01% sodium azide. Bring the solution to a pH of 7.5 with sodium hydroxide.</li>
	</ol>
	</li>
	<li>Place the cleared sample in RIMS with a refractive index of 1.46 - 1.48 and 1% agarose. Insert the sample into borosilicate glass tubing and allow the agarose to solidify at room temperature (23 &#176;C).</li>
	<li>Attach a 3-D motorized translational stage to the borosilicate glass tubing to control the movement and orientation of the sample within the 3-D printed ABS chamber (see Supplemental Figure 1).</li>
</ol>
4. Adult Heart Imaging
<ol>
	<li>Inject 8.7 x 1012 viruses of type adeno-associated virus vector 9 (AAV9) containing a cardiac-specific troponin T promoter (cTnT) and green fluorescent protein (GFP) in a 100-&#956;L volume into the tail vein of a 2-month wild type (C57BL/6) mouse. 
	Note: The AAV9 was developed according to the technique described by Yuan et al.13. This causes GFP to bind to the renal outer medullary potassium channel (ROMK) producing AAV9-cTnT-ROMK-GFP (see Table of Materials).</li>
	<li>Dissect the adult mouse hearts at 7.5 months of age according to the technique described by the NHLBI11. Prepare the samples using steps 3.1 - 3.3.</li>
	<li>Connect the motor controller to the motor actuator. Attach the actuator to the borosilicate glass tubing to control the movement and orientation of the sample within the 3-D printed ABS chamber.</li>
	<li>Position the sample so that it is in the center of the Gaussian beam created by the dual illumination system. Acquire images using the sCMOS camera at a rate of 100 fps.</li>
	<li>Using the motor controller, move the sample 1 mm in the axial direction and acquire images at each 1-mm increment with the sCMOS camera. Continue until the entire sample has been imaged. 
	Note: The system is controlled by a custom-developed instrument-control software.</li>
	<li>Stack the acquired images using a visualization software (see Table of Materials). Develop 3-D images using these image stacks and the visualization software14. Deconvolve the PSF from step 2.6 with the acquired image stack. Set a pixel threshold intensity value to observe the contours of the heart and add pseudo-color to the images based on this gray-scale intensity.</li>
</ol>

<ol><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537058/">Clarifying tissue clearing.</a> Cell. 162 (2), 246-257 (2015).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4-Dimensional+light-sheet+microscopy+to+elucidate+shear+stress+modulation+of+cardiac+trabeculation%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.</a> Journal of Clinical Investigation. 126 (5), 1679-1690 (2016).</li>
<li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li>
<li>Huisken, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+plane+illumination+microscopy+techniques+in+developmental+biology%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Selective plane illumination microscopy techniques in developmental biology.</a> Development. 136 (12), 1963-1975 (2009).</li>
<li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovascular Research. 91 (2), 279-288 (2011).</li>
<li>High, F. A., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+multifaceted+role+of+Notch+in+cardiac+development+and+disease%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">The multifaceted role of Notch in cardiac development and disease.</a> Nature Reviews Genetics. 9 (1), 49-61 (2008).</li>
<li>Sachinidis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+specific+differentiation+of+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Cardiac specific differentiation of mouse embryonic stem cells.</a> Cardiovascular Research. 58 (2), 278-291 (2003).</li>
<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light-sheet+fluorescence+imaging+to+localize+cardiac+lineage+and+protein+distribution%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Light-sheet fluorescence imaging to localize cardiac lineage and protein distribution.</a> Scientific Reports. 7, 42209(2017).</li>
<li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nature Protocols. 9 (7), 1682-1697 (2014).</li>
<li>Robbins, N., Thompson, A., Mann, A., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+excision+of+murine+aorta%3B+a+versatile+technique+in+the+study+of+cardiovascular+disease%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease.</a> Journal of Visualized Experiments. (93), e52172(2014).</li>
<li>National Heart, L., Blood Institute, <a target="_blank" href="http://community.parentprojectmd.org/profiles/blogs/standard-operating-procedures-for-duchenne-animal-models">Standard Operating Procedures (SOP's) for Duchenne Animal Models</a>. , (2015).</li>
<li>Sung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simplified+three-dimensional+tissue+clearing+and+incorporation+of+colorimetric+phenotyping%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping.</a> Scientific Reports. 6, 30736(2016).</li>
<li>Yuan, Z., Qiao, C., Hu, P., Li, J., Xiao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+adeno-associated+virus+vector+producer+cell+line+method+for+scalable+vector+production+of+different+serotypes%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.</a> Human Gene Therapy. 22 (5), 613-624 (2011).</li>
<li>FEI, <a target="_blank" href="https://www.fei.com/software/amira-user-guide/">Amira User's Guide</a>. , Konrad-Zuse-Zentrum fur Informationstechnik. Berlin. 59-138 (2017).</li>
<li>Gao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+imaging+of+3D+dynamics+in+thickly+fluorescent+specimens+beyond+the+diffraction+limit%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Noninvasive imaging of 3D dynamics in thickly fluorescent specimens beyond the diffraction limit.</a> Cell. 151 (6), 1370-1385 (2012).</li>
<li>Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+and+fast+live+imaging+with+two-photon+scanned+light-sheet+microscopy%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Deep and fast live imaging with two-photon scanned light-sheet microscopy.</a> Nature Methods. 8 (9), 757-760 (2011).</li>
<li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+light-sheet+imaging+with+virtual+reality+to+recapitulate+developmental+cardiac+mechanics%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics.</a> JCI Insight. 2 (22), (2017).</li>
</ol>

The authors have nothing to disclose.

The LSFM system and technique described here utilizes a dual-sided illumination beam combined with an optical clearing to image the mouse heart at post-natal and adult stages of its development. A traditional single illumination beam suffers from photon scattering and absorption through thicker and larger tissue samples1,3. The dual-sided beams provide a more even illumination of the sample, thereby minimizing the effect of striping and other artifacts that are o...

Light-sheet fluorescence microscopy was a technique first developed in 1903 and is used today as a method to study gene expression and also to produce 3-D or 4-D models of tissue samples1,2,3. This imaging method uses a thin sheet of light to illuminate a single plane of a sample so that only that plane is captured by the detector. The sample can then be moved in the axial-direction to capture each layer, one section at a time, and render a 3-D model after the post-processing of the acquired images4. However, due to the absorption and scattering of photons, LSFM has been limited to samples that are either a few microns thick or are optically transparent1.
The limitations of LSFM have led to extensive studies of organisms that have tissues that are optically transparent, such as the zebrafish. Studies involving cardiac development and differentiation are often conducted on zebrafish since there are conserved genes between humans and zebrafish5,6. Although these studies have led to advances in cardiac research related to cardiomyopathies6,7, there is still a need to conduct similar research on higher-level organisms such as mammals.
Mammalian cardiac tissue presents a challenge due to the thickness and opacity of the tissue, the absorption due to hemoglobin in red blood cells, and the striping that occurs due to single-sided illumination of the sample under traditional LSFM methods1,8. To compensate for these limitations, we proposed to use dual-sided illumination and a simplified version of the CLARITY technique9 combined with a refractive index matching solution (RIMS). Therefore, this system allows for the imaging of a sample that is greater than 10 x 10 x 10 mm3 while maintaining a good quality resolution in the axial and lateral planes8.
This system was first calibrated using fluorescent beads arranged in different configurations within the glass tubing. Then, the system was used to image post-natal and adult murine hearts. First, the post-natal mouse heart was imaged at 7 days (P7) to reveal the ventricular cavity, the thickness of the ventricular wall, the valve structures, and the presence of trabeculation. Secondly, a study was conducted to identify cells that would differentiate into cardiomyocytes by using a post-natal mouse heart at 1 day (P1) with Cre-labeled cardiomyocytes and yellow fluorescent protein (YFP). Finally, adult mice at 7.5 months were imaged to observe the presence of renal outer medullary potassium (ROMK) channels after gene therapy8.

<table><tbody><tr><td>CW Laser</td><td>Laserglow Technologies</td><td>LMM-GBV1-PF3-00300-05</td><td>Excitation of fluorophores</td></tr><tr><td>Neutral density filter</td><td>Thorlabs</td><td>NDC-50C-4M</td><td>Controls amount of light entering system</td></tr><tr><td>Achromatic beam expander</td><td>Thorlabs</td><td>GBE05-A</td><td>Expands the beam of light</td></tr><tr><td>Mechanical slit</td><td>Thorlabs</td><td>VA100C</td><td>Controls width of beam</td></tr><tr><td>Beam splitter</td><td>Thorlabs</td><td>BS013</td><td>Forms dual-illumination beam</td></tr><tr><td>Stereo microscope with 1X objective lense</td><td>Olympus</td><td>MVX10</td><td>Used for observation of sample</td></tr><tr><td>ORCA-Flash4.0 LT sCMOS camera</td><td>Hamamatsu Photonics</td><td>C11440-42U</td><td>Used to capture Images</td></tr><tr><td>Acrylonitrile butadiene styrene (ABS)</td><td>Stratasys</td><td>uPrint</td><td>Material used to 3-D print a sample holder</td></tr><tr><td>Fluorescent polystyrene beads</td><td>Spherotech Inc</td><td>PP-05-10</td><td>Used for imaging system calibration</td></tr><tr><td>Borosilicate glass tubing</td><td>Corning</td><td>Pyrex 7740</td><td>Tubing for sample embedding</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-4</td><td>Fill for sample chamber</td></tr><tr><td>Phosphate-buffered saline</td><td>Fisher Scientific</td><td>BP39920</td><td>Rinse solution for mouse hearts</td></tr><tr><td>Paraformaldehyde</td><td>Electron microscopy sciences</td><td>RT-15700</td><td>First incubation solution</td></tr><tr><td>Acrylamide</td><td>Wako Chemicals</td><td>AAL-107</td><td>Mixed with 2,2&#039;-Azobis dihydrochloride for second incubation solution for mouse hearts</td></tr><tr><td>2,2&#039;-Azobis dihydrochloride</td><td>Wako Chemicals</td><td>VA-044</td><td>Mixed with Acrylamide for second incubation solution for mouse hearts</td></tr><tr><td>Sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma Aldrich</td><td>71725</td><td>Mixed with Boric acid for third incubtion solution for mouse hearts</td></tr><tr><td>Boric acid</td><td>Fischer Scientific</td><td>A74-1</td><td>Mixed with Sodium dodecyl sulfate for third incubtion solution for mouse hearts</td></tr><tr><td>Sigma D2158</td><td>Sigma Aldrich</td><td>D2158</td><td>Mixed with PB, Tween-20, and Sodium azide as a refractive index matching solution</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>11332465001</td><td>Mixed with Sigma D2158, PB, and Sodium azide as a refractive index matching solution</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002</td><td>Mixed with Sigma D2158, PB, and Tween-20 as a refractive index matching solution</td></tr><tr><td>Adeno-associated virus vector 9 with a cardiac-specific Troponin T promoter tagged with GFP</td><td>Vector Biolabs</td><td>VB2045</td><td>Expresses GFP when bound to ROMK</td></tr><tr><td>DC Servo Motor Actuator</td><td>Thorlabs</td><td>Z825B</td><td>Used for movement of sample in axial direction within light sheet</td></tr><tr><td>K-Cube Brushed DC Servo Motor Controller</td><td>Thorlabs</td><td>KDC101</td><td>Connects to motor actuator and controls movement of the actuator</td></tr><tr><td>Amira</td><td>FEI Software</td><td>N/A</td><td>Visualization software for producing 2-D and 3-D images</td></tr></tbody></table>

light-sheet fluorescence microscopy for the study of the murine heart

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

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

The technique described here used a dual-sided illumination beam combined with an optical clearing of mouse heart tissue samples to achieve a deeper imaging depth and a larger imaging volume with sufficient imaging resolution (Figure 1). To calibrate the system, fluorescent beads were placed inside the glass tubing and within the imaging system. The beads were then imaged and visualized in the x-y plane, y-z plane, and in the x-z plane, to obtain the point sp...

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

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

light-sheet-fluorescence-microscopy-for-the-study-of-the-murine-heart

Research

JoVE Journal

Bioengineering

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.

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

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

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

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

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.

Mesoscopic Optical Imaging of Whole Mouse Heart

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

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 Imaging to Reveal Cardiac Structure in Rodent Hearts

Three-dimensional (3D) reconstruction of cardiac damage in reperfused acute myocardial infarction (repAMI) enables faithful quantification and co-localization of associated disease-affecting patterns. Here, an automatable light sheet-guided imaging approach is provided for contemporaneous measurement of cardiac damage, area at risk, and immune cell response.

Simultaneous 3D Analysis of Cardiac Damage and Immune Response in Reperfused Acute Myocardial Infarction Using Light Sheet Fluorescence Microscopy

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

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

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

Learn light-sheet fluorescence microscopy for cardiac imaging using dual-sided illumination and optical clearing in murine hearts. This method enables rapid 3D visualization of heart structure and gene therapy effects.

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

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