Name: light-sheet fluorescence microscopy for the study of the murine heart
Uploaded: 2018-09-15T14:00:00
Description: Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:967px;" width="967">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">CW Laser</td>
			<td style="width:136px;">Laserglow Technologies</td>
			<td style="width:393px;">LMM-GBV1-PF3-00300-05</td>
			<td style="width:191px;">Excitation of fluorophores</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Neutral density filter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">NDC-50C-4M</td>
			<td style="width:191px;">Controls amount of light entering system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Achromatic beam expander</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">GBE05-A</td>
			<td style="width:191px;">Expands the beam of light</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Mechanical slit</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">VA100C</td>
			<td style="width:191px;">Controls width of beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Beam splitter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">BS013</td>
			<td style="width:191px;">Forms dual-illumination beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Stereo microscope with 1X objective lense</td>
			<td style="width:136px;">Olympus</td>
			<td style="width:393px;">MVX10</td>
			<td style="width:191px;">Used for observation of sample</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">ORCA-Flash4.0 LT sCMOS camera</td>
			<td style="width:136px;">Hamamatsu Photonics</td>
			<td style="width:393px;">C11440-42U</td>
			<td style="width:191px;">Used to capture Images</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Acrylonitrile butadiene styrene (ABS)</td>
			<td style="width:136px;">Stratasys</td>
			<td style="width:393px;">uPrint</td>
			<td style="width:191px;">Material used to 3-D print a sample holder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Fluorescent polystyrene beads</td>
			<td style="width:136px;">Spherotech Inc</td>
			<td style="width:393px;">PP-05-10</td>
			<td style="width:191px;">Used for imaging system calibration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Borosilicate glass tubing</td>
			<td style="width:136px;">Corning</td>
			<td style="width:393px;">Pyrex 7740</td>
			<td style="width:191px;">Tubing for sample embedding</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Glycerol</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP229-4</td>
			<td style="width:191px;">Fill for sample chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Phosphate-buffered saline</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP39920</td>
			<td style="width:191px;">Rinse solution for mouse hearts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Paraformaldehyde</td>
			<td style="width:136px;">Electron microscopy sciences</td>
			<td style="width:393px;">RT-15700</td>
			<td style="width:191px;">First incubation solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Acrylamide</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">AAL-107</td>
			<td style="width:191px;">Mixed with 2,2&#39;-Azobis dihydrochloride for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">2,2&#39;-Azobis dihydrochloride</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">VA-044</td>
			<td style="width:191px;">Mixed with Acrylamide for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">71725</td>
			<td style="width:191px;">Mixed with Boric acid for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Boric acid</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td style="width:393px;">A74-1</td>
			<td style="width:191px;">Mixed with Sodium dodecyl sulfate for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Sigma D2158</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">D2158</td>
			<td style="width:191px;">Mixed with PB, Tween-20, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Tween-20</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>11332465001</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium azide</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>S2002</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Tween-20 as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Adeno-associated virus vector 9 with a cardiac-specific Troponin T promoter tagged with GFP</td>
			<td style="width:136px;">Vector Biolabs</td>
			<td style="width:393px;">VB2045</td>
			<td style="width:191px;">Expresses GFP when bound to ROMK</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">DC Servo Motor Actuator</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">Z825B</td>
			<td style="width:191px;">Used for movement of sample in axial direction within light sheet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">K-Cube Brushed DC Servo Motor Controller</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">KDC101</td>
			<td style="width:191px;">Connects to motor actuator and controls movement of the actuator</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Amira</td>
			<td style="width:136px;">FEI Software</td>
			<td style="width:393px;">N/A</td>
			<td style="width:191px;">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 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...

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

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

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. ...

The main advantage of this technology is that it allows for rapid imaging of either the mouse heart. And it can be used to observe the presence of air channels after gene therapy. Although this method can provide insight into development cardiology, it can also be applied to other systems, such as neurology and pulmonology.Generally, individuals will struggle with this technique because mounting an imaging of the adult mouse heart can be difficult, and it's different from other, traditional techniques, such as confocal and inverted microscopy. Place a continuous wave laser with three wavelengths of 405 nanometers, 473 nanometers, and 532 nanometers. Then, affix mirror one and align it with the mirror plane at 45 degrees to the beam.This will direct the laser toward the beam splitter, which forms the dual-sided illumination setup. Next, pass the beam through a 50 millimeter diameter neutral density filter, a beam expander, and a 25 millimeter diameter pin hole, all positioned 150 millimeters from each other. Pass the beam through a 50-50 beam splitter, placed 150 millimeters from the pinhole.Next, place mirror two 150 millimeters from the beam splitter, at 90 degrees to the forward beam, and align it so that its mirror plane is at a 45 degree angle to the beam. Then, place mirror three 100 millimeters from mirror two, and align it so that it's mirror plane is at a 45 degree angle to the beam reflected from mirror two. Use this reflected beam to form one side of the dual illumination light sheet.On the far side of the beam splitter, place mirror five so that it's mirror plane is at 45 degrees to the beam that is emitted in a forward direction. Use the beam emitted from mirror five to form the second side of the dual illumination light sheet. Next, set up the dual-sided illumination system in systemic fashion.Place cylindrical lens two 150 millimeters away from mirror three, and another identical cylindrical lens 150 millimeters away from mirror five, on the other side of the dual illumination setup. Next, place two mirrors, each in line with the cylindrical lenses, at distances of 50 millimeters to reflect the beam at 90 degrees. On both sides of the light sheet, form an achromatic doublet from a pair of lenses, with the first lens being placed 100 millimeters from the previous mirror, and having a one inch diameter and a focal length of 100 millimeters.The second lens should be placed 160 millimeters from lens one, with a diameter of one inch, and a focal length of 60 millimeters. Then, place the illumination objectives one and two 150 millimeters from the previous achromatic doublets, so that they are in line with the beam. The beam emitted from the objectives forms the light sheet for imaging the samples.First, prepare the fluorescent bead sample by diluting a 0.53 micrometer bead solution, one to 150, 000, in a pre-warmed refractive index matching solution, with one percent low melt pointing agarose. Next, use a piece of borosilicate glass tubing with an inner diameter of 12 millimeters, and an outer diameter of 18 millimeters, to a length of 30 millimeters. Pipette the bead agarose solution in the borosilicate tubing and allow the agarose to solidify at room temperature.Now, fill a 3-D printed ABS chamber with a 99.5 percent gylcerol solution. Place the borosilicate glass tubing, containing the beads, inside the chamber. 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.Then, use custom designed software to acquire images using the sCMOS camera, at a rate of 30 frames per second. Using the motor controller, move the sample one millimeter in the lateral direction, and acquire images at each one millimeter increment, until the entire sample has been imaged. Stack the acquired images using a visualization software, and measure the point spread function of the system using these bead images.Use a refractive index matching solution, with a pH of 7.5, and dissolve one percent agarose into the matching solution. Place a cleared adult mouse heart sample into the refractive index matching solution, with one percent agarose dissolved. Then, insert the sample into borosilicate glass tubing, and allow the agarose to solidify at room temperature.Attach a 3-D motorized translational stage to the borosilicate glass tubing to control the movement and orientation of the sample within a 3-D printed ABS chamber. Position the sample so that it is in the center of the Gaussian beam, created by the dual illumination system. Then, set the acquisition rate of the sCMOS camera to 30 frames per second.Now, using the motor controller, move the sample one millimeter in the axial direction and acquire images at each one millimeter increment. Continue until the entire sample has been imaged. Stack the acquired images using a visualization software.Develop the 3-D images using these image stacks. To accomplish this, deconvolve the point spread function determined previously, and utilize it for the acquired image stacks. Finally, 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.At day one post-natal, one can visualize the valves, atrium, ventricle, pectinate muscle, and trabeculation, among other features. At day seven post-natal, the features are even more defined. The atrium, ventricle, ventricular cavity dimensions, and ventricle wall thickness are all evident.To study cardiomyocyte differentiation within the heart, heterozygous knock-in mice were cre-labeled to show cardiomyocytes. The labeled cells are shown here, in each plane direction. The three planes were merged, to create a 3-D rendering of the heart.The red circled region within the inset shows two translucent mouse hearts after undergoing the clarity technique and being inserted into the borosilicate glass tubing. In addition to studying post-natal mice, the imaging system can also be used to study the adult mouse heart. Here, GFP tagged ROMK channels are shown at 7.5 months.These were mainly found in the ventricular wall. Once mastered, this technique can be done in one to two minutes. When attempting this procedure, it is important to remove all the bubbles around the sample in the tube.Other methods, such as auto-segmentation of the image stacks, can be performed to answer additional questions related to cardiovascular injury and regeneration. After its development, this technique was used by researchers to study the cardiac architecture in post-natal and adult developmental stages in mice.

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Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Light-Sheet Microscopy and Tissue Clearing for Comprehensive Cardiac Imaging

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures 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 ...