Light-sheet Fluorescence Microscopy for the Study of the Murine Heart
Summary
This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| CW Laser | Laserglow Technologies | LMM-GBV1-PF3-00300-05 | Excitation of fluorophores |
| Neutral density filter | Thorlabs | NDC-50C-4M | Controls amount of light entering system |
| Achromatic beam expander | Thorlabs | GBE05-A | Expands the beam of light |
| Mechanical slit | Thorlabs | VA100C | Controls width of beam |
| Beam splitter | Thorlabs | BS013 | Forms dual-illumination beam |
| Stereo microscope with 1X objective lense | Olympus | MVX10 | Used for observation of sample |
| ORCA-Flash4.0 LT sCMOS camera | Hamamatsu Photonics | C11440-42U | Used to capture Images |
| Acrylonitrile butadiene styrene (ABS) | Stratasys | uPrint | Material used to 3-D print a sample holder |
| Fluorescent polystyrene beads | Spherotech Inc | PP-05-10 | Used for imaging system calibration |
| Borosilicate glass tubing | Corning | Pyrex 7740 | Tubing for sample embedding |
| Glycerol | Fisher Scientific | BP229-4 | Fill for sample chamber |
| Phosphate-buffered saline | Fisher Scientific | BP39920 | Rinse solution for mouse hearts |
| Paraformaldehyde | Electron microscopy sciences | RT-15700 | First incubation solution |
| Acrylamide | Wako Chemicals | AAL-107 | Mixed with 2,2'-Azobis dihydrochloride for second incubation solution for mouse hearts |
| 2,2'-Azobis dihydrochloride | Wako Chemicals | VA-044 | Mixed with Acrylamide for second incubation solution for mouse hearts |
| Sodium dodecyl sulfate | Sigma Aldrich | 71725 | Mixed with Boric acid for third incubtion solution for mouse hearts |
| Boric acid | Fischer Scientific | A74-1 | Mixed with Sodium dodecyl sulfate for third incubtion solution for mouse hearts |
| Sigma D2158 | Sigma Aldrich | D2158 | Mixed with PB, Tween-20, and Sodium azide as a refractive index matching solution |
| Tween-20 | Sigma Aldrich | 11332465001 | Mixed with Sigma D2158, PB, and Sodium azide as a refractive index matching solution |
| Sodium azide | Sigma Aldrich | S2002 | Mixed with Sigma D2158, PB, and Tween-20 as a refractive index matching solution |
| Adeno-associated virus vector 9 with a cardiac-specific Troponin T promoter tagged with GFP | Vector Biolabs | VB2045 | Expresses GFP when bound to ROMK |
| DC Servo Motor Actuator | Thorlabs | Z825B | Used for movement of sample in axial direction within light sheet |
| K-Cube Brushed DC Servo Motor Controller | Thorlabs | KDC101 | Connects to motor actuator and controls movement of the actuator |
| Amira | FEI Software | N/A | Visualization software for producing 2-D and 3-D images |
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