光片荧光显微术在小鼠心脏研究中的应用
Summary
本研究采用双侧光照光片荧光显微镜 (LSFM) 技术, 结合光清除法对小鼠心脏进行研究。
Abstract
光片荧光显微技术已广泛应用于从微米到毫米尺度的高轴向分辨率的快速图像采集。传统的光片技术涉及在样品组织中使用单一光照束定向正交。使用单一光照光束制作的大型样品的图像含有条纹或工件, 由于组织的散射和吸收, 导致分辨率降低。本研究采用双侧光照光束和简化的小鼠心脏清晰度光清除技术。这些技术可以通过去除心脏的脂质和产生一个大的成像领域, 大于 10 x 10 x 10 毫米3, 更深层次的成像。因此, 这一策略使我们能够量化的心室尺寸, 跟踪心脏谱系, 并本地化的心脏特异性蛋白和离子通道的空间分布, 从产后到成年小鼠心脏有充分的对比度和分辨率。
Introduction
光片荧光显微镜是1903年首次开发的一种技术, 目前作为一种研究基因表达的方法, 也用于生产3维或4维组织样本1,2,3的模型。这种成像方法使用一层薄薄的光来照亮样本的单个平面, 以便只有该平面被探测器捕获。然后, 可以在轴向方向移动样本以捕获每一层, 一次一节, 并在获得的图像4后处理后呈现一个3维模型。然而, 由于光子的吸收和散射, LSFM 仅限于几微米厚或光学上透明1的样品。
LSFM 的局限性导致了对生物体的广泛研究, 这些有机体具有光学上透明的组织, 如斑马鱼。有关心脏发育和分化的研究往往是对斑马鱼, 因为有保守的基因之间的人和斑马鱼5,6。尽管这些研究已经导致了与心肌6,7相关的心脏研究的进展, 但仍有必要对哺乳动物等高级生物进行类似的研究。
哺乳动物的心脏组织提出了一个挑战, 由于组织的厚度和不透明, 红细胞中血红蛋白的吸收, 以及在传统的 LSFM 方法下, 由于样品单面光照产生的条带.1,8. 为了弥补这些限制, 我们建议使用双侧照明和简化版的清晰度技术9结合折射率匹配解决方案 (轮辋)。因此, 该系统允许对大于 10 x 10 x 10 毫米3的样本进行成像, 同时在轴向和侧面平面上保持良好的质量分辨率8。
该系统是第一次校准使用荧光珠安排在不同的配置在玻璃管。然后, 该系统用于图像产后和成年鼠心脏。首先, 产后鼠心脏被成像7天 (P7), 以揭示心室腔, 心室壁的厚度, 瓣膜结构, 和 trabeculation 的存在。其次, 研究了在1天 (P1) 用培养的心肌细胞和黄色荧光蛋白 (YFP), 通过使用产后小鼠心脏, 来识别将会分化为细胞的干细胞。最后, 7.5 月的成年小鼠在基因治疗后观察到肾外髓质钾 (ROMK) 通道的存在,8。
Protocol
Representative Results
Discussion
这里描述的 LSFM 系统和技术利用双侧照明光束和光清除相结合, 在产后和成人阶段对鼠标心脏进行图像处理。传统的单一光照光束受光子散射和吸收通过更厚和更大的组织样本1,3。双面光束为样品提供了更均匀的光照, 从而最大限度地减少了条带和其他工件在单面光照产生的图像中经常看到的效果。该技术还可以通过调整双高斯光束8的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望向加州大学洛杉矶分校的邵阮和淳浩表示感谢, 他们为小鼠提供了图像样本。这项研究得到了 NIH HL118650 (粽子 k Hsiai) 的资助, HL083015 (粽子 k Hsiai), HD069305 (到北卡罗来纳州和粽子 k Hsiai), HL111437 (粽子 k. Hsiai 和北卡罗来纳州), HL129727 (粽子 k Hsiai), 德克萨斯大学系统明星资助 (Juhyun李)。
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 |
References
- Richardson, D. S., Lichtman, J. W. Clarifying tissue clearing. Cell. 162 (2), 246-257 (2015).
- Lee, J., et al. 4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation. Journal of Clinical Investigation. 126 (5), 1679-1690 (2016).
- Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 305 (5686), 1007-1009 (2004).
- Huisken, J., Stainier, D. Y. Selective plane illumination microscopy techniques in developmental biology. Development. 136 (12), 1963-1975 (2009).
- Bakkers, J. Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovascular Research. 91 (2), 279-288 (2011).
- High, F. A., Epstein, J. A. The multifaceted role of Notch in cardiac development and disease. Nature Reviews Genetics. 9 (1), 49-61 (2008).
- Sachinidis, A. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovascular Research. 58 (2), 278-291 (2003).
- Ding, Y., et al. Light-sheet fluorescence imaging to localize cardiac lineage and protein distribution. Scientific Reports. 7, 42209 (2017).
- Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nature Protocols. 9 (7), 1682-1697 (2014).
- Robbins, N., Thompson, A., Mann, A., Blomkalns, A. L. Isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease. Journal of Visualized Experiments. (93), e52172 (2014).
- National Heart, L., Blood Institute, . Standard Operating Procedures (SOP’s) for Duchenne Animal Models. , (2015).
- Sung, K., et al. Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping. Scientific Reports. 6, 30736 (2016).
- Yuan, Z., Qiao, C., Hu, P., Li, J., Xiao, X. A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes. Human Gene Therapy. 22 (5), 613-624 (2011).
- FEI, . Amira User’s Guide. , 59-138 (2017).
- Gao, L., et al. Noninvasive imaging of 3D dynamics in thickly fluorescent specimens beyond the diffraction limit. Cell. 151 (6), 1370-1385 (2012).
- Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nature Methods. 8 (9), 757-760 (2011).
- Ding, Y., et al. Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics. JCI Insight. 2 (22), (2017).
