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Bioingegneria

光片成像揭示啮齿动物心脏的心脏结构

Published: March 29, 2024
doi:
10.3791/66707
, , , ,
1Department of Bioengineering,The University of Texas at Dallas, 2Center for Imaging and Surgical Innovation,The University of Texas at Dallas, 3Hamon Center for Regenerative Science and Medicine,UT Southwestern Medical Center

Summary

该协议利用先进的光片显微镜以及适应的组织透明化方法来研究啮齿动物心脏中复杂的心脏结构，为理解心脏形态发生和重塑具有巨大潜力。

Abstract

光片显微镜 （LSM） 在理解心脏复杂的三维 （3D） 结构方面发挥着关键作用，为基本的心脏生理学和病理反应提供了重要的见解。在此，我们深入研究了LSM技术的开发和实施，以阐明小鼠模型中心脏的微结构。该方法将定制的 LSM 系统与组织透明化技术相结合，可减轻心脏组织内的光散射以进行体积成像。将传统的 LSM 与图像拼接和多视图反卷积方法相结合，可以捕获整个心脏。为了解决轴向分辨率和视场 （FOV） 之间固有的权衡问题，我们进一步引入了轴向扫描光片显微镜 （ASLM） 方法，以最大限度地减少离焦光并在整个传播方向上均匀地照亮心脏。同时，iDISCO等组织透明化方法增强了光的穿透力，促进了深层结构的可视化，并确保了对整个心脏心肌的全面检查。所提出的LSM与组织透明化方法的结合为研究人员解析啮齿动物心脏心脏结构提供了一个有前途的平台，为理解心脏形态发生和重塑具有巨大的潜力。

Introduction

心力衰竭仍然是全球死亡的主要原因，主要是由于成熟心肌细胞缺乏再生能力1。心脏错综复杂的结构在其功能中起着至关重要的作用，并提供了对发育过程的见解。对心脏结构的深刻理解对于阐明心肌梗死后心脏形态发生和重塑的基本过程至关重要。最近的进展表明，新生小鼠可以在受伤后恢复心脏功能，而成年小鼠则缺乏这种再生能力2。这为研究小鼠模型中与结构和功能异常相关的线索奠定了基础。传统的成像方法，如共聚焦显微镜，具有技术局限性，包括穿透深度受限、点扫描方案缓慢以及长时间暴露在激光下造成的照片损伤。这些阻碍了完整心脏的全面三维 （3D） 成像。在这种情况下，光片显微镜 （LSM） 作为一种强大的解决方案出现，具有高速成像、减少光损伤和卓越的光学切片能力等优势 3,4,5。LSM 的独特功能使其成为一种有前途的方法，可以克服传统技术的局限性，为心脏发育和重塑过程提供前所未有的见解 6,7,8。

在该协议中，我们引入了一种成像策略，该策略将先进的LSM与适应的组织透明化方法9相结合，允许对整个小鼠心脏进行成像，而无需特定的标记和机械切片。我们进一步提出，传统的LSM成像可以通过多视图反卷积10或轴向扫描光片显微镜（ASLM）技术11,12,13,14,15来增强，以提高轴向分辨率。此外，将图像拼接与这两种方法中的任何一种相结合都可以有效地克服空间分辨率和视场（FOV）之间的权衡，从而推进成年小鼠心脏的成像。结合多种组织透明化方法，包括疏水性、亲水性和基于水凝胶的方法，可以更深入地穿透光来捕获整个心脏的形态 16,17,18,19。

虽然多种透明化方法与当前的 LSM 系统兼容，但目标是通过用与其折射率非常匹配的介质替换脂质来最大限度地减少光子散射并增强光在组织（如心脏）中的穿透力。iDISCO被选为代表20,21，并因其快速处理和高透明度而适用于该协议中的自发荧光成像（图1A）。总的来说，先进的LSM方法与组织透明化技术的整合为揭示啮齿动物心脏中复杂的心脏解剖结构提供了一个有前途的框架，为推进我们对心脏形态发生和发病机制的理解具有巨大的潜力。

Protocol

动物协议和实验已在德克萨斯大学达拉斯分校机构动物护理和使用委员会 （IACUC #21-03） 的监督下获得批准和进行。本研究使用C57BL6小鼠，包括出生后第1天（P1）的新生儿和8周大的成人。男性和女性之间没有观察到差异。所有数据采集和图像后处理均使用具有研究或教育许可的开源软件或平台进行。这些资源可根据作者的合理要求提供。 1. 样品制备和组织清理（6 – 10 天?…

Representative Results

LSM 已被证明可以促进心脏研究 31,32,33,34,35,36,37，因为与其他光学成像方法（如明场和点扫描技术）相比，LSM 具有最小的光损伤风险、高空间分辨率和光学切片 6,8,38,39,40 </su…

Discussion

成像、计算和组织透明化方法的进步为广泛研究心脏结构和功能提供了无与伦比的机会。这对于使用完整的啮齿动物心脏模型加深我们对心脏形态发生和发病机制的理解具有很大的潜力。与使用类似方法对斑马鱼心脏进行体内研究相比 40,41,42,43，先进的 LSM 技术和组织透明化方法的集成使我们能够克服在对啮齿?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢德克萨斯大学西南医学中心的Eric Olson博士团队慷慨地分享动物模型。我们感谢德克萨斯大学达拉斯分校的D-incubator成员提供的所有建设性意见。这项工作得到了 NIH R00HL148493 （Y.D.）、R01HL162635 （Y.D.） 和 UT Dallas STARS 计划 （Y.D.） 的支持。

Materials

1% Agarose
Low melting point agarose Thermo Fisher 16520050
Deionized water – –
Chemicals for tissue clearing 
5-Amino-1,3,3-trimethylcyclohexanemethylamine, mixture of cis and trans Sigma-Aldrich 118184
D.E.R.™ 332 Sigma-Aldrich 31185
D.E.R.™ 736 Sigma-Aldrich 31191
Dibenzyl ether (DBE) Sigma-Aldrich 33630
Dichloromethane (DCM) Sigma-Aldrich 270997
Fluorescent beads Spherotech FP-0556-2
Hydrogen peroxide (H2O2) Sigma-Aldrich 216736
Methanol Sigma-Aldrich 439193
Paraformaldehyde (PFA) Thermo Fisher 47392
Phosphate Buffered Saline (PBS) Sigma-Aldrich 79383
Potassium Chloride (KCl) Sigma-Aldrich P3911
Software and algorithms
Amira Thermo Fisher Scientific 2021.2
BigStitcher Hörl et al.22
Fiji-ImageJ Schindelin et al.20 1.54f
HCImage Live Hamamatsu Photonics 4.6.1.2
LabVIEW National Instruments Corporation 2017 SP1
Key components of the customized light-sheet system
0.63 – 6.3X Zoom body Olympus MVX-ZB10 
10X Illumination objective Nikon MRH00105
1X detection objective Olympus MV PLAPO 1X/0.25 
473nm DPSS Laser Laserglow Technologies LRS-0473-PFM-00100-05
532nm DPSS laser Laserglow Technologies LRS-0532-PFM-00100-05
589 nm DPSS laser Laserglow Technologies LRS-0589-GFF-00100-05
BNC connector National Instrument BNC-2110
Cylindrical lens Thorlabs ACY254-050-A
DC-Motor Controller, 4 axes Physik Instrumente C-884.4DC
ETL Optotune EL-16-40-TC-VIS-5D-1-C
ETL Cable Optotune CAB-6-300
ETL Lens Driver Optotune EL-E-4i
Filter Chroma ET525/30
Filter Chroma ET585-40
Filter Chroma ET645-75
Filter wheel  Shutter Instrument LAMBDA 10-B
Motorized translation stage Physik Instrumente L-406.20DG10
Motorized translation stage Physik Instrumente L-406.40DG10
Motorized translation stage Physik Instrumente M-403.4PD
NI multifunction I/O National Instrument PCIe-6363
sCMOS camera Hamamatsu C13440-20CU
Stepper motor Pololu 1474
Tube lens Olympus MVX-TLU
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Tags

Keywords: Light-sheet ImagingCardiac StructureRodent HeartsMyocardial MicrostructuresTrabeculationCardiac DevelopmentCardiac RemodelingLight-sheet Microscopy3D StructureCardiac PhysiologyCardiac PathologyImage StitchingMultiview DeconvolutionAxially Swept Light-sheet MicroscopyIDISCO
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Almasian, M., Saberigarakani, A., Zhang, X., Lee, B., Ding, Y. Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts. J. Vis. Exp. (205), e66707, doi:10.3791/66707 (2024).
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