Faculty of Dentistry, National University of Singapore, Singapore, Singapore.
Department of Mechanical Engineering, National University of Singapore, Singapore, Singapore.
Tissue Eng Part B Rev. 2022 Aug;28(4):926-937. doi: 10.1089/ten.TEB.2021.0140. Epub 2022 Jan 5.
The role of angiogenesis in health and disease have gained considerable momentum in recent years. Visualizing angiogenic patterns and associated events of surrounding vascular beds in response to therapeutic and laboratory-grade biomolecules has become a commonplace in regenerative medicine and the biosciences. To achieve high-quality imaging for elucidating the molecular mechanisms of angiogenesis, the two-photon excitation fluorescence (2PEF) microscopy, or multiphoton fluorescence microscopy is increasingly utilized in scientific investigations. The 2PEF microscope confers several distinct imaging advantages over other fluorescence excitation microscopy techniques-for the observation of in-depth, three-dimensional vascularity in a variety of tissue formats, including fixed tissue specimens and vasculature in live specimens. Understanding morphological and subcellular changes that occur in cells and tissues during angiogenesis will provide insights to behavioral responses in diseased states, advance the engineering of physiologically relevant tissue models, and provide biochemical clues for the design of therapeutic strategies. We review the applicability and limitations of the 2PEF microscope on the biophysical and molecular-level signatures of angiogenesis in various tissue models. Imaging techniques and strategies for best practices in 2PEF microscopy will be reviewed. Impact Statement Deep live tissue imaging provides unique opportunities to study angiogenesis and associated events in real-time. In contrast to cross-sectional data provided by conventional methods, two-photon microscopy enables high-resolution tissue imaging, data acquisition over time, real-time visualization of angiogenic events, and reduces the number of animal models used in scientific research. This review provides insights on different two-photon microscopy methods and its application in live and deep tissue imaging of angiogenesis on and tissues. We believe that the current trends in imaging can transform the investigation of angiogenesis, cancer research, and biofabrication of vascularized tissues.
近年来，血管生成在健康和疾病中的作用备受关注。可视化治疗和实验室级生物分子引起的血管床的血管生成模式和相关事件，已成为再生医学和生物科学中的常见做法。为了实现阐明血管生成分子机制的高质量成像，双光子激发荧光（2PEF）显微镜或多光子荧光显微镜在科学研究中越来越多地被应用。与其他荧光激发显微镜技术相比，2PEF 显微镜具有几个独特的成像优势——可用于观察各种组织形式（包括固定组织标本和活体标本中的血管）的深层三维血管结构。了解血管生成过程中细胞和组织发生的形态和亚细胞变化，将为了解疾病状态下的行为反应提供线索，推进生理相关组织模型的工程设计，并为治疗策略的设计提供生化线索。我们回顾了 2PEF 显微镜在各种组织模型中的血管生成的生物物理和分子水平特征的适用性和局限性。将回顾 2PEF 显微镜的最佳实践成像技术和策略。影响陈述 深层活体组织成像为实时研究血管生成和相关事件提供了独特的机会。与传统方法提供的横截面数据相比，双光子显微镜能够实现高分辨率组织成像、随时间采集数据、实时可视化血管生成事件，并减少在科学研究中使用的动物模型数量。本综述介绍了不同的双光子显微镜方法及其在 和 组织的血管生成的活体和深层组织成像中的应用。我们相信，目前成像技术的发展趋势可以改变血管生成、癌症研究和血管化组织生物制造的研究方式。
