Two-Photon Fluorescence Microscopy and Applications in Angiogenesis and Related Molecular Events

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 have become a commonplace in regenerative medicine and the biosciences. To aid imaging investigations in angiogenesis, the two-photon excitation fluorescence microscopy (2PEF), 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 in vivo 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.