Title page for ETD etd-05162012-235327

Type of Document Dissertation
Author Hofmann, Matthias Colin
Author's Email Address hofmann@vt.edu
URN etd-05162012-235327
Title Localized Excitation Fluorescence Imaging (LEFI)
Degree PhD
Department Electrical and Computer Engineering
Advisory Committee
Advisor Name Title
Xu, Yong Committee Chair
Baumann, William T. Committee Member
Soker, Shay Committee Member
Wang, Anbo Committee Member
Wang, Ge Committee Member
  • Deep Tissue Imaging
  • Blood Vessel
  • Fiber Optics
Date of Defense 2012-05-04
Availability unrestricted
A major limitation in tissue engineering is the lack of nondestructive methods to assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot “see” through thick and optically opaque tissue constructs. To address this deficiency, we developed a scanning fiber imaging method capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque vascular scaffold, contained in a bioreactor. This imaging modality is based on local excitation of fluorescent cells, acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells, stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled endothelial cells on the luminal surface through a ~500 µm thick tubular scaffold at cell-level resolutions and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution of the order of 20-30 µm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable nondestructive monitoring of endothelial cells seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.
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