Florescence lifetime imaging microscopy of migrating cells in response to geometrical constrains in vitro and non invasive wound healing in vivo

• Main Authors: Gitanjal Deka, 黛卡
• Other Authors: Fu-Jen Kao
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/02886777759910353537

博士 === 國立陽明大學 === 生醫光電研究所 === 102 === Fluorescence lifetime imaging (FLIM) is a unique technique for molecular imaging. Molecules with inseparable fluorescence emission band or the same molecule at different conformation can be distinguished by their different lifetime signatures. Cellular metabolic coenzyme, NADH (Reduced Nicotinamide adenine dinucleotide) is such a molecule which has two different lifetime signature when bound to proteins and floating freely in cell cytosole. It binds to metabolic enzymes to produce ATP in the process of glycolysis and electron transport system. Hence the relative quantity of enzyme-bound and free NADH can be the signature of metabolic activity of cells. With the help of Ti: sapphire femto second laser induced two photon excitation, the components of functional states of NADH in a cell can me assessed by FLIM. NADH-FLIM has been employed to monitor the relative metabolic activity of migrating cells through guided paths of different widths. Prior to migration, cells were grown in Nd: YVO4 laser engraved two dimensional micro-domains over a specially designed platform. The platform is comprised of a cell adhesive bottom glass surface coated with thin gold layer as thermal transducer at the middle, and a cell repellent polymer top layer. Micro-domains are engraved on the platform, subsequently exposing specific cell adhesive areas by ablating the gold-polymer coating photo-thermally. Experimental results indicate that the proposed approach is applicable under culture conditions with higher engraving speed. Based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical constrains were studied. Our results further demonstrated that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. NADH-FLIM technique is used to study metabolic activity of the migrating cells in response to these geometrical constrains. We compared the metabolic activity of migrating cells with that of static ones. Additionally, we also compared the cellular metabolic activity of cell at the leading edge with that were just started to migrate. It has been argued that collective cell migration is guided by leader cells and has been followed by “follower” cells. Our results elucidated that leader cells have higher metabolic activity than that of the follower cells; and cells far away from the path in the pool of cells have the lowest metabolic activity and is similar to cells in the pool without paths. Multiphoton microscopy based on FLIM is also used to study normal wound healing process none invasively on live rat model. Skin wound healing consists of sequence of fundamental cellular and physiological activities. Where a series of cell lineages are triggered, followed by certain cell migration to the wound site and collagen regeneration to remodel the damaged tissues. NADH from cells and collagen from extracellular matrix at the time of healing are imaged by fluorescence and second harmonic generation microscopy (SHG) in the same microscope setup respectively. We have determined the NADH free to enzyme bound ratio in each day of healing to evaluate relative changes in metabolic activity during wound healing. Our results indicates that at the first week the metabolic activity is relatively higher than the normal skin, however it gradually decreases, and by the time healing completes it become comparable to normal skin. In addition to that SHG imaging revealed that collagen undergoes degradation initially, but regenerated after one week and fill up the wound gape to form the scar tissue. Use of fluorescence lifetime imaging and SHG microscopy provide possibilities for platform to study clinically challenge problems, such as wound healing, cancer invasion. The micro engraving along with cell migration study opens up possibilities to study other cell migration related problems in vitro, such as cancer metastasis, embryonic development, migrating cells during wound healing. Our methodology also demonstrates a possible alternative way of diagnosing wound healing.