Browsing by Subject "fluorescent"
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Item Measurement and model assessment of fluorescence lifetime sensing in multiply scattering media(Texas A&M University, 2005-08-29) Kuwana, EddyThe generation and propagation of fluorescence light within biological tissue offers the potential for biomedical diagnostics and analyte sensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the tissue??s biochemical environment, providing quantitative in vivo information of the confined tissue site. The impact of light propagation and decay kinetics upon the measured signals is important for consideration, simply because tissue scatters light, giving rise to nanosecond photon time-of-flights that are comparable to fluorescence relaxation kinetics. The goal of this study is to develop a time-dependent model describing (i) the generation of fluorescence from dyes exhibiting multi-exponential or more complex kinetics and (ii) its propagation in scattering media. In the preliminary study, fluorescence lifetime spectroscopy is investigated in tissue-like scattering solution. Two fluorescent dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes and each exhibits single-exponential decay kinetics, were employed. Measurements of phase-modulation as a function of modulation frequency were made at varying concentration ratios of the two dyes to experimentally simulate fluorescence multi-exponential decay kinetics in non-scattering and scattering solutions. The results suggest that frequency-domain measurements of fluorescent decay kinetics along with models of light propagation may be enhanced by scatter in order to probe kinetics more sensitively than in non-scattering solutions. The next study involved fluorescence lifetime sensing in scattering and non-scattering solutions with a pH sensitive dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1), which is known to exhibit multi-exponential decay kinetics. The results demonstrate accurate pH sensing in scattering solution via fluorescence kinetics using a simplified propagation model incorporating an average lifetime. Finally, fluorescence lifetime sensing in immobilized systems were investigated. C-SNAFL-1 was immobilized in poly(ethylene glycol) (PEG) microparticles that were immersed in buffered polystyrene solutions. The results demonstrate the ability to perform pH sensing with fluorescence lifetime without the confounding effect of fluorophore loading or the use of 'reference' measurement within multiply scattering systems. In addition, the stability of the immobilized fluorescence sensor and the reliability of fluorescence lifetime measurement verify the prospect of this technology for implantable purposes.Item Synthesis and Characterization of Novel Fluorescent Injectable Micro-Carriers for Tissue Regeneration(2014-04-28) Arora, AkshiSpecific problem: Our previous study showed that the nanofibrous poly-l-lactic acid (NF-PLLA) microspheres are excellent cell carriers for tissue regeneration. However, these injectable microspheres are not fluorescent biomaterials. Incorporation of fluorescent chromophore into NF-PLLA microspheres will allow imaging for proper delivery of scaffold at the specified site and monitor time related degradation in the scaffold, and tissue regeneration by live fluorescent imaging, without the need of sacrificing the animals or undertaking elaborate histological procedures. To date, there is no report on the synthesis of fluorescent PLLA. In this research, we aim to develop an injectable fluorescent PLLA scaffold for tissue regeneration by using Eosin Y (EY) fluorophore as initiator. Method: The fluorescent polymer (PLLA-EY) was synthesized by ring-opening polymerization (ROP) of l-lactide by bulk polymerization method using stannous octoate Sn(Oct)_(2) catalyst and EY fluorophore initiator, at four different monomer/initiator (M/I) molar ratios (20:1,100:1,200:1,400:1). The PLLA-EY polymer was characterized by FT-IR, UV-visible spectrophotometry and molecular weight (MW). The smooth walled (SW) and nanofibrous (NF) microspheres were fabricated from PLLA-EY 200:1 and 400:1 from methods previously described. These were characterized by SEM, confocal, in vitro biodegradation in PBS (pH change and SEM) and cytotoxicity testing (MTS assay) on dental pulp stem cells (DPSCs). Results: EY initiator generated free radicals causing ROP of l-lactide and incorporation of EY in the PLLA polymer chain. FT-IR and UV-vis spectra confirmed incorporation of EY in the polymer. Increasing the M/I ratio increased MW of PLLA-EY polymer. Microspheres formed from PLLA-EY were auto-fluorescent and increasing the polymer MW resulted in more well-defined nanofibers. Both short term (7d) and long term (21d) cytotoxicity results confirmed non-toxicity of the fluorescent polymer to DPSCs. NF microspheres formed small aggregates with cellular extensions between the DPSCs. Biodegradation of NF microspheres was not seen until 6 weeks in PBS solution under SEM. Conclusion: Fluorescent PLLA-EY polymer and its microparticles can be manufactured, and appear to be very promising candidates for dental pulp regeneration. Future studies should evaluate the ability to track the polymer and their microparticles in vivo, and their ability to accommodate cell adhesion, proliferation, differentiation and in vivo implantation.