| Intensive studies have been conducted to understand the sequelae of stroke and its injury mechanism. A technique, intrinsic/extrinsic optical signal imaging (IOSI/EOSI), that can monitor a moderate area in brain has been adopted by many neuroscientists to study the systematic response of functional cortical areas. The application of extrinsic dyes in EOSI increases signal-noise-ratio (SNR) and reduces the imaging time; however, the cyto-toxicity and the leaching effect of fluorescent dyes limit the use of various dyes in EOSI. Encapsulation of fluorescence sensors in EOSI can provide bio-compatible sensors that have controllable sizes for intracellular or extracellular loadings.;The objective of this dissertation project is to design a biocompatible sensor encapsulation process for EOSI imaging and to build a platform for future stroke-induced Hypoxia/Ischemia/Reperfusion (H/I/R) injury research. A core-shell structure based upon biocompatible sol-gel sub-micron chitosan-alginate sphere for encapsulation of DAF-2 fluorescent dyes, which are sensitive to nitric oxide (NO), is proposed and tested. This sphere has controllable diameter from 200 nm to 4mum, and nanometer-scale thickness Layer-by-Layer (LbL) self-assembled polyelectrolyte films have been coated onto the surface of this sphere to control the inner-core environments and prevent the leaching of fluorescent dyes. A suitable reference dye, R-PE, was immobilized in the core, and DAF-2 was immobilized in the polyelectrolyte walls by electrostatic force attraction. A wide-field EOSI system has been constructed to test the optical properties of these sensors and for future imaging purpose. A NO standard generated by SNAP was used to calibrate the NO nanosensor under the fluorescence spectrometer and the wide-field EOSI system. Comparisons between the NO nanosensor and amperometric electrodes indicated that the NO nanosensor had a sensitivity similar to that of a glass carbon electrodes modified for NO. With the NO nanosensor, the EOSI system is able to image NO concentrations within physiological levels.;In vitro experiment of the NO nanosensor in mouse brain tissue slice shows that there are differences in NO productions in hippocampus CAI area with or without NO precursor L-arginine in the perfusate. The results also show that there are regional NO production differences, and that regional differences could be explored in future research. |
