Change Detection On The Intracellular Calcium Concentration Of Acetylcholine

This dissertation consists of three parts:In the preface, first of all, laser scanning confocal microscope (LSCM) and epifluorescence microscopy were introduced briefly. The detail operation principles of LSCM and epifluorescence microscopy were delivered. Secondly, Ca²⁺, as a kind of cellular second messagers, calcium channel and cholinoceptor which was a sort of receptor operated calcium channel were reviewed. Then, acetylcholine and methods for detections were introduced briefly.In the chapter one, we developed a novel method for analysis of acetylcholine (Ach) by increased fluorescent intensity of intracellular Ca²⁺ signaling. In this method, Fluo-3AM was applied to calcium fluorescent probe, and laser scanning confocal microscope (LSCM) and epifluorescence microscopy were carried out to detect laser induced fluorescent (LIF) intensity by acetylcholine stimulated cell. Firstly, Fluo-3AM was loaded at room temperature for 30 min. Then, after Fluo-3AM was changed into Fluo-3 by the intracellular esterases, we can observe a very large fluorescence intensity increase in response to Ca²⁺ binding. The operation mode of LSCM was chosen to continuous scanning the acetylcholine stimulated cell. The acquired images and anlytical results indicated that increased intracellular Ca²⁺ concentration was stimulated by acetylcholine. The experimental condition was defined with epifluorescence microscopic imaging, which was acquired by EMCCD and MateMorph soft. The line range for the concentration of Ach form 5.0×10^-13 mol/L to1.0×10^-11 mol/L, and the limit of detection was 3.2×10^-13 mol/L.In the chapter two, measurement of acetylcholine (Ach) with the technology of microfluidic chips combined with cellular biosensor. A PDMS microfluidic chips about 100 μm column diameter was fabricated. Cells were separated to single one by...