Fluorescence Bleaching Restore Mobility, Determination Of Macrophage Fc Receptors

In the chapter one of this dissertation, the detail review of single molecule detection(SMD), especially of using optical methods was delivered. The review included the significance, general methods, technique key of SMD, single molecule manipulation, the selection and labeling methods of fluorescent probes, the fluorescent character of single molecule, the discriminating of a single fluorophore behavior, etc. The theories and applications of confocal laser scanning fluorescence microscopy(CLSFM) and total internal reflection fluorescence microscopy(TIRFM), the nanomanipulation, such as optical tweezers, patch clamp, and microneedle, the fluorescence resonance energy transfer(FRET) and fluorescence recovery after photobleaching(FRAP) detection technologies , the fluorescence characters and tag methods of green fluorescent protein(GFP), phycobiliprotein and quantum does(QD) are all introduced in detail. Additionally, the applications and developments of SMD in molecule motors, signal conduction, dynamics, enzymology, transmembrance transportation and ion channels are summarized. 146 reference have been referred.In the chapter two, we studied the percentage of fluorescence recoverery and mobile fraction of Fc receptor in the membrane of macrophage cell(Mφ) mainly. First of all, the macrophage cell was distilled from the ascites in little rat. Then cells were incubated in 37℃ with goat anti-rat IgG tagged with Alexa 488. Through comparing the different conditions, such as the solutions treatment, the opsonin concentrations and the incubation times, we found the best conditions were the concentration of goat anti-rat IgG tagged with Alexa 488 was 4×10^-6 g/mL, and the incubation time was 30 min. We researched the distributing of Fc in cell membrane by CLSFM, and found it was not well proportioned. Under the certain conditions, such as laser intensity of photobleaching and scanning, times of photobleaching and recovery, the fluorescence recovery after photobleaching technology was used in examining the fluorescence recovery ratio and calculating the mobile fraction of FcR. The change of fluorescent intensity in local area of Mφ was analyzed too.In the chapter three, the feasibility of 4X 10"6 s/mL goat anti-rat IgG conjunct Alexa 488 as opsonin of Zymosan using fluorescence double labeling was discussed by the method ; r double fluorescence labeling, followed by the determination of phagocytic ratio of M 4>.