Epifluorescence Microscopy Technique High-throughput Analysis Of Single Cell Esterase

This thesis consists of three parts: Ⅰ .Determination of esterase activity by epi-fluorescence microscopy. Ⅱ. Determination of esterase activity by high-throughput single-cell epi-fluorescence spectroscopy. Ⅲ. A high-throughput fluorescence spectroscopy method with a microfluidic chip for single-cell fluorcscence.In Chapter Ⅰ ,the esterase activity in the extracts of MGC803 was determined by cpi-fluorcscence microscopy. In this part FDA was used as the enzyme substrate. A enzyme catalytic reaction occurred.The resultant was excited by a 488 nm argon-ion laser.The fluorescence intensity of the resultant was recorded by the EMCCD.The suitable consistens of the FDA and the pH of PBS were studied.The time of the reaction was determined.The esterase activity could be quantified using the calibration curve.In Chapter Ⅱ,A high-throughput.single-cell.fluorescence spectroscopy method was presented. In this part FDA was used as the enzyme substrate. FDA can penetrate to the cell freely tlirough the membrane and react under the catalysis of esterase. After the cells were incubated in the reaction chamber, the attached cells were studied by the fluorescence microscope. The suitable consistens of the FDA and the time of the reaction were studied. The esterase activity of a single living cell was quantified by a high-throughput.single-cell.fluorescence spectroscopy method.In Chapter Ⅲ, A high-throughput fluorescence spectroscopy method with a microfluidic chip was presented for analysis of single cells.In this part, the microfluidic chip was made of PDMS. The PDMS channel was fixed on the cover glass. FDA was used as the enzyme substrate. A high-throughput single-cell fluorescence was detected by epi-fluorescence microscopy on the chip.