Fabricate Scanning Laser Induced Fluorescence Microscope And Image The Substrate Via Negative Feedback Mode

In chapter one of this thesis, several scanning microscope was reviewed briefly. We mostly introduced the principles, application, speciality and disadvantage of scanning electron microscope (SEM), scanning tunneling microscope (STM), atomic force microscope (AFM), scanning near-field optical microscope (SNOM), scanning electrochemical microscope (SECM) and confocal laser scanning microscope (CLSM). In the chapter 72 references were cited.In chapter two , we devised a novel scanning probe microscope: scanning laser induced fluorescence microscope (SLIFM), and assembled the new apparatus by ourselves. The apparatus comprised scanning laser induced fluorescence system, high sensitively detecting fluorescence system, software system to control scanning system, collect fluorescence signal and analyse data. We immobilized one end of a optical fiber on the focus of the object lens of a microscope, the other end used as optical probe was fixed in a three-dimensional manipulator which compised a three-dimensional motor and a piezoelectricity drivers. The three-dimensional manipulator controlling by software could make the optical probe move over the object we investigated in X-Y-Z directions by micron yardstrick. We used photomultiplier tube(PMT) as the detecting apparatus and utilized CHI910B software to analyse signals. Using this apparatus we obtained the approach curve when the fiber move to the bottom of a detection pool filled with fluorescence matter, charactered the patterns of the solution surfaces and the HRP spots immobilized on the glass substrate. We also could got the size of the micropool by the solution surfaces' pattern, which was the same as the micropool's trim size.In chapter three we fabricated a kind of "enzyme probe" which was the optical fiber modified with HRP and created the negative feedback mode of the scanning laser induced fluorescence microscope based on the enzyme catalyzing reactants in the solution. This optical probe, which was modified optical fiber and could detect substrate, was never reported about other scanning microscope. We modified HRP on the optical fiber's end surface via silanization to fabricate the enzyme probe conducting light. HRP modified on the fiber could convert the substrates: ADHP and H₂O₂ to product: resorufin. By the catalyzing reaction we obtained the approach curve of the negative feedback mode when the enzyme probe move to the bottom of a detection pool filled with HRP's substrates, and charactered the etched figures on the glass, we also could measure the height and width of the figures on the glass via the approach curve and line scanning curve and the data was same to the objects' full size.