Study On The Application Of Solid Immersion Lens In Fluorescence Microscopy

Currently, fluorescence microscopy is one of the most important tools in biomedical, analytical chemistry, surface physics, and material sciences research. With the development of science & technology into micro & nano scale, the demand for high spatial resolution of fluorescence microscopy is increasing. The advantages of fluorescence microscope include high sensitivity, high signal to noise ratio, high speciality and so on. However, due to the diffraction limite, the resolution of fluorescence microscopy is limited toλ/ 2. To meet the requirement of micro & nano scale study, the resolution of fluorescence microscopy has to be increased continuously. Futhermore, the fluorescence is so weak that the detectors can not distinguish fluorescent molecule. Thereby increasing the fluorescence collection efficiency of fluorescence microscopy is also very necessary. Applying solid immersion lens in fluorescence microscopy can not only improve the resolution of the system, but also improve the fluorescence collection efficiency. Meanwhile, the solid immersion lens also has the advantages of simply fabrication, easy to use, and good environmental compatibility. In the thesis, the application of solid immersion lens in the fluorescence microscope is intensively studied. This is helpful for the application of solid immersion lens in single-molecule tracking technology.The major works in the thesis are the followings:1. The principle, classification and applications of fluorescence microscopy and solid immersion lens are introduced. The advantages and disadvantages of applying the solid immersion lens in the fluorescence microscopy are analyzed.2. The influence of applying three kinds of solid immersion lens in fluorescence microscopy on the resolution, fluorescence collection efficiency and magnification are analyzed.3. Hemisphere and super hemisphere solid immersion lens for fluorescence microscopy are designed and manufactured. Then the methods of its fabrication process are discussed.4. The surface roughness of solid immersion lens and their application to fluorescence microscopy are evaluated. The results show that using hemisphere and super hemisphere solid immersion lens, respectively, the fluorescence collection efficiency can be increased 2.54 times and 5.5 times; and the microscope magnification were increased 2.06 times and 7.9 times, respectively; and in that case, the system resolution has been improved.5. The influence of changing the radius, thickness and coating the bottom of solid immersion lens on the resolution, fluorescence collection efficiency and magnification are analyzed. It is showed that larger radius makes the fluorescence collection efficiency decrease, but will not change the resolution; It is also show that solid immersion lens with larger thickness makes the resolution, fluorescence collection efficiency and magnification increase, but if the thickness is larger than that of the Weierstrass super hemisphere solid immersion lens, the fluorescence collection efficiency and resolution begin to gradually decrease. Finally, coating the bottom of the solid immersion lens can achieve higher fluorescence collection efficiency without affecting the resolution.6. The content of the thesis is summaried, and the future work is prospected.