Development Of High Resolution Phase Contrast Components In Biological Microscopy Systems

With the deep research of biomedicine,phase contrast microscope has been widely used in cell,microorganism and histopathology.The phase contrast element is placed on the back focal plane of the objective lens of the phase contrast microscope system,and the phase information of the object is converted into amplitude information,which greatly improves the distinguishability of small transparent objects.Therefore,improving the accuracy of the phase contrast element is an inevitable way to improve the phase contrast imaging.Based on the principle of the Zernike phase contrast method and the design theory of phase contrast plates,this paper uses a wide-angle depolarization property of metallic material Ag to design a phase shift film with low polarization effect to suppress the polarization effect caused by stray light at different angles,which solves the problem of imaging errors caused by the conventional phase contrast plate.The refractive index and physical thickness of the material are the direct factors affecting the thickness of the phase.Based on the thin-film transmittance theory and dispersion model,the subject inverts the optical constants of the thin-film material and optimizes the process to fit the optical constants of the material.Based on the design theory of the film system and the obtained optical constants,the phase-shifting film was designed using a quantitative method.By adjusting the thickness of the Al2O3 film layer,the phenomenon of ring edge breaking in the stripping process was overcome,and the adhesion of the Ag film was ensured,The physical thickness of the Al2O3 film is finally determined to be 15 nm.Photolithography and physical vapor deposition were used to develop a phase contrast plate with a new thin film structure that can be used in phase contrast microscopes.During the phase-shifting film plating process,TFCalc software was used to perform reverse inversion analysis to optimize the deposition process of the Ag film and Al2O3 film.By controlling the physical thickness of the dielectric film and the metal film,the phase and transmittance of the phase contrast plate were adjusted respectively to improve phase contrast;by optimizing the photolithography and thin film preparation process parameters,a phase contrast ring structure with neat pattern edges and a ring width of 0.722 mm was obtained.Finally,a phase contrast plate with a ring width that meets the system requirements,an amplitude transmission of 18.03%,and a phase shift amount of 0.49? was obtained.The application environment of the phase contrast plate was tested accordingly,and its reliability was tested using a phase contrast microscope system.Observation of the cells confirmed that the phase contrast plate developed by this project has good imaging results and the internal structure of the cells is relatively clear.