The Application Of Adaptive Optical Fluorescence Closed-loop Technology In Confocal Imaging

Confocal fluorescence microscopy has advantages with high resolution, three-dimensional imaging. However, due to influence of aberration, three-dimensional resolution of microscope, fluorescence excitation efficiency, SNR of fluorescence image would degrade with the increase of the imaging depth. So aberrations have greatly limited the application of confocal fluorescence microscope for thick tissue samples imaging and in vivo imaging. Undering this background, theme of this study is the application of wavefront sensorless adaptive optics in confocal microscopy. The theories of adaptive optical confocal fluorescence microscope, wavefront correction algorithm and experimental system have been studied, and in vivo imaging with aberration correction have been realized.First, theories of confocal fluorescence microscopy are studied. Three-dimensional intensity point spread function and optical transfer function of microscope have been derived, which are foundation of aberration correction analysis. The impact of pinhole size on resolution and fluorescence intensity have been simulated, and the results are adopted in experimental system. We also have analyzed influence of the aberrations on lateral and axial resolution of confocal microscope. For two types of aberration, we have studied both stochastic parallel gradient descent(SPGD) algorithm and modal method for accuracy and speed of aberration correction with simulation computing. At the same time, impacts of image noise on the SPGD convergence speed have been studied.We have completed adaptive optics confocal fluorescence microscope experimental system. Based on theoretical analysis, we identified key parameters of the system. Timing control and experimental software design were completed. Using simulated fluorescence target, aberration correctiton experiments have been completed and results verify the validity of SPGD algorithm in confocal microscope. We also have compared images quality after aberration correction with SPGD algorithm and direct slope method. The results show that SPGD algorithm has similar correction accuracy compared with wavefront sensor aberration correction. SPGD convergence rate has been studied undering different imaging SNR. Finally, three-dimensional resolution of microscope was calibrated using fluorescent beads with and without adaptive oprics.Eventually, the confocal fluorescence microscopy based on adaptive optics has been applied to in vivo in mice vascular imaging. For in vivo imaging, the image blur caused by animals breathing would lead to SPGD algorithm can’t converge. We brought out the pyramid KLT algorithm to track image location while in vivo imaging. In vivo imaging and aberration correction experiments for mice ear and skull bone marrow blood vessels have been completed respectively. The three-dimensional maps of blood vessels after correction and fluorescence images show that adaptive optics based on SPGD algorithm could correct aberration, improve resolution of the system and fluorescence intensity, enhance image quality effectively while in vivo imaging for confocal fluorescence microscopy.