| Two-photon excitation plays important roles in biological science.Compared with single photon excitation,two-photo excitation has a lot of advantages.To begin with,the two-photon excitation uses a longer excitation wavelength,it has larger penetration depth in biological tissues than that of single photon confocal imaging.Secondly,because the two-photon excitation is a nonlinear process,the excitation requires high photon density.Therefore,the excitation is spatially constrained and the out of focus excitation is significantly suppressed.This provides better optical sectioning capability for the thick sample imaging.The suppressed out-of-focus excitation and lower photon energy also reduce photobleaching and photodamage and thus increases tissue viability which is crucial for long-term biological imaging.Besides,since the excitation wavelength and fluorescence wavelength of two photon excitation are far from each other in the spectrum,the excitation light and the fluorescence are easier to be separately detected.With these advantages,two photon fluorescence microscopy is widely used in biomedical research.However,there are still problems and deficiencies such as the limited resolution and field of view in the existing methods.Further optimization of the performance of the system is still of great significance.In this dissertation,based on the theory of nonlinear optics,point spread function engineering,fluorescence emission difference method and pupil segmentation scanning,in-depth studies of two-photon microscopy methods and techniques are presented.The main contents and innovations of this dissertation are as follows:1.The effect of fluorescence emission difference method combined with fluorescence saturation effect on imaging resolution and contrast of two-photon microscopy is studied in depth.Theoretical analysis and simulation are carried out and the two-photon FED microscope system is constructed for the first time.The experimental results show that the proposed method can improve the imaging resolution of the two-photon microscope system to the order of 100 nanometers and improve the imaging contrast.2.Parallel detection and pixel reassignment are introduced into two-photon microscopy.The application of pixel reassignment theory and algorithm in two-photon microscopic imaging is simulated and experimentally verified.The optical fibers in the fiber bundle are arranged in a circular manner,and the optical fibers in the peripheral profile deviate from the optical axis of the system,which can detect higher frequency information and improve the imaging resolution.A two-photon microscopy with de-scan detection path is designed and built.The image with enhanced resolution and contrast can be obtained by processing the information obtained by parallel detection.This parallel detection combined with pixel reassignment method only needs one scan for imaging and ensures the imaging speed on the premise of improving the imaging quality of the system.3.The axial FED is studied in depth and used to improve the axial resolution in the two-photon microscopy system,and the corresponding imaging system is constructed.In this system,the axial splitting point spread function is obtained by phase modulation of the illumination beam,and the axial resolution of the system is successfully improved by FED,and the optical sectioning ability of the system is improved.4.The problem that the inherent short coherence length of the commonly used femtosecond pulses fundamentally restricts the achievable field-of-view of optogenetic modulation is studied in depth.By using pupil segmentation scanning,the pixels on the spatial light modulator(SLM)are fully utilized.Multiple holograms are quickly readout and delivered by galvo scanners.Two-photon excitation over 1.3 mm field-of-view within 1.3 milliseconds is achieved.The method is scalable and compatible with the commonly used two-photon sources and imaging systems in neuroscience research. |
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