Research On Large Area High Speed Nonlinear Optical Microscopy And Its Application

Nonlinear optical microscopy (NOM) based on two-photon absorption and second harmonic generation (SHG) has provide a powerful tool for biology research due to its high resolution, greater penetration depth, inherent optical sectioning ability and lower photo-damage. However, the small field of view (FOV), lower imaging speed and bulk imaging devices of conventional nonlinear optical microscope confine its application. In this dissertation, the research focus on the challenges mentioned above.The large area nonlinear optical microscope has been constructed and an intensive study of its application has been made. Auto fluorescence and SHG images of the lung tissue of C57BL/6 mice at different time points after tail vein injection of B16 cells have been achieved by home-built large area imaging system. Qualitative differences in morphologic features and quantitative measurement of the fluorescence and SHG images demonstrated that large area NOM succeed in monitoring the progression of pulmonary melanoma metastasis. In addition, morphological features of an intact cerebellum and brain stem coronal slice have been characterized by large area nonlinear optical microscope. The results demonstrate that large area NOM not only can provide the detailed structures of cerebellum and brain stem, but can be used to measure the density of neuron in the cerebellum.To monitor the cell dynamically, the video-rate nonlinear optical microscope with four detection channels has been built. The problems of hardware, such as polygon mirror controlling, trigger signal shaping and voltage attenuation and so on have been solved. A well structured LabVIEW program framework has been designed, which can achieve huge amounts of data acquiring and storing. The parameters of the systemn have been measured. The lateral and axial resolutions are 0.42μn and 1.6μm (XLPLN25×WMP/1.05 NA, Olympus), respectively. The imaging speed of single FOV scanning, large area imaging and axial imaging are 30frame/s, 10frame/s and 6section/s, respectively. In addition, multicolor imaging for cells and dynamic monitoring the dendrite cells in the chamber have been achieved. The results demonstrate that video-rate NOM combined with multi-channel detection can provide a new observation tool for the cell-cell interaction research in vivo.For meeting the requirement of in vivo imaging, fiber coupler for nonlinear optical microscopy has also been studied. Combined with the requirements of fiber-optic NOM, we propose that the fiber coupler for NOM should have following characters:delivering ultrashort pulses with low distortion, collecting backscattering fluorescence with high efficiency, and opposite coupling efficiencies at visible wavelength and at near infrared wavelength for separating the excitation laser and the fluorescence signal. Owing to large mode area, endlessly single mode and lower nonlinear effect of large mode area endlessly single mode photonic crystal fiber (LMA-ESM-PCF), the characteristics of LMA-ESM-PCF coupler working at laser wavelength of 800nm and fluorescence wavelength of 400nm-650nm have been discussed by using of fully vectorial effective index method and the weak-coupling theory. In addition, the tunable coupling efficiency, a unique feature of side-polished LMA-ESM-PCF coupler, has also been well studied. The numerical results demonstrate that properly controlling the polishing depth and the angle between two side-polished fibers can accomplish a LMA-ESM-PCF coupler to simultaneously deliver the near infrared laser for excitation and split the fluorescence for detection.