Extending The Imaging Area Of Two-photon Microscopy

Traditional two-photon microscopy(TPM)is capable of three-dimensional imaging neural dynamics with subcellular resolution,but it is difficult to extend into multi-brain and even whole-brain imaging due to the limited imaging field of view(FOV).To extend the FOV,the scanning angle of the galvanometer scanner increases,which induce a rapidly increasing of off-axis aberration.Hence the imaging performance of TMP could greatly decrease.In this thesis,a large FOV TPM system with a FOV of 2.45×2.45 mm~2 and micron-level resolution is proposed,which is based on the theory of the Abbe's optical invariant and designed by optimizing the off-axis aberration.We have performed the following research in this thesis:1.We propose an off-axis parabolic mirror(OAPM)afocal scanning system,which can improve the image quality in the marginal area of TPM.Compared with the traditional double lenses afocal scanning system,the OAPM afocal scanning system consists of a pair of parabolic mirrors.The two-axial galvanometer scanner is located at the front focal spot and back focal spot of the two parabolic mirrors,respectively.Simulation results by ZEMAX show that the OAPM afocal scanning system can effectively optimize the off-axis aberrations represented by astigmatism,field curvature,distortion at the edge of FOV,then improve the imaging quality.2.A segment scanning adaptive optics technique is proposed to correct off-axis aberrations generated during the large-FOV scanning process based on adaptive optics technology.The 1-?m-diameter fluorescent beads experiment used to verify the accuracy for correcting the off-axis aberration.The result shows that the fluorescence signals and the resolution at the edge of the FOV can effectively be increased.3.A large-FOV of 2.45×2.45 mm~2TPM system was build up.Some biological samples in virto and in vivo were imaged,includes the nerve cells of the thy-1 e GFP mouse brain slices in vitro,the microglial of the CX3CR1-GFP mouse brain,and the vasculature of Balb/c mouse in vivo.The imaging results showed that our imaging system could extend the FOV of TPM from 0.5×0.5 mm2to 2.45×2.45 mm~2 with micro-resolution successfully.The proposed large FOV TPM has a broad application prospect in the field that requires high resolution and large FOV,such as providing a useful technique for the study of multiple brain regions.