Research On Large Field-of-view,High-resolution Laser Scanning Flat-field Microscopy

Understanding the mechanism of brain is a milestone in the history of human science and technology.The key to occupying this milestone is to study the structure and functional network of the brain.Commercial optical microscopy has the ability of cell resolution and plays an important role in the study of brain structure and function.However,the Space Bandwidth Product of existing optical microscopes is limited,and the large field of view and high resolution can't be taken into account at the same time,which greatly limits their application scenarios.Although many research groups have developed large field-of-view high-resolution microscopes,most of these microscopes have serious field curvature.So they are mainly used for functional brain imaging,and are difficult to be used for fine brain structure imaging.In this paper,a new method of large field-of-view high resolution laser scanning flat-field microscopy is proposed.The systematic research work is carried out from the aspects of principle design,field curvature correction and experimental verification.The highest Space Bandwidth Product commercial objective was being used to design a large filed-of-view high resolution laser scanning microscopic imaging system.The interaction of key design parameters in scanning imaging was studied.A relay lens was designed to adapt for high Spatial Bandwidth Product objective,and its LagrangeHelmholtz invariant is five times as much as that of conventional relay lens.The large aperture and large angle scanning system was studied and successfully applied to the large field-of-view high resolution scanning system.A method for automatic measurement of field curvature was developed.The field curvature of the large field-of-view high resolution laser scanning microscopic imaging system was quantitatively measured and analyzed.Aiming at the serious problem of field curvature in large field of view imaging,a new method of field curvature correction using remote focusing technology was proposed.Based on this method,a large field-of-view high resolution laser scanning flat-field microscopic imaging system was built,and the field curvature correction parameters needed for flat-field imaging were measured.The causes of distortion in imaging system was analyzed,and the automatic measurement method of distortion correction parameters was described.The measured Distortion Correction Parameters were finally used to realize distortion-free imaging.The synchronization control of remote focusing unit,scanning system and detector is realized.The real-time image reconstruction algorithm in flat-field imaging process is described.The method of column pixel redundancy was proposed to solve the problem of blurred reconstructed image caused by slow response of remote focusing unit.We show that the large field-of-view high resolution laser scanning microscopic imaging system is capable of capturing a 5 mm × 5 mm field-of-view image with 1.1 ?m lateral resolution and 19.1 ?m axial resolution.