Research On Nonlinear Optical Fiber Endoscope For Neuronal Calcium Imaging In Awake Mice

Understanding structure and function of the brain is the most challenging scientific frontiers in the 21 st century.The neuroscience aims at understanding how nervous system codes and processes information at the neural circuits and cell level.In recent years,optical combined with calcium indicator and transgenic technology has gradually become the main method of neuron activity recording.Researchers are able to investigate a specific type of neurons in real time record,and study neural circuits at a cellular level.However,the traditional desktop nonlinear optical microscope is not flexible,which is not suitable for brain calcium imaging in awake animals.There are two main aspects in developing a miniature microscope for brain calcium imaging in awake animals.On one hand,how to achieve efficient fluorescence excitation and collection,including excitation types and pulse delivery,as well as probe design.On the other hand,how to develop a micro scanning mechanism,including precise control,frame rate increase and distortion correction.We have optimized the structure and drive control of the existing piezoelectric resonant fiber scanner.Firstly,we propose a four-plate regime with lower dissipation,which afford to drive large-diameter special fiber.Secondly,we design a set of motion braking signal,which shortens fiber retracing period,resulting in higher frame rate.Thirdly,we reconstruct images by using measured scanning trace,relieving the distortion.By using optimized scanner,a 350-?m-diameter fiber has been driven to achieve scanning imaging at 8 fps without distortion,which provides a micro scanning mechanism for neuron calcium activity observation and recording.Based on the scanning mechanism above,we design and develop a prototype of nonlinear optical fiber endoscope for head-mounted mice.The structure of the prototype is simple due to the use of single high efficient fiber.Femtosecond laser pulse has been delivered without distortion after dispersion pre-compensation,which promotes efficiency of excitation.The prototype shows excellent imaging performance in optical properties test,which meets the demand of various types of imaging.It provides a reliable microscopic imaging system for subsequent calcium imaging in awake mice.We also design an encapsulation,which is suitable for head-mounted and easy to assemble and weighs less than 1 g.A wheel system has been set up to simulate the experiment mouse close to nature.Besides improving the frame rate,series problems such as effective imaging period,effective scanning lines and pixel dwell time all have been optimized.Monitoring and recording a group of neurons cell calcium activities in awake mice has been achieved.In this thesis,aiming at neuronal calcium imaging in awake mice,we firstly optimized the scanner structure and drive control,which enables driving large-diameter special fiber at high frame rate.Then,based on that large-diameter special fiber,we designed a prototype which has simple structure.Finally,we also design a head-mounted encapsulation,to achieve continuous calcium imaging in awake mice.Our study provides a new imaging tool for neurobiological research.