Micro Light Stimulation And High-Quality Focus Recovery In Large Field-of-View Within Scattering Medium

In recent years,the rapid development of optogenetics has enabled neuroscientists to further explore the structure of the brain and neural system,establishing the relationship with animals’behavior characteristics in vivo experiment.But there are two significant challenges in vivo experiments in the field of optogenetics.First,the free movement of animals is limited by the optical fiber connection between light stimulation devices and light sources.And it makes the long-term observation inaccessible.The other is the heterogeneous distribution of the refractive index in the biological tissue makes it difficult to locate target cells accurately and stimulate with enough power.However,the correction field of view of the conventional adaptive optical method is limited,resulting in decelerating the correcting rates and the non-real-time recording of the image signals while light stimulating.Thus,we design and produce a wireless miniatured light stimulation system based on the infrared remote control.The transmitter consists of an infrared LED and the constant current driver chip(LM3410X),synchronously controlled by a signal generator.And the miniatured receiver circuit includes an ultra-low power output comparator(TLV3691),a blue LED for light stimulation and a customized fiber coupling device.The output power at the end of the fiber is 1.45 mW and the stable remote control range is 1 m.So this system can realize a long-distance wireless control in an experimental box.And we have evaluated our system performance by animal behavior experiments.At the same time,this paper proposes the large area focus optimization method with multiple guide stars,and the simulation results show that the method can enhance the resolution of fluorescence imaging and achieve the target optimization in large field.Firstly,we theoretically analyze the principle of the coherent optical adaptive technique for adaptive optics correction.After studying the influence of the number of guide stars on the correction effect,we can draw a conclusion that the correction effect of five guide stars in the large field of view is the best.Then we compare the average correction effect for a random phase mask in the large area after the correction of our method and other conventional adaptive optical correction method.Finally,we use a random phase mask and a 120-μm-thick mouse brain tissue to mimic the scattering medium,and a single layer of randomly distributed fluorescent beads(5 μm diameters in average)as the object,and the confocal system was used to obtain the fluorescent images.The fluorescence imaging and the corrected field of view of different methods are compared.The research shows that under the condition of the mouse brain tissue as the scattering media,the new method’s corrected field of view is-251 times of that after the traditional pupil adaptive correction method and is～7.3 times of that after the conjugate adaptive correction method with a single guide star.Eventually,the wireless miniatured light stimulation system can realize a remote controlling light stimulating in the experimental box and it does not affect the mouse’s free movement.The focus optimization and the fluorescent imaging quality improvement in a large area is implemented rapidly by the wavefront correction method with multiple guide stars,thus it paves the way to real-time focus optimization while high-resolution bioimaging in vivo experiment.