Research On Spatial Selective Optical Irradiation System Combined With Laser Speckle Blood Flow Imaging

Optical stimulation can be used for regulating the status of biological tissues.For instance,optogenetic techniques can regulate the activity of specific type of neuron,and photochemical effects can be used to produce cerebral ischemia.These methods provide important tools for studying the function of neural circuits and the mechanism of cerebral ischemic injury,which attract extensive attentions in recent years.However,there is still a lack of flexible control of the location,shape,size and number of regions of interest of the optical illumination in current optogenetic and photothrombosis studies,as well as the simutaneously real-time evaluation of changes in brain function during optical stimulation,which is conducive to the studies of neurovascular coupling and pathologics of cerebral ischemia.In this disseration,a method for spatially selective optical irradiation based on wavefront modulation was developed.Combined with laser speckle contrast imaging,the response of cerebral blood flow induced by the activation of specific neurons was investigated by using photogenetic technique.Combined with photochemical method,spatially selective photothrombosis method with real-time feedback of blood flow imaging was developed.The main contents of this disseratoin were as follows:(1)The error distribution of phase modulation method based on LC-SLM was analyzed.It is pointed out that the attenuation of the included angle between the error vector and the target vector is accounted for the stagnation of the iterative algorithm.A method is proposed to reduce this iteration error by adding an image background.Under the condition that the intensity ratio of target area to background area is 10,the iteration error can be reduced from 0.012 to 0.002 and the uniformity of the output rises from 0.7 to 0.9.Based on above method,an optimal iterative method based on image mask is proposed,which can improve the efficiency of the output without adding the background of the target image.The contrast of the modulated image is increased from 10 to 60 compared to the G-S algorithm after 500 iterations,which greatly improves the quality of the modulated image.(2)A set of spatial selective optical irradiation based on LC-SLM combined with blood flow imaging was proposed,which combined wavefront modulation technology and laser speckle contrast imaging(LSCI),and the software of the integrated system was developed.Cerebral blood flow was obtained by LSCI,and LC-SLM was used to generate irradiation patterns targeting designated locations in the cortex by phase modulation method(photogenetic stimulation: 473 nm,photochemical ischemia: 532 nm).(3)Selective photoactivation experiments were conducted on optogenetic mice(VGAT-Ch R2)by the developed system,and cerebral blood flow distribution was recorded using LSCI to study the response evoked by specific neuron activation.Photostimulation was delivered at 10 Hz with 1 s duration.The cerebral blood flow index at the stimulation area increased by about 30% under a stimulation power of 0.5 m W.Experiment of simultaneous stimulation at different locations were performed.The alterations of cerebral blood flow show locality.The changes of cerebral blood flow outside the range of stimulation were not obvious.In addition,experiments of the different multiple stimulation positions have been carried out.The results indicated that the neurovascular regulation may not scale linearly in response to multiple stimulations.(4)Real-time feedback of cerebral blood flow imaging for spatial selective photochemical ischemia was achieved by our system.The photosensitizer(Rose Bengal,10mg/ml)was injected through the tail vein.532 nm laser source was used for spatial selective illumination to generate thrombus in the brain,resulting in ischemia.The formation of thrombus and dynamic characteristics of cerebral blood flow were observed.This method could control the degree of ischemia by monitoring and controlling the time of illumination through real-time blood flow imaging.The results show that the proposed method can accurately embolize a single vessel or several different vessels.