On Randomly Scanning Optical Stimulation System

The combination of genetic engineering and photostimulation has boosted anemergence of a new technology–optogenetics, which provides a fast, non-invasive, andselective control of neuronal activity. However, the normal wide-field light illuminationused in the most fluorescence microscopes results in Optogenetic activation of all the cellsor the whole neural circuits in the field of view (FOV). A laser scanning microscopeequipped with galvanometer-driven scanning mirrors has been oftern used to achieveselective Optogenetic activation of multiple cells. Nevertheless, the scanning speed andthe targeting positions are limited because of the inherent inertia of the scanning mirrors.Recently, the spatial light modulator (SLM) was introduced into the microscope to achieveselective activation in a non-inertial way. However, the relatively lower refresh rate andlower diffraction efficiency of SLM have limited the application of this technology. Incontrast, acousto-optical deflector (AOD) with the advantage of fast random addressinghas been used to deliver laser non-inertia to multiple pre-selected sites for light activationof caged compounds in cultured neurons. The excellent spatial selection also makes AODsuitable for selectively stimulation of multiple cells or neurons that express light-sensitivechannels.In the first part of present study, a random-access photostimulation system forselective stimulation based on a pair of perpendicularly oriented AODs has beendeveloped in order to achieve much faster laser activation among multiple positions.Related works are summarized as follows:(1) To achive the scanning of laser beam, a pair of AODs were orientedperpendicularly in the laser light path. The scanning beam was focused onto samples byobjective for the light stimulation. General optical devices and adjusting brackets wereused in the system to couple with a commercial microscope for observing the samples andselecting interested cells or neurons. Mechanical parts of this system were designed ccording to the concept of modular design, which made all optical parts and electroniccomponents were encapsulated into a small box. This complete all-in-a-box allows thissystem easily attached to different brands of commercial microscopes. Meanwhile, anAOD controller box has been built with a standard data sampling card, electronic circuitsfor data transmission and communication with computer and other peripherals. Oneapplication routine was developed using LabVIEW software for controlling thephotostimulation process and the codes of the program is accessibly by the end users.(2) The performances of this system were then tested. It possesses a highrandom-addressing rate up to10μs per site. The spatial resolution and stimulation rangeof the system are1.38and164μm respectively, under40×objective. The fluctuation oflaser power in this system is less than5%, and spatial targeting error is less than1%.These assessment data indicate that the system is applicable for rapid and stable laserstimulation of mutltiple postions.In the second part of this study, we further analysed the performances of laeractrivation of light-sensitive channels in cultures, brain slices or intact Drosophila brain,collaborating with groups of Drs. Zhang Xiaohui and Wang Zuoren at the Institute ofNeuroscience, Chines Academy of Sciences. The results are summarized as follows:(1) To evaluate the efficacy of stimulating cells, light-sensitive channels ChIEFs wereexpressed in HEK293cells and hippocampal neurons in cultures. Our results show thatthis system is able to activate cells or induce a neuron to fire action potentials in10milliseconds stimulation. The spatial resolution of this system was correlated with laserpower and could achieve single-cell activation by using relatively low laser power. Inaddition, the spatial resolution can be substantially improved equipped with two-photonlaser. Region stimulation mode could increase the amplitude and the slope of inducedcurrent, and evoke much high-frequency action potentials after spot stimulation mode andregion stimulation mode were analysized in detail. The random scanning way in regionmode could activate cells more effectively than the two other. The ultra-fast multi-site stimulation features of the system provide a flexible solution for manipulating neuronalactivity at high efficiency and multiple modes.(2) Finally, we demonstrated the versatility of this high-resolution system indissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo.These data indicate that this system could be applied to varied species with different cellsize for mapping the synaptic connectivity and studying the neuronal informationprocessing. These results prove this system will be widely used in neuroscience.