| Two-photon laser scanning microscopy has been widely used to observe neuronal activity in living mammals with high resolution at the cellular or subcellular level,.However,with the continuous development of neuroscience research,the limitations of two-photon laser scanning technology also begin to emerge.The main limitation is that the observation area is only limited to a single small field(<1mm diameter).This is because previous neuroscientific studies tend to focus on a single brain region.However,in mammals,the cerebral cortex is distributed with brain regions responsible for various functions.In addition to the cortex,there are also many brain regions located in the brain that play an important role in biological activities,such as the hippocampus and striatum.Since biological activity is the common action of a series of complex neural activities,involving the coordination and interaction of many different brain regions,it is particularly important to observe the neuronal activity of multiple brain regions at the same time to understand the neuronal mechanism of the brain.Here we present a novel imaging technique that allows simultaneous recording of calcium signal activity in different brain regions at a maximum diameter of 12 mm,with a single field of view in each brain region of around 200 ?m in diameter,achieving imaging quality at single-cell resolution with at least 10 Hz frame rate.In order to further demonstrate the feasibility of the band technology,we have studied a set of reasonable biological imaging experiments related to the implementation of the technology.For example,we have shown the simultaneous recording of neuronal activity in the primary visual cortex,the primary motor cortex,and the CA1 area of the hippocampus in mice under different physiological states of anesthesia and wakefulness.The technology is characterized by the combination of the optimized self-focusing lens and the low-power air objective lens,so that the field of view on the 2-dimensional plane can be extended to the 3-dimensional space.In other words,this technique can achieve real-time imaging of different regions in both horizontal and vertical directions.Taking the above example as an example,the longitudinal distance between the hippocampus and the cortical brain region is already more than 1mm,but the application of this technology can well realize the purpose of real-time observation.The main research contents of this works are as follows:1.A fast two-photon laser scanning microscope was built based on two fast scanning schemes,and the two fast scanning systems were finally integrated into a twophoton microscope.A set of system is a fast scanning system based on galvanometer and resonance mirror as scanner,mainly including X scanner(resonance mirror),Y scanner(galvanometer)and scanning lens and tube lens.The propagation process of the beam is as follows: the laser is emitted from the femtosecond light source module,arrives at the position of X/Y scanner,is scanned by X/Y scanner,enters the scanning lens and focuses on the working plane of the scanning lens,and then enters the fluorescence microscopic objective through the tube lens and excites the fluorescent material.The maximum speed is 1000Hz(600×24 pixel size),and the maximum scanning resolution is 4096×4096 pixels(frame rate is up to 6Hz).Another set based on two-axis acoustic optical deflector(AOD)fast scanning system,fter the design of dispersion compensation for acousto-optic crystals,four transducers are proposed to improve the acousto-optic quality coefficient and diffraction efficiency the system maximum frame rate of 10000 Hz(250×40 pixels size),because the system has high scanning speed compared with the galvanometer scanning system and higher signal-tonoise ratio as well,the maximum signal-to-noise increased by 175%.While at the same level of the signal,we found 10000 Hz has lower fluorescence photobleaching.This can be achieved for a longer time observation of neural activity.Based on the above two kinds of fast scanning systems,although the scanning speed of the galvanometer system is not as good as the latter,it can provide a wider field of view with the combination of low-power air objective lens.Therefore,we choose to use the galvanometer and resonance mirror as the scanning system as the scanner of multi-brain region two-photon microscopy system.The experimental results show that it can meet the requirements of various biological experiments.2.A rapid multi-brain region two-photon laser scanning microscope system was built,which for the first time combined multiple grin lenses with low magnification air objective to simultaneously record the activity of the second layer neurons of cortex in the three brain regions.Through the combination of different air objectives and selffocusing objectives,we carefully analyzed the main optical parameters of different combinations – resolution and simulated the energy coupling of NA matching and mismatching.At the same time,in order to maximize the energy utilization,we also designed a NA matching self-focusing lens,and compared the differences between the two situations in terms of experiment and calculation.In addition,in order to further observe the activity of neurons in different brain regions,we designed another grin lens through the combinations of different grin lenses and low magnification air objective.We recorded the calcium signals in three brain regions with different horizontal and vertical distribution for the first time successfully.3.Research and design of multi-brain area biological imaging experiment method and related experimental device design.Due to the originality of this technology,corresponding experimental methods are needed for the biological neuroimaging experiments based on this system,especially the mouse fixation device with the optical system and the surgical method.Different from conventional craniotomy,in order to achieve simultaneous imaging of multiple brain regions,specific fixed chambers are needed to be fixed on the skull of mice,and corresponding sites should be designed in advance in the chamber according to the location of the brain to be imaged,and craniotomies of multiple sites should be carried out according to that.This process is more complicated than the conventional experiment.In order to better implement the experiment through this system,we explored relevant surgical experimental techniques and procedures,designed and formulated corresponding surgical experimental steps,and ensured the success rate and experimental quality of the experiment.the technical structure is simple,just on the two-photon microscope of traditional simply transform,can be more easily modified on conventional two photon microscope,this will greatly help the researchers in the field of neuroscience for in vivo mammal activity of neurons in different regions of the observation and research,to promote the related research provides powerful technology. |
PDF Full Text Request