Fast Light Field Volumetric Imaging Of Freely Moving C.elegans

An important topic in neuroscience research is to explore how large-scale neurons in the nervous system cooperate with each other to form neural circuits and ultimately guide on biological individuals to generate complex thoughts and behaviors.The solution of this problem will help improve human beings' in-depth understanding of various complex physiological activities of different lives,and also will actually help the exploration of the treatment of some neurological diseases.Solving this problem requires the microscopic imaging observation methods to achieve high resolution while maintaining the advantages of high-speed threedimensional imaging,thus the simultaneous recording of large-scale neuron populations in organisms can be achieved.Compared with traditional methods such as confocal imaging and light-sheet fluorescence microscopy that rely on sequential scanning to collect threedimensional information of objects,light field microscopy is an acquisition method that does not require scanning,just capturing a single two-dimensional light field image and reconstructing complete three-dimensional information.Due to only limited by the frame rate of the acquisition camera,this method could achieve a higher three-dimensional imaging throughput.This inspires us to combine the high-throughput advantage of light field microscopy with a real-time feedback tracking system,which will help realize the simultaneous recording of real-time behavior and neural activity of free-moving biological samples,so as to facilitate subsequent exploration of the internal connections between neural activities and behaviors.Based on the practical problems discussed above,this article will apply the advantage of high-speed three-dimensional imaging of light field microscopy to the exploration of the relationship between neural activity and behavior control of freely moving samples.On the one hand,relying on commercial microscope to design and build an in situ light field-wide field detection microscopic imaging system,and the point spread function light-field image of the system was captured with its three-dimensional reconstruction being performed and compared with in situ wide-field acquisition to prove the high-speed and high-resolution threedimensional imaging performance of the system.Based on the above system,this article further used the microfluidic chip technology to carry out the long-term acquisition of dual-channel light field data on semi-free moving C.elegans.Furtherly the behavior and neural activity analysis of the acquisition process was visualized to study the inner relationship between neural activity and behavior.On the other hand,this article designed and built a commercial microscope-based integrated fluorescence light field-behavior detection optical system that simplifies the complexity of the system.Based on this design,we developed a real-time searching and tracking system for freely moving C.elegans,which can complete the entire acquisition process of long-term automatic sample searching,contour recognition and tracking under single high-magnification objective detection.The stable tracking and light field image acquisition through combined with light field detection proved the potential of the integrated fluorescence light field-behavior detection imaging system on capturing the neural activity and real-time behavior of free-moving biological samples,and lays a technical foundation for the research of exploring complex physiological activities of organisms.