Three-dimensional Multifocal Photostimulation Based On Light-sheet Microscopy

The neuronal circuits are complex distributed in space and difficult to access. Wide field of view, high spatiotemporal resolution and low photobleaching make the light-sheet microscopy stand out in the recording methods for neural activity in large populations of neurons. However, in order to figure out the role of some specific neurons during the neural activity, these neurons should be manipulated while the neuronal circuits are imaged. Spatial light modulator can generate multiple focus accurately and efficiently in the three-dimensional space at the same time, which can be used to stimulate specific cells involved in neural activity. A system was developed, based on light sheet microscopy, to study the optogenetics in zebrafish, which can precisely stimulate specific neurons in the space and to image the whole-brain activity simultaneously.Algorithm based on Debye theory was adopted to realize the three dimensional pattern. The methods for multiple focus generation using spatial light modulator were analyzed. We carried on the transverse and axial sizes of the focus both in the theoretical calculation and experimental analysis. The accuracy of the system was verified by stimulating fluorescent beans precisely.We built the light sheet microscopy and assessed its performance. To prove the advantages of the system, we applied it to zebrafish's in vivo whole-brain imaging. Multifocal photostimulation module was coupled into light sheet microscopy to manipulate specific neurons and record the neural activity simultaneously. The function of the system was confirmed by stimulating and imaging the fluorescent beans. After discussion, we found that the system needs to axially decouple the imaging and stimulation planes when both optical pathways are combined into the same microscope objective.The last but not the least, we described the theory of remote-focusing technique which avoids the spherical aberration effectively. We introduce the technique to improve the system and verify the accuracy of the system. Optogenetics in zebrafish were studied by the final system, photoactivation of specific neurons and whole-brain imaging were achieved.