A Preliminary Study On Three-Dimensional Imaging Based On Sliding Thin Sectioning Of Large-Volume Resin Samples

The brain is one of the most complex organs in human.In recent years,the whole brain optical microscopy technology has been widely used in acquiring mouse whole brain’s structural information with single neuron resolution,which is helpful to understand the brain function mechanism in the mouse.In order to study the advanced brain functions such as thinking,learning,and cognition,it is necessary to obtain information from non-human primate brain samples,which are more evolutionarily close to human beings.The volume of these samples is much larger than a mouse brain,e.g.,the volume of a rhesus monkey brain is about 200 times that of a mouse brain.Obtaining high-resolution data of rhesus monkey brain usually leads to a large increase in imaging time.At the same time,it requires long-term stable preservation of samples and the stability of the imaging system.This article presents a scheme of sliding thin sectioning technology combined with wide-field structure illumination microscopy,i.e.,a sliding cutting machine quickly acquires the sample’s smooth surface and optical micro-imaging for obtaining fine structures over the large sample.The system is divided into two parts: mechanical cutting and optical imaging.In the mechanical cutting part,the sliding sectioning technology based on long-edge tungsten carbide knife is used to solve the time-consuming problem of cutting large volume samples with a diamond knife,and realize fast thin sectioning of large area surface.In the optical microscopic imaging part,structural light illumination and mosaic imaging technology are used to realize the high-resolution imaging of large area surface.At the same time,real-time re-dyeing of sample cytoarchitecture information and activation of GFP signal were realized by droplet dyeing device.Through the design of dropping ring,high numerical aperture water immersion objective imaging was used without immersing samples,so that the system resolution reached the sub-micron level.Finally,high-resolution three-dimensional continuous imaging of large volume samples is realized by acquisition workstation’s control of the high precision three-dimensional stage,area array camera,digital micromirror device,and other devices.The system designed in this article finally realizes automatic sectioning imaging of large volume samples such as rhesus monkey brain.In terms of cutting,the surface roughness at the cutting speed of 200 mm/min by the tungsten carbide knife is within 2 μm,which satisfies the imaging requirements of the sample surface.And when cutting a large area surface of "50 mm × 50 mm" compared with a diamond knife,it reduces 94% of the cutting time.In terms of imaging,the voxel resolution of the system has reached "0.32 μm × 0.32 μm × 5 μm ",which satisfies the level of neuron resolution.The imaging ability of the system was verified by the continuous acquisition of large samples such as mustela putorius furo whole brain and rhesus monkey half brain.