Research On Serial Thin Sectioning Of Resin Block For High-resolution Three Dimensional Imaging Of Large Samples

With the development of the study on brain-wide neural network structure of the mammal, it is necessary to exploit the imaging technology which can provide neuronal processes or even synapses resolutions throughout a whole brain. A lot of new imaging methods and systems have been developed, among which the microscopic imaging based on resin embedding and ultra-thin sectioning has been considered one of the most realistic techniques for determining fine connectivity architectures of a brain-wide neural network. As this kind of technology is used for centimeter sized sample such as a whole brain, the processability and stability of thin sectioning greatly affect the qualities of neural network structure imaging and large data acquisition with micron resolutions and long-term work. However, few systemic studies have been conducted on the serial ultra-thin sectioning, especailly on large-sized samples.Micro-optical Sectioning Tomography (MOST) is representative one of microscopic imaging techniques based on resin embedding and ultra-thin sectioning, which focuses on the high resolution imaging of whole mouse brain stained with various kinds of techniques. MOST achieves large volumetric data acquisition through embeding the whole mouse brain in resin and automatic process of0.5-1μm-thick thin sectioning and imaging. This paper used MOST system as experimental platform, and established a quantitative evaluation and research methods of serial-thin-sectioning performance in3D high-resolution imaging of large samples and researched the MOST micron-sectioning of large sample made of Spurr resin representing epoxy resins.Since the direct measurement of the sections of the large sample is difficult and unreliable, we provided the basic idea of performance evaluation based on processed surface topography measurement, and described the technology choice, overall program, evaluation and optimization criteria. Section image quality and the accuracy of three-dimensional reconstruction are directly related to the surface waviness, surface roughness and section thickness. These three basic performances were studied through the acquisition and measurement of surface waviness curve,3D topography and step height. The specific research on the MOST system indicated that MOST system should avoid the F300section speed which would enhance vibration and surface waviness in micron sectioning of Spurr resin. And the Shore hardness of sample should be80. Based on these conditions, we could obtain the high quality surface with Rq below20nm and Rz below200nm. By adjust the roll angle of knife blade, the thickness variation range of the sections could be controlled within10%.Thin sectioning of large sample has to experience that the whole slice decomposes to several sectioned strip, which would cause the problem on the lateral registration of the strip and the boundary loss, and hence effect the accuracy and integrity of the three-dimensional image data. We firstly proposed the roll angle measurement of knife blade based on surface sawtooth height, and evaluated the lateral registration of strips. Strip boundary loss was studied by using scanning electron microscopy and evaluated by the nick width on boundary. From the experiment on the MOST micron sectioning of Spurr resin, it shows that large sawtooth height of cause large nick width on boundary and large dislocation of lateral stripes. Soft samples are more likely to have a larger boundary nick. By adjusting the roll angle of knife blade, the sawtooth height is controlled within200-300nm. Besides, hard sample(recommend1#Shore hardness,83.5) should be used. Under these conditions, the registration deviation of lateral strips can be control within20%to30%, and the nick width on the boundary can be control below the lateral resolution of MOST system (0.71μm). By the measurement of the micron step height and the sawtooth height, we calculated the average thickness in MOST micron sectioning of Spurr resin, and the deviation between the actual value and the set value of1μm was less than3%. Thin sections of the large sample should be operated under water, in which case the water may soften the resin and make it expansion which may affect the performance of the section. For studying and optimizing the sectioning performance under water environment, a quantitative research method of the surface expansion with a short-time immersion based on surface-step-height measurement was firstly proposed. And this method was applied to studied the hygroscopic expansion characteristics of Spurr resin. According to the hygroscopic expansion properties of Spurr resin, the immersion duration of each sectioned strip should be both constant and as short as possible, normally within4minutes. When the water immersion duration is longer than8minutes, the mechanical properties of sectioned surface may degenerate and cause serious data loss. Using Shore hardness measurement to study hardness changes of the Spurr resin after water immersion, it is considered that the section performance of Spurr resin could avoid degenerating under the25℃water temperature and the surface renewal rate above1mm/24h.Through the experimental study, we obtained a serial of condition parameters to optimize the sectioning performance. And it showed that when MOST system is running under the optional parameter conditions, we can achieve high accuracy and long-time stability of micron sectioning and the performance indicators is better than the lateral and axial resolution of current system, which can meet the requirements of data acquisition of the brain-wide neural network structure. Comprehensively considering the slice performance and the influence of water environment, we designed specail sectioning-control scheme for Spurr resin specimens during MOST long-time data acquisition, and developed a set of processing standards for sectioning performance monitoring of MOST data acquisition.