Enhanced Knife-Edge Scanning Microscopy for Sub-micrometer Imaging of Whole Small Animal Organs

奖项摘要#1256086：用于对整个小型动物组织进行亚微米成像的增强型刀口扫描显微镜这个由德克萨斯农工大学脑网络实验室颁发的国家科学基金会奖，将有助于改进和改造刀口扫描显微镜，这是一种独一无二的显微镜仪器，将其转化为更广泛应用于生物研究的系统。KESM能够以亚微米分辨率(即非常精细的细节，小于单个细胞的大小)对整个小动物器官进行切片和成像。构建了一台KESM样机，并通过扫描小鼠全脑、章鱼脑和小鼠肺等多种生物器官的亚微米分辨率成功地展示了该仪器的性能。该项目将大大增强这一原型，并将其转变为一个强大的系统，可以由其他研究小组和行业合作伙伴轻松复制和操作。该项目将建造的增强仪器至少涉及生物学研究中的两个主要新兴方向：(1)“组学”(对某些生物实体的总体集合的研究，例如基因基因组学)和(2)多尺度建模(对一系列尺度的系统进行建模，例如从细胞到整个器官)。随着基因组学在20世纪90年代S及以后的巨大成功，生物研究正日益走向各种形式的组学。许多组学研究依赖于整个器官水平的解剖学信息(例如，连接组学，研究大脑中的完整接线图)或基因组信息(例如，基因表达水平)。多尺度建模也已成为生物学研究的一种主要方法过程。在组学和多尺度建模的许多项目中，来自整个生物器官的亚微米显微数据是必不可少的，但现有的工具无法满足当前的需求。预计KESM将填补这一空白。该仪器的具体改进包括：(1)增强的成像(更高分辨率的光学和照相机、荧光成像[遗传学和分子研究的关键])，(2)增强的机械控制(更严格、更精确、更高分辨率的运动控制)，(3)增强的切割(振动刀，实时监控)，以及(4)增强的稳健性(质量监控、校准)。由此产生的总体改进可概括为：(1)成像分辨率提高3倍，(2)操作稳健性提高10倍，(3)成像速度提高10倍(与竞争方法相比)，(4)新的荧光成像能力。更广泛的影响：(1)对研究界的影响：增强的KESM将使研究人员能够获得高分辨率的全器官数据，用于对各种类型的生物器官进行多尺度、组学研究。(2)对教育的影响：整个生物器官的微观图集，如项目小组开发的基于网络的KESM小鼠脑图集，将作为各级学生和教育工作者(K-12、本科生、研究生和普通公众)的教育资源。作为该项目的一部分，研究生和本科生将在多学科环境(神经科学和计算机科学)中接受培训。(3)仪器传播计划：项目组将与一家初创公司合作，简化KESM仪器的系统集成和制造，以便更广泛地传播。该仪器的设计和操作说明将提供给生物研究界，供那些希望建造自己的仪器的人使用。

Award Abstract #1256086: Enhanced Knife-Edge Scanning Microscopy for Sub-micrometer Imaging of Whole Small Animal OrgansThis NSF award, made to the Brain Networks Laboratory at Texas A&M University, will help enhance and transform the Knife-Edge Scanning Microscope (KESM), a "one of a kind" microscopy instrument, into a more widely available system for biological research. KESM is capable of sectioning and imaging whole small animal organs at submicrometer resolution (that is, in very fine detail, smaller than the size of a single cell). A prototype KESM instrument was constructed, and its capability was successfully demonstrated by scanning diverse biological organs including whole mouse brains, octopus brains, and the mouse lung at submicrometer resolution. This project will enhance this prototype significantly, and transform it into a robust system that can be replicated and operated with ease by other research groups and industry partners. The enhanced instrument to be built by this project addresses at least two major emerging directions in biological research: (1) "omics" (the study of a total collection of some biological entity, for example, genomics for genes) and (2) multi-scale modeling (modeling systems across a range of scales, for example from the cell up to the whole organ). With the tremendous success of genomics in the 1990's and beyond, biological research is increasingly moving toward various forms of omics. Much of the omics research depends on anatomical information (e.g., connectomics, studying the complete wiring diagram in the brain) or genomic information (e.g., gene expression levels) at the whole-organ-level. Multi-scale modeling has also become a major methodological process in biological research. In many projects in omics and multi-scale modeling, sub-micrometer microscopy data from whole biological organs are essential yet available tools are unable to meet the current demand. KESM is expected to fill this gap. The specific enhancements of the instrument include: (1) enhanced imaging (higher resolution optics and camera, fluorescence imaging [key to genetic and molecular studies]), (2) enhanced mechanical control (more rigid, accurate, and higher resolution motion control), (3) enhanced cutting (vibrating knife, real time monitoring), and (4) enhanced robustness (quality monitoring, calibration). The overall resulting improvement can be summarized as follows: (1) 3X improvement in imaging resolution, (2) 10X improvement in robustness of operation, (3) 10X improvement in imaging speed (compared to competing methods), (4) new fluorescence imaging capability. Broader impacts: (1) Impact on the research community: The enhanced KESM will allow researchers to obtain high resolution, whole-organ data for multi-scale, omics investigation of various types of biological organs. (2) Impact on education: Microscopic atlases of whole biological organs, such as the web-based KESM mouse brain atlas developed by the project team, will serve as an educational resource for students and educators at all levels (K-12, undergraduate, graduate, and general public). As part of this project, graduate and undergraduate students will be trained in a multidisciplinary environment (neuroscience and computer science). (3) Instrument dissemination plan: The project team will collaborate with a start-up company, to streamline system integration and manufacturing of the KESM instrument for broader dissemination. The design of the instrument and the operational instructions will be made available to the biological research community for those who wish to build their own instrument.