Automation and Control of Micro-scale Biological Tasks: Single Cell Surgery

在生物科学领域，过去十年见证了人们对单细胞生物学的兴趣与日俱增，同时这项工作也使这项工作成为可能，工程研究中开发工具进行单细胞手术的兴趣与日俱增。这些单细胞手术过程包括：(I)去除(活组织)细胞器，如细胞核、线粒体，以确定细胞器的性质；(Ii)注入细胞内感应的化学标记；(Iii)将RNA、DNA转移到细胞内以创造转基因生物；(Iv)在人类干细胞治疗中对干细胞系的细胞成分进行活组织检查；(V)卵裂球或滋养外胚层活组织检查(人类胚胎细胞)；(V)植入前遗传学诊断(PGD)，以在儿童发育之前检测遗传病。在加拿大和世界各地的研究实验室和体外受精(IVF)诊所，对这一过程的需求都在不断增加。目前，这些任务是由熟练的技术人员使用显微镜和微型吸管进行的。使用明场显微镜，细胞在这种显微镜下看起来是透明的。由于景深非常窄(不到几微米)，只有很薄的一片细胞被对焦。因此，感兴趣的对象在图像平面上方或下方都是极不对焦的。这种仅有几微米的有限焦深，再加上操作员疲劳和潜在的人类污染，对细胞手术的吞吐量和成功率造成了严重的负面影响。通过将自动化制造原理应用于细胞手术任务，结合实时3D图像反馈来控制自动化过程，找到了这些问题的解决方案。取消人工操作员将导致提高吞吐量和成功率。*为了克服2D图像反馈在细胞手术中的局限性，我们建议研究和开发一种用于细胞手术的实时3D成像系统，该系统将连续提取细胞手术所需的3D信息，如细胞器质心位置、细胞膜位置等。*研究计划的目标*这项拟议的研究旨在利用自动化的机器人生物操作系统来研究单细胞手术的自动化。*1.开发机器人驱动的微吸管自动细胞手术的自动化策略。*2.开发基于3D视觉的方法，以促进细胞的定位和后续处理。*3.在加工前对细胞进行细胞器鉴定。这一成像将识别感兴趣的细胞器及其位置。*4.利用带有视觉反馈的MEMS器件开发电磁方法，以双向旋转悬浮细胞，用于成像和后续的细胞手术。*5.利用3D图像信息开发基于机器人的小细胞细胞器自动活检和细胞注射控制方法。*****

Within the biological sciences, the last decade has witnessed growing interest in single cell biology, and simultaneously enabling this work, increasing interest within engineering research to develop tools to carry out single cell surgery. These single cell surgery processes include: (i) removal (biopsy) of cell organelles e.g. nucleus, mitochondria, for organelle property characterization, (ii) injection of chemical markers for intracellular sensing, (iii) transfer of RNA, DNA into the cell to create transgenic organisms, (iv) biopsy of cell components for stem cell lines in human stem cell therapy and (v) blastomere or trophectoderm biopsy (human embryonic cell) Preimplantation Genetic Diagnosis (PGD) to detect genetic disease prior to the development of a child. Increasing demand for such processes is seen in both research laboratories and in-vitro fertilization (IVF) clinics in Canada and worldwide.******Currently, these tasks are carried out by highly skilled technicians, using microscopes, with micro-pipettes. Bright field microscopes are used, under which cells appear transparent. Due to the very narrow depth of field (less than a few microns), only a very thin slice of the cell is in focus. Hence, objects of interest above or below the image plane, are extremely out of focus. This limited depth of focus of only a few µm. coupled with operator fatigue and potential human contamination, contributes to a significant negative impact on throughput and success rate of cell surgery. A solution to these issues is found through the application of automated manufacturing principles to cell surgery tasks, coupled with real-time 3D image feedback to control the automation processes. Elimination of human operators will result in increased throughput and success rates. ******To overcome the limitations of 2D image feedback in cell surgery, we propose to investigate and develop a real-time 3D imaging system for cell surgery which will continuously extract 3D information essential for cell surgery, e.g. organelle centroid location, cell membrane location, and other quantities. ******Objectives of the Research Program***This proposed research aims to investigate automation of single cell surgery with an automated robotic bio-manipulation system. ***1. Develop automation strategies for robotic actuated micro-pipette automated cell surgery.***2. Develop 3D vision based methodologies to facilitate the location and subsequent processing of cells. ***3. Cell organelle identification will be carried out on cells prior to processing. This imaging will identify the cell organelle of interest, and its location. ***4. Develop electromagnetic approaches using MEMS device with vision feedback, to rotate suspended cells in two direction rotations, for imaging and subsequent cell surgery. ***5. Develop robotic based control methods for automated cell organelle biopsy and cell injection for small cells using 3D image information. *****