A spectrochemical imaging system for rapid and nondisruptive screening for pluripotency and differentiation in cultures of embryonic stem cells

胚胎干细胞（ESC）为解决临床和实验室环境中许多重要的生物医学问题提供了巨大的潜力。利用这些细胞设计新的细胞疗法来治疗许多目前使人衰弱和昂贵的人类疾病（如糖尿病和帕金森氏病），以及不可修复的损伤（如脊髓麻痹），这种潜力引起了极大的兴奋。然而，在我们目前的状态和新的治疗应用之间还有许多障碍需要克服。阻碍这一领域进展的基本技术障碍之一是缺乏快速、无损的方法来区分生长培养中的ESC细胞和其他（已分化的）细胞。我们建议基于一种称为相干反斯托克斯拉曼散射（CARS）显微镜的光学技术，开发这样一种快速、无损、非接触的筛选方法。这种非线性光学方法允许在培养物中原位成像ESCs，并利用CARS信号携带的丰富化学信息来区分干细胞和分化细胞。具体来说，我们建议i)开发一个强大的光学元件系统，用于有效的曝光和收集CARS光谱，以及先进的信号处理方法，以帮助解释ESC的CARS光谱；ii)进行第一次涉及人类ESC的光谱化学成像研究，以识别和表征对伴随分化的细胞变化（和/或这种变化的速度）最敏感的光谱特征；（三）准备将这项技术安装在现有的生物成像核心设施中，干细胞生物学家和其他医学科学家可以使用该设施。该项目将为加速人类胚胎干细胞疗法的发展奠定基础，并将促进这些细胞的广泛应用，以确定刺激或抑制体内特定再生事件的新药。

Embryonic stem cells (ESC) offer tremendous potential for addressing a host of important biomedical problems, both in clinical and laboratory settings. This potential has generated extraordinary excitement over the use of these cells to devise novel cellular therapies for many currently debilitating and costly human diseases (e.g., diabetes and Parkinson's disease), as well as irreparable injuries (e.g., spinal cord paralysis). There are, however, many hurdles to overcome between our present state and new therapeutic applications. One of the fundamental technological obstacles that dramatically slows progress in this area is the lack of rapid, nondestructive assays for distinguishing ESC from other (differentiated) cells within a growing culture. We are proposing to develop such a rapid, nondestructive, non-contact screening methodology based on an optical technique called coherent anti-Stokes Raman scattering (CARS) microscopy. This nonlinear optical method permits imaging of ESCs in situ within a culture and exploits the rich chemical information carried by the CARS signal to distinguish stem cells from differentiating cells. Specifically, we are proposing i) to develop a robust optical component system for efficient exposure and collection of CARS spectra and advanced signal-processing methods to aid the interpretation of CARS spectra of ESC; ii) to undertake the first spectrochemical imaging investigation involving human ESC to identify and characterize the spectral features most sensitive to cellular changes (and/or the rate of such changes) accompanying differentiation; iii) to prepare this technology for installation at an existing bioimaging core facility that provides access by stem cell biologists and other medical scientists. This project will lay the foundation for accelerated progress toward developing human embryonic stem cell-based therapies and will facilitate the broader use of these cells to identify new drugs that stimulate or repress particular regenerative events in vivo.