Incubator and heating insert for live cell studies on zeiss confocal microscope

申请资金用于一台先进显微镜的细胞培养箱，一台Bioscope II，一台蔡司共聚焦和原子力显微镜（AFM）的组合。申请人对活细胞的纳米级研究需要为这些细胞提供与显微镜载物台相容的存活环境的能力，在显微镜载物台中温度、湿度和CO2被控制，使得实验可以进行延长的时间段。所要求的蔡司系统是为我们的显微镜定制设计的，因此是唯一的报价。我们希望建立在我们的发展的新方法的图案化活细胞在微流体环境中，并探测他们在高空间（几十纳米），时间（秒）和光谱分辨率，下降到近单分子水平的光学和电化学技术的进展。图案化的生物活性区域已经在PDMS中的微流体通道中产生，所述PDMS是光学透明的并且对CO2和O2是可渗透的。我们已经证明，在我们目前的环境中，我们只能在短时间内（约30分钟）保持细胞活力，并通过短期借用相同的系统证明了所需设备的适用性。借来的设备通常不能借出。我们现在迫切需要一个能够保持细胞长时间存活的系统。我们将使用该系统来研究一些膜介导的过程。i）诺顿：a）细胞粘附和运动性以及通过改性PDMS基底的弹性波纹检测的与拉伸应力的相关性; B）活细胞膜上分子的运输，例如阿片类物质与受体的相互作用; ii）Lagugné-Labarthet：开发用于膜蛋白研究的表面增强拉曼光谱（Sers）; iii）Ding：例如，结合共聚焦和扫描电化学显微镜（SECM）研究神经元细胞对非热微波的响应。所有这些研究都需要延长细胞在微流体环境中的寿命，因此需要常规使用所需的设备;我们目前和未来的研究计划严重依赖于它。

Funding is requested for a cell incubator for an advanced microscope, a Bioscope II, a combination Zeiss confocal and atomic force microscope (AFM). The applicants' nanoscale studies on live cells require the ability to provide a viable environment for these cells compatible with the microscope stage(s) in which temperature, humidity and CO2 are controlled, so that experiments can be undertaken for extended periods of time. The requested Zeiss system is custom-designed for our microscope and hence is the only one quoted. We wish to build upon progress in our development of new methods of patterning live cells in a microfluidic environment and of probing them at high spatial (few 10s of nm), temporal (seconds) and spectroscopic resolutions, down to near-single molecule levels by optical and electrochemical techniques. Patterned bioactive regions have been created in microfluidic channels in PDMS which is optically transparent and permeable to CO2 and O2. We have demonstrated that we are only able to maintain cell viability for short time periods (~ 30 min) in our current environment, and have demonstrated the suitability of the requested equipment via a short-term loan of an identical system. The borrowed equipment is not routinely available for loan. We now urgently need routine access to a system capable of keeping the cells alive for extended periods of time (hours). We will use the system to study a number of membrane-mediated processes. i) Norton: a) cell adhesion and motility and correlation with tensile stresses detected by elastic rippling of the modified PDMS substrates; b) trafficking of molecules at membranes of live cells, for example the interaction of opioids with receptors; ii) Lagugné-Labarthet: development of surface enhanced Raman spectroscopy (SERS) for studies on membrane proteins; iii) Ding: combined confocal and scanning electrochemical microscopy (SECM) studies, for example, of the response of neuronal cells to non-thermal microwaves. All this research requires extending the lifetime of cells in the microfluidic environment, and hence the need for the routine use of the requested equipment; our present and future research plans are critically dependent upon it.