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

点击翻译按钮获取中文摘要

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.