COMPUTER INTERFACED SCANNING FLUORESCENCE LIFETIME MICROSCOPE APPLIED TO DIRECTED EVOLUTION METHODOLOGIES AND METHODS FOR LIGHT-MEDIATED PATTERNING IN CELL SELECTION

摘要

This invention provides a method for screening large numbers of individual cells or colonies of cells using scanning microscopy coupled with fluorescence lifetime measurement and analysis, using time-correlated single photon counting. Both the imaging of the fluorescence lifetime data from cells and/or colonies on a surface and the analysis of this data are controlled and performed in an automated and rapid manner using a computer. This screening method, or any imaging technique with suitable resolution, can then be used with either light-mediated patterned cell growth methodologies, as further provided by this invention, or mechanical methods to select individual cells or colonies based on their fluorescent properties. The invention further provides an automated method for selecting cells that exhibit desired characteristics. This method utilizes a computer-controlled scanning microscope system to focus a laser beam onto a surface upon which cells immobilized on the timescale of the procedure. The cells that are illuminated in this way are killed or their growth is inhibited. The focused laser beam is scanned across the surface and turned on and off during the scanning process such that only non-irradiated cells survive, resulting in a patterned growth of cells. In an alternative embodiment of this patterned growth cell selection method, a computer-controlled projection device, such as a micro-mirror array or a liquid crystal display system, is used to project an image onto the cells. Cells onto which this image is projected are killed or their growth is inhibited, again resulting in a patterned growth of cells. By employing this invention, cells can be selected with high spatial resolution, and large numbers of cells can be processed. This invention permits selection of desirable cells in directed evolution techniques, since cells can be selected with great resolution at sub-visual sizes, allowing a vast number of cells to be processed at once, without the need for antibiotic resistance markers or growth on selective media lacking required nutrients. Further, cell patterning can be used with essentially any cell type, including yeast and mammalian cells, using appropriately selected or modified chemical sensitizers.