Microflow time-resolved cytometry for FRET and fluorescent protein development

总结

Summary This application requests supplemental support for the purchase of a fluorescence lifetime imaging microscope (FLIM). A new microscope of this type will significantly advance the aims of the project: R01GM129859 Microflow time-resolved Flow Cytometry for FRET and Fluorescence Protein Development. FLIM systems have unique abilities to image cells whereby the image is a rendering of the fluorescence lifetimes measured across cells at a high resolution. The fluorescence lifetime is the average time a fluorophore spends in the excited state; it carries a significant amount of information about the fluorophore such as the microenvironment, near-neighbor proximity, and movement of intracellular molecules. This will support the parent R01 project because the objective for this work is to design and adapt fluorescence lifetime flow cytometry onto a microchip platform for cell screening and eventual sorting. The aims will develop a novel microflow cytometer that will, when completely vetted, incorporate many unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple- fluorescence lifetime values per cell, and electro-optical design for eventual cell sorting based on fluorescence lifetime measurements. Concurrently with the design and construction of the microflow cytometer two major applications of this tool are planned/underway. These are 1) the quantification of Förster resonance energy transfer (FRET) events inside of mammalian cells, and 2) the enrichment of near-infrared fluorescent proteins based on photo-kinetic data. Therefore a microscope (FLIM) with the unique ability to measure fluorescence decay kinetics is essential for 4 reasons: 1) it will enable us to independently cross-validate our cytometry measurements thereby providing resolution and precision that are orders of magnitude greater for reliable optimization; 2) it will significantly enhance the collaborative applications that we are exploring with our novel cytometer and current aims; 3) it will greatly expand the applicability of our cytometer for other fields thus broadening the biomedical impact while supporting future grant applications; and 4) it will become a tool for training and development of students and collaborating faculty at New Mexico State University, a Hispanic Serving Institution.