We propose to maintain the momentum of new discoveries using multicolor fluorescence cell analysis and sorting by developing a new instrument to provide more light generation per cell, better light collection, improved signal evaluation and optimized sorting capability. We recently developed a FACS instrument able to measure up to 11 fluorescent reagents, and experiments using 8 and 9 colors are being performed routinely. The move to higher order multicolor analysis has been very successful in producing new biological results, and it has increased the dye choices and flexibility in work using fewer colors. However, in multicolor experiments the data quality in individual measurement channels is often not as good as what we obtain with comparable single color stains on these channels. We have analyzed the signals and concluded that, while there are systematic problems such as logamp scaling inaccuracy and mismatches between logamps which degrade the accuracy of spectral overlap corrections, the fundamental problem is lack of sufficient light in some of the single measurements and, more generally, lack of sufficient light to accurately evaluate and correct spectral overlaps. Further work on new dyes and different laser sources will help to decrease spectral overlaps between dyes, but efforts to increase the number of usable reagents and to use particular probes whose spectra crowd other dyes of interest will assure that spectral overlap and spectral overlap corrections continue to be limiting factors in flow cytometry. To address these limitations we will develop a FACS yielding at least 20 times the signal levels we obtain with the current system. Initially, we will build separate analytical and sorting prototyping systems. Improved signal evaluation electronics and procedures will be developed. A series of new developments will also be carried out to facilitate high quality cell sorting. Based on developments in these systems, we will assemble a combined biologically useful FACS. We will begin to carry out biological experiments at as early a stage in the work as possible, and the complete instrument will be made available to the full array of users in our FACS service center.
