Monitoring the fluorescence intensity and anisotropy decay of individual cells within a population

Biological systems are heterogeneous in general and when stained with fluorescent molecules they emit signals that may consist of a number of lifetimes and rotational correlation times that result from the system heterogeneity and define the measured fluorescence intensity and anisotropy decays, respectively. All currently used instruments which measure these parameters lack the ability to re-measure specific cells, and give only an average value over the entire population yielding poor statistics. In this work a special setup was designed and built comprising a stroboscopic laser-based time domain Model C-720T fluorescence lifetime apparatus from PTI, Inc., and a modified Cellscan system. The Cellscan apparatus, has the unique ability to perform repeated measurements on individual cells within a population. Using this new system, different solutions (fluorescein, labeled beads and acridine orange with different concentrations of DNA) were measured and analyzed. Combining kinetic measurements along with the application of appropriate analysis procedures for the time-resolved fluorescence anisotropy data enabled the examination of the dynamics of specific portions of a given macromolecule. To the best of our knowledge, this is the first time fluorescence intensity and anisotropy decays of individual cells within a population are being monitored.