Optical methods for exploring dynamics of single copies of green fluorescent protein

Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system. Cytometry 36:232–238, 1999. © 1999 Wiley‐Liss, Inc.