We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L͞S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pK a ‫؍‬ 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When Because of the rapidly increasing number of applications of green fluorescent protein (GFP) and its mutants as noninvasive fluorescent markers in molecular biology (1, 2), considerable interest has developed in its biochemical and optical properties. Understanding its photophysics on the basis of recently solved crystal structures (3, 4) should allow the design of mutants tailored to specific needs. The chromophore of wild-type GFP is formed autocatalytically from Ser-65, Tyr-66, and Gly-67 (5) and has two major absorption maxima, at Ϸ390 nm and Ϸ480 nm. Structural analysis (6, 7) and quantum mechanical calculations (8) suggest that they represent chromophores with protonated and deprotonated tyrosyl hydroxyl groups, respectively. The hydroxyl group is part of an intricate network of hydrogen bonds that, depending on the specific local arrangement, favors either the protonated form (wild-type) or the deprotonated form [mutants GFP-S65T and GFP-F64L͞S65T(ϵ EGFP)], with both forms present in a fixed ratio over a certain pH range (about 8 to 11 for EGFP). Interestingly, excitation at either 390 nm or 490 nm leads to similar emission spectra, peaking around 505 nm. This photophysical pattern has been rationalized by an excited-state proton transfer from the hydroxyl group to the hydrogen bond network, leading to a deprotonated chromophore from which actual emission occurs, even when excitation is at 390 nm (9, 10). At lower pH values, however, GFP fluorescence is very sensitive to proton concentration, decreasing to zero below pH 4 (11-14). An objective of this paper is to report observations of the dynamics of the molecular fluorescence fluctuations, which reveal the underlying molecular processes.The protonation state of individual molecules, however, is not fixed over time, but changes rapidly due to the intrinsic dynamics of the molecular system, leading to fluctuations of their photophysical properties. As the concept of protein substates suggests (15) We applied FCS to study the dynamics of the fluctuations of the protonation state of GFP and find that at low pH protonation of the chromophore from the bulk solution occurs, whereas at high pH an internal protonation process takes over. These processes are distinct from the above-mentioned excited-state proton transfer and might pr...

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.

Tryptophan and serotonin were imaged with infrared illumination by three-photon excitation (3PE) of their native ultraviolet (UV) fluorescence. This technique, established by 3PE cross section measurements of tryptophan and the monoamines serotonin and dopamine, circumvents the limitations imposed by photodamage, scattering, and indiscriminate background encountered in other UV microscopies. Three-dimensionally resolved images are presented along with measurements of the serotonin concentration ( approximately 50 mM) and content (up to approximately 5 x 10(8) molecules) of individual secretory granules.

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.

Fluorescence correlation spectroscopy (FCS) is a sensitive and widely used technique for measuring diffusion. FCS data are conventionally modeled with a finite number of diffusing components and fit with a least-square fitting algorithm. This approach is inadequate for analyzing data obtained from highly heterogeneous systems. We introduce a Maximum Entropy Method based fitting routine (MEMFCS) that analyzes FCS data in terms of a quasicontinuous distribution of diffusing components, and also guarantees a maximally wide distribution that is consistent with the data. We verify that for a homogeneous specimen (green fluorescent protein in dilute aqueous solution), both MEMFCS and conventional fitting yield similar results. Further, we incorporate an appropriate goodness of fit criterion in MEMFCS. We show that for errors estimated from a large number of repeated measurements, the reduced chi(2) value in MEMFCS analysis does approach unity. We find that the theoretical prediction for errors in FCS experiments overestimates the actual error, but can be empirically modified to serve as a guide for estimating the goodness of the fit where reliable error estimates are unavailable. Finally, we compare the performance of MEMFCS with that of a conventional fitting routine for analyzing simulated data describing a highly heterogeneous distribution containing 41 diffusing species. Both methods fit the data well. However, the conventional fit fails to reproduce the essential features of the input distribution, whereas MEMFCS yields a distribution close to the actual input.