Marcel Ameloot

Photophysics of the fluorescent Ca2+ indicator Fura-2

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.

Practical Time-Resolved Fluorescence Spectroscopy: Avoiding Artifacts and Using Lifetime Standards

In this chapter we describe how artifacts can be avoided in the two most commonly used time-resolved fluorometries, namely the single-photon timing and the multifrequency phase-modulation techniques. The most frequently encountered artifacts (inner filter effect, autofluorescence, polarization effects, color effect, photobleaching, deoxygenation, pulse pile-up, and linearity of the time response in the time-to-amplitude converter) are described in detail and remedies are presented to avoid these pitfalls. An extensive list of fluorescence lifetime standards is presented, which allows the spectroscopist to calibrate and test time-resolved instruments for systematic errors.

The Effect of Protein Binding on the Calibration Curve of the pH Indicator BCECF

The fluorescent dye BCECF (2’,7’-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein) is very popular as a dual excitation probe to monitor intracellular pH. The spectral characteristics are known to be different in vitro and in vivo. Upon introduction into the cellular environment its absorption shows a red shift of about 5 nm.1 This spectral shift may exhibit a pronounced effect on pH vs BCECF fluorescence calibration curves obtained in ratiometric mode. It is not clear whether the spectral shift of BCECF is due to binding to cellular proteins or to other environmental effects.2 Measurements of the translational mobility of BCECF in the cell cytoplasm provide contradictory results. It has been reported that BCECF is transiently bound to intracellular components of low mobility3 although this was not observed by others.2

Model testing capacity of global analysis of single-photon timing data

The performance of global (simultaneous) analysis of multiexponential fluorescence decay surfaces using reference convolution is investigated in a systematic way using simulated and experimental data sets. It is shown that the increased model discrimination ability and the more accurate parameter recovery by global analysis as compared to single curve analysis originate from combining decay traces with differing contributions of the decay components. Simultaneous analysis of decay traces in which the contributions of the components are changed as much as possible is the most beneficial. Consequently, including more decay traces in the global decay surface does not necessarily lead to a better model distinction capability. For decay surfaces collected as a function of the emission (excitation) wavelength, the decays with minimal overlap between the emission (absorption) spectra associated with the decay components will contribute the most to the improved model discrimination and parameter recovery.

Determination of the rate constants of the fluorescent pH probe C.SNAFL-1 in the excited state

A proper interpretation of the signals from fluorescent indicators in imaging technology implies a knowledge of the processes in the excited state. This work focuses on the pH probe C.SNAFL-1. The kinetics of the excited-state processes are investigated by global compartmental analyses of fluorescence decay surfaces obtained by time-correlated single photon counting. Within the pH range 5-12 only two species have to be considered in the ground and the excited state. The two fluorescent decay times do not depend on pH. Therefore, the process of protonation in the excited state is very slow as compared to the deactivation rate of the excited states. A proper identifiability study has been performed to determine the rate constant of the deprotonation process. The rate of deprotonation is also small and its upper value is estimated to be 0.05 ns-1. It can be concluded that there is negligible interference of the excited-state reaction on the determination of intracellular pH by C.SNAFL-1.

Evidence for reversible excited-state process in tryptophan zwitterion

This report gives evidence that the biexponential fluorescence decay of tryptophan zwitterion in H2O solution is due to the occurrence of a reversible two-state excited-state process whereby the corresponding ground-state species are excited. The rate constants are within the intervals: 0<k01<0.57(ns)-1, 0.76(ns)-1<k21<1.33(ns)-1, 0<k02<0.58(ns)-1, 0.77(ns)-1<k12<1.35(ns)-1. These limits were calculated using the values for S1 equals k01 + k21 (1.33+/- 0.01(ns)-1), S2 equals k02 + k12(1.35+/- 0.01(ns)-1), and P equals k21k12(1.03+/- 0.01(ns)-2). The emission spectra of the two excited-state species can be uniquely determined and are different from those associated with the decay times. These results were obtained by repetitive global compartmental analyses of the fluorescence decay surface of tryptophan zwitterion measured over the entire emission spectrum as a function of quencher concentration. This new and powerful analysis method is applicable to all biexponential protein fluorescence decays.

Photophysical study of the Ca2+ indicator Fura-2 and the K+ indicator PBFI

The fluorescent indicators Fura-2 and PBFI are widely used for the determination of intracellular concentrations of Ca2+ and K+, respectively. To investigate the complex forming reaction between Fura-2 and Ca2+, and between PBFI and K+ in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surfaces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution: (1) for Fura-2: k01 equals 1.2 X 109 s-1, k21 equals 1.0 X 1011 M-1x-1, k02 equals 5.5 X 108s-1, k12 equals 2.2 X 107s-1 (2) for PBFI: k01 equals 1.1 X 109s-1, k21 equals 2.7 X 108M-1s-1, k02 equals 1.8 X 109s-1, k12 equals 1.4 X 109s-1 k01 and k02 denote the deactivation rate constants of the free and bound forms of the indicator, respectively k21 represents the bimolecular rate constant of binding of the cation by the indicator whereas k12 is the rate constant of dissociation of the cation:indicator complex. For both probes the effect of the excited-state reaction can be neglected in the determination of Kd and/or the ion concentration.

Compartmental analysis of the fluorescence decay surface of intramolecular two-state excited-state processes with added quencher

The fluorescence decay analysis of intramolecular two-state excited-state processes with added quencher is discussed in terms of compartments. The kinetic equations specifying the fluorescence decay and the time-course of the two excited-state species concentrations are expressed in terms of the rate constants and the spectroscopic parameters b1 and c1. b1 and c1 are respectively the relative absorbance and the normalized spectral emission weighting factor of species 1. The report investigates what has to be known beforehand to determine all relevant parameters. The results of this identifiability study indicate that the following conditions have to be satisfied in order to make an intramolecular two-state excited-state system with added quencher identifiable. First, at least three different quencher concentrations must be used. Second, the two rate constants of quenching must be different. Third, at least one parameter must be known. This parameter can be (1) one rate constant which is not a rate constant of quenching, (2) one b1 value or, (3) one c1 value. In each of these cases an alternative set of system parameters is mathematically possible. A unique solution is guaranteed when the fluorescence decays of a quenched model compound are included in the compartmental analysis.

Investigation of ground state interaction through fluorescence decay surfaces of excited state reactions

fluorescence decay surfaces of excited state reactions can be globally analyzed directly in terms of reaction rates and species associated spectra [Beechem et al., Chem. Phys. Letters 120 (1985) 466]. The identifiability of two-state excited state reactions has been investigated for properly normalized decay curves assuming that the ratio of the ground state absorbances is known [Ameloot et al, Chem. Phys. Letters 129 [1986] 21 1]. It is demonstrated in this paper that the condition of proper normalization is not always required. In addition, it is shown that the ratio of the absorbances of the species in the ground state can be obtained from fluorescence decay surfaces. The required experimental design is indicated.