Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related... In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectros - copy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later. FCS can be used for monitoring reaction kinetics, based on fluctuations at thermodynamic equilibrium, rather than on relaxation measurements following perturbations. In this review, we more specifically discuss FCS measurements on photodynamic, electronic state transi - tions in fluorophore molecules, and on proton exchange dynamics in solution and on biomembranes. In the latter case, FCS measurements have proven capable of casting new light on the mechanisms of proton exchange at biological membranes, of central importance to bioenergetics and signal transduction. Finally, we describe the transient-state (TRAST) spectroscopy/ imaging technique, sharing features with both relaxation (T-jump) and equilibrium fluctuation (FCS) techniques. TRAST is broadly applicable for cellular and molecular studies, and we briefly outline how TRAST can provide unique information from fluorophore blinking kinetics, reflecting e.g., cellular metabolism, rare molecular encounters, and molecular stoichiometries. Keywords T-jump · Fluctuations · Fluorescence correlation spectroscopy · Proton exchange · Photodynamics · Triplet state T‑jump and relaxation kinetics fields. A variety of methods have been developed applying such pulses to observe the relaxation of a chemical equilib- In 1967, Manfred Eigen, together with Ronald G.W. Norrish rium from one state into another one (Eigen and de Maeyer and George Porter, were awarded the Nobel Prize in Chem- 1963). The Stockholm story in this field can be said to have istry, for their “studies of extremely fast chemical reactions, begun when Rudolf Rigler came to Göttingen to work with effected by disturbing the equilibrium by means of very short Manfred Eigen on a temperature-jump (T-jump) apparatus. pulses of energy”. In the developed relaxation methods, chem- In this apparatus, fast heat generation could be accomplished ical equilibria can be disturbed by intensive pulses, chang- by fast capacitor discharge, and subsequent observation of ing physical quantities like temperature, pressure, or electric changes in light absorption compared to the equilibrium state was then available. In these absorption measurements, the difference in transmitted light was measured at very high light Special Issue: Chemical Kinetics, Biological Mechanisms and intensities in order to increase the signal-to-noise ratios with Molecular Evolution. detectors allowing dynode switching (Rabl 1973). With his experience in fluorescence spectroscopy Rigler * Jerker Widengren jerker@biomolphysics.kth.se (1969) started to develop a T-jump machine with fluores - cence detection. In order to make fluorescence measure - Department of Medical Biochemistry and Biophysics, ments a reality it was necessary to increase the total intensity Karolinska Institute, Stockholm, Sweden of the emitted fluorescence. The construction of the fluo - Experimental Biomolecular Physics/ Department of Applied rescence T-jump cell with the high aperture “fish eyes” was Physics, Royal Institute of Technology (KTH), Stockholm, of key importance for providing adequate signal intensities. Sweden Vol.:(0123456789) 1 3 480 European Biophysics Journal (2018) 47:479–492 It was produced in the outstanding workshop of the Max- Baldwin, a previous postdoc of Manfred Eigen in Göttingen. In Planck Institute for Biophysical Chemistry, by the master this letter, Elson told Rigler about the results of their paper on mechanic Wolfgang Simm. FCS, which was published in 1972 (Magde et al. 1972). At that The first working fluorescence T-jump machine (Rigler time, Ehrenberg and Rigler (1972) were involved in presenting et  al. 1974) became quite popular and its reputation also for the first time a coherent description of the theory of rota- attracted scientists outside of Göttingen. One day, Jean tional motion in the excited state using pulsed excitation. Ehren- Pierre Changeux from the Institut Pasteur in Paris arrived berg and Rigler (1974) transposed this problem and its theory with a bag of isolated nicotinic acetyl choline receptor, to be into the observation of fluctuations between the ground and studied with the new fluorescence T-jump machine. How - excited state of fluorescent molecules, providing the description ever, the ensuing experiments took an unexpected end. Due of excitation anti-bunching followed by rotational relaxation of to un-controlled conductivity in the solution of the receptor the fluorescent species. the electric pulse energy was dissipated fully into the meas- The theory and first experimental realizations of the FCS urement cavity and the valuable T-jump cell was destroyed. technique were thus in this way introduced during the years This moment of despair however led into new ideas for how 1972–1974 (Magde et al. 1972, 1974; Ehrenberg and Rigler to retrieve the information sought from the T-jump meas- 1974; Elson and Magde 1974). Although showing great poten- urements. Thus this at the time unfortunate happening gave tial, the applicability of FCS was however strongly reduced at rise to the idea of using instead fluctuations in the equilib - this time due to methodological constraints. In the measurements rium state, and coupled fluctuations in the optical signals, (Magde et al. 1972), the excitation beam was focused in a sam- to follow the reaction. Together with Leo de Mayer and ple cell, and the fluorescence collected by a parabolic reflector. Klaus Gnädig, the first experiments were undertaken dur - High background intensities, dominated by Raman scattering of ing 1968/1969, observing the association kinetics of acridin the water molecules, and proportional to the size of the relatively dyes and DNA for which data were available from T-jump large (> pL) detection volume, made it impossible to reduce the measurements (Ramstein et al. 1980). fluorophore concentrations without the background dominat- ing over the fluorescence signal. As a consequence, the aver - age number of fluorescent molecules in the detection volume Fluorescence correlation spectroscopy (FCS) had to be high, leading to lower relative fluorescence intensity fluctuations. Long measurement times were therefore required Pioneer work in the 1970s to distinguish and analyze these fluctuations, in turn imposing strict requirements on stability and absence of systematic noise The first attempts to use fluorescence as the readout in fluctua- in the optical and electronic parts of the instrumentation. The tion experiments illustrated that such analyses of systems under limited instrumental stability over long times, in combination thermodynamic equilibrium can oe ff r an attractive alternative with long time-range photochemical degradation, restricted the to relaxation measurements, studying the response in a sample possible applications of FCS. Since FCS relies on the ability following some external perturbation. This general concept of to detect and analyze fluctuations in the detected fluorescence fluctuation analysis was introduced already more than 100 years signal stemming from dynamic events of single molecules, a ago (Svedberg 1911; von Schmolukowski 1914; Chandrasekhar major figure-of-merit for FCS measurements is the number of 1943) and over the years several techniques have been developed fluorescence photons that can be detected per molecule and time monitoring fluctuations in the number of particles or molecules of (the fluorescence brightness) (Koppel 1974). Consequently, high a specic fi type within a x fi ed sample volume. Well-known exam - background light levels, low detection quantum yields, as well ples include the dynamic light scattering technique, exploiting the as the low capacity of the computers to analyze the fluctuation light scattering intensity from the particles of interest (Schaefer data, limited the applicability of FCS measurements at the time. 1973; Berne and Pecora 1975), and the voltage clamp approach (Hodgkin et al. 1949), where fluctuation analysis of electrical Breakthrough in the 1990s currents over sections of cellular or artificial membranes is per - formed (with the later-developed patch clamp technique (Neher In the 1990s, more than 15 years after its first demonstra- and Sakmann 1976) as its single-molecule counterpart). tion, the prerequisites for FCS measurements had greatly Fluorescence correlation spectroscopy (FCS) thus belongs to improved. Detection of individual fluorophore molecules, an established category of fluctuation spectroscopy techniques, first in solid crystals under cryo-temperatures by absorption but with clear advantages coming with the use of the highly (Moerner and Kador 1989) and then by fluorescence (Orrit sensitive and specific fluorescence intensity signal as the fluc- and Bernard 1990), led further to single-molecule detection tuating quantity. When Rudolf Rigler returned to the Karolinska in aqueous solution under room temperature (Shera et al. Institute in 1970 to build up his own group, he received a letter 1990). At the same time, the introduction of small, diffraction- from Elliot Elson, who learned relaxation kinetics from Robert limited observation volumes in FCS measurements, confocal 1 3 European Biophysics Journal (2018) 47:479–492 481 epi-illumination, highly sensitive avalanche photodiodes for fluorophores with known diffusion coefficients, D for the fluorescence detection and very selective band-pass filters to fluorescent species studied can be determined. Equation  2 discriminate the uo fl rescence from the background, made it is based on the assumption that F(t) can be considered a possible to improve signal-to-background ratios in FCS-meas- stationary and ergodic process (essentially that its average urements by several orders of magnitude (Rigler and Widen- and variance is constant over time, and that the average of gren 1990; Rigler et al. 1993). This development formed the F(t) from one or a few fluorescent molecules recorded over basis for the FCS measurements as they are performed today. a longer time should be the same as an instant average of In an FCS measurement, in its most simple realization, very many fluorescent molecules). Further assumptions are fluorescence intensity fluctuations arise from translational that the FCS measurement is performed at equilibrium in diffusion, as the fluorescent molecules are diffusing into and an ideal solution, with no photobleaching and in a sample out of a focused laser beam in an open confocal detection volume ≫ than the confocal detection volume. volume (Fig. 1a). For fluorescent molecules with a concen- tration, c(r ̄, t) , the detected fluorescence intensity can then FCS for monitoring of reaction kinetics be written as (Rigler et al. 1993; Widengren et al. 1995): FCS is not limited to analyses of the number and diffusion 𝜎 I (r̄) exc F(t)= 𝛷 𝛷 CEF(r̄)k c(r̄, t)dV properties of fluorescent molecules. Even for a standard FCS F D 10 𝜎 I (r̄)+ k exc 10 instrument with a single-point, stationary detection volume, (1) a wide range of processes can be studied, spanning a time = 𝛷 𝛷 W(r̄)c(r̄, t)dV. F D range from sub-nanoseconds to seconds. In principle, any Here, Ф , σ and k denote the f luorescence quantum process at equilibrium conditions, which reflects itself as a F 10 yield, excitation cross section, and deactivation rate of change of the detected fluorescence F (t), can be measured, the fluorescent molecules. Ф and CEF(r ̄) signify the given that it occurs within the dwell time of the fluorescent detection quantum yield and the collection efficiency molecules in the detection volume (  ≈  ∕4D ). Cor rela- function of the instrument. I (r ̄) denotes the excitation tion analyses can also be performed on fluorescence intensi- exc intensity of the laser. W(r ̄) is the molecular detection ties recorded in e.g., different spectral channels [fluorescence efficiency. cross-correlation spectroscopy, FCCS (Ricka and Binkert From the fluctuations in F (t), denoted δF(t), informa- 1989; Schwille et al. 1997)], spatial locations [image cor- tion can be retrieved about the translational diffusion coef- relation spectroscopy, ICS (Brinkmeier et al. 1999; Petersen ficients, D, and the average number of molecules, N, resid- et al. 1993; Digman et al. 2005)], or based on fluorescence ing simultaneously in the detection volume. Assuming that lifetime changes [fluorescence lifetime correlation spectros- W(r ̄) has a Gaussian distribution is both the radial and copy, FLCS (Benda et al. 2006)]. It is beyond the scope of axial dimensions, and in the absence of any other kinetic this review to discuss the full range of molecular dynamic process than translational diffusion affecting the fluores- processes that can be studied by FCS. In this review, we cent molecules, the time-dependent normalized intensity will rather discuss how FCS can be used to study reaction autocorrelation function (ACF) can be written (Magde kinetics at thermodynamic equilibrium, as an alternative to et al. 1972, 1974): relaxation experiments, for reactions which generate changes < F(t)F(t + 𝜏 ) > < [< F > +𝛿 F(t)][< F > +𝛿 F(t + 𝜏 )] > G(𝜏 )= = 2 2 < F > < F > (2) <𝛿 F(t)𝛿 F(t + 𝜏 ) > 1 1 1 = F(t) is stationary = + { } + 1 =   1. 2 2 2 < F > N 1 + 4D𝜏 𝜔 1 + 4D𝜏 𝜔 1 2 G(τ) gives a measure of how the fluorescence intensity in the fluorescence brightness, Q , of the molecules. In its detected at a certain time, F(t), is related to that detected at easiest realization, as depicted in Fig. 1a, the (reversible) a correlation time, τ, later, i.e., to F(t + τ). In Eq. 2, brackets reaction to be studied involves one fluorescent species, takes denote average over the measurement time, which should place on a time scale much faster than the passage times of be long enough to secure convergence of G(τ). ω and ω the molecules through the detection volume, and switches 1 2 signify the distances from the center of the detection volume the fluorescence completely on and off, yielding high con- in the radial and axial dimensions, respectively, at which trast fluorescence intensity fluctuations, superimposed on W(r ̄) has decreased by a factor of e . With knowledge about those due to translational diffusion of the molecules in and ω and ω , e.g., from FCS calibration measurements with out of the detection volume. The ACF recorded from such 1 2 1 3 482 European Biophysics Journal (2018) 47:479–492 Fig. 1 Monitoring of reaction kinetics at thermodynamic equi- to and from the dark, long-lived, triplet state (T ). In the ACF of the librium by FCS. a Principal drawing of the type of reactions that recorded fluorescence intensity (Eq.  2), two relaxation processes will can be studied. Fluorescent molecules undergoing Brownian diffu- show up, as described by Eqs. 3 and 4. With the full amplitude of the sion through a confocal detection volume (lower left) also undergo a ACF normalized to unity, the amplitude B corresponds to the aver- reaction, switching the fluorescence of the molecules on and off with age fraction of the fluorescent molecules in the detection volume rates k and k (upper part). This reaction takes place on a faster which are in the triplet state. τ denotes the relaxation time for the 1 −1 T time scale than the diffusion of the fluorescent molecules into and out singlet–triplet state transition and τ is related to the average dwell of the detection volume. As a result, fluctuations in the fluorescence time of the fluorescent molecules in the detection volume. c Exam- intensity, F(t), can be detected, with the faster time-scale fluctua- ples of FCS curves, recorded from the fluorophore Rh6G in water, tions caused by the reaction superimposed on the slower fluctuations with different excitation intensities, I , applied. Increasing I , leads exc exc caused by diffusion (lower right). b Principal drawing of an FCS to higher B and shorter τ values in the FCS curves. From the I - T exc curve, recorded from fluorescent molecules undergoing fluorescence dependence of B and τ , the transition rates to and from T can be T 1 on–off blinking in the microsecond time-range, caused by transitions determined a sample (Fig. 1b) can then be separated into two factors. chemical relaxation time(s) and/or the diffusion coefficients The first factor, G (τ), depends on the transport properties of all fluorescent species can be considered equal (Palmer (diffusion or flow) of the molecules and the second, R (τ), and Thompson 1987). For a fluorescent species studied by depends only on the reaction rate constants (Palmer and FCS, and undergoing chemical reactions, the second factor Thompson 1987): can more generally be expressed as: G()= G ()R()+ 1. (3) Q Q X (𝜏 ) i j ij i,j=1 R(𝜏 )= . (5) In this particular case, the separate reaction-related factor ∑ Q C i=1 i can be written as: R()= 1 − B + B exp(−k ) (4) Here, Q is the u fl orescence brightness coec ffi ient of state 1 − B i and X (τ) is the solution to the following set of differential ij where B denotes the average fraction of the fluorescent mol- equations and initial conditions: ecules in the detection volume which are in the dark state. k = 1∕ is the relaxation rate of the reaction, and is given B B dX (𝜏 )∕d𝜏 = T X (𝜏 ) by the sum of the fluorescence on and off rates, k and k . In ik ij jk 1 −1 (6) j=1 a more general case, the separation of G(τ) into two factors as in Eq. 3 is possible for reactions in which the diffusion of X (0)= C 𝛿 , ik i ik the reactants and product molecules is much slower than the 1 3 European Biophysics Journal (2018) 47:479–492 483 where δ  = 1 if i = k, and δ  = 0 if i ≠ k. X (τ) describes photo-induced dark states, such as triplet, photo-isomerized, ik ik ij the probability of finding a molecule in state j at time τ, and photo-oxidized states reduce the fluorescence brightness given that it was in state i at time 0. M is the number of of the fluorophore molecules studied, a major figure-of-merit species participating in the chemical reaction, and T rep- for FCS measurements (Koppel 1974). Some of these states ij resents the corresponding matrix of the kinetic rate coef- may also act as precursor states for photobleaching, and the ficients. In Eq.  5, it can be noted that in FCS measurements, blinking caused by these transitions may cause problems in each different species analyzed is weighted by the square FCS and in single-molecule experiments, in that they may of its fluorescence brightness. For an ACF recorded from shadow other molecular processes of interest, taking place a sample containing several different fluorescent species, it in the same time range. On the other side, based on the gen- can therefore be strongly misleading to interpret the inverse eral approach to study reaction kinetics via changes in the amplitude of the ACF as the true average number of fluores- fluorescence brightness Q (Eqs. 3–7), FCS has also turned cent molecules (1/G(0) = N in Eq. 2). More generally, for M out to be a very suitable tool to study these transitions. Fig- different species, with brightnesses Q and average numbers ure  1c shows FCS curves recorded from the fluorophore N (and disregarding fluorescence anti-bunching) (Magde rhodamine 6G (Rh6G) in air-saturated aqueous solution, et al. 1974; Widengren and Mets 2001): and how the average population of the dark, lowest triplet state of Rh6G, given by the relative amplitude B, as well as � � ∑ 2 N Q the singlet–triplet state relaxation time, τ , corresponding i i i=1 1∕G(0)= . (7) 2 to τ in Eq. 4, vary with the excitation irradiance within the N Q i=1 i confocal detection volume in the FCS experiment. From the excitation irradiance dependence observed in the FCS curves When applicable, it is very convenient to treat the kinet- (a so-called FCS power series), the transition rate constants ics of a chemical reaction separately from the translational to and from T can be determined in a straightforward man- diffusion in the fluctuation analysis, as given by Eq.  2. This ner (Widengren et al. 1995). Similarly, a whole range of treatment applies to a rather broad range of chemical reac- photo-induced dark transient states can be kinetically char- tions such as inter- or intra-molecular dynamics, influenced acterized, including photo-ionized (Widengren et al. 1997) by fluorescence quenching (Bonnet et al. 1998; Chattopad- and photo-isomerized states (Widengren and Schwille 2000; hyay et al. 2002). Moreover, for a reaction that under certain Widengren and Seidel 2000), as well as the influence of conditions does not fulfill the criteria, it is sometimes pos- chemical additives and environmental conditions on these sible to modify the conditions. For instance, the dwell times transitions (Widengren et al. 1995, 1997, 2007; Widengren can be retarded with respect to the chemical relaxation times and Schwille 2000). Likewise, the overall photostabilities of by expanding the detection volume, or the reactions under fluorophores under excitation conditions required for FCS study can be speeded up, e.g., by using higher concentrations and other forms of ultrasensitive fluorescence spectroscopy of un-labelled reactants (Widengren et al. 1995, 1999, 2007; and imaging can be studied (Widengren et al. 2007; Egg- Widengren and Rigler 1997). eling et al. 1998; van den Berg et al. 2001). Interestingly, in In the sections below, we will discuss two realizations of such studies, compounds known in the fluorescence spec- this FCS approach to monitor reaction kinetics, generating troscopy field as fluorescence quenchers, such as potas- changes in Q of the fluorescent species studied. First, it will sium iodide, may under excitation conditions for FCS and be shown how FCS can be used to monitor a range of photo- single-molecule fluorescence spectroscopy turn out to act induced transitions in fluorophores. Second, how monitoring as anti-fading compounds (Chmyrov et al. 2010). Similar of ion-sensitive fluorophores by FCS offers an alternative transitions as in organic fluorophore molecules can also be way of monitoring proton exchange kinetics and how this found in green fluorescent proteins (GFPs). In GFPs, the approach can be used to investigate protonation kinetics at transitions are however far less influenced by environmental biological membranes. parameters, since the fluorescently active unit is located in the inner part of the GFPs, shielded from the surroundings FCS for photodynamic characterization by a tight beta sheet barrel structure (Widengren et al. 1999; of fluorescent species Haupts et al. 1998). Compared to e.g., transient-state absorption/flash pho - Photophysical properties of the fluorescent molecules under tolysis (Van Amerongen and Van Grondelle 1995; Korobov study set the fundamental limits for the overall perfor- and Chibisov 1978) and phosphorescence studies (Jovin and mance of virtually all forms of fluorescence spectroscopy Vaz 1989) the FCS approach offers some advantages. For and imaging, where high sensitivities, read-out rates and/ triplet state studies, it uses the highly sensitive fluorescence or resolutions are required. Similarly, these properties also readout to monitor the triplet state, rather than the faint, set the ultimate limits for FCS measurements. Population of easily quenched, phosphorescence signal from the triplet 1 3 484 European Biophysics Journal (2018) 47:479–492 state itself. Thereby, a favorable combination of a high sig- Naturally, if there is no difference in brightness of the nal level (given by the readout of fluorescence photons) and fluorophore upon protonation (Q  = 1), B will be zero and an outstanding environmental sensitivity (given by the long no relaxation can be observed in the ACFs. For many pH- lifetimes of the transient states) can be obtained. Quenching sensitive fluorophores Q is small (1–2%), and then only mar- of the triplet states of the fluorophores by oxygen or other ginally affects the relaxation amplitudes. With knowledge compounds will not ruin the read-out signal. Compared to of Q it can also be properly corrected for. Alternatively, flash photolysis the experimental realization is relatively higher-order correlation analyses of δF(t) can be applied simple and more easily applicable to a broader range of to resolve Q, as recently demonstrated (Abdollah-Nia et al. samples. 2017). As an additional alternative, ratio-metric pH-sensitive dyes can be used, for which the excitation and/or the emis- FCS for studies of proton exchange dynamics sion spectrum changes upon protonation. For such dyes, in solution and on biomembranes two or several Q values can be included, effective for dif- ferent laser excitation wavelengths and/or detection within Monitoring blinking rates and the fractions of fluorescent different wavelength bands. If the excitation/emission in and non-fluorescent fluorophores by FCS, as outlined above one wavelength band increases upon protonation of the dye, (Eqs. 3–7), can also be applied to characterize ion exchange to it normally decreases in another wavelength band. FCCS and from ion-sensitive fluorophores at thermodynamic equi- measurements, recording the cross-correlation of intensities librium (Widengren and Rigler 1997; Widengren et al. 1999). recorded at die ff rent excitation and/or emission wavelengths, In such measurements, taking as an example a pH-sensitive then typically display negative B relaxation amplitudes dye in a buffered aqueous solution, which is non-fluorescent in (Persson et al. 2009). its protonated form, the recorded FCS curves can be described The FCS-based approach for ion exchange studies can by Eqs. 3 and 4 (Fig. 1b). The amplitude B then corresponds offer selective advantages over other techniques for meas- to the fraction of non-fluorescent protonated fluorophores, uring local ion concentrations, and in particular exchange and the relaxation rate k to the sum of the protonation and kinetics of ions on a local scale. We have exploited these de-protonation rates of the fluorophores (Fig.  2a, c). With advantages in a series of papers to study proton exchange at −pK knowledge of the pK (and K = 10 ) of the fluorophore, biological membranes (Brändén et al. 2006; Öjemyr et al. a a + −pH the local pH (and H = 10 ) can then be determined from 2009; Sanden et al. 2010; Xu et al. 2016; Sjöholm et al. the relaxation amplitude B in the ACFs (Eq. 4). If the fluoro- 2017). Proton gradients across biological membranes act phore becomes non-fluorescent upon protonation (Widengren as driving forces for many energy-consuming cellular pro- and Rigler 1997; Widengren et al. 1999): cesses, not the least ATP synthesis by ATP synthase in the mitochondria. To generate the gradients, proton transport at and across membranes is required and involves a series of B = , (8a) membrane-spanning proteins in the inner membranes of the [H ] + K mitochondria. The underlying mechanisms for this proton transport has been subject to extensive research (Medvedev if it is fluorescent in the protonated form and becomes non- fluorescent upon de-protonation: and Stuchebrukhov 2011), but is nonetheless not completely understood. One of the key questions concerns the nature of B = . coupling between proton generators, such as cytochrome C (8b) [H ] + K oxidase (CytcO), pumping protons across the membrane, and proton consumers, such as ATP synthase, using the pro- Moreover, in a buffered aqueous solution, k directly ton gradients across the membrane to drive the ATP synthe- reflects and depends linearly on the local buffer concentra- sis (Medvedev and Stuchebrukhov 2011). Both the outlet of tion (Widengren et al. 1999). the generator and inlet of the consumer proteins are located Dyes used for FCS studies of proton (or ion exchange) do on the same side of the membrane, but the proteins are spa- not have to be completely non-fluorescent upon protonation tially separated. A major question is how the generated pro- (or de-protonation). However, the relaxation amplitude B teins get to the consumers before they are dissociated from will get smaller the less distinct the brightness difference the membrane surface. A major experimental technique for upon protonation is. Introducing Q as the relative brightness these molecular proton exchange studies is the laser-induced of the dimmer form of the dye, protonated or de-protonated, proton pulse approach (Gutman 1986) and other relaxation the amplitudes in Eqs. 8a and 8b will change into (Widen- techniques. Basically, a light flash directed to one side of gren and Schwille 2000): a membrane releases protons from caged compounds in B(1 − Q) the membrane, and the response from pH-sensitive fluoro- B = . (9) 1 + Q (1 − B) phores is then monitored on the other side of the membrane. 1 3 European Biophysics Journal (2018) 47:479–492 485 Fig. 2 Protonation kinetics in solution and at biological mem- into and out of the detection volume of the FCS instrument. d Exam- branes, studied by FCS. Proton exchange to and from pH-sensitive ples of FCS curves recorded from the pH-sensitive fluorophore Fluo- fluorophores results in fluorescence blinking, which can be analyzed rescein in water at different pH. Three relaxations can be observed in by FCS as outlined in Fig.  1, and by use of Eq.  4. For fluorophores the FCS curves: singlet–triplet state relaxation in the µs time range free in solution (a), FCS measurements show that the protonation on- (with amplitude T, marked red, reflecting the fraction of fluorophores rate is two orders of magnitude slower than when the fluorophores being in the triplet state while passing the detection volume), proton are located close to a lipid membrane of a small unilamellar vesicle exchange in a time range of 1–100 µs (with the amplitude P, marked (SUV) (b). Apart from direct exchange of protons between the fluo- blue, related to the fraction of fluorophores in the protonated, close-to rophore and the bulk (red arrows), the membrane–water interface also non-fluorescent state of Fluorescein), and diffusion relaxation beyond exchange protons with the bulk (thin black arrows), and protons at the 100 µs (with the detection volume expanded well beyond the diffrac- surface may migrate along the surface (thick black arrows) and reach tion limit in this series of measurements). e Illustration of a mem- the fluorophore, before dissociating from the membrane surface into brane acting as a proton collecting antenna, having a radius given by the bulk. c The proton exchange to and from fluorophores, located at how far a proton can diffuse along the surface before the probability the membranes of SUVs, were measured on SUVs freely diffusing to find it on the surface equals the average value on the surface The FCS approach above can offer complementary points measured protonation dynamics reflect the conditions in the of view on these relaxation techniques. In particular, in media the protons pass on their way from light absorbing FCS measurements protons associating to and dissociating proton emitters to the pH-sensitive fluorophores. from pH-sensitive fluorophores are observed at equilibrium Based on these selective advantages of the FCS approach conditions. No perturbation into some, often strongly un- we have investigated the principal role of biological mem- physiological, initial condition is required. Moreover, while branes for proton uptake of membrane incorporated proteins protonation kinetics measurements by proton pulse tech- (Fig. 2a–e). Interestingly, we found that the protonation rate niques require 1–5% of the lipids in the membranes to be of the pH-sensitive dye fluorescein increased by two orders 11 13 −1 −1 labeled (Serowy et al. 2003), FCS is typically performed at of magnitude (from ~ 10 to ~ 10 M  s ) when labeled about 1000-fold lower concentrations. Thereby, the influence to a lipid in a membrane of a small unilamellar vesicle of buffering effects due to ions binding to the fluorophores (SUV, Fig. 2b), compared to when free in solution (Fig. 2a) themselves, or to any light absorbing proton emitter, can (Brändén et al. 2006). In comparison, FCS measurements be avoided. Finally, the proton exchange seen in the FCS in membranes with negatively charged head-groups instead measurements reflects the conditions in the immediate sur - of zwitterionic head-groups only changed the protonation roundings of individual fluorescent probes, in contrast to rate by a factor of two (Brändén et al. 2006). This finding proton pulse measurements (Serowy et al. 2003), where the gives direct evidence that the membrane-water interface can 1 3 486 European Biophysics Journal (2018) 47:479–492 Fig. 3 Illustration of how fluorophore photodynamics is moni- imaging. The beam of a continuous wave (CW) laser is passed tored in transient state (TRAST) spectroscopy/imaging. a Elec- through an acousto-optical modulator, generating rectangular excita- tronic state model for a fluorophore, comprising the ground and tion pulse trains with varying pulse durations, w. These are fed into excited singlet states (S and S ), the lowest triplet state (T ) and a a microscope and via its objective onto the sample. The fluorescence 0 1 1 photo-oxidized state ( R ). Following onset of constant excitation, generated by the excitation pulse trains is collected by the same τ , τ and τ denote the relaxation times for the population of the objective and recorded by a camera in the image plane. By recording AB T R singlet states, T and R , respectively . b For the same fluorophore, images of the average fluorescence, F , for different duration, w, of 1 W subject to rectangular excitation pulses, the graph illustrates how the excitation pulses, TRAST curves can be generated within regions the average fluorescence, F , can vary with the duration, w, of the of interest in the image, and TRAST images can be obtained, map- excitation pulses. This so-called TRAST curve reflects the popula- ping e.g., τ and τ , or the underlying transition rates to and from T T R 1 tion build-up of the electronic states in (a) upon onset of excitation, and R and the relaxation times τ , τ and τ . c Principle setup for TRAST AB T R act as a proton collecting antennae (PCA), with an area far et  al. 2006), and protonation dynamics measurements by exceeding the size (or the physical cross section for protona- FCS on lipid nanodiscs, with diameters around 10 nm, show tion) of the fluorophore itself. In follow-up studies, the same that the protonation on-rate of fluorescein remains as high enhancement of more than two orders of magnitude in the as in SUVs (diameter of ~ 30 to 100 nm), even when incor- protonation on-rate was also found when the fluorophore porated into the small membrane areas of the nanodiscs. label was placed on the proton generator CytcO, when free A reasonable interpretation is that the density of protonat- in solution, compared to when in the membrane of an SUV able groups bound to the membrane surface can sustain a (Öjemyr et al. 2009). The PCA effect is less observable at higher local proton concentration at the surface than in the lower pH (< 7), when the proton association rate via the bulk solution, and a higher proton exchange rate of a surface membrane becomes comparable to that of direct protona- group (Fluorescein) with the surface than with the bulk solu- tion of the fluorophore from the bulk solution (Fig.  2, left tion (even if the diffusion coefficient of protons in bulk water bottom) (Sanden et al. 2010). The radius of the PCA, R , is considerably higher than along the membrane) (Brändén PCA is related to how far a proton generated at the membrane et al. 2006). The dependence of the protonation on-rate on surface can diffuse from its site of generation before the the buffer concentration in the bulk water and Monte-Carlo probability to find a proton equals the average value on the simulations of the proton exchange to and from a fluoro- surface (Gutman and Nachliel 1995). From proton pulse phore at a nanodisc of different size further confirmed that experiments, the R of lipid membranes has been deter-both R and D along lipid membranes are 2–3 orders PCA PCA S mined to be as large as tens of micrometers, and the diffusion of magnitude smaller, when measured by FCS (Xu et al. coefficients of the protons, D , along the membrane-water 2016), as compared to proton pulse data (Medvedev and −5 2 interfaces as high as 5 × 10  cm /s (Medvedev and Stuche- Stuchebrukhov 2011). The underlying reason for this quite brukhov 2011; Serowy et al. 2003). In contrast, from FCS significant difference can likely be attributed to the fact that experiments D is two orders of magnitude lower (Brändén FCS measures the very local proton exchange kinetics, in 1 3 European Biophysics Journal (2018) 47:479–492 487 the very surroundings of individual fluorophores (nanom- versatile means to analyze photodynamic events, this limits eter scale), while proton pulse measurements monitor the the application range and throughput of FCS measurements, propagation of proton pulses over distances beyond millim- and makes parallel readouts and imaging difficult. eters. The longer distance measurements capture an aver- To overcome these limitations, we have introduced age migration behavior of the protons, including “hopping” an approach, so-called transient-state (TRAST) imaging between the lipid head group region of the membrane, the (Sanden et al. 2007, 2008), where the fluorescent sample membrane-water interface and the bulk water. In contrast, is subject to a modulated excitation (Fig. 3). Depending FCS measurements capture the local exchange dynamics at on the excitation pulse train characteristics (e.g., pulse the lipid head group region and membrane-water interface duration, separation and height) long-lived photo-induced only. These results thus emphasizes the ability of FCS to transient states (e.g., triplet, photo-isomerized and photo- monitor the proton exchange at the very local scale, and also ionized states) of the fluorophores in the sample will be provide experimental support to recent theoretical studies populated to different extents. Figures  3a, b illustrate how showing that proton diffusion along membrane surfaces is the fluorescence intensity varies with time after onset of time- and length-scale dependent (Wolf et al. 2014; Yamash- excitation, and how equilibration between the ground and ita and Voth 2010). the excited singlet states (S and S ) typically takes place 0 1 within nanoseconds after onset of excitation, between the singlet and triplet (T ) states within microseconds, and Transient‑state (TRAST) spectroscopy/ finally how a population of photo-oxidized (or –reduced) imaging to exploit the information content states ( R ) build up and equilibrate within milliseconds of fluorescence blinking kinetics after onset of excitation. Upon transient-state popula- tion build-up in the sample, the fluorescence intensity Concept for TRAST will drop in a corresponding manner. By systematically varying the excitation pulse train characteristics, e.g., by As discussed above, fluorophore blinking, caused by popula- use of an acousto-optical modulator (AOM), and regis- tion dynamics of photo-induced, long-lived, non-fluorescent tering how the plain time-averaged fluorescence intensity + − ̇ ̇ R R triplet (T ), photo-oxidized ( ), photo-reduced ( ) or changes with e.g., the pulse width (a so-called TRAST photo-isomerized states (Fig. 3a) is of central importance to curve, Fig. 3c) the population kinetics of the fluorophore all forms of fluorescence-based ultrasensitive and ultrahigh- transient states can then be retrieved. As with FCS, the resolution spectroscopy/imaging. While blinking has to be TRAST technique combines the high detection sensitivity suppressed in single-molecule fluorescence studies (reduces of the fluorescence readout with the high environmental brightness and obscures observation of other dynamic pro- sensitivity of the dark transient states, but does not rely cesses), blinking is an absolute prerequisite for all forms of on a high fluorescent brightness of the molecules studied, super-resolution microscopy [see e.g., (Eggeling et al. 2015) on a high time resolution, or on a certain concentration of and (Blom and Widengren 2017) for reviews]. Moreover, the sample. This makes TRAST imaging a broadly appli- −6 the long lifetimes of these non-fluorescent states, ~ 10 to cable approach, applicable e.g., on live cells, and allows −3 −9 10 s, compared to ~ 10 s for the fluorescence lifetime of transient states to be imaged by e.g., a standard CCD cam- the excited singlet state, make these states highly environ- era. For live cell studies by light microscopy in general, ment sensitive. These states thus represent a whole set of photo-toxic and photo-sensitizing effects from both endog- additional parameters, which in a very sensitive manner can enous and exogenous (fluorophore) compounds have to be reflect microenvironments as well as biomolecular dynam- considered. These effects depend on several parameters, ics and interactions of fluorescent molecules (Widengren such as the concentration of these compounds in the cells, 2010). This possibility seems not to have been fully realized the wavelength of the irradiating light, the overall excita- in the fluorescence field until more recently (Weidemann tion light dose, and the distribution of the light dose in et al. 2014; Kawai et al. 2015; Mahoney et al. 2015; Bag and time (Magidson and Khodjakov 2013; Tinevez et al. 2012; Wohland 2014; Querard et al. 2015). Logg et al. 2009; Schneckenburger et al. 2012). TRAST FCS-based monitoring of these states combines high imaging requires excitation irradiances high enough to detection sensitivity by the fluorescence readout, with high induce population of the transient states. However, using environmental sensitivity, given by the long lifetimes of high triplet yield dyes (Spielmann et al. 2014; Mücksch the transient states. However, FCS measurements require et  al. 2015), or dyes for isomerization  (Chmyrov et  al. fluorescent molecules with high brightness and in low con- 2015), high populations of dark transient states can be centrations, and highly sensitive detectors with high time reached with lower excitation intensities, and live cell resolution. Although FCS provides a very powerful and TRAST imaging can be performed with overall excitation 1 3 488 European Biophysics Journal (2018) 47:479–492 light doses almost an order of magnitude lower than esti- TRAST, examples of biomolecular and cellular mated maximum tolerable light doses for maintaining cell information that can be retrieved viability (100 J/cm at 514 nm irradiation) (Schnecken- burger et al. 2012; Wagner et al. 2010). Since TRAST does In Fig. 4, a few examples are given for how TRAST imag- not rely on fluctuation measurements of single molecules, ing can be applied. Figure 4a shows a fluorescence inten- molecular brightness is not a strongly limiting issue, and sity image (top) and a corresponding image (bottom) of the low fluorescence signals can be compensated by higher T -to-S rate. This rate is almost completely determined by 1 0 concentrations of fluorescent molecules in the sample, as quenching from dissolved molecular oxygen (Widengren an alternative to increased excitation irradiances. Thereby, et al. 1995; Spielmann et al. 2014). The latter image is gen- also relatively low brightness compounds, such as auto- erated by taking consecutive fluorescence intensity images fluorescent species (Hevekerl et al. 2016), can be studied. with different excitation pulse trains (pulse durations) Fig. 4 Examples of how TRAST imaging/spectroscopy can be number of independently blinking emitters on the diffusing units. applied. a Fluorescence (top) and TRAST (bottom) images (100 From the difference in the blinking amplitude in FCS and TRAST, ×  100  µm) of cultured cells from a breast cancer cell line (MCF-7), the absolute numbers of fluorophores per diffusing unit can be deter - loaded with the fluorophore EosinY. The TRAST image shows the mined. See main text for further information. c Model system used deactivation rate of the T state, k , obtained from TRAST curves to demonstrate how low-frequency collisional interactions can be 1 t extracted pixel-wise from multiple fluorescence intensity images, monitored via triplet state quenching. Quenching of the triplets states recorded with different excitation modulation. b Concept for determi- of Lissamine rhodamine B, labeled to DOPE lipids (DOPE-RH), nation of molecular stoichiometry based on FCS and TRAST meas- by lipids labeled with the triplet state quencher TEMPO (DOPC- urements. Because of the synchronous blinking of the fluorophores TEMPO) was studied in the membrane of DOPC SUVs. S denotes in a TRAST experiment, the fluctuation amplitude will not change the quenching radius of interaction (≈ 0.2 nm), and R (≈ 20 nm) the with several fluorophores on the diffusing units studied. In contrast, radius of the SUVs. See main text for further details in an FCS experiment, the blinking amplitude will decrease with the 1 3 European Biophysics Journal (2018) 47:479–492 489 applied. The variation of the fluorescence intensity from the Figure  4c illustrates the principal use of TRAST to excitation modulation (TRAST curves) can then be gener- monitor low-frequency collisional interactions. In a proof- ated in a pixel-wise manner. By fitting these curves to a of-principle study (Strömqvist et al. 2010), we monitored photodynamic model of the fluorophores (Fig.  3a), an image the quenching of long-lived triplet states of Lissamine Rho- of the oxygen-dependent T -to-S rate can be obtained. Live damine B dyes, labeled on the head group of lipids. Dye- 1 0 cell imaging with this approach allowed the local oxygen labeled lipids (DOPE-RH) were placed in the membranes of concentration within cells to be determined, and charac- small unilamellar vesicles (SUVs) with radius R ≈ 20 nm. teristic differences in oxygen consumption between nor - In the SUV membranes we also added varying amounts of mal and cancer cells to be detected (Spielmann et al. 2014; lipids (from 0.15% up to 8% of the lipids) labelled with the Mücksch et al. 2015). Such differences cannot be resolved triplet quencher TEMPO (DOPC-TEMPO). Both the S and by conventional fluorescence imaging parameters, but may T states can be quenched upon interaction between DOPE- be captured by room temperature phosphorescence (RTP). RH and DOPC-TEMPO in the SUV membranes (when Like TRAST, RTP benefits from long lifetimes, to probe within a radius of interaction, S, of a few Ångström to each subtle changes in environmental conditions (accessibili- other). However, T states have typically at least three orders ties of quenchers, polarities etc.), or to reveal structural and of magnitude longer lifetimes (µs-ms) than S states (ns), and dynamic information of biological macromolecules (Cioni thus a correspondingly longer time to be influenced by the and Strambini 2002). However, while TRAST is based on environment (or quencher collisions). As a result, quenching the readout of a strong fluorescence signal, the RTP signal of S , observed as a relative change of the fluorescence life- is weak and susceptible to dynamic quenching by oxygen time of a few percent, could only be observed for the higher and trace impurities. Moreover, while TRAST can be based DOPC-TEMPO concentrations, while quenching of T could on a broad range of different fluorophores and even auto- be observed also for the lowest DOPC-TEMPO concentra- fluorescent compounds, specific RTP probes are scarce and tions. In other words, interactions between dye-labeled and cannot easily be loaded into cells (Yu et al. 2017). In contrast triplet quencher-labeled molecules can be analyzed via the to RTP, TRAST is also not limited to imaging of triplet state triplet state population kinetics, at collisional frequencies parameters only. Also other transitions can be imaged, e.g., too low to be reflected as fluorescence intensity or lifetime viscosity-dependent trans–cis isomerization of lipophilic changes (Strömqvist et al. 2010). This illustrates how tran- cyanine dyes, providing images of the microfluidity of cel- sient state monitoring by TRAST (or FCS) can combine lular membranes (Chmyrov et al. 2015). With a different the environmental sensitivity of long-lived transient states, readout than in established fluorescence-based approaches with the detection sensitivity of the fluorescence signal, and based on anisotropy, polarity-sensitive dyes, or excimer retrieve information not within reach by conventional fluo- formation, this TRAST approach offers complementary rescence readouts. information and can capture different aspects of membrane microviscosity and fluidity of the cellular membranes. Figure 4b illustrates a concept to determine molecular Conclusions stoichiometries, by comparing the fluorescence fluctuations under continuous excitation using FCS, when all the fluo- Molecular relaxation and kinetics measurements have played rophores on a diffusing unit are blinking independently of a key role in reaching a fundamental understanding of a each other, with those occurring under square-pulsed excita- broad range of biological processes. In this review, we have tion using TRAST spectroscopy, when all fluorophores are given a historical view of how our research in the develop- blinking in a synchronized manner. Thereby, the number ment and use of fluorescence correlation spectroscopy and of fluorophores per molecule can be determined (Hevekerl related techniques for such measurements has its roots and and Widengren 2015). In the recorded FCS curves, the fast has further evolved from the pioneering work of Manfred relaxation amplitude, B, of a transient state, is lowered with Eigen and his colleagues and coworkers. Although now the number of independently blinking emitters, N, on a dif- 50 years have passed since Eigen, Norrish, and Porter were fusing unit by B/N. In contrast, in TRAST measurements awarded the Nobel Prize for their achievements in relaxa- the N emitters are blinking in synchrony due to the excita- tion kinetics measurements, novel molecular fluctuation and tion modulation, and the corresponding relaxation ampli- relaxation techniques are still under development. This field tude is still B. By dividing the TRAST amplitude with the is thus still not exhausted and will not be in many years to FCS amplitude N can thus be obtained directly. The FCS come. Biology is far too intricate. Mother Nature has left and TRAST measurements can be done consecutively in many riddles for us to solve, and not unlikely with molecular the same setup. 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Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

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Life Sciences; Biochemistry, general; Biological and Medical Physics, Biophysics; Cell Biology; Neurobiology; Membrane Biology; Nanotechnology
ISSN
0175-7571
eISSN
1432-1017
D.O.I.
10.1007/s00249-017-1271-1
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Abstract

In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectros - copy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later. FCS can be used for monitoring reaction kinetics, based on fluctuations at thermodynamic equilibrium, rather than on relaxation measurements following perturbations. In this review, we more specifically discuss FCS measurements on photodynamic, electronic state transi - tions in fluorophore molecules, and on proton exchange dynamics in solution and on biomembranes. In the latter case, FCS measurements have proven capable of casting new light on the mechanisms of proton exchange at biological membranes, of central importance to bioenergetics and signal transduction. Finally, we describe the transient-state (TRAST) spectroscopy/ imaging technique, sharing features with both relaxation (T-jump) and equilibrium fluctuation (FCS) techniques. TRAST is broadly applicable for cellular and molecular studies, and we briefly outline how TRAST can provide unique information from fluorophore blinking kinetics, reflecting e.g., cellular metabolism, rare molecular encounters, and molecular stoichiometries. Keywords T-jump · Fluctuations · Fluorescence correlation spectroscopy · Proton exchange · Photodynamics · Triplet state T‑jump and relaxation kinetics fields. A variety of methods have been developed applying such pulses to observe the relaxation of a chemical equilib- In 1967, Manfred Eigen, together with Ronald G.W. Norrish rium from one state into another one (Eigen and de Maeyer and George Porter, were awarded the Nobel Prize in Chem- 1963). The Stockholm story in this field can be said to have istry, for their “studies of extremely fast chemical reactions, begun when Rudolf Rigler came to Göttingen to work with effected by disturbing the equilibrium by means of very short Manfred Eigen on a temperature-jump (T-jump) apparatus. pulses of energy”. In the developed relaxation methods, chem- In this apparatus, fast heat generation could be accomplished ical equilibria can be disturbed by intensive pulses, chang- by fast capacitor discharge, and subsequent observation of ing physical quantities like temperature, pressure, or electric changes in light absorption compared to the equilibrium state was then available. In these absorption measurements, the difference in transmitted light was measured at very high light Special Issue: Chemical Kinetics, Biological Mechanisms and intensities in order to increase the signal-to-noise ratios with Molecular Evolution. detectors allowing dynode switching (Rabl 1973). With his experience in fluorescence spectroscopy Rigler * Jerker Widengren jerker@biomolphysics.kth.se (1969) started to develop a T-jump machine with fluores - cence detection. In order to make fluorescence measure - Department of Medical Biochemistry and Biophysics, ments a reality it was necessary to increase the total intensity Karolinska Institute, Stockholm, Sweden of the emitted fluorescence. The construction of the fluo - Experimental Biomolecular Physics/ Department of Applied rescence T-jump cell with the high aperture “fish eyes” was Physics, Royal Institute of Technology (KTH), Stockholm, of key importance for providing adequate signal intensities. Sweden Vol.:(0123456789) 1 3 480 European Biophysics Journal (2018) 47:479–492 It was produced in the outstanding workshop of the Max- Baldwin, a previous postdoc of Manfred Eigen in Göttingen. In Planck Institute for Biophysical Chemistry, by the master this letter, Elson told Rigler about the results of their paper on mechanic Wolfgang Simm. FCS, which was published in 1972 (Magde et al. 1972). At that The first working fluorescence T-jump machine (Rigler time, Ehrenberg and Rigler (1972) were involved in presenting et  al. 1974) became quite popular and its reputation also for the first time a coherent description of the theory of rota- attracted scientists outside of Göttingen. One day, Jean tional motion in the excited state using pulsed excitation. Ehren- Pierre Changeux from the Institut Pasteur in Paris arrived berg and Rigler (1974) transposed this problem and its theory with a bag of isolated nicotinic acetyl choline receptor, to be into the observation of fluctuations between the ground and studied with the new fluorescence T-jump machine. How - excited state of fluorescent molecules, providing the description ever, the ensuing experiments took an unexpected end. Due of excitation anti-bunching followed by rotational relaxation of to un-controlled conductivity in the solution of the receptor the fluorescent species. the electric pulse energy was dissipated fully into the meas- The theory and first experimental realizations of the FCS urement cavity and the valuable T-jump cell was destroyed. technique were thus in this way introduced during the years This moment of despair however led into new ideas for how 1972–1974 (Magde et al. 1972, 1974; Ehrenberg and Rigler to retrieve the information sought from the T-jump meas- 1974; Elson and Magde 1974). Although showing great poten- urements. Thus this at the time unfortunate happening gave tial, the applicability of FCS was however strongly reduced at rise to the idea of using instead fluctuations in the equilib - this time due to methodological constraints. In the measurements rium state, and coupled fluctuations in the optical signals, (Magde et al. 1972), the excitation beam was focused in a sam- to follow the reaction. Together with Leo de Mayer and ple cell, and the fluorescence collected by a parabolic reflector. Klaus Gnädig, the first experiments were undertaken dur - High background intensities, dominated by Raman scattering of ing 1968/1969, observing the association kinetics of acridin the water molecules, and proportional to the size of the relatively dyes and DNA for which data were available from T-jump large (> pL) detection volume, made it impossible to reduce the measurements (Ramstein et al. 1980). fluorophore concentrations without the background dominat- ing over the fluorescence signal. As a consequence, the aver - age number of fluorescent molecules in the detection volume Fluorescence correlation spectroscopy (FCS) had to be high, leading to lower relative fluorescence intensity fluctuations. Long measurement times were therefore required Pioneer work in the 1970s to distinguish and analyze these fluctuations, in turn imposing strict requirements on stability and absence of systematic noise The first attempts to use fluorescence as the readout in fluctua- in the optical and electronic parts of the instrumentation. The tion experiments illustrated that such analyses of systems under limited instrumental stability over long times, in combination thermodynamic equilibrium can oe ff r an attractive alternative with long time-range photochemical degradation, restricted the to relaxation measurements, studying the response in a sample possible applications of FCS. Since FCS relies on the ability following some external perturbation. This general concept of to detect and analyze fluctuations in the detected fluorescence fluctuation analysis was introduced already more than 100 years signal stemming from dynamic events of single molecules, a ago (Svedberg 1911; von Schmolukowski 1914; Chandrasekhar major figure-of-merit for FCS measurements is the number of 1943) and over the years several techniques have been developed fluorescence photons that can be detected per molecule and time monitoring fluctuations in the number of particles or molecules of (the fluorescence brightness) (Koppel 1974). Consequently, high a specic fi type within a x fi ed sample volume. Well-known exam - background light levels, low detection quantum yields, as well ples include the dynamic light scattering technique, exploiting the as the low capacity of the computers to analyze the fluctuation light scattering intensity from the particles of interest (Schaefer data, limited the applicability of FCS measurements at the time. 1973; Berne and Pecora 1975), and the voltage clamp approach (Hodgkin et al. 1949), where fluctuation analysis of electrical Breakthrough in the 1990s currents over sections of cellular or artificial membranes is per - formed (with the later-developed patch clamp technique (Neher In the 1990s, more than 15 years after its first demonstra- and Sakmann 1976) as its single-molecule counterpart). tion, the prerequisites for FCS measurements had greatly Fluorescence correlation spectroscopy (FCS) thus belongs to improved. Detection of individual fluorophore molecules, an established category of fluctuation spectroscopy techniques, first in solid crystals under cryo-temperatures by absorption but with clear advantages coming with the use of the highly (Moerner and Kador 1989) and then by fluorescence (Orrit sensitive and specific fluorescence intensity signal as the fluc- and Bernard 1990), led further to single-molecule detection tuating quantity. When Rudolf Rigler returned to the Karolinska in aqueous solution under room temperature (Shera et al. Institute in 1970 to build up his own group, he received a letter 1990). At the same time, the introduction of small, diffraction- from Elliot Elson, who learned relaxation kinetics from Robert limited observation volumes in FCS measurements, confocal 1 3 European Biophysics Journal (2018) 47:479–492 481 epi-illumination, highly sensitive avalanche photodiodes for fluorophores with known diffusion coefficients, D for the fluorescence detection and very selective band-pass filters to fluorescent species studied can be determined. Equation  2 discriminate the uo fl rescence from the background, made it is based on the assumption that F(t) can be considered a possible to improve signal-to-background ratios in FCS-meas- stationary and ergodic process (essentially that its average urements by several orders of magnitude (Rigler and Widen- and variance is constant over time, and that the average of gren 1990; Rigler et al. 1993). This development formed the F(t) from one or a few fluorescent molecules recorded over basis for the FCS measurements as they are performed today. a longer time should be the same as an instant average of In an FCS measurement, in its most simple realization, very many fluorescent molecules). Further assumptions are fluorescence intensity fluctuations arise from translational that the FCS measurement is performed at equilibrium in diffusion, as the fluorescent molecules are diffusing into and an ideal solution, with no photobleaching and in a sample out of a focused laser beam in an open confocal detection volume ≫ than the confocal detection volume. volume (Fig. 1a). For fluorescent molecules with a concen- tration, c(r ̄, t) , the detected fluorescence intensity can then FCS for monitoring of reaction kinetics be written as (Rigler et al. 1993; Widengren et al. 1995): FCS is not limited to analyses of the number and diffusion 𝜎 I (r̄) exc F(t)= 𝛷 𝛷 CEF(r̄)k c(r̄, t)dV properties of fluorescent molecules. Even for a standard FCS F D 10 𝜎 I (r̄)+ k exc 10 instrument with a single-point, stationary detection volume, (1) a wide range of processes can be studied, spanning a time = 𝛷 𝛷 W(r̄)c(r̄, t)dV. F D range from sub-nanoseconds to seconds. In principle, any Here, Ф , σ and k denote the f luorescence quantum process at equilibrium conditions, which reflects itself as a F 10 yield, excitation cross section, and deactivation rate of change of the detected fluorescence F (t), can be measured, the fluorescent molecules. Ф and CEF(r ̄) signify the given that it occurs within the dwell time of the fluorescent detection quantum yield and the collection efficiency molecules in the detection volume (  ≈  ∕4D ). Cor rela- function of the instrument. I (r ̄) denotes the excitation tion analyses can also be performed on fluorescence intensi- exc intensity of the laser. W(r ̄) is the molecular detection ties recorded in e.g., different spectral channels [fluorescence efficiency. cross-correlation spectroscopy, FCCS (Ricka and Binkert From the fluctuations in F (t), denoted δF(t), informa- 1989; Schwille et al. 1997)], spatial locations [image cor- tion can be retrieved about the translational diffusion coef- relation spectroscopy, ICS (Brinkmeier et al. 1999; Petersen ficients, D, and the average number of molecules, N, resid- et al. 1993; Digman et al. 2005)], or based on fluorescence ing simultaneously in the detection volume. Assuming that lifetime changes [fluorescence lifetime correlation spectros- W(r ̄) has a Gaussian distribution is both the radial and copy, FLCS (Benda et al. 2006)]. It is beyond the scope of axial dimensions, and in the absence of any other kinetic this review to discuss the full range of molecular dynamic process than translational diffusion affecting the fluores- processes that can be studied by FCS. In this review, we cent molecules, the time-dependent normalized intensity will rather discuss how FCS can be used to study reaction autocorrelation function (ACF) can be written (Magde kinetics at thermodynamic equilibrium, as an alternative to et al. 1972, 1974): relaxation experiments, for reactions which generate changes < F(t)F(t + 𝜏 ) > < [< F > +𝛿 F(t)][< F > +𝛿 F(t + 𝜏 )] > G(𝜏 )= = 2 2 < F > < F > (2) <𝛿 F(t)𝛿 F(t + 𝜏 ) > 1 1 1 = F(t) is stationary = + { } + 1 =   1. 2 2 2 < F > N 1 + 4D𝜏 𝜔 1 + 4D𝜏 𝜔 1 2 G(τ) gives a measure of how the fluorescence intensity in the fluorescence brightness, Q , of the molecules. In its detected at a certain time, F(t), is related to that detected at easiest realization, as depicted in Fig. 1a, the (reversible) a correlation time, τ, later, i.e., to F(t + τ). In Eq. 2, brackets reaction to be studied involves one fluorescent species, takes denote average over the measurement time, which should place on a time scale much faster than the passage times of be long enough to secure convergence of G(τ). ω and ω the molecules through the detection volume, and switches 1 2 signify the distances from the center of the detection volume the fluorescence completely on and off, yielding high con- in the radial and axial dimensions, respectively, at which trast fluorescence intensity fluctuations, superimposed on W(r ̄) has decreased by a factor of e . With knowledge about those due to translational diffusion of the molecules in and ω and ω , e.g., from FCS calibration measurements with out of the detection volume. The ACF recorded from such 1 2 1 3 482 European Biophysics Journal (2018) 47:479–492 Fig. 1 Monitoring of reaction kinetics at thermodynamic equi- to and from the dark, long-lived, triplet state (T ). In the ACF of the librium by FCS. a Principal drawing of the type of reactions that recorded fluorescence intensity (Eq.  2), two relaxation processes will can be studied. Fluorescent molecules undergoing Brownian diffu- show up, as described by Eqs. 3 and 4. With the full amplitude of the sion through a confocal detection volume (lower left) also undergo a ACF normalized to unity, the amplitude B corresponds to the aver- reaction, switching the fluorescence of the molecules on and off with age fraction of the fluorescent molecules in the detection volume rates k and k (upper part). This reaction takes place on a faster which are in the triplet state. τ denotes the relaxation time for the 1 −1 T time scale than the diffusion of the fluorescent molecules into and out singlet–triplet state transition and τ is related to the average dwell of the detection volume. As a result, fluctuations in the fluorescence time of the fluorescent molecules in the detection volume. c Exam- intensity, F(t), can be detected, with the faster time-scale fluctua- ples of FCS curves, recorded from the fluorophore Rh6G in water, tions caused by the reaction superimposed on the slower fluctuations with different excitation intensities, I , applied. Increasing I , leads exc exc caused by diffusion (lower right). b Principal drawing of an FCS to higher B and shorter τ values in the FCS curves. From the I - T exc curve, recorded from fluorescent molecules undergoing fluorescence dependence of B and τ , the transition rates to and from T can be T 1 on–off blinking in the microsecond time-range, caused by transitions determined a sample (Fig. 1b) can then be separated into two factors. chemical relaxation time(s) and/or the diffusion coefficients The first factor, G (τ), depends on the transport properties of all fluorescent species can be considered equal (Palmer (diffusion or flow) of the molecules and the second, R (τ), and Thompson 1987). For a fluorescent species studied by depends only on the reaction rate constants (Palmer and FCS, and undergoing chemical reactions, the second factor Thompson 1987): can more generally be expressed as: G()= G ()R()+ 1. (3) Q Q X (𝜏 ) i j ij i,j=1 R(𝜏 )= . (5) In this particular case, the separate reaction-related factor ∑ Q C i=1 i can be written as: R()= 1 − B + B exp(−k ) (4) Here, Q is the u fl orescence brightness coec ffi ient of state 1 − B i and X (τ) is the solution to the following set of differential ij where B denotes the average fraction of the fluorescent mol- equations and initial conditions: ecules in the detection volume which are in the dark state. k = 1∕ is the relaxation rate of the reaction, and is given B B dX (𝜏 )∕d𝜏 = T X (𝜏 ) by the sum of the fluorescence on and off rates, k and k . In ik ij jk 1 −1 (6) j=1 a more general case, the separation of G(τ) into two factors as in Eq. 3 is possible for reactions in which the diffusion of X (0)= C 𝛿 , ik i ik the reactants and product molecules is much slower than the 1 3 European Biophysics Journal (2018) 47:479–492 483 where δ  = 1 if i = k, and δ  = 0 if i ≠ k. X (τ) describes photo-induced dark states, such as triplet, photo-isomerized, ik ik ij the probability of finding a molecule in state j at time τ, and photo-oxidized states reduce the fluorescence brightness given that it was in state i at time 0. M is the number of of the fluorophore molecules studied, a major figure-of-merit species participating in the chemical reaction, and T rep- for FCS measurements (Koppel 1974). Some of these states ij resents the corresponding matrix of the kinetic rate coef- may also act as precursor states for photobleaching, and the ficients. In Eq.  5, it can be noted that in FCS measurements, blinking caused by these transitions may cause problems in each different species analyzed is weighted by the square FCS and in single-molecule experiments, in that they may of its fluorescence brightness. For an ACF recorded from shadow other molecular processes of interest, taking place a sample containing several different fluorescent species, it in the same time range. On the other side, based on the gen- can therefore be strongly misleading to interpret the inverse eral approach to study reaction kinetics via changes in the amplitude of the ACF as the true average number of fluores- fluorescence brightness Q (Eqs. 3–7), FCS has also turned cent molecules (1/G(0) = N in Eq. 2). More generally, for M out to be a very suitable tool to study these transitions. Fig- different species, with brightnesses Q and average numbers ure  1c shows FCS curves recorded from the fluorophore N (and disregarding fluorescence anti-bunching) (Magde rhodamine 6G (Rh6G) in air-saturated aqueous solution, et al. 1974; Widengren and Mets 2001): and how the average population of the dark, lowest triplet state of Rh6G, given by the relative amplitude B, as well as � � ∑ 2 N Q the singlet–triplet state relaxation time, τ , corresponding i i i=1 1∕G(0)= . (7) 2 to τ in Eq. 4, vary with the excitation irradiance within the N Q i=1 i confocal detection volume in the FCS experiment. From the excitation irradiance dependence observed in the FCS curves When applicable, it is very convenient to treat the kinet- (a so-called FCS power series), the transition rate constants ics of a chemical reaction separately from the translational to and from T can be determined in a straightforward man- diffusion in the fluctuation analysis, as given by Eq.  2. This ner (Widengren et al. 1995). Similarly, a whole range of treatment applies to a rather broad range of chemical reac- photo-induced dark transient states can be kinetically char- tions such as inter- or intra-molecular dynamics, influenced acterized, including photo-ionized (Widengren et al. 1997) by fluorescence quenching (Bonnet et al. 1998; Chattopad- and photo-isomerized states (Widengren and Schwille 2000; hyay et al. 2002). Moreover, for a reaction that under certain Widengren and Seidel 2000), as well as the influence of conditions does not fulfill the criteria, it is sometimes pos- chemical additives and environmental conditions on these sible to modify the conditions. For instance, the dwell times transitions (Widengren et al. 1995, 1997, 2007; Widengren can be retarded with respect to the chemical relaxation times and Schwille 2000). Likewise, the overall photostabilities of by expanding the detection volume, or the reactions under fluorophores under excitation conditions required for FCS study can be speeded up, e.g., by using higher concentrations and other forms of ultrasensitive fluorescence spectroscopy of un-labelled reactants (Widengren et al. 1995, 1999, 2007; and imaging can be studied (Widengren et al. 2007; Egg- Widengren and Rigler 1997). eling et al. 1998; van den Berg et al. 2001). Interestingly, in In the sections below, we will discuss two realizations of such studies, compounds known in the fluorescence spec- this FCS approach to monitor reaction kinetics, generating troscopy field as fluorescence quenchers, such as potas- changes in Q of the fluorescent species studied. First, it will sium iodide, may under excitation conditions for FCS and be shown how FCS can be used to monitor a range of photo- single-molecule fluorescence spectroscopy turn out to act induced transitions in fluorophores. Second, how monitoring as anti-fading compounds (Chmyrov et al. 2010). Similar of ion-sensitive fluorophores by FCS offers an alternative transitions as in organic fluorophore molecules can also be way of monitoring proton exchange kinetics and how this found in green fluorescent proteins (GFPs). In GFPs, the approach can be used to investigate protonation kinetics at transitions are however far less influenced by environmental biological membranes. parameters, since the fluorescently active unit is located in the inner part of the GFPs, shielded from the surroundings FCS for photodynamic characterization by a tight beta sheet barrel structure (Widengren et al. 1999; of fluorescent species Haupts et al. 1998). Compared to e.g., transient-state absorption/flash pho - Photophysical properties of the fluorescent molecules under tolysis (Van Amerongen and Van Grondelle 1995; Korobov study set the fundamental limits for the overall perfor- and Chibisov 1978) and phosphorescence studies (Jovin and mance of virtually all forms of fluorescence spectroscopy Vaz 1989) the FCS approach offers some advantages. For and imaging, where high sensitivities, read-out rates and/ triplet state studies, it uses the highly sensitive fluorescence or resolutions are required. Similarly, these properties also readout to monitor the triplet state, rather than the faint, set the ultimate limits for FCS measurements. Population of easily quenched, phosphorescence signal from the triplet 1 3 484 European Biophysics Journal (2018) 47:479–492 state itself. Thereby, a favorable combination of a high sig- Naturally, if there is no difference in brightness of the nal level (given by the readout of fluorescence photons) and fluorophore upon protonation (Q  = 1), B will be zero and an outstanding environmental sensitivity (given by the long no relaxation can be observed in the ACFs. For many pH- lifetimes of the transient states) can be obtained. Quenching sensitive fluorophores Q is small (1–2%), and then only mar- of the triplet states of the fluorophores by oxygen or other ginally affects the relaxation amplitudes. With knowledge compounds will not ruin the read-out signal. Compared to of Q it can also be properly corrected for. Alternatively, flash photolysis the experimental realization is relatively higher-order correlation analyses of δF(t) can be applied simple and more easily applicable to a broader range of to resolve Q, as recently demonstrated (Abdollah-Nia et al. samples. 2017). As an additional alternative, ratio-metric pH-sensitive dyes can be used, for which the excitation and/or the emis- FCS for studies of proton exchange dynamics sion spectrum changes upon protonation. For such dyes, in solution and on biomembranes two or several Q values can be included, effective for dif- ferent laser excitation wavelengths and/or detection within Monitoring blinking rates and the fractions of fluorescent different wavelength bands. If the excitation/emission in and non-fluorescent fluorophores by FCS, as outlined above one wavelength band increases upon protonation of the dye, (Eqs. 3–7), can also be applied to characterize ion exchange to it normally decreases in another wavelength band. FCCS and from ion-sensitive fluorophores at thermodynamic equi- measurements, recording the cross-correlation of intensities librium (Widengren and Rigler 1997; Widengren et al. 1999). recorded at die ff rent excitation and/or emission wavelengths, In such measurements, taking as an example a pH-sensitive then typically display negative B relaxation amplitudes dye in a buffered aqueous solution, which is non-fluorescent in (Persson et al. 2009). its protonated form, the recorded FCS curves can be described The FCS-based approach for ion exchange studies can by Eqs. 3 and 4 (Fig. 1b). The amplitude B then corresponds offer selective advantages over other techniques for meas- to the fraction of non-fluorescent protonated fluorophores, uring local ion concentrations, and in particular exchange and the relaxation rate k to the sum of the protonation and kinetics of ions on a local scale. We have exploited these de-protonation rates of the fluorophores (Fig.  2a, c). With advantages in a series of papers to study proton exchange at −pK knowledge of the pK (and K = 10 ) of the fluorophore, biological membranes (Brändén et al. 2006; Öjemyr et al. a a + −pH the local pH (and H = 10 ) can then be determined from 2009; Sanden et al. 2010; Xu et al. 2016; Sjöholm et al. the relaxation amplitude B in the ACFs (Eq. 4). If the fluoro- 2017). Proton gradients across biological membranes act phore becomes non-fluorescent upon protonation (Widengren as driving forces for many energy-consuming cellular pro- and Rigler 1997; Widengren et al. 1999): cesses, not the least ATP synthesis by ATP synthase in the mitochondria. To generate the gradients, proton transport at and across membranes is required and involves a series of B = , (8a) membrane-spanning proteins in the inner membranes of the [H ] + K mitochondria. The underlying mechanisms for this proton transport has been subject to extensive research (Medvedev if it is fluorescent in the protonated form and becomes non- fluorescent upon de-protonation: and Stuchebrukhov 2011), but is nonetheless not completely understood. One of the key questions concerns the nature of B = . coupling between proton generators, such as cytochrome C (8b) [H ] + K oxidase (CytcO), pumping protons across the membrane, and proton consumers, such as ATP synthase, using the pro- Moreover, in a buffered aqueous solution, k directly ton gradients across the membrane to drive the ATP synthe- reflects and depends linearly on the local buffer concentra- sis (Medvedev and Stuchebrukhov 2011). Both the outlet of tion (Widengren et al. 1999). the generator and inlet of the consumer proteins are located Dyes used for FCS studies of proton (or ion exchange) do on the same side of the membrane, but the proteins are spa- not have to be completely non-fluorescent upon protonation tially separated. A major question is how the generated pro- (or de-protonation). However, the relaxation amplitude B teins get to the consumers before they are dissociated from will get smaller the less distinct the brightness difference the membrane surface. A major experimental technique for upon protonation is. Introducing Q as the relative brightness these molecular proton exchange studies is the laser-induced of the dimmer form of the dye, protonated or de-protonated, proton pulse approach (Gutman 1986) and other relaxation the amplitudes in Eqs. 8a and 8b will change into (Widen- techniques. Basically, a light flash directed to one side of gren and Schwille 2000): a membrane releases protons from caged compounds in B(1 − Q) the membrane, and the response from pH-sensitive fluoro- B = . (9) 1 + Q (1 − B) phores is then monitored on the other side of the membrane. 1 3 European Biophysics Journal (2018) 47:479–492 485 Fig. 2 Protonation kinetics in solution and at biological mem- into and out of the detection volume of the FCS instrument. d Exam- branes, studied by FCS. Proton exchange to and from pH-sensitive ples of FCS curves recorded from the pH-sensitive fluorophore Fluo- fluorophores results in fluorescence blinking, which can be analyzed rescein in water at different pH. Three relaxations can be observed in by FCS as outlined in Fig.  1, and by use of Eq.  4. For fluorophores the FCS curves: singlet–triplet state relaxation in the µs time range free in solution (a), FCS measurements show that the protonation on- (with amplitude T, marked red, reflecting the fraction of fluorophores rate is two orders of magnitude slower than when the fluorophores being in the triplet state while passing the detection volume), proton are located close to a lipid membrane of a small unilamellar vesicle exchange in a time range of 1–100 µs (with the amplitude P, marked (SUV) (b). Apart from direct exchange of protons between the fluo- blue, related to the fraction of fluorophores in the protonated, close-to rophore and the bulk (red arrows), the membrane–water interface also non-fluorescent state of Fluorescein), and diffusion relaxation beyond exchange protons with the bulk (thin black arrows), and protons at the 100 µs (with the detection volume expanded well beyond the diffrac- surface may migrate along the surface (thick black arrows) and reach tion limit in this series of measurements). e Illustration of a mem- the fluorophore, before dissociating from the membrane surface into brane acting as a proton collecting antenna, having a radius given by the bulk. c The proton exchange to and from fluorophores, located at how far a proton can diffuse along the surface before the probability the membranes of SUVs, were measured on SUVs freely diffusing to find it on the surface equals the average value on the surface The FCS approach above can offer complementary points measured protonation dynamics reflect the conditions in the of view on these relaxation techniques. In particular, in media the protons pass on their way from light absorbing FCS measurements protons associating to and dissociating proton emitters to the pH-sensitive fluorophores. from pH-sensitive fluorophores are observed at equilibrium Based on these selective advantages of the FCS approach conditions. No perturbation into some, often strongly un- we have investigated the principal role of biological mem- physiological, initial condition is required. Moreover, while branes for proton uptake of membrane incorporated proteins protonation kinetics measurements by proton pulse tech- (Fig. 2a–e). Interestingly, we found that the protonation rate niques require 1–5% of the lipids in the membranes to be of the pH-sensitive dye fluorescein increased by two orders 11 13 −1 −1 labeled (Serowy et al. 2003), FCS is typically performed at of magnitude (from ~ 10 to ~ 10 M  s ) when labeled about 1000-fold lower concentrations. Thereby, the influence to a lipid in a membrane of a small unilamellar vesicle of buffering effects due to ions binding to the fluorophores (SUV, Fig. 2b), compared to when free in solution (Fig. 2a) themselves, or to any light absorbing proton emitter, can (Brändén et al. 2006). In comparison, FCS measurements be avoided. Finally, the proton exchange seen in the FCS in membranes with negatively charged head-groups instead measurements reflects the conditions in the immediate sur - of zwitterionic head-groups only changed the protonation roundings of individual fluorescent probes, in contrast to rate by a factor of two (Brändén et al. 2006). This finding proton pulse measurements (Serowy et al. 2003), where the gives direct evidence that the membrane-water interface can 1 3 486 European Biophysics Journal (2018) 47:479–492 Fig. 3 Illustration of how fluorophore photodynamics is moni- imaging. The beam of a continuous wave (CW) laser is passed tored in transient state (TRAST) spectroscopy/imaging. a Elec- through an acousto-optical modulator, generating rectangular excita- tronic state model for a fluorophore, comprising the ground and tion pulse trains with varying pulse durations, w. These are fed into excited singlet states (S and S ), the lowest triplet state (T ) and a a microscope and via its objective onto the sample. The fluorescence 0 1 1 photo-oxidized state ( R ). Following onset of constant excitation, generated by the excitation pulse trains is collected by the same τ , τ and τ denote the relaxation times for the population of the objective and recorded by a camera in the image plane. By recording AB T R singlet states, T and R , respectively . b For the same fluorophore, images of the average fluorescence, F , for different duration, w, of 1 W subject to rectangular excitation pulses, the graph illustrates how the excitation pulses, TRAST curves can be generated within regions the average fluorescence, F , can vary with the duration, w, of the of interest in the image, and TRAST images can be obtained, map- excitation pulses. This so-called TRAST curve reflects the popula- ping e.g., τ and τ , or the underlying transition rates to and from T T R 1 tion build-up of the electronic states in (a) upon onset of excitation, and R and the relaxation times τ , τ and τ . c Principle setup for TRAST AB T R act as a proton collecting antennae (PCA), with an area far et  al. 2006), and protonation dynamics measurements by exceeding the size (or the physical cross section for protona- FCS on lipid nanodiscs, with diameters around 10 nm, show tion) of the fluorophore itself. In follow-up studies, the same that the protonation on-rate of fluorescein remains as high enhancement of more than two orders of magnitude in the as in SUVs (diameter of ~ 30 to 100 nm), even when incor- protonation on-rate was also found when the fluorophore porated into the small membrane areas of the nanodiscs. label was placed on the proton generator CytcO, when free A reasonable interpretation is that the density of protonat- in solution, compared to when in the membrane of an SUV able groups bound to the membrane surface can sustain a (Öjemyr et al. 2009). The PCA effect is less observable at higher local proton concentration at the surface than in the lower pH (< 7), when the proton association rate via the bulk solution, and a higher proton exchange rate of a surface membrane becomes comparable to that of direct protona- group (Fluorescein) with the surface than with the bulk solu- tion of the fluorophore from the bulk solution (Fig.  2, left tion (even if the diffusion coefficient of protons in bulk water bottom) (Sanden et al. 2010). The radius of the PCA, R , is considerably higher than along the membrane) (Brändén PCA is related to how far a proton generated at the membrane et al. 2006). The dependence of the protonation on-rate on surface can diffuse from its site of generation before the the buffer concentration in the bulk water and Monte-Carlo probability to find a proton equals the average value on the simulations of the proton exchange to and from a fluoro- surface (Gutman and Nachliel 1995). From proton pulse phore at a nanodisc of different size further confirmed that experiments, the R of lipid membranes has been deter-both R and D along lipid membranes are 2–3 orders PCA PCA S mined to be as large as tens of micrometers, and the diffusion of magnitude smaller, when measured by FCS (Xu et al. coefficients of the protons, D , along the membrane-water 2016), as compared to proton pulse data (Medvedev and −5 2 interfaces as high as 5 × 10  cm /s (Medvedev and Stuche- Stuchebrukhov 2011). The underlying reason for this quite brukhov 2011; Serowy et al. 2003). In contrast, from FCS significant difference can likely be attributed to the fact that experiments D is two orders of magnitude lower (Brändén FCS measures the very local proton exchange kinetics, in 1 3 European Biophysics Journal (2018) 47:479–492 487 the very surroundings of individual fluorophores (nanom- versatile means to analyze photodynamic events, this limits eter scale), while proton pulse measurements monitor the the application range and throughput of FCS measurements, propagation of proton pulses over distances beyond millim- and makes parallel readouts and imaging difficult. eters. The longer distance measurements capture an aver- To overcome these limitations, we have introduced age migration behavior of the protons, including “hopping” an approach, so-called transient-state (TRAST) imaging between the lipid head group region of the membrane, the (Sanden et al. 2007, 2008), where the fluorescent sample membrane-water interface and the bulk water. In contrast, is subject to a modulated excitation (Fig. 3). Depending FCS measurements capture the local exchange dynamics at on the excitation pulse train characteristics (e.g., pulse the lipid head group region and membrane-water interface duration, separation and height) long-lived photo-induced only. These results thus emphasizes the ability of FCS to transient states (e.g., triplet, photo-isomerized and photo- monitor the proton exchange at the very local scale, and also ionized states) of the fluorophores in the sample will be provide experimental support to recent theoretical studies populated to different extents. Figures  3a, b illustrate how showing that proton diffusion along membrane surfaces is the fluorescence intensity varies with time after onset of time- and length-scale dependent (Wolf et al. 2014; Yamash- excitation, and how equilibration between the ground and ita and Voth 2010). the excited singlet states (S and S ) typically takes place 0 1 within nanoseconds after onset of excitation, between the singlet and triplet (T ) states within microseconds, and Transient‑state (TRAST) spectroscopy/ finally how a population of photo-oxidized (or –reduced) imaging to exploit the information content states ( R ) build up and equilibrate within milliseconds of fluorescence blinking kinetics after onset of excitation. Upon transient-state popula- tion build-up in the sample, the fluorescence intensity Concept for TRAST will drop in a corresponding manner. By systematically varying the excitation pulse train characteristics, e.g., by As discussed above, fluorophore blinking, caused by popula- use of an acousto-optical modulator (AOM), and regis- tion dynamics of photo-induced, long-lived, non-fluorescent tering how the plain time-averaged fluorescence intensity + − ̇ ̇ R R triplet (T ), photo-oxidized ( ), photo-reduced ( ) or changes with e.g., the pulse width (a so-called TRAST photo-isomerized states (Fig. 3a) is of central importance to curve, Fig. 3c) the population kinetics of the fluorophore all forms of fluorescence-based ultrasensitive and ultrahigh- transient states can then be retrieved. As with FCS, the resolution spectroscopy/imaging. While blinking has to be TRAST technique combines the high detection sensitivity suppressed in single-molecule fluorescence studies (reduces of the fluorescence readout with the high environmental brightness and obscures observation of other dynamic pro- sensitivity of the dark transient states, but does not rely cesses), blinking is an absolute prerequisite for all forms of on a high fluorescent brightness of the molecules studied, super-resolution microscopy [see e.g., (Eggeling et al. 2015) on a high time resolution, or on a certain concentration of and (Blom and Widengren 2017) for reviews]. Moreover, the sample. This makes TRAST imaging a broadly appli- −6 the long lifetimes of these non-fluorescent states, ~ 10 to cable approach, applicable e.g., on live cells, and allows −3 −9 10 s, compared to ~ 10 s for the fluorescence lifetime of transient states to be imaged by e.g., a standard CCD cam- the excited singlet state, make these states highly environ- era. For live cell studies by light microscopy in general, ment sensitive. These states thus represent a whole set of photo-toxic and photo-sensitizing effects from both endog- additional parameters, which in a very sensitive manner can enous and exogenous (fluorophore) compounds have to be reflect microenvironments as well as biomolecular dynam- considered. These effects depend on several parameters, ics and interactions of fluorescent molecules (Widengren such as the concentration of these compounds in the cells, 2010). This possibility seems not to have been fully realized the wavelength of the irradiating light, the overall excita- in the fluorescence field until more recently (Weidemann tion light dose, and the distribution of the light dose in et al. 2014; Kawai et al. 2015; Mahoney et al. 2015; Bag and time (Magidson and Khodjakov 2013; Tinevez et al. 2012; Wohland 2014; Querard et al. 2015). Logg et al. 2009; Schneckenburger et al. 2012). TRAST FCS-based monitoring of these states combines high imaging requires excitation irradiances high enough to detection sensitivity by the fluorescence readout, with high induce population of the transient states. However, using environmental sensitivity, given by the long lifetimes of high triplet yield dyes (Spielmann et al. 2014; Mücksch the transient states. However, FCS measurements require et  al. 2015), or dyes for isomerization  (Chmyrov et  al. fluorescent molecules with high brightness and in low con- 2015), high populations of dark transient states can be centrations, and highly sensitive detectors with high time reached with lower excitation intensities, and live cell resolution. Although FCS provides a very powerful and TRAST imaging can be performed with overall excitation 1 3 488 European Biophysics Journal (2018) 47:479–492 light doses almost an order of magnitude lower than esti- TRAST, examples of biomolecular and cellular mated maximum tolerable light doses for maintaining cell information that can be retrieved viability (100 J/cm at 514 nm irradiation) (Schnecken- burger et al. 2012; Wagner et al. 2010). Since TRAST does In Fig. 4, a few examples are given for how TRAST imag- not rely on fluctuation measurements of single molecules, ing can be applied. Figure 4a shows a fluorescence inten- molecular brightness is not a strongly limiting issue, and sity image (top) and a corresponding image (bottom) of the low fluorescence signals can be compensated by higher T -to-S rate. This rate is almost completely determined by 1 0 concentrations of fluorescent molecules in the sample, as quenching from dissolved molecular oxygen (Widengren an alternative to increased excitation irradiances. Thereby, et al. 1995; Spielmann et al. 2014). The latter image is gen- also relatively low brightness compounds, such as auto- erated by taking consecutive fluorescence intensity images fluorescent species (Hevekerl et al. 2016), can be studied. with different excitation pulse trains (pulse durations) Fig. 4 Examples of how TRAST imaging/spectroscopy can be number of independently blinking emitters on the diffusing units. applied. a Fluorescence (top) and TRAST (bottom) images (100 From the difference in the blinking amplitude in FCS and TRAST, ×  100  µm) of cultured cells from a breast cancer cell line (MCF-7), the absolute numbers of fluorophores per diffusing unit can be deter - loaded with the fluorophore EosinY. The TRAST image shows the mined. See main text for further information. c Model system used deactivation rate of the T state, k , obtained from TRAST curves to demonstrate how low-frequency collisional interactions can be 1 t extracted pixel-wise from multiple fluorescence intensity images, monitored via triplet state quenching. Quenching of the triplets states recorded with different excitation modulation. b Concept for determi- of Lissamine rhodamine B, labeled to DOPE lipids (DOPE-RH), nation of molecular stoichiometry based on FCS and TRAST meas- by lipids labeled with the triplet state quencher TEMPO (DOPC- urements. Because of the synchronous blinking of the fluorophores TEMPO) was studied in the membrane of DOPC SUVs. S denotes in a TRAST experiment, the fluctuation amplitude will not change the quenching radius of interaction (≈ 0.2 nm), and R (≈ 20 nm) the with several fluorophores on the diffusing units studied. In contrast, radius of the SUVs. See main text for further details in an FCS experiment, the blinking amplitude will decrease with the 1 3 European Biophysics Journal (2018) 47:479–492 489 applied. The variation of the fluorescence intensity from the Figure  4c illustrates the principal use of TRAST to excitation modulation (TRAST curves) can then be gener- monitor low-frequency collisional interactions. In a proof- ated in a pixel-wise manner. By fitting these curves to a of-principle study (Strömqvist et al. 2010), we monitored photodynamic model of the fluorophores (Fig.  3a), an image the quenching of long-lived triplet states of Lissamine Rho- of the oxygen-dependent T -to-S rate can be obtained. Live damine B dyes, labeled on the head group of lipids. Dye- 1 0 cell imaging with this approach allowed the local oxygen labeled lipids (DOPE-RH) were placed in the membranes of concentration within cells to be determined, and charac- small unilamellar vesicles (SUVs) with radius R ≈ 20 nm. teristic differences in oxygen consumption between nor - In the SUV membranes we also added varying amounts of mal and cancer cells to be detected (Spielmann et al. 2014; lipids (from 0.15% up to 8% of the lipids) labelled with the Mücksch et al. 2015). Such differences cannot be resolved triplet quencher TEMPO (DOPC-TEMPO). Both the S and by conventional fluorescence imaging parameters, but may T states can be quenched upon interaction between DOPE- be captured by room temperature phosphorescence (RTP). RH and DOPC-TEMPO in the SUV membranes (when Like TRAST, RTP benefits from long lifetimes, to probe within a radius of interaction, S, of a few Ångström to each subtle changes in environmental conditions (accessibili- other). However, T states have typically at least three orders ties of quenchers, polarities etc.), or to reveal structural and of magnitude longer lifetimes (µs-ms) than S states (ns), and dynamic information of biological macromolecules (Cioni thus a correspondingly longer time to be influenced by the and Strambini 2002). However, while TRAST is based on environment (or quencher collisions). As a result, quenching the readout of a strong fluorescence signal, the RTP signal of S , observed as a relative change of the fluorescence life- is weak and susceptible to dynamic quenching by oxygen time of a few percent, could only be observed for the higher and trace impurities. Moreover, while TRAST can be based DOPC-TEMPO concentrations, while quenching of T could on a broad range of different fluorophores and even auto- be observed also for the lowest DOPC-TEMPO concentra- fluorescent compounds, specific RTP probes are scarce and tions. In other words, interactions between dye-labeled and cannot easily be loaded into cells (Yu et al. 2017). In contrast triplet quencher-labeled molecules can be analyzed via the to RTP, TRAST is also not limited to imaging of triplet state triplet state population kinetics, at collisional frequencies parameters only. Also other transitions can be imaged, e.g., too low to be reflected as fluorescence intensity or lifetime viscosity-dependent trans–cis isomerization of lipophilic changes (Strömqvist et al. 2010). This illustrates how tran- cyanine dyes, providing images of the microfluidity of cel- sient state monitoring by TRAST (or FCS) can combine lular membranes (Chmyrov et al. 2015). With a different the environmental sensitivity of long-lived transient states, readout than in established fluorescence-based approaches with the detection sensitivity of the fluorescence signal, and based on anisotropy, polarity-sensitive dyes, or excimer retrieve information not within reach by conventional fluo- formation, this TRAST approach offers complementary rescence readouts. information and can capture different aspects of membrane microviscosity and fluidity of the cellular membranes. Figure 4b illustrates a concept to determine molecular Conclusions stoichiometries, by comparing the fluorescence fluctuations under continuous excitation using FCS, when all the fluo- Molecular relaxation and kinetics measurements have played rophores on a diffusing unit are blinking independently of a key role in reaching a fundamental understanding of a each other, with those occurring under square-pulsed excita- broad range of biological processes. In this review, we have tion using TRAST spectroscopy, when all fluorophores are given a historical view of how our research in the develop- blinking in a synchronized manner. Thereby, the number ment and use of fluorescence correlation spectroscopy and of fluorophores per molecule can be determined (Hevekerl related techniques for such measurements has its roots and and Widengren 2015). In the recorded FCS curves, the fast has further evolved from the pioneering work of Manfred relaxation amplitude, B, of a transient state, is lowered with Eigen and his colleagues and coworkers. Although now the number of independently blinking emitters, N, on a dif- 50 years have passed since Eigen, Norrish, and Porter were fusing unit by B/N. In contrast, in TRAST measurements awarded the Nobel Prize for their achievements in relaxa- the N emitters are blinking in synchrony due to the excita- tion kinetics measurements, novel molecular fluctuation and tion modulation, and the corresponding relaxation ampli- relaxation techniques are still under development. This field tude is still B. By dividing the TRAST amplitude with the is thus still not exhausted and will not be in many years to FCS amplitude N can thus be obtained directly. The FCS come. Biology is far too intricate. Mother Nature has left and TRAST measurements can be done consecutively in many riddles for us to solve, and not unlikely with molecular the same setup. 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European Biophysics JournalSpringer Journals

Published: Dec 19, 2017

References

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