Applications of Fluorescence Correlation Spectroscopy to the Study of Nucleic Acid Conformational Dynamics

In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

The remarkable revival of fluorescence correlation spectroscopy (FCS), stimulated by major technical improvements, has led to novel insights into dynamical processes occurring in biological macromolecules over a large time span [2.1–2.3]. The application of FCS has enabled the retrieval of information on the properties of single biomolecules as distinguished from the collective properties of a whole molecular ensemble [2.4,2.5]. The FCS technique is not confined to clear solutions of single purified biomolecules, since the next challenging step to apply the technique to living cells has already been taken [2.6,2.7]. To date, most FCS experiments have been restricted to fluorescent dye molecules, which possess the ability to emit a large number of fluorescence photons before being destroyed. The application of FCS to natural fluorophores has been extremely limited, since the fluorescent moiety usually does not have favorable properties for FCS. The famous green fluorescent protein (GFP) [2.8] was the first example where FCS provided very specific dynamic information on a natural fluorophore [2.9,2.10]. However, in the latter two references the fluorophore in GFP was engineered to yield more brightness and a higher absorption cross section in the blue-green spectral range. The natural occurrence of GFP is limited to a few species of marine organisms.KeywordsFluorescence Correlation SpectroscopyAntenna ProteinFluorescence PhotonAutocorrelation TraceTriplet LifetimeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In some applications the dye distribution within fluorescently labeled nanoparticles and its stability over long periods of time are important issues. In this article we study numerically and experimentally the applicability of fluorescence correlation spectroscopy (FCS) to resolve such questions. When the size of fluorescently labeled particles is comparable to or larger than the confocal volume, the effective confocal volume seen in FCS experiments is increasing. Such an effect has already been studied for uniformly labeled spherical particles. In this work we analyze the form of the FCS correlation functions (CFs) for core-labeled and shell-labeled core-shell particles. For shell-labeled particles an additional fast decay was found both in simulations and in experiments on custom-made surface-labeled particles. Universal scaling of FCS correlation times based on the squared ratio of the labeled part radius of gyration to the Gaussian radius of the beam profile was found. Recipes based on the analysis of simulated CFs, proposed for interpretation of experimental results, were successfully applied to the FCS results on suspensions of large core-labeled and surface-labeled particles.

In recent years, fluorescence correlation spectroscopy has been increasingly applied for the study of polymer dynamics on the nanometer scale. The core idea is to extract, from a measured autocorrelation curve, an effective mean-square displacement function that contains information about the underlying conformational dynamics. This Letter presents a fundamental study of the applicability of fluorescence correlation spectroscopy for the investigation of nanoscale conformational and diffusional dynamics. We find that fluorescence correlation spectroscopy cannot reliably elucidate processes on length scales much smaller than the resolution limit of the optics used and that its improper use can yield spurious results for the observed dynamics.

Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy

Self-propelled chemical motors are chemically powered micro- or nanosized swimmers. The energy required for these motors' active motion derives from catalytic chemical reactions and the transformation of a fuel dissolved in the solution. While self-propulsion is now well established for larger particles, it is still unclear if enzymes, nature's nanometer-sized catalysts, are potentially also self-powered nanomotors. Because of its small size, any increase in an enzyme's diffusion due to active self-propulsion must be observed on top of the enzyme's passive Brownian motion, which dominates at this scale. Fluorescence correlation spectroscopy (FCS) is a sensitive method to quantify the diffusion properties of single fluorescently labeled molecules in solution. FCS experiments have shown a general increase in the diffusion constant of a number of enzymes when the enzyme is catalytically active. Diffusion enhancements after addition of the enzyme's substrate (and sometimes its inhibitor) of up to 80% have been reported, which is at least 1 order of magnitude higher than what theory would predict. However, many factors contribute to the FCS signal and in particular the shape of the autocorrelation function, which underlies diffusion measurements by fluorescence correlation spectroscopy. These effects need to be considered to establish if and by how much the catalytic activity changes an enzyme's diffusion. We carefully review phenomena that can play a role in FCS experiments and the determination of enzyme diffusion, including the dissociation of enzyme oligomers upon interaction with the substrate, surface binding of the enzyme to glass during the experiment, conformational changes upon binding, and quenching of the fluorophore. We show that these effects can cause changes in the FCS signal that behave similar to an increase in diffusion. However, in the case of the enzymes F1-ATPase and alkaline phosphatase, we demonstrate that there is no measurable increase in enzyme diffusion. Rather, dissociation and conformational changes account for the changes in the FCS signal in the former and fluorophore quenching in the latter. Within the experimental accuracy of our FCS measurements, we do not observe any change in diffusion due to activity for the enzymes we have investigated. We suggest useful control experiments and additional tests for future FCS experiments that should help establish if the observed diffusion enhancement is real or if it is due to an experimental or data analysis artifact. We show that fluorescence lifetime and mean intensity measurements are essential in order to identify the nature of the observed changes in the autocorrelation function. While it is clear from theory that chemically active enzymes should also act as self-propelled nanomotors, our FCS measurements show that the associated increase in diffusion is much smaller than previously reported. Further experiments are needed to quantify the contribution of the enzymes' catalytic activity to their self-propulsion. We hope that our findings help to establish a useful protocol for future FCS studies in this field and help establish by how much the diffusion of an enzyme is enhanced through catalytic activity.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. By time-correlation of the fluorescence fluctuations induced by molecules diffusing through a focused light, FCS can quantitatively evaluate the concentration, diffusion coefficient, and interaction of the molecules in vitro or in vivo. In this review, the basic principle and implementation of FCS are introduced. Then, the advances of FCS variants are reviewed, covering dual-color FCCS, multi-focus FCS, pair correlation function (pCF), scanning FCS, focus-reduced FCS, SPIM-FCS, and inverse-FCS. Besides, the applications of FCS are demonstrated with the measurement of local concentration, hydrodynamic radius, diffusion coefficient, and the interaction of different molecules. Lastly, a discussion is given by summarizing the pros and cons of different FCS techniques, as well as the outlooks and perspectives of FCS.

Fluorescence correlation spectroscopy (FCS) is an ideal analytical tool for studying concentrations, propagation, interactions and internal dynamics of molecules at nanomolar concentrations in living cells. FCS analyzes minute fluorescence-intensity fluctuations about the equilibrium of a small ensemble (<10(3)) of molecules. These fluctuations act like a 'fingerprint' of a molecular species detected when entering and leaving a femtoliter-sized optically defined observation volume created by a focused laser beam. In FCS the fluorescence fluctuations are recorded as a function of time and then statistically analyzed by autocorrelation analysis. The resulting autocorrelation curve yields a measure of self-similarity of the system after a certain time delay, and its amplitude describes the normalized variance of the fluorescence fluctuations. By fitting the curves to an appropriate physical model, this method provides precise information about a multitude of measurement parameters, including diffusion coefficients, local concentration, states of aggregation and molecular interactions. FCS operates in real time with diffraction-limited spatial and sub-microsecond temporal resolution. Assessing diverse molecular dynamics within the living cell is a challenge well met by FCS because of its single-molecule sensitivity and high dynamic resolution. For these same reasons, however, intracellular FCS measurements also harbor the large risk of collecting artifacts and thus producing erroneous data. Here we provide a step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell. A discussion of advantages and disadvantages of one-photon versus two-photon excitation for FCS is available in Supplementary Methods online.

Decision letter: The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model

Fluorescence correlation spectroscopy (FCS) has been extensively applied to study the kinetics and photophysics of molecules as well as interactions between molecules by extracting information from the fluctuation of signals. In particular, single molecule applications of FCS promise the greatest amounts of information. Ideally, one would like to carry out FCS in real-time; however, due to the time-consuming nature of the correlation process, performing the correlation in real-time is totally nontrivial. Generally an expensive hardware correlator or a TCSPC board is required for this purpose. Recently highly-efficient algorithms based on multi-tau method have been proposed to build up a software correlator. In this work, we set forth an innovative algorithm capable of realizing the real-time correlation, without turning to the multi-tau method. This algorithm takes advantage of the low count rate generally existing in the FCS experiments, directly using the time interval between each photon its adjacent photon to efficiently update the correlation function. Based on this efficiency, it is possible to build a low-cost software correlator with just an ordinary counter board. We practically demonstrate the feasibility by setting up this correlator to measure the diffusion motion of rhodamine 6G in water using FCS. The algorithm was validated by duplicating the signal from the photon detector and sending it to both the ordinary counter board with our software correlator and a commercial correlator simultaneously. The perfect coincidence of the correlation curves from these two correlators and the real-time display of the correlation function indicate the validity and practicability of our approach.

The fluorescence correlation spectroscopy (FCS) has become a powerful tool that entails the information about molecules at very minute concentrations in the biological system. With the advances in the laser technology and confocal microscopy, the applications of FCS have been extended to the studies of protein dynamics in living cells as well as drug-screening. Two assumptions are made in FCS: the biological system under study is in its equilibrium state and the molecules diffuse within the system freely according to Brownian motions. Fluorescence intensity fluctuations occur when the fluorescent molecule moving in and out of the confocal microscopy defined detection volume in which bursts of photons are emitted. Based on the assumptions above, the statistical-physics-based autocorrelation function of the fluorescence intensity fluctuations was formulated by Aragon and Pecora, which provides the information about the dynamics of the fluorescent macromolecules in the system. However, in this manuscript, we demonstrate that the temporal autocorrelation function of Aragon and Pecora was inadequately derived due to the fact that the process of the positions of a molecule in the system over time is not a stationary Gaussian process. Efforts are made here to derive a new version of the autocorrelation function of the temporal fluorescence intensity fluctuation. The fit of the new autocorrelation function will be compared with that of Aragon and Pecora.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation–modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.

The function of living cells is based on chemical reactions. It has been shown that the velocity of these reactions is limited by the molecular transport in the cell. Therefore also the spatial organization of a cell plays a major role.&#13;\n In order to investigate such transport processes, fluorescence correlation spectroscopy (FCS) is often used in combination with fluorescently labeled proteins. In FCS a small subvolume of the cell (~1µm³) is observed with a laser-based microscope. The fluctuations of the fluorescence, emitted from this subvolume, are acquired. An autocorrelation analysis of these fluctuations reveals the concentrations and diffusion coefficients of the labeled particles.&#13;\nUsually, FCS is implemented using a confocal microscope, which can observe only a single spot at any time. &#13;\nFor this thesis, FCS was extended to an imaging method, by combining it with light sheet fluorescence microscopy (SPIM). &#13;\nThis relatively new widefield microscopy technique allows to observe an arbitrarily positionable, thin plane (diameter: 1-3µm) in the cell.&#13;\n By using a fast electron-multiplying charge-coupled device camera, the combination of SPIM and FCS allowed to map the motion also of relatively small autofluorescent proteins in living cells.&#13;\n&#13;\nAt first, the setup of a light sheet microscope is described. This microscope was designed and optimized for SPIM-FCS measurements in living cells.&#13;\nSeveral test measurements show the applicability of SPIM-FCS to in vitro samples and to all larger compartments of a living cell (nucleus, cytoplasm, cellular membrane).&#13;\nAfterwards, the usability of several commercially available cameras as image sensor for SPIM-FCS measurements is assessed. At the time of writing, EM-CCD cameras offer the best trade-off between photosensitivity and achievable temporal resolution (~ 500µs). In addition to these linear cameras, also the use of single-photon avalanche diode (SPAD) arrays is investigated. These offer a significantly better temporal resolution (1-10µs) than current EM-CCD cameras, which would render them the ideal image sensor for SPIM-FCS. However, they do not yet reach the photo-sensitivity of EM-CCDs. Two different SPAD arrays were characterized in detail and first successful SPIM-FCS measurements of solute fluorescent molecules could be demonstrated.&#13;\n&#13;\nIn a second step, SPIM-FCS was extended by a cross-correlation analysis (SPIM-FCCS), which allowed for the first time to map the interactions of differently labeled cytosolic molecules in living cells. For this purpose, the cross-correlation function between the fluorescence fluctuations from two different color channels is analyzed. A non-zero amplitude of this cross-correlation function is found only, if the differently labeled molecules interact and move together.&#13;\n&#13;\nFinally, the methods developed during this project were applied to different cellular systems. The mapping of the mobility of inert tracer molecules of different sizes allowed to measure the viscosity of the cytoplasm in different cells. A position-dependence of this mobility could only be found in the nucleoli. In addition, an important step in the remodelling cycle of the keratin intermediate filament system was investigated. As a third application, SPIM-F(C)CS measurements of different chromatin-associated proteins demonstrated the dynamics in the cellular nucleus. Mobility maps of labeled histone proteins revealed the organization of chromatin in interphase nuclei. In addition, the activity of the nuclear receptor RXR and a transcription factor were mapped.

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive tool in biochemistry and biophysics. It directly obtains physical parameters such as the average number of fluorescent molecules and their diffusion time in a tiny detection area. It also provides other useful information such as the brightness of molecules. Ultimately, it can give precise information about molecular interactions in the aqueous condition. In FCS experiments, the fluctuation of fluorescence emission intensity from the tiny detection area is monitored as a function of time. The monitored fluorescence fluctuation signals are transformed to an autocorrelation curve according to the autocorrelation calculator unit and the curves are then fitted to an appropriate physical model. This protocol outlines an FCS example involving a shift of the autocorrelation curve.

Fluorescence Correlation Spectroscopy (FCS) is a powerful single molecule technique for the study of the stability and the association dynamics of supramolecular systems and, in particular, of host–guest inclusion complexes. With FCS the host–guest binding equilibrium constant is determined analysing the variation in the diffusion coefficient of the fluorescent guest or host with no need for a change in the photophysical properties of the fluorescent probe. FCS gives also access to the association/dissociation rate constants of the host–guest inclusion providing that the fluorescence intensity of host or guest changes upon complexation. These rate constants can be compared with that of a diffusion-controlled process estimated from the same FCS experiment allowing for a better understanding of the association dynamics. The results show that cyclodextrin cavities act as “hard” cages which put geometric and orientational restrictions on the inclusion of a hydrophobic guest, whereas micelles behave as “soft” cages without geometrical requirements. In our contribution to this special issue we review briefly the application of FCS to the study of host–guest inclusion complexes with an emphasis on practical aspects and relevant bibliographic references.