Quantifying intracellular dynamics using fluorescence fluctuation spectroscopy

Originally developed for the field of physical chemistry, fluorescence fluctuation spectroscopy (FFS) has evolved to a family of methods to quantify concentrations, diffusion rates and interactions of fluorescently labelled molecules. The possibility to measure at the nanomolar concentration level and to combine these techniques with microscopy allow to study biological processes with high sensitivity in the living cell. In this review, the basic principles, challenges and recent developments of the most common FFS methods are being discussed and illustrated by intracellular applications.     

Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy

We introduce a new analysis technique for fluorescence fluctuation data. Time-integrated fluorescence cumulant analysis (TIFCA) extracts information from the cumulants of the integrated fluorescence intensity. TIFCA builds on our earlier FCA theory, but in contrast to FCA or photon counting histogram (PCH) analysis is valid for arbitrary sampling times. The motivation for long sampling times lies in the improvement of the signal/noise ratio of the data. Because FCA and PCH theory are not valid in this regime, we first derive a theoretical model of cumulant functions for arbitrary sampling times. TIFCA is the first exact theory that describes the effects of sampling time on fluorescence fluctuation experiments. We calculate factorial cumulants of the photon counts for various sampling times by rebinning of the original data. Fits of the data to models determine the brightness, the occupation number, and the diffusion time of each species. To provide the tools for a rigorous error analysis of TIFCA, expressions for the variance of cumulants are developed and tested. We demonstrate that over a limited range rebinning reduces the relative error of higher order cumulants, and therefore improves the signal/noise ratio. The first four cumulant functions are explicitly calculated and are applied to simple dye systems to test the validity of TIFCA and demonstrate its ability to resolve species.     

Cumulant analysis in fluorescence fluctuation spectroscopy

A novel technique for the analysis of fluorescence fluctuation experiments is introduced. Fluorescence cumulant analysis (FCA) exploits the factorial cumulants of the photon counts and resolves heterogeneous samples based on differences in brightness. A simple analytical model connects the cumulants of the photon counts with the brightness epsilon and the number of molecules N in the optical observation volume for each fluorescent species. To provide the tools for a rigorous error analysis of FCA, expressions for the variance of factorial cumulants are developed and tested. We compare theory with experiment by analyzing dye mixtures and simple fluorophore solutions with FCA. A comparison of FCA with photon-counting histogram (PCH) analysis, a related technique, shows that both methods give identical results within experimental uncertainty. Both FCA and PCH are restricted to data sampling times that are short compared to the diffusion time of molecules through the observation volume of the instrument. But FCA theory, in contrast to PCH, can be extended to treat arbitrary sampling times. Here, we derive analytical expressions for the second factorial cumulant as a function of the sampling time and demonstrate that the theory successfully models fluorescence fluctuation data.     

Brownian dynamics simulations of fluorescence fluctuation spectroscopy

We have developed a program for the simulation of the fluorescence fluctuations as detected from highly diluted samples of (bio)molecules. The model is applied to translational diffusion and takes into account the hydrodynamic interactions. The solution concentration is kept constant by assuming periodic boundary conditions and spans here the range 0.5< C < 10 nM. We show that the fluorescence correlation functions can be accurately computed on systems of limited size (a few molecules per simulation box) by simulating for a total time approximately 100-300 times the diffusion relaxation time of the fluorescence autocorrelation function. The model is applied also to the simulation of the scanning fluorescence correlation spectroscopy (FCS) and of the photon counting histograms for the confocal collection configuration. Scanning FCS simulations of highly diluted samples (C approximately equals 0.5 nM) show anticorrelation effects in the autocorrelation functions of the fluorescence signal that are less evident for higher concentrations. We suggest here that this effect may be due to the non-uniform occupancy of the scanning area by the fluorophores.     

The photon counting histogram in fluorescence fluctuation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution.     

Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.     

Stochastic fractal behavior in concentration fluctuation and fluorescence correlation spectroscopy.

Fluctuations in the concentration of Brownian particles in one and two dimensions, or any reasonable measurement of the concentration such as in fluorescence correlation spectroscopy, is shown to be a stochastic fractal with a long tail. Being singular at omega = 0, the power spectrum of the fluctuation S(omega) approximately omega-1/2 for diffusion in one dimension, approximately log omega in two dimensions, but non-singular in three dimensions. This discovery provides one simple physical mechanism for possible long-memory fractal behavior, and its implications to various biological processes are discussed.     

The photon counting histogram in fluorescence fluctuation spectroscopy with non-ideal photodetectors

Fluorescence fluctuation spectroscopy utilizes the signal fluctuations of single molecules for studying biological processes. Information about the biological system is extracted from the raw data by statistical methods such as used in fluctuation correlation spectroscopy or photon counting histogram (PCH) analysis. Since detectors are never ideal, it is crucial to understand the influence of photodetectors on signal statistics to correctly interpret the experimental data. Here we focus on the effects of afterpulsing and detector dead-time on PCH statistics. We determine the dead-time and afterpulse probability for our detectors experimentally and show that afterpulsing can be neglected for most experiments. Dead-time effects on the PCH are concentration-dependent and become significant when more than one molecule is present in the excitation volume. We develop a new PCH theory that includes dead-time effects and verify it experimentally. Additionally, we derive a simple analytical expression that accurately predicts the effect of dead-time on the molecular brightness. Corrections for non-ideal detector effects extend the useful concentration range of PCH experiments and are crucial for the interpretation of titration and dilution experiments.     

SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy

Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.     

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.     

DNA-induced polymerization of HIV-1 integrase analyzed with fluorescence fluctuation spectroscopy

Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 microm(-1) under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 +/- 0.6 x 10(8) m(-1). Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.     

Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy

Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.     

Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy

Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.     

Visualization of mismatch repair complexes using fluorescence microscopy

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients.During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5′ and 3′ nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed.Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.     

Endothelin receptor in virus-like particles: ligand binding observed by fluorescence fluctuation spectroscopy

The functional analysis of transmembrane receptor proteins is frequently hampered by the difficulty to produce sufficiently homogeneous receptor preparations that preserve the physiological biomembrane integration of the receptor protein. To improve the receptor protein density in the lipid bilayer and to maintain the physiological lipid-protein environment, a novel method has been established that enables the selective integration of transmembrane receptors into a virus-like particle (VLiP). Here we have studied the binding of tetramethylrhodamine-labeled endothelin-1 (TMR-ET-1) to VLiP-integrated endothelin A receptor (ET(A)R) by fluorescence fluctuation spectroscopy. The concentration of TMR-ET-1 was determined by fluorescence correlation spectroscopy (FCS). These measurements also confirmed that the free ligand is monomeric in solution in our experiments. Fluorescence intensity distribution analysis (FIDA) was used to quantify the fraction of ligands bound to ET(A)Rs in the VLiPs. For the interaction between ET-1 and VLiP-integrated ET(A)Rs, K(D) values of 0.5 nM and 0.3 nM were determined from ligand and receptor titration experiments, respectively. For comparison, a FIDA analysis was also carried out with ET(A)Rs in membrane fragments derived from an ET(A)R-overexpressing mammalian cell line, which yielded a similar K(D) of 0.2 nM. In addition, we examined the binding competition of a set of reference compounds to VLiP-ET(A)Rs in the presence of ET-1 and obtained K(i) values similar to those reported in the literature. Our results demonstrate that integration into VLiPs does not change the binding properties of the ET(A)Rs. FIDA analysis of VLiP-integrated receptors shows great promise for highly miniaturized and fast compound testing in the pharmaceutical industry.     

A peripheral pacemaker drives the circadian rhythm of synaptic boutons in Drosophila independently of synaptic activity

Circadian rhythms in the morphology of neurons have been demonstrated in the fly Drosophila melanogaster. One such rhythm is characterized by changes in the size of synaptic boutons of an identified flight motor neuron, with larger boutons during the day compared with those at night. A more detailed temporal resolution of this rhythm shows here that boutons grow at a time of increased locomotor activity during the morning but become gradually smaller during the day and second period of increased locomotor activity in the evening. We have experimentally manipulated the synaptic activity of the fly during short periods of the day to investigate whether changes in bouton size might be a consequence of the different levels of synaptic activity associated with the locomotion rhythm of the fly. In the late night and early morning, when the flies normally have an intense period of locomotion, the boutons grow independently of whether the flies are active or completely paralyzed. Bouton size is not affected by sleep-deprivation during the early night. The cycle in bouton size persists for 2 days even in decapitated flies, which do not move, reinforcing the notion that it is largely independent of synaptic activity, and showing that a pacemaker other than the main biological clock can drive it.     

Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.     

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.