Cross talk free fluorescence cross correlation spectroscopy in live cells

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.     

Scanning image correlation spectroscopy

Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio-temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross-correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio-temporal map of molecular interactions.     

Fluorescence correlation spectroscopy in living cells

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.     

Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy

We report detailed studies of the dynamics of the mitochondrial reticulum in live cells using two independent experimental techniques: Fourier imaging correlation spectroscopy and digital video fluorescence microscopy. When both methods are used to study the same system, it is possible to directly compare measurements of preaveraged statistical dynamical quantities with their microscopic counterparts. This approach allows the underlying mechanism of the observed rates to be determined. Our results indicate that the dynamics of the reticulum structure is composed of two independent contributions, each important on very different time and length scales. During short time intervals (1-15 sec), local regions of the reticulum primarily undergo constrained thermally activated motion. During long time intervals (>15 sec), local regions of the reticulum undergo long-range "jump" motions that are associated with the action of cytoskeletal filaments. Although the frequency of the jumps depend on the physiological state of the cells, the average jump distance ( approximately 0.8 microm) is unaffected by metabolic activity. During short time intervals, the dynamics appear to be spatially heterogeneous, whereas the cumulative effect of the infrequent jumps leads to the appearance of diffusive motion in the limit of long time intervals.     

Multiplexed FRET to image multiple signaling events in live cells

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.     

Raman spectroscopy and imaging of beta-carotene in live corpus luteum cells

Raman spectroscopy has been used to identify and locate beta-carotene within individual living luteal cells. The cells were either freshly prepared or cultured; the latter was incubated in the presence or absence of beta-carotene in the form of enriched bovine high-density lipoprotein. Luteal cells were investigated using several Raman spectroscopic and imaging techniques. These techniques did not give accurate concentration levels of beta-carotene within parts of the cell but illustrated the distribution of the molecule. Freshly prepared luteal cells were found to contain an appreciable concentration of beta-carotene. Over a period of several days, the concentration gradually reduced to a nearly undetectable level; similar results were found for cells cultured in the absence of the beta-carotene. For cells cultured in the presence of beta-carotene, the molecular concentration was maintained for as long as 2 weeks. The Raman spectra of fragmented cells showed that the beta-carotene is predominantly localised in the lipid-rich cell components, with the concentration highest in the microsomal fraction. The Raman imaging techniques revealed that beta-carotene was spread over the entire volume of the luteal cells with higher levels occurring at distinct sites, including the surface.     

Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells

Imaging products of gene expression in live cells will provide unique insights into the biology of cells. Molecular beacons make attractive probes for imaging mRNA in live cells as they can report the presence of an RNA target by turning on the fluorescence of a quenched fluorophore. However, when oligonucleotide probes are introduced into cells, they are rapidly sequestered in the nucleus, making the detection of cytoplasmic mRNAs difficult. We have shown that if a molecular beacon is linked to a tRNA, it stays in the cytoplasm and permits detection of cytoplasmic mRNAs. Here we describe two methods of linking molecular beacons to tRNA and show how the joint molecules can be used for imaging an mRNA that is normally present in the cytoplasm in live cultured cells. This protocol should take a total of 4 d to complete.     

Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy

We present an extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS). Using simulations of laser scanning microscopy image time series, we investigate the effect of spatiotemporal sampling, particle density, noise, sampling frequency, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS. We show that the recovery of transport coefficients is usually limited by spatial sampling, while the measurement of accurate number densities is restricted by background noise in an image series. We also demonstrate that photobleaching of the fluorophore causes a consistent overestimation of diffusion coefficients and flow rates, and a severe underestimation of number densities. We derive a bleaching correction equation that removes both of these biases when used to fit temporal autocorrelation functions, without increasing the number of fit parameters. Finally, we image the basal membrane of a CHO cell with EGFP/alpha-actinin, using two-photon microscopy, and analyze a subregion of this series using TICS and apply the bleaching correction. We show that the photobleaching correction can be determined simply by using the average image intensities from the time series, and we use the simulations to provide good estimates of the accuracy and precision of the number density and transport coefficients measured with TICS.     

Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 microm(-2) within a 121 x 98 pixel subregion of a single cell.     

Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (L_o) and liquid disordered (L_d) phases. The abundance of the L_o phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the L_d or gel (S_o) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (E_Arr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the S_o–L_d phase are static and large while they are small and dynamic in the L_o–L_d phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313 K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity.     

Automated detection and recognition of live cells in tissue culture using image cytometry

An automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid-state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line. To distinguish cells from other detected objects, a recognition algorithm utilizing 18 characteristic cell signal features was developed. The algorithm first tests whether a set of feature values falls within specified upper and lower bounds, and then applies a linear discriminant function to the remaining data to further discriminate cells from debris. False-positive errors of 5% or less were achieved with this method, whereas 15-35% of cells were misclassified as debris.     

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.     

Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy

Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble beta-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.     

Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.     

Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.     

Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction

Image correlation spectroscopy allows sensitive measurement of the spatial distribution and aggregation state of fluorescent membrane macro molecules. When studying a single population system (i.e., aggregates of similar brightness), an accurate measure can be made of the aggregate number per observation area, but this measurement becomes much more complex in a distributed population system (i.e., bright and faint aggregates). This article describes an alternate solution that involves extraction of the bright aggregate population information. This novel development for image correlation spectroscopy, termed intensity subtraction analysis, uses sequential uniform intensity subtraction from raw confocal images. Sequential intensity subtraction results in loss of faint aggregate fluctuations that are smaller in magnitude than fluctuations due to the brightest aggregates. The resulting image has correlatable fluctuations originating from only the brightest population, permitting quantification of this population's distribution and further cross-correlation measurements. The feasibility of this technique is demonstrated using fluorescent microsphere images and biological samples. The technique is further used to examine the spatial distribution of a plasma-membrane-labeled fluorescent synthetic ganglioside, and to cross-correlate this probe with various membrane markers. The evidence provided demonstrates that bright aggregates of the fluorescent ganglioside are associated with clathrin-coated pits, membrane microvilli, and detergent-resistant membranes.     

Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells

Fluorescence correlation spectroscopy (FCS) allows the study of interactions of fluorescently labeled ligand with receptors in living cells at single-molecule detection sensitivity. From the autocorrelation functions of fluorescence intensity fluctuations, the diffusion time of molecules through the confocal volume is analyzed, and from that, the molecular weights of free and bound molecules can be calculated. We have applied FCS to study the receptor diversity for the neuropeptide galanin (GAL) in cultured cells. FCS measurement of the fluorophore rhodamine-labeled GAL (Rh-GAL) has been performed in 0.2-fL confocal volume elements of the laser beam. The analysis of autocorrelation functions of Rh-GAL in solution above cells and at cell membranes demonstrates that the diffusion time of unbound Rh-GAL is 0.16 ms, whereas diffusion times of membrane-bound Rh-GAL are 22 and 700 ms. Because both of the diffusion times (22 and 700 ms) are much longer as compared to that of unbound Rh-GAL, they correspond to slow-diffusing complexes when Rh-GAL is bound to the cell membranes. Addition of excess nonlabeled GAL is accompanied by competitive displacement. Full saturation of the GAL binding is obtained at nanomolar concentrations. Scatchard analysis of binding data reveal one binding process, assuming one binding site per Rh-GAL (n = 1). On the other hand, the appearance of two diffusion times, 22 and 700 ms, suggests the existence of two subpopulations of GAL receptor complexes or two subtypes of GAL receptor not detected before. This makes an important point that FCS permits the identification of receptors, which were not possible to detect before by conventional binding techniques. The inhibitory effect of pertussis toxin on the GAL binding considers a G-protein-involved allosteric system, important for the clarification of essential steps in the G-protein-related signal transduction. This study is of pharmaceutical significance, since it will provide insights into how FCS can be used as a rapid technique for studying ligand-receptor interactions in living cells, which is one step forward for large-scale drug screening in cell cultures.     

Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy

We investigate the challenges and limitations that are encountered when studying membrane protein dynamics in vivo by means of fluorescence correlation spectroscopy (FCS). Based on theoretical arguments and computer simulations, we show that, in general, the fluctuating fluorescence has a fractal dimension D(0) >or= 1.5, which is determined by the anomality alpha of the diffusional motion of the labeled particles, i.e., by the growth of their mean square displacement as (Deltax)(2) approximately t(alpha). The fractality enforces an initial power-law behavior of the autocorrelation function and related quantities for small times. Using this information, we show by FCS that Golgi resident membrane proteins move subdiffusively in the endoplasmic reticulum and the Golgi apparatus in vivo. Based on Monte Carlo simulations for FCS on curved surfaces, we can rule out that the observed anomalous diffusion is a result of the complex topology of the membrane. The apparent mobility of particles as determined by FCS, however, is shown to depend crucially on the shape of the membrane and its motion in time. Due to this fact, the hydrodynamic radius of the tracked particles can be easily overestimated by an order of magnitude.     

Effect of viscoelasticity on the analysis of single-molecule force spectroscopy on live cells

Single-molecule force spectroscopy is used to probe the kinetics of receptor-ligand bonds by applying mechanical forces to an intermediate media on which the molecules reside. When this intermediate media is a live cell, the viscoelastic properties can affect the calculation of rate constants. We theoretically investigate the effect of media viscoelasticity on the common assumption that the bond force is equal to the instantaneous applied force. Dynamic force spectroscopy is simulated between two cells of varying micromechanical properties adhered by a single bond with a constant kinetic off-rate. We show that cell and microvilli deformation, and hydrodynamic drag contribute to bond forces that can be 28-90% lower than the applied force for loading rates of 10(3)-10(7) pN/s, resulting in longer bond lifetimes. These longer bond lifetimes are not caused by changes in bond kinetics; rather, they are due to the mechanical response of the intermediate media on which the bonds reside. Under the assumption that the instantaneous bond force is equal to the applied force--thereby ignoring viscoelasticity--leads to 14-39% error in the determination of off-rates. We present an approach that incorporates viscoelastic properties in calculating the instantaneous bond force and kinetic dissociation parameter of the intermolecular bond.