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.     

Recent applications of fluorescence correlation spectroscopy in live systems

Fluorescence correlation spectroscopy (FCS) is a widely used technique in biophysics and has helped address many questions in the life sciences. It provides important advantages compared to other fluorescence and biophysical methods. Its single molecule sensitivity allows measuring proteins within biological samples at physiological concentrations without the need of overexpression. It provides quantitative data on concentrations, diffusion coefficients, molecular transport and interactions even in live organisms. And its reliance on simple fluorescence intensity and its fluctuations makes it widely applicable. In this review we focus on applications of FCS in live samples, with an emphasis on work in the last 5 years, in the hope to provide an overview of the present capabilities of FCS to address biologically relevant questions.     

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.     

Looking into live cells with in-cell NMR spectroscopy

In-cell NMR spectroscopy has gained recent popularity since it provides means to analyze the conformational and functional properties of proteins inside living cells and at atomic resolution. High-resolution in-cell NMR spectroscopy was originally established in bacterial cells and based on a rationale that relies on protein over-expression and sample analysis within the same cellular environment. Here, we review in-cell NMR approaches in Xenopus laevis oocytes and evaluate potential future applications in other eukaryotic cell types.     

Transport and accumulation of PVP-Hypericin in cancer and normal cells characterized by image correlation spectroscopy techniques

PVP-Hypericin (PVP: polyvinylpyrrolidone) is a potent anti-cancer photosensitizer for photodynamic diagnosis (PDD) and therapy (PDT). However, cellular targets and mechanisms involved in the cancer-selectivity of the photosensitizer are not yet fully understood. This paper gives new insights into the differential transport and localization of PVP-Hypericin in cancer and normal cells which are essential to unravel the mechanisms of action and cancer-selectivity. Temporal (TICS) and spatiotemporal (STICS) image correlation spectroscopy are used for the assessment of PVP-Hypericin diffusion and/or velocity in the case of concerted flow in human cervical epithelial HeLa and human lung carcinoma A549 cells, as well as in human primary dendritic cells (DC) and human peripheral blood mononuclear cells (PBMC). Spatiotemporal image cross-correlation spectroscopy (STICCS) based on organelle specific fluorescent labeling is employed to study the accumulation of the photosensitizer in nucleus, mitochondria, early-endosomes and lysosomes of the cells and to assess the dynamics of co-migrating molecules. Whereas STICS and TICS did not show a remarkable difference between the dynamics of PVP-Hypericin in HeLa, A549 and DC cells, a significantly different diffusion rate of the photosensitizer was measured in PBMC. STICCS detected a stationary accumulation of PVP-Hypericin within the nucleus, mitochondria, early endosomes and lysosomes of HeLa and A549 cells. However, significant flow due to the directed motion of the organelles was detected. In contrast, no accumulation in the nucleus and mitochondria of DC and PBMC could be monitored.     

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.     

Accessing molecular dynamics in cells by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffraction-limited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.     

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.     

NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor-fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm.     

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.     

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.     

The Sendai virus membrane fusion mechanism studied using image correlation spectroscopy.

The mechanism of Sendai virus membrane fusion to cultured cell membranes was studied. Viral lipids were labeled with the lipophilic dye, 4-(4-(dihexadecylamino)styryl-N-methylquinolinium iodine) (DiQ), and viral proteins were labeled using fluorescein isothiocyanate (FITC). The redistribution of these probes from the virus to cultured cells was followed using the technique of image correlation spectroscopy. This technique assayed the intensity change and the redistribution of these probes as fusion progressed from a more to less aggregated state. The lipid probe DiQ dispersed into the membrane of the target membrane at both 22 and 37 degrees C, while the FITC-labeled proteins dispersed only at 37 degrees C. Simultaneous labeling of virus with both of these probes showed that at 37 degrees C their redistribution proceeded at different rates. These data were consistent with the formation of a hemifusion intermediate during the fusion process.     

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.