Literature Fluorescence Correlation Spectroscopy (FCS)

Anhut, T., K. Hassler, T. Lasser, K. Koenig and R. Rigler (2005). Fluorescence Correlation Spectroscopy on Dielectric Surfaces in Total Internal Reflection Geometries. Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications III, San Jose, CA, USA, SPIE.
External Literature Link Download PDF

Aragon, S. R. and R. Pecora (1976). "Fluorescence Correlation Spectroscopy as a Probe of Molecular Dynamics." Journal of Chemical Physics 64(4): 1791-1803.
Download PDF

Banks, D. S. and C. Fradin (2005). "Anomalous Diffusion of Proteins Due to Molecular Crowding." Biophysical Journal 89: 2960-2971.
Download PDF

We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent characterizing the dependence of the mean-square displacement of the tracer proteins on time, r²(t)" src="/math/rang.gif" border=0 t. We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive ( < 1) even at low obstacle concentration. The anomalous diffusion exponent decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, reaches a limit value of _l 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion.

Bieschke, J., A. Giese, W. Schulz-Schaeffer, I. Zerr, S. Poser, M. Eigen and H. Kretzschmar (2000). "Ultrasensitive Detection of Pathological Prion Protein Aggregates by Dual-Color Scanning for Intensely Fluorescent Targets." PNAS 97(10): 5468-5473.
External Literature Link Download PDF

Campbell, R. E., O. Tour, A. E. Palmer, P. A. Steinbach, G. S. Baird, D. A. Zacharias and R. Y. Tsien (2002). "A Monomeric Red Fluorescent Protein." Proceedings of the National Academy of Sciences of the United States of America 99(12): 7877-7882.
External Literature Link Download PDF

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ?25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

Chambenoit, O., Y. Hamon, D. Marguet, H. Rigneault, M. Rosseneu and G. Chimini (2001). "Specific Docking of Apolipoprotein a-I at the Cell Surface Requires a Functional Abca1 Transporter."
Download PDF

Chanda, B., R. Blunck, L. C. Faria, F. E. Schweizer, I. Mody and F. Bezanilla (2005). "A Hybrid Approach to Measuring Electrical Activity in Genetically Specified Neurons." 8(11): 1619-1626.
External Literature Link Download PDF

Chen, I., M. Howarth, W. Lin and A. Y. Ting (2005). "Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes Using Biotin Ligase." Nat Methods 2: 99-104.
External Literature Link Download PDF

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.

Chen, I. and A. Y. Ting (2005). "Site-Specific Labeling of Proteins with Small Molecules in Live Cells." Current Opinion in Biotechnology 16(1 SPEC. ISS.): 35-40.
Download PDF

The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest. Recently developed methods for labeling proteins with small-molecule probes in cells employ special protein or peptide handles that recruit small-molecule ligands, harness enzymes to catalyze small-molecule conjugation or hijack the cell's protein translation machinery. © 2005 Elsevier Ltd. All rights reserved.

Chen, Y., L.-N. Wei and J. D. Muller (2005). "Unraveling Protein-Protein Interactions in Living Cells with Fluorescence Fluctuation Brightness Analysis." Biophys. J. 88(6): 4366-4377.
External Literature Link Download PDF

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Costantino, S., J. W. D. Comeau, D. L. Kolin and P. W. Wiseman (2005). "Accuracy and Dynamic Range of Spatial Image Correlation and Cross-Correlation Spectroscopy." Biophys. J. 89(2): 1251-1260.
External Literature Link Download PDF

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 {micro}m-2 within a 121 x 98 pixel subregion of a single cell.

Web Links:
PubMed
Scopus

Demaurex, N. (2005). "Calcium Measurements in Organelles with Ca2+-Sensitive Fluorescent Proteins." Cell Calcium 38(3-4): 213-222.
External Literature Link

The recent improvement in the design and use of genetically encoded fluorescent Ca2+ indicators should foster major progress in three aspects of Ca2+ signaling. At the subcellular level, ratiometric probes with expanded dynamics are now available to measure accurately the local Ca2+ changes occurring within specific cell compartments. These tools will allow to determine precisely the role of organelles and of cellular microdomains in Ca2+ handling. At the cellular level, the permanent labeling offered by the genetic probes enables large-scale, long-term Ca2+ measurements with robotic multiplexing technologies such as fluorescence plate readers or automated microscopes. This opens the way to large-scale pharmacological or genetic screens based on organelle-specific functional assays. At the whole animal level, probes with improved dynamics and reduced interference with endogenous proteins will allow to generate transgenic animals bearing Ca2+ sensitive indicators in specific cells and tissues. With this approach, Ca2+ signals can be recorded in neurons, heart, and pancreas of live animals during physiological and pathological stimulations. In this chapter, I will review the progress made in the design and use of genetic Ca2+ indicators, and discuss how these new tools provide an opportunity to challenge several unsolved questions in Ca2+ signaling.

Digman, M. A., C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz and E. Gratton (2005). "Measuring Fast Dynamics in Solutions and Cells with a Laser Scanning Microscope." Biophys. J. 89(2): 1317-1327.
External Literature Link Download PDF

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

Digman, M. A., P. Sengupta, P. W. Wiseman, C. M. Brown, A. R. Horwitz and E. Gratton (2005). "Fluctuation Correlation Spectroscopy with a Laser-Scanning Microscope: Exploiting the Hidden Time Structure." Biophys. J. 88(5): L33-36.
External Literature Link Download PDF

Images obtained with a laser-scanning microscope contain a time structure that can be exploited to measure fast dynamics of molecules in solution and in cells. The spatial correlation approach provides a simple algorithm to extract this information. We describe the analysis used to process laser-scanning images of solutions and cells to obtain molecular diffusion constant in the microsecond to second timescale.

Dittrich, P. S., S. P. Schafer and P. Schwille (2005). "Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level." Biophys. J. 89(5): 3446-3455.
External Literature Link Download PDF

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with{lambda} = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser ({lambda} = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at {lambda} = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s-1 for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.

Doi, N., H. Takashima, M. Kinjo, K. Sakata, Y. Kawahashi, Y. Oishi, R. Oyama, E. Miyamoto-Sato, T. Sawasaki, Y. Endo and H. Yanagawa (2002). "Novel Fluorescence Labeling and High-Throughput Assay Technologies for in Vitro Analysis of Protein Interactions." Genome Research 12(3): 487-492.
Download PDF

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

Elson, E. L. (2001). "Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells." Traffic 2(11): 789-796.
External Literature Link Download PDF

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.

Elson, E. L. (2004). "Quick Tour of Fluorescence Correlation Spectroscopy from Its Inception." Journal of Biomedical Optics 9(5): 857-864.
External Literature Link Download PDF

Elson, E. L. and D. Magde (1974). "Fluorescence Correlation Spectroscopy. I. Conceptual Basis and Theory." Biopolymers 13(1): 1-27.
Download PDF

Evanko, D. (2005). "Focus on Fluorescence Imaging." Nature Methods 2(12): 901.
Download PDF

Flusberg, B. A., E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung and M. J. Schnitzer (2005). "Fiber-Optic Fluorescence Imaging." 2(12): 941-950.
External Literature Link Download PDF

Gosch, M., H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, A. Magnusson and S. Hrd (2005). "Parallel Dual-Color Fluorescence Cross-Correlation Spectroscopy Using Diffractive Optical Elements." Journal of Biomedical Optics 10(5): 054008-7.
External Literature Link Download PDF

Griesbeck, O. (2004). "Fluorescent Proteins as Sensors for Cellular Functions." Current Opinion in Neurobiology 14(5): 636-641.
External Literature Link Download PDF

Griesbeck, O., G. S. Baird, R. E. Campbell, D. A. Zacharias and R. Y. Tsien (2001). "Reducing the Environmental Sensitivity of Yellow Fluorescent Protein. Mechanism and Applications." Journal of Biological Chemistry 276(31): 29188-29194.
External Literature Link Download PDF

Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 &°C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK? (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 &°C and in organelles. The halide resistance is explained by a 2.2-A? x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca2+] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

Web Links:
PubMed
Scopus

Haupts, U., S. Maiti, P. Schwille and W. W. Webb (1998). "Dynamics of Fluorescence Fluctuations in Green Fluorescent Protein Observed by Fluorescence Correlation Spectroscopy." PNAS 95(23): 13573-13578.
External Literature Link Download PDF

Haustein, E. and P. Schwille (2004). "Single-Molecule Spectroscopic Methods." Current Opinion in Structural Biology 14(5): 531-540.
External Literature Link Download PDF

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms.

Hebert, B., S. Costantino and P. W. Wiseman (2005). "Spatiotemporal Image Correlation Spectroscopy (Stics) Theory, Verification, and Application to Protein Velocity Mapping in Living Cho Cells." Biophys. J. 88(5): 3601-3614.
External Literature Link Download PDF

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 {micro}m/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of {micro}m/min) and diffusion of interacting{alpha} 5 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.

Heim, N. and O. Griesbeck (2004). "Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein." J Biol Chem 279: 14280-6.
External Literature Link Download PDF

Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K(d)s ranging from 470 nm to 29 microm, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.

Hess, S. T., E. D. Sheets, A. Wagenknecht-Wiesner and A. A. Heikal (2003). "Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells." Biophys. J. 85(4): 2566-2580.
External Literature Link Download PDF

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within [~]10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 {+/-} 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the {beta}-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.

Hess, S. T. and W. W. Webb (2002). "Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy." Biophys. J. 83(4): 2300-2317.
External Literature Link Download PDF

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 {micro}s to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as ~80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (~4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.

Howarth, M., K. Takao, Y. Hayashi and A. Y. Ting (2005). "Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase." Proc Natl Acad Sci U S A 102: 7583-8.
External Literature Link Download PDF

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody-antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse-chase labeling.

Hwang, L. C. and T. Wohland (2004). "Dual-Color Fluorescence Cross-Correlation Spectroscopy Using Single Laser Wavelength Excitation." ChemPhysChem 5(4): 549-551.
External Literature Link Download PDF

Spotlight on interactions: The authors show that single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS) is possible using only one laser beam to excite a combination of labels, in work performed with studied FRET (fluorescence resonance energy transfer) dyes and quantum dots. They demonstrate that equilibrium binding and stoichiometry can easily be determined (see graphic; QR = quantum red dye, BF = fluorescein-labeled biotin). SW-FCCS is a promising tool for high-throughput screening applications as well as for the measurement of molecular interactions.

Hwang, L. C. and T. Wohland (2005). "Single Wavelength Excitation Fluorescence Cross-Correlation Spectroscopy with Spectrally Similar Fluorophores: Resolution for Binding Studies." Journal of Chemical Physics 122(11): 1-11.
External Literature Link Download PDF

It was shown recently that fluorescence cross-correlation spectroscopy (FCCS) can be performed using a single laser wavelength for excitation (SW-FCCS) [L. C. Hwang and T. Wohland, Chem. Phys. Chem 5, 549 (2004).]. This method simplifies the FCCS setup since it does not require the simultaneous alignment of two lasers to the same focal spot. But up to now the method was shown to work only with dyes possessing large Stokes' shifts, and thus was limited to the use of quantum dots and tandem dyes. In this work we show that standard organic dyes with overlapping emission spectra, for instance fluorescein and tetramethylrhodamine, can be used as fluorescent pairs in SW-FCCS. As a biological model system for ligand-receptor interactions we studied the binding of biotin to streptavidin. To investigate the applicability of SW-FCCS for binding studies we adapt the existing FCCS theory for SW-FCCS and calculate limits for the measurement of dissociation constants in dependence on sample concentration, sample purity, and spectral cross talk between the different detection channels. © 2005 American Institute of Physics.

Kapuscinski, J. (1995). "Dapi: A DNA-Specific Fluorescent Probe." Biotech Histochem 70: 220-33.
External Literature Link Download PDF

DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

Kask, P., K. Palo, N. Fay, L. Brand, U. Mets, D. Ullmann, J. Jungmann, J. Pschorr and K. Gall (2000). "Two-Dimensional Fluorescence Intensity Distribution Analysis: Theory and Applications." Biophys. J. 78(4): 1703-1713.
External Literature Link Download PDF

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.

Kim, S. A., K. G. Heinze, K. Bacia, M. N. Waxham and P. Schwille (2005). "Two-Photon Cross-Correlation Analysis of Intracellular Reactions with Variable Stoichiometry." Biophys. J. 88(6): 4319-4336.
External Literature Link Download PDF

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated, and autophosphorylated forms of the enzyme. Key binding parameters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.

Kim, S. A. and P. Schwille (2003). "Intracellular Applications of Fluorescence Correlation Spectroscopy: Prospects for Neuroscience." Current Opinion in Neurobiology 13(5): 583-590.
External Literature Link Download PDF

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high spatial resolution. The introduction of dual-color cross-correlation, imaging, two-photon excitation, and coincidence analysis coupled with fluorescence correlation spectroscopy has expanded the utility of the technique to encompass a wide range of promising applications in living cells that may provide unprecedented insight into understanding the molecular mechanisms of intracellular neurobiological processes.

Kohler, R. H., P. Schwille, W. W. Webb and M. R. Hanson (2000). "Active Protein Transport through Plastid Tubules: Velocity Quantified by Fluorescence Correlation Spectroscopy." Journal of Cell Science 113(22): 3921-3930.
External Literature Link Download PDF

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 ?m/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

Kunst, B. H., A. Schots and A. J. Visser (2002). "Detection of Flowing Fluorescent Particles in a Microcapillary Using Fluorescence Correlation Spectroscopy." Anal Chem 74: 5350-7.
External Literature Link Download PDF

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.

Web Links:
PubMed
Scopus

Larson, D. R., W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. W. Wise and W. W. Webb (2003). "Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo." Science 300(5624): 1434-1436.
External Literature Link Download PDF

The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

Lukyanov, K. A., D. M. Chudakov, S. Lukyanov and V. V. Verkhusha (2005). "Photoactivatable Fluorescent Proteins." Nature Reviews Molecular Cell Biology 6(9).
Download PDF

The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.

Magde, D. and E. L. Elson (1974). "Fluorescence Correlation Spectroscopy. Ii. An Experimental Realization." Biopolymers 13: 29-61.
Download PDF

Magde, D. and E. L. Elson (1978). "Fluorescence Correlation Spectroscopy. Iii. Uniform Translation and Laminar Flow." Biopolymers 17: 361-376.
Download PDF

Maiti, S., H. Ulrich and W. W. Webb (1997). "Fluorescence Correlation Spectroscopy: Diagnostics for Sparse Molecules." Proc Natl Acad Sci U S A 94: 11753-11757.
Download PDF

Malvezzi-Campeggi, F., M. Jahnz, K. G. Heinze, P. Dittrich and P. Schwille (2001). "Light-Induced Flickering of Dsred Provides Evidence for Distinct and Interconvertible Fluorescent States." Biophys. J. 81(3): 1776-1785.
External Literature Link Download PDF

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (Matz et al., 1999), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.

Masuda, A., K. Ushida and T. Okamoto (2005). "New Fluorescence Correlation Spectroscopy Enabling Direct Observation of Spatiotemporal Dependence of Diffusion Constants as an Evidence of Anomalous Transport in Extracellular Matrices." Biophys. J. 88(5): 3584-3591.
External Literature Link Download PDF

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (Dobs), the size of the effective confocal volume (Veff) for FCS sampling (sampling volume) was continuously changed on a scale of 300-500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the Dobs of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in Veff (220-340 nm in the half-axis), because the network structure of HA of 7-33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount ([~]0.1 wt %) of HA in ECM. Because the change in Dobs along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.

Meseth, U., T. Wohland, R. Rigler and H. Vogel (1999). "Resolution of Fluorescence Correlation Measurements." Biophys. J. 76(3): 1619-1631.
External Literature Link Download PDF

The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution.

Miyamoto-Sato, E., N. Nemoto, H. Yanagawa and K. Kobayashi (2000). "Specific Bonding of Puromycin to Full-Length Protein at the C-Terminus." Nucleic Acids Research 28(5): 1176-1182.
Download PDF

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 ?M) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0.04-1.0 ?M bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

Nagai, T., S. Yamada, T. Tominaga, M. Ichikawa and A. Miyawaki (2004). "Expanded Dynamic Range of Fluorescent Indicators for Ca(2+) by Circularly Permuted Yellow Fluorescent Proteins." Proc Natl Acad Sci U S A 101: 10554-9.
External Literature Link Download PDF

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

Nagy, A., J. Wu and K. M. Berland (2005). "Observation Volumes and Gamma-Factors in Two-Photon Fluorescence Fluctuation Spectroscopy." Biophysical Journal 89: 2077-2090.
Download PDF

Nagy, A., J. Wu and K. M. Berland (2005). "Characterizing Observation Volumes and the Role of Excitation Saturation in One-Photon Fluorescence Fluctuation Spectroscopy." Journal of Biomedical Optics 10(4): 044015-9.
External Literature Link Download PDF

Nemoto, N., E. Miyamoto-Sato and H. Yanagawa (1999). "Fluorescence Labeling of the C-Terminus of Proteins with a Puromycin Analogue in Cell-Free Translation Systems." FEBS Letters 462(1-2): 43-46.
Download PDF

We have developed a new method for the C-terminus-specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C-terminus of mature proteins in cell-free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50-95%. Here we demonstrate a protein-protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy.

Web Links:
PubMed
Scopus

Octobre, G., C. Lemercier, S. Khochbin, M. Robert-Nicoud and C. Souchier (2005). "Monitoring the Interaction between DNA and a Transcription Factor (Mef2a) Using Fluorescence Correlation Spectroscopy." Comptes Rendus - Biologies 328(12): 1033-1040.
External Literature Link Download PDF

Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant KD for the full-length MEF2A was estimated. © 2005 Acade?mie des sciences. Published by Elsevier SAS. All rights reserved.

Pederson, T. (2001). "Protein Mobility within the Nucleus--What Are the Right Moves?" Cell 104(5): 635-638.
External Literature Link Download PDF

Piston, D. W., G. H. Patterson and S. M. Knobel (1999). "Quantitative Imaging of the Green Fluorescent Protein (Gfp)." Methods in cell biology 58: 31-48.
Download PDF

Qian, H. and E. L. Elson (1991). "Analysis of Confocal Laser-Microscopy Optics for 3-D Fluorescence Correlation Spectroscopy." Applied Optics 30(10): 1185-1195.
Download PDF

Rigler, R., Z. Foldes-Papp, F.-J. Meyer-Almes, C. Sammet, M. Volcker and A. Schnetz (1998). "Fluorescence Cross-Correlation: A New Concept for Polymerase Chain Reaction." Journal of Biotechnology 63: 97-109.
Download PDF

Rigler, R. and U. Mets (1993). Diffusion of Single Molecules through a Gaussian Laser Beam. Laser Spectroscopy of Biomolecules, Jyvaskyla, Finland, SPIE.
External Literature Link Download PDF

Rigler, R., U. Mets, J. Widengren and P. Kask (1993). "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background: Analysis of Translational Diffusion." European Biophysics Journal 22(3): 169-175.
External Literature Link Download PDF FCS User Club

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Rizzo, M. A., G. H. Springer, B. Granada and D. W. Piston (2004). "An Improved Cyan Fluorescent Protein Variant Useful for Fret." Nat Biotechnol 22: 445-9.
External Literature Link Download PDF

Many genetically encoded biosensors use Forster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Web Links:
PubMed
Scopus

Saffarian, S. and E. L. Elson (2003). "Statistical Analysis of Fluorescence Correlation Spectroscopy: The Standard Deviation and Bias." Biophysical Journal 84: 2030-2042.
Download PDF

Sakai, R., V. Repunte-Canonigo, C. D. Raj and T. Knopfel (2001). "Design and Characterization of a DNA-Encoded, Voltage-Sensitive Fluorescent Protein." European Journal of Neuroscience 13(12): 2314-2318.
External Literature Link Download PDF

Schenk, A., S. Ivanchenko, C. Rocker, J. Wiedenmann and G. U. Nienhaus (2004). "Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy." Biophys. J. 86(1): 384-394.
External Literature Link Download PDF

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.

Schwartz, J. W., G. Novarino, D. W. Piston and L. J. DeFelice (2005). "Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter." J. Biol. Chem. 280(19): 19177-19184.
External Literature Link Download PDF

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 {micro}s. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

Schwille, P. (2001). "Cross-Correlation Analysis in Fcs." Springer Series in Chemical Physics 65: 360-378.
External Literature Link