In vivo quantitative studies of dynamic intracellular processes using fluorescence correlation spectroscopy

It has been a significant challenge to quantitatively study the dynamic intracellular processes in live cells. These studies are essential for a thorough understanding of the underlying mechanisms regulating the signaling pathways and the transitions between cell cycle stages. Our studies of Cdc20, an important mitotic checkpoint protein, throughout the cell cycle demonstrate that fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes. In this study, Cdc20 is found to be present primarily in a large complex (>1 Mda) during interphase with a diffusion constant of 1.8+/-0.1 microm2/s and a concentration of 76+/-24 nM, consistent with its association with the APC/C. During mitosis, however, a proportion of Cdc20 dissociates from APC/C at a rate of 12 pM/s into a soluble pool with a diffusion constant of 19.5+/-5.0 microm2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (approximately 40 nM) during chronic activation of the mitotic checkpoint but disappears during mitotic exit at a rate of 31 pM/s. The observed changes in the biochemical assembly states of Cdc20 closely correlate to the known temporal pattern of the activity of APC/CCdc20 in mitosis. Photon counting histograms reveal that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/CCdc20 throughout the cell cycle are discussed in light of our experimental observations.     

High-pressure fluorescence correlation spectroscopy

We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300 MPa. The fluctuation amplitude and the diffusion coefficient show a small pressure dependence. The changes of these parameters, which are on the order of 10%, are due to the pressure changes of the viscosity and the density of the aqueous medium.     

Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy.

The hybridization of fluorescently tagged 18mer deoxyribonucleotides with complementary DNA templates was analysed by fluorescence correlation spectroscopy (FCS) in a droplet under an epi-illuminated fluorescence microscope at the level of single molecules. The interaction can be monitored by the change in the translational diffusion time of the smaller (18mer) primer when binding to the bigger (7.5 kb) DNA containing the complementary sequence. The hybridization process in the presence of template M13mp18 ssDNA was monitored in a small volume (2 x 10(-16)I) at various temperatures. The Arrhenius plot of the association rate constant shows that the activation energy was 38.8 kcal/mol, but the hybridization process may involve several components. The titration experiment suggested that approximately 2 primers can be associated with one template DNA at 40 degrees C. Results of a simple homology search for the sequences complementary to the primer indicate the existence of additional sites of lower specificity.     

Scanning fluorescence correlation spectroscopy. II. Application to virus glycoprotein aggregation.

Scanning fluorescence correlation spectroscopy is a new approach to measuring changes in the state of aggregation of cell membrane proteins. Measurements of the mean number of aggregates of virus glycoproteins from Sindbis virus and vesicular stomatitis virus agree with the findings of a recent fluorescence photobleaching recovery study on the same systems (Johnson, D.C., M.J. Schlesinger, and E.L. Elson, 1981, Cell, 23:423-431). Sindbis Virus glycoproteins are immobilized and cannot be induced to aggregate further by antibody cross linking. In this study, we find that Sindbis virus glycoprotein is more highly aggregated than vesicular stomatitis virus glycoprotein, which can be patched further with antibody. These measurements demonstrate the potential of scanning fluorescence correlation spectroscopy in studies of aggregation problems in membranes of cultured cells.     

Supercritical angle fluorescence correlation spectroscopy

We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.     

Position-sensitive scanning fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.     

On the statistics of fluorescence correlation spectroscopy

I present a detailed statistical analysis of fluorescence correlation spectroscopy (FCS) which is a natural extension of an early work. This analysis more realistically takes account of the following issues. (1) A spatial Gaussian laser excitation of fluorescence, (2) the effect of a small number of fluorescent molecules in the observation volume, (3) the shot noise effect due to random emission of fluorescent photons, and (4) a hyperbolic form for the fluorescence autocorrelation function obtained in the case of diffusion. Based on these assumptions, the results differ from the earlier work in several respects, in particular, the dependence of the signal-to-noise ratio on sample concentration and the understanding of shot noise in fluorescence fluctuation moments.     

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.     

Detection method for quantifying global DNA methylation by fluorescence correlation spectroscopy

A method for quantifying global DNA methylation using fluorescence correlation spectroscopy (FCS) has been established. The single-molecule methylation assay (SMMA) is based on two methodologies. One methodology, FCS, estimates the translational diffusion coefficient of molecules in solution, whereas the other methodology uses the high affinity of methyl-CpG-binding domain protein 2 (MBD2) to bind specifically to methylated DNA. We studied the specific binding rates of fluorescence-labeled MBD2 and methylated DNA from biological samples using the automated FCS system. Using a standard curve with methylated control DNA, we developed the SMMA index to assess the global DNA methylation level of the biological samples. A marked decrease in the SMMA index was observed when human leukemia cell lines (U937 and K562) were cultured with DNA demethylating agents. Our findings clearly indicate the applicability of SMMA as a simple and rapid tool for quantifying global DNA methylation. SMMA may prove useful for genome-wide comparative methylation analyses of malignancies and as an indicator of the demethylation effects of epigenetic drugs.     

Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors

Transport parameters determine the access of drugs to tumors. However, technical difficulties preclude the measurement of these parameters deep inside living tissues. To this end, we adapted and further optimized two-photon fluorescence correlation microscopy (TPFCM) for in vivo measurement of transport parameters in tumors. TPFCM extends the detectable range of diffusion coefficients in tumors by one order of magnitude, and reveals both a fast and a slow component of diffusion. The ratio of these two components depends on molecular size and can be altered in vivo with hyaluronidase and collagenase. These studies indicate that TPFCM is a promising tool to dissect the barriers to drug delivery in tumors.     

Theory of sample translation in fluorescence correlation spectroscopy.

New applications of the technique of fluorescence correlation spectroscopy (FCS) require lateral translation of the sample through a focused laser beam (Peterson, N.O., D.C. Johnson, and M.J. Schlesinger, 1986, Biophys. J., 49:817-820). Here, the effect of sample translation on the shape of the FCS autocorrelation function is examined in general. It is found that if the lateral diffusion coefficients of the fluorescent species obey certain conditions, then the FCS autocorrelation function is a simple product of one function that depends only on transport coefficients and another function that depends only on the rate constants of chemical reactions that occur in the sample. This simple form should allow manageable data analyses in new FCS experiments that involve sample translation.     

Quantifying intracellular dynamics using fluorescence fluctuation spectroscopy

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

Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.     

Recent advances in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy is a method in which fluctuations in the fluorescence arising from a very small sample volume are correlated to obtain information about the processes giving rise to the fluctuations. Recent progress has been made in methodologies such as two-photon excitation, photon counting histogram analysis, cross-correlation, image correlation and evanescent excitation. Fluorescence correlation spectroscopy techniques have been applied to several biological processes, including fluorescent protein photodynamics, binding equilibria and kinetics, protein oligomerization, nucleic acid interactions, and membrane and intracellular dynamics.     

Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.

Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods.