Fluorescence Correlation Methods Research Articles

Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence Correlation Methods.

Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.

Variably Sized and Multi-Colored Silica-Nanoparticles Characterized by Fluorescence Correlation Methods for Cellular Dynamics.

Controlling the uptake of nanoparticles into cells so as to balance therapeutic effects with toxicity is an essential unsolved problem in the development of nanomedicine technologies. From this point of view, it is useful to use standard nanoparticles to quantitatively evaluate the physical properties of the nanoparticles in solution and in cells, and to analyze the intracellular dynamic motion and distribution of these nanoparticles at a single-particle level. In this study, standard nanoparticles are developed based on a variant silica-based nanoparticle incorporating fluorescein isothiocyanate (FITC) or/and rhodamine B isothiocyanate (RITC) with a variety of accessible diameters and a matching fluorescent cobalt ferrite core-shell structure (Fe2O4/SiO2). The physical and optical properties of the nanoparticles in vitro are fully evaluated with the complementary methods of dynamic light scattering, electron microscopy, and two fluorescence correlation methods. In addition, cell uptake of dual-colored and core/shell nanoparticles via endocytosis in live HeLa cells is detected by fluorescence correlation spectroscopy and electron microscopy, indicating the suitability of the nanoparticles as standards for further studies of intracellular dynamics with multi-modal methods.

Fluorescence Correlation Methods for Determining Absolute Numbers of Molecules from Microscopy Images

Fluorescence Correlation Methods for Determining Absolute Numbers of Molecules from Microscopy Images

Segmental dynamics of polymer by rotational fluorescence correlation microscopy.

The segmental dynamics of polymers is known to be closely related to the glass transition where the glass transition is the single most important parameter in its application. In this study, we designed an efficient and reliable experimental method to study the ensemble segmental dynamics of polymers by probing rotation of fluorescent molecules in the polymer matrix using a home-built microscope setup. The rotational dynamics of fluorescent molecules was analyzed using a fluorescence correlation method that extracts information through orthogonally polarized fluorescence images. From fluorescence intensities, autocorrelation functions (ACFs) were obtained in many areas simultaneously and by averaging several ACFs, well-defined ACF and precise experimental values were obtained from a single measurement movie. The robustness of the method and optimal experimental conditions were investigated by performing experiments with various probe concentrations, frame rates, and measurement lengths. By employing a home-built vacuum chamber, a wide temperature range was achieved, and we demonstrate the versatility and efficiency of imaging rotational FCM (fluorescence correlation microscopy) by probing segmental dynamics of different polymeric systems with glass transition temperature that differ by ≈100 K and with fragility ranging from 49 to 131. The imaging rotational FCM covers dynamics up to 4 orders of magnitude near the glass transition, and it was found that the rapidity of the stretching exponent β variation with temperature correlates with the fragility of polymers.

Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution.

We propose a hypothesis that explains two apparently contradicting observations for the heterogeneity of the unfolded proteins. First, the line confocal method of the single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy revealed that the unfolded proteins possess broad peaks in the FRET efficiency plot, implying the significant heterogeneity that lasts longer than milliseconds. Second, the fluorescence correlation method demonstrated that the unfolded proteins fluctuate in the time scale shorter than 100ns. To formulate the hypothesis, we first summarize the recent consensus for the structure and dynamics of the unfolded proteins. We next discuss the conventional method of the sm-FRET spectroscopy and its limitations for the analysis of the unfolded proteins, followed by the advantages of the line confocal method that revealed the heterogeneity. Finally, we propose that the structural heterogeneity formed by the local clustering of hydrophobic residues modulates the distribution of the long-range distance between the labeled chromophores, resulting in the broadening of the peak in the FRET efficiency plot. A clustering of hydrophobic residues around the chromophore might further contribute to the broadening. The proposed clusters are important for the understanding of protein folding mechanism.

Spot size variation FCS in simulations of the 2D Ising model

Spot variation fluorescence correlation spectroscopy (svFCS) was developed to study the movement and organization of single molecules in plasma membranes. This experimental technique varies the size of an illumination area while measuring correlations in time using standard fluorescence correlation methods. Frequently, this data is interpreted using the assumption that correlation measurements reflect the dynamics of single molecule motions, and not motions of the average composition. Here, we explore how svFCS measurements report on the dynamics of components diffusing within simulations of a 2D Ising model with a conserved order parameter. Simulated correlation functions report on both the fast dynamics of single component mobility and the slower dynamics of the average composition. Over a range of simulation conditions, a conventional svFCS analysis suggests the presence of anomalous diffusion even though single molecule motions are nearly Brownian in these simulations. This misinterpretation is most significant when the surface density of the fluorescent label is elevated, therefore we suggest future measurements be made over a range of tracer densities. Some simulation conditions reproduce qualitative features of published svFCS experimental data. Overall, this work emphasizes the need to probe membranes using multiple complimentary experimental methodologies in order to draw conclusions regarding the nature of spatial and dynamical heterogeneity in these systems.

Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase

Abstract AzoR is a homodimeric, flavin mononucleotide (FMN)-containing, NADH-dependent azoreductase from Escherichia coli . In this paper, we investigated the effect of the concentration of both AzoR and R59G on the spectral behavior of the bound FMN using two-dimensional fluorescence correlation spectra. Two cross peaks (530, 490) and (580, 530) were observed from the dilution-induced 2D asynchronous correlation map of wt AzoR, while only one cross peak appeared at (600, 530) for R59G mutant. This result indicated that the mutation at site 59 influenced the formation of dilution-induced intermediates. The specific activity of both AzoR and R59G mutant was unaffected by dilution when the enzyme concentration is below 1 μmol/L, which suggested that no significant dissociation of FMN occurred at low concentrations. Additionally, in order to explore the origin of these intermediates, we carried out a 2D correlation analysis using excitation wavelength-dependent fluorescence emission spectroscopy. The results showed that there coexisted two types of FMN that emitted fluorescence at 530 nm and 500 nm, respectively. Taken together, these results suggested that the 2D method is a very powerful method to identify the heterogeneous distribution of the bound FMN in solution.

A correlative approach at characterizing nanoparticle mobility and interactions after cellular uptake

The interactions of nanoparticles with human cells are of large interest in the context of nanomaterial safety. Here, we use live cell imaging and image-based fluorescence correlation methods to determine colocalization of 88 nm and 32 nm silica nanoparticles with endocytotic vesicles derived from the cytoplasmic membrane and lysosomes, as well as to quantify intracellular mobility of internalized particles, in contrast to particle number quantification by counting techniques. In our study, A549 cells are used as a model for human type II alveolar epithelial cells. We present data supporting endocytotic uptake of the particles and subsequent active transport to the perinuclear region. The presence of particles in lamellar bodies is proposed as a potential exocytosis route.

Comparison of Different Fluorescence Fluctuation Methods for their Use in FRET Assays: Monitoring a Protease Reaction

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head.

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Number analysis of fluorescence correlation spectroscopy for the cleaving process of fluorescence labeled DNA

The cleaving process of rhodamine labeled DNA with T7-exonuclease was monitored using the fluorescence correlation method. The fluorescence correlation function, formulated so as to take into account the fluorescence efficiency of the fragment, was derived to allow analysis of the two-component system. The reaction was well represented by the two-component model, allowing monitoring of the progressive cleavage of DNA. The analysis yielded not only the size of the polymer fragment but also the efficiency of the incorporation of rhodamine and the ratio of its quantum yield in DNA and in solution.

Femtosecond vibrational relaxation measurement of azulene using temporally incoherent light

The vibrational relaxation time within the first excited singlet state of azulene in ethylene glycol was measured by the two-photon fluorescence correlation method using temporally incoherent light. The relaxation time depends on the excitation wavelength for the S 1 state and is found to be 50 fs at 6170 Å and 300 fs at 6480 Å.

A variant method of correlation spectroscopy

The authors describe a mask correlation spectrometer operating in tandem with a gas filter analyzer and intended for the monitoring of iodine and other toxic gases in the atmosphere. An algorithm is constructed to demonstrate that in the proposed fluorescence-correlation method the recorded signal is proportional to the concentration of the analyzed path gas and to assess the sensitivity thresholds of the method. The authors also pursue the analogy between the fluorescence-correlation and gas-filter methods and compare their signal-to-noise ratios. Experimental data for the determination of iodine are presented.