Photon Counting Histogram Research Articles

Peak intensity analysis as a method for estimation of fluorescent probe binding to artificial and natural nanoparticles: Tetramethylrhodamine uptake by isolated mitochondria

A modified version of fluorescence correlation spectroscopy (FCS) closely related to the photon counting histogram (PCH) method, which is used in the case of a mixture of molecules with similar diffusion coefficients, was applied here for analyzing the binding of the potential-sensitive dye tetramethylrhodamine ethyl ester, TMRE, to isolated mitochondria both in energized and deenergized states. Fluorescence time traces of suspensions of TMRE-doped mitochondria representing sequences of peaks of different intensity appeared to be similar to those of fluorescent beads and TMRE-doped latex particles. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. The statistics of the brightness of identical fluorescent particles reflecting their random walk through the confocal volume was described by a simple analytical equation which enabled us to perform the peak intensity analysis (PIA) of TMRE-doped mitochondria. The validity of PIA was tested with fluorescent beads of different sizes and TMRE-doped latex particles. Mitochondrial energization in the presence of TMRE led to the increase in the number and the intensity of the peaks in fluorescence time traces, the PIA of which allowed us to determine mitochondrial membrane potential and additionally a number of mitochondrial particles per ml of the suspension. The value of the membrane potential on a single mitochondrion was estimated to be about 180 mV in agreement with the data related to mitochondrial suspensions. Importantly, the PIA method required less than 1 microgram of mitochondrial protein per measurement.

SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy.

Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.

Paxillin Dynamics Measured during Adhesion Assembly and Disassembly by Correlation Spectroscopy

Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.

Fluorescence Fluctuation Analysis of Arabidopsis thaliana Somatic Embryogenesis Receptor-Like Kinase and Brassinosteroid Insensitive 1 Receptor Oligomerization

Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.

Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor.

The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein transmembrane signaling complexes. Our approach helps in limiting the alteration of the data due to out-of-focus effects and in minimizing the overestimation of the molecular brightness. In addition to a careful design of the cellular model, it gives reliable estimates of diffusion coefficients and oligomerization of GPI-anchored proteins, in steady-state conditions, at low expression levels, and in live, unperturbed cells.

3P-033 フォトンカウンティングヒストグラム法と蛍光相関分光法を用いたAβ多量体形成の解析(蛋白質・機能,第46回日本生物物理学会年会)

3P-033 フォトンカウンティングヒストグラム法と蛍光相関分光法を用いたAβ多量体形成の解析(蛋白質・機能,第46回日本生物物理学会年会)

Concentration Effects in “Single-Molecule” Spectroscopy

A concentration as low as 0.1 molecule per observation volume may not be small enough for single-molecule Förster resonance energy transfer (FRET) efficiency measurements of molecules diffusing through a laser spot. This result follows from a rigorous theory that takes many molecules into account. We consider the distributions of the number of photons (photon counting histograms) and show that multiple-molecule effects are pronounced at large photon counts even at low concentrations. FRET efficiency distributions reveal multiple-molecule effects at large threshold values. This might be misinterpreted as multiple conformational states. Multiple-molecule effects strongly depend on the brightness of fluorophores. A simple test is suggested to determine parameters for which the single-molecule description is applicable.

Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.

Size Distribution of Linear and Helical Polymers in Actin Solution Analyzed by Photon Counting Histogram

Actin is a ubiquitous protein that is a major component of the cytoskeleton, playing an important role in muscle contraction and cell motility. At steady state, actin monomers and filaments (F-actin) coexist, and actin subunits continuously attach and detach at the filament ends. However, the size distribution of actin oligomers in F-actin solution has never been clarified. In this study, we investigated the size distribution of actin oligomers using photon-counting histograms. For this purpose, actin was labeled with a fluorescent dye, and the emitted photons were detected by confocal optics (the detection volume was of femtoliter (fL) order). Photon-counting histograms were analyzed to obtain the number distribution of actin oligomers in the detection area from their brightness, assuming that the brightness of an oligomer was proportional to the number of protomers. We found that the major populations at physiological ionic strength were 1–5mers. For data analysis, we successfully applied the theory of linear and helical aggregations of macromolecules. The model postulates three states of actin, i.e., monomers, linear polymers, and helical polymers. Here we obtained three parameters: the equilibrium constants for polymerization of linear polymers, K l = (5.2 ± 1.1) × 10 6 M −1, and helical polymers, K h = (1.6 ± 0.5) × 10 7 M −1; and the ratio of helical to linear trimers, γ = (3.6 ± 2.3) × 10 −2. The excess free energy of transforming a linear trimer to a helical trimer, which is assumed to be a nucleus for helical polymers, was calculated to be 2.0 kcal/mol. These analyses demonstrate that the oligomeric phase at steady state is predominantly composed of linear 1–5mers, and the transition from linear to helical polymers occurs on the level of 5–7mers.

A Comparison between Photon Counting Histogram and Fluorescence Intensity Distribution Analysis

Photon counting histogram (PCH) and fluorescence intensity distribution analysis (FIDA) are two methods that were developed independently but reported almost simultaneously. Both of them have been successfully applied to fluorescence fluctuation spectroscopy (FFS). Though publications have indicated that they are theoretically equivalent, they are still commonly considered as different methods, especially in their ways to treat the point spread functions (PSFs). In this paper, the two methods are examined in detail for a direct comparison. After a direct proof of the theoretical equivalence, the authors further point out that PCH and FIDA are completely equivalent in the way of modeling PSFs; that is, any modeling approach developed from one of them can always be applied to the other. It is also demonstrated that simplified FIDA and PCH formulas in the form of power series can be applied for fast and precise numerical calculations. The two methods are also compared for their merits in the calculation efficiency.

Dual-Color Photon Counting Histogram Analysis of mRFP1 and EGFP in Living Cells

We investigate the potential of dual-color photon counting histogram (PCH) analysis to resolve fluorescent protein mixtures directly inside cells. Because of their small spectral overlap, we have chosen to look at the fluorescent proteins EGFP and mRFP1. We experimentally demonstrate that dual-color PCH quantitatively resolves a mixture of EGFP and mRFP1 in cells from a single measurement. To mimic the effect of protein association, we constructed a fusion protein of EGFP and mRFP1 (denoted EGFP-mRFP1). Fluorescence resonant energy transfer within the fusion protein alters the dual-channel brightness of the fluorophores. We describe a model for fluorescence resonant energy transfer effects on the brightness and incorporate it into dual-color PCH analysis. The model is verified using fluorescence lifetime measurements. Dual-color PCH analysis demonstrated that not all of the expressed EGFP-mRFP1 fusion proteins contained a fluorescent mRFP1 molecule. Fluorescence lifetime and emission spectra measurements confirmed this surprising result. Additional experiments show that the missing fluorescent fraction of mRFP1 is consistent with a dark state population of mRFP1. We successfully resolved this mixture of fusion proteins with a single dual-color PCH measurement. These results highlight the potential of dual-color PCH to directly detect and quantify protein mixtures in living cells.

Evaluation of a femtosecond fiber laser for two‐photon fluorescence correlation spectroscopy

This work evaluates a femtosecond fiber laser for use in two-photon fluorescence fluctuation spectroscopy. Fiber lasers present an attractive alternative to Ti:Sapphire systems because of their compact size and portability. Autocorrelation of the second harmonic generation signal from the laser demonstrates that its stability is sufficient for two-photon fluorescence correlation spectroscopy. Fluorescence correlation spectroscopy autocorrelation traces were well fit by a Gaussian-Lorentzian squared model with a beam waist near the diffraction limit for the 810 nm wavelength. A photon counting histogram collected with this system also fit nicely to a single-species model, further demonstrating the quality of the focal shape. The authors conclude that the output from the femtosecond fiber laser is sufficiently stable and has a high enough quality beam shape for fluctuation fluorescence methods, and thus represents an effective, compact, readily portable two-photon excitation source.

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 μm 2/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 μm 2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (∼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/C Cdc20 in mitosis. Photon counting histograms reveal that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/C Cdc20 throughout the cell cycle are discussed in light of our experimental observations.

Extended Photon Counting Histogram and Fluorescence Intensity Distribution Analysis Approaches for Optically Biased Photon Counting Statistics

A gradient potential induced by a strongly focused laser beam can perturb the diffusion dynamics of particles in solutions and result in biased photon counting statistics in fluorescence fluctuation spectroscopy (FFS). In this paper, the theories of the photon counting histogram (PCH) and fluorescence intensity distribution analysis (FIDA) approaches are extended independently to fit the biased experimental data and retrieve the unbiased parameters, i.e., the average number of the sample particles in the focal volume N, their brightness epsilon, and polarizability alpha. The extended theories are tested using Monte Carlo simulations for single- and double-component systems. It is also proved numerically and analytically that the extended PCH and FIDA approaches are completely equivalent. Practical implementations and possible applications of extended PCH and FIDA are discussed.

Quantitative determination of the single-molecule detection regime in fluorescence fluctuation microscopy by means of photon counting histogram analysis

Fluorescence fluctuation experiments are performed in single-molecule detection regime if the fluorescence of at most one molecule is registered at a time. Although the significance of such experiments for investigations of complex nonergodic systems like those met in the biosciences has been stressed out by many scientists, the quantitative and accurate determination of the single-molecule detection regime received rather little attention. In this work we present a method based on the photon counting histogram (PCH) analysis, which enables the determination of the average number N of molecules within the observation volume, for which only the fluorescence of individual molecules is detected at a time. Thus, the accurate design of fluorescence fluctuation experiments performed in single-molecule detection regime is possible. Demonstrative fluorescence fluctuation experiments based on two-photon excitation are performed on diluted solutions of coumarin 153, in order to verify the potential of the PCH analysis in experiments on the single-molecule detection level. If the mean number N of molecules within the excitation volume is larger than 0.048, the probability to simultaneously detect the fluorescence of two or more molecules is no longer negligible, i.e., no single-molecule detection regime. If the mean number N of molecules is lower than 0.0057, the detection limit of the method is reached, i.e., the fluorescence signal cannot be distinguished from the background. Consequently, the concentration of coumarin 153 characteristic for the single-molecule detection regime lies in the range 13-110 pmol/l for the given experimental conditions. We also investigate the influence of the molecular brightness, i.e., detected photons per fluorophore molecule and sampling time, on the single-molecule detection regime.

A Three-State Mechanism for DNA Hairpin Folding Characterized by Multiparameter Fluorescence Fluctuation Spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.

1P239 Size distribution of actin oligomers in physiological actin solutions analyzed by photon counting histogram(8. Muscle contraction and muscle protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P239 Size distribution of actin oligomers in physiological actin solutions analyzed by photon counting histogram(8. Muscle contraction and muscle protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Detecting and characterising returns in a pulsed ladar system

A new multi-spectral laser radar (ladar) system based on the time-correlated single photon counting, time-of-flight technique has been designed to detect and characterise distributed targets at ranges of several kilometres. The system uses six separated laser channels in the visible and near infrared part of the electromagnetic spectrum. The authors present a method to detect the numbers, positions, heights and shape parameters of returns from this system, used for range profiling and target classification. The algorithm has two principal stages: non-parametric bump hunting based on an analysis of the smoothed derivatives of the photon count histogram in scale space, and maximum likelihood estimation using Poisson statistics. The approach is demonstrated on simulated and real data from a multi-spectral ladar system, showing that the return parameters can be estimated to a high degree of accuracy.

Cytochrome c conformations resolved by the photon counting histogram: watching the alkaline transition with single-molecule sensitivity.

We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0-9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5-10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry.

The Dual-Color Photon Counting Histogram with Non-Ideal Photodetectors

Dual-color photon counting histogram (PCH) analysis utilizes the photon counts in two detection channels to distinguish species by differences in brightness and color. Here we modify the existing dual-color PCH theory, which assumes ideal detectors, to include the non-ideal nature of the detector. Specifically, we address the effects of deadtime and afterpulsing. Both effects modify the shape of the dual-color PCH and thus potentially lead to incorrect values for the brightness and number of molecules if an ideal model is assumed. We use the modified theory to predict the effects of detector non-idealities on dual-color PCH as a function of concentration and brightness. In addition, we introduce a method based on moment analysis to determine the error in brightness due to non-ideal detector effects. We verify our theory experimentally by measuring a dye solution as a function of concentration and brightness. We determine the deadtime and afterpulse probability of our detectors and show that both effects play an important role in the analysis of dual-color PCH experiments. We demonstrate that resolving a mixture of CFP and YFP requires taking non-ideal detector effects into account. These corrections are also crucial for cellular measurements, as shown for GFP and RFP in mammalian cells.