Photon Counting Histogram Research Articles

Gradient Distribution of LFA-1 cluster Size in the Immunological Synapse

Coordinated rearrangement of membrane receptors into clusters upon ligand binding has emerged as an important regulator of cellular signaling. However, due to cell membrane complexity and constant change in membrane morphology, probing protein clusters that are below optical resolution in live cells has long been a challenging task. Here, we use a hybrid live cell-supported membrane interface to enable high resolution fluorescence fluctuation measurements. We explore the cluster distribution of LFA-1:ICAM-1 complexes that form a ring that spans ∼10µm in diameter in a mature immunological synapse(IS). IS formation is triggered by exposing primary T cells to a supported membrane that is functionalized with activating peptide-MHC and ICAM-1-YFP. We scan across the IS with a focused laser beam to monitor the photobleaching and fluorescence fluctuations of LFA-1 bound ICAM-1-YFP molecules within the optical area. By changing the excitation laser from continuous to a pulsed form and applying photon counting histogram analysis, we are able to distinguish between the fluorescent signals of large clusters from those of small clusters. Instead of an even distribution, our results indicate that LFA-1:ICAM-1 complexes at the IS exhibit two differently clustered populations. One population shows a strong association to the T cell cytoskeleton and thus displays low lateral mobility. Interestingly, the fraction and the size of clusters of the cytoskeleton-associated complexes increase toward the center of the IS. The second population of LFA-1:ICAM-1 complexes exhibits random diffusion and a small cluster number that is no more than 3 on average. Therefore, more compartmentalized domains exist beyond the classical picture of three concentric zones at the IS. This supports the frictional force coupling model as the mechanism of IS formation, i.e. larger clusters experience larger transport force toward the center of the IS.

Global analysis of autocorrelation functions and photon counting distributions

In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. The global analysis approach is described and tested with experimental fluorescence fluctuation data of enhanced green-fluorescent protein (eGFP) and dimeric eGFP (two eGFP molecules connected by a six amino acid long linker) in aqueous buffer. Brightness values and diffusion constants are recovered with good precision elucidating novel excited-state and motional properties of both proteins.

2C1524 アミロイドβ多量体形成解析のための新規フォトンカウンティングヒストグラム法の開発(蛋白質_機能1,第49回日本生物物理学会年会)

2C1524 アミロイドβ多量体形成解析のための新規フォトンカウンティングヒストグラム法の開発(蛋白質_機能1,第49回日本生物物理学会年会)

Characterization of Brightness and Stoichiometry of Bright Particles by Flow-Fluorescence Fluctuation Spectroscopy

Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations.

Amyloid Beta Oligomer Studied by Newly Developed Single Molecule Analysis Method

Amyloid beta (Ab) is 4 kDa peptide which forms aggregates such as oligomers and fibrils. They have been considered to cause Alzheimer's diseases (AD). Recent results have suggested that soluble Ab oligomers are the causative agent of AD since such oligomers are more cytotoxic than fibrils. It was also suggested that Ab oligomers affect not only cell death but also early stage of cell dysfunction and cause memory loss.However, the formation mechanism of these soluble oligomers is still unknown. In this study, we developed new single molecule analysis method that can analyze Ab oligomer distribution. For this purpose, we combined total internal reflection fluorescence microscopy (TIRFM) with photon counting histogram (PCH) (Terada, N. et al. (2007) Biophys. J. 92, 2162). Using TIRFM, fluorescent intensity of monomer is obtained from discrete photobleaching. Using PCH method, the number of protomers in oligomers and concentrations are obtained from histograms of photons from fluorescent molecules diffusing through the confocal volume.A model Ab oligomer was prepared by sonication treatment of Ab fibrils made from FITC labeled Ab monomer. The number of Ab monomer in a single Ab oligomer was estimated by TIRFM, which agrees well with the results of PCH analysis. Thus we concluded that PCH can be applied to analyze Ab oligomer formation. Next, Ab oligomers were formed at physiological Ab concentration (LeVineIII, H. (2004) Anal. Biochem. 335, 81). Oligomer formation was observed with TIRFM analysis. PCH analysis is now in pregress.

Receptor-Ligand Interactions in the Plasma Membrane of Live Cells Resolved in Space and Time by N&B Analysis

3898-Pos Receptor-Ligand Interactions in the Plasma Membrane of Live Cells Resolved in Space and Time by N&B Analysis Christian Hellriegel 1 , Valeria R. Caiolfa 2,1 , Nicolai Sidenius 3 , Enrico Gratton 4 , Moreno Zamai 2,1 . CNIC, Madrid, Spain, 2 San Raffaele Scientific Institute, Milano, Italy, IFOM- Italian Cancer Research Foundation FIRC, Milano, Italy, Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, CA, USA. In this presentation we show how we push the Number and Brightness analysis (N&B) to the limits of applicability. We demonstrate that by N&B we can observe how a GFP labeled membrane receptor (namely uPAR) dimerizes upon ligand binding in live cells. We show how we obtain real time, spatially and temporally resolved images of the molecular reorganization of uPAR in the cell membrane. These results are backed by extensive simulations, and by well-defined live cell calibration experiments (using monomeric and dimeric GFP-uPAR constructs). N&B quantifies the amplitudes of fluorescence intensity fluctuations as individ- ual fluorescent species diffuse in and out of a pixel in a series of images. The basic idea is that the amplitude fluctuations of a diffusing molecule labeled with two dyes (e.g. a dimer, or a bound ligand-receptor pair) will be twice as large as the amplitudes of a molecule with only one dye (i.e. a monomer, or the unbound ligands and receptors), simply because the doubly labeled object is twice as bright as the individual one. N&B is related to fluctuation spectroscopy such as fluorescence correlation spectroscopy FCS and photon counting histogram, PCH. These methods can re- solve molecule-molecule interactions, but are usually restricted to the acquisi- tion at one specific pixel. N&B was described recently for 2-photon scanning microscopy. There, N&B was typically used to distinguish between mobile molecules and large aggregates in cells, using time-sequences of about 50- 100 frames (typically 512x512 pixels at 4s/frame). However, when attempting to distinguish between monomers and dimers, as the smallest possible increment of molecule-molecule interactions, the experi- menter is confronted with low signal-to-noise ratios and long-term perturba- tions (cell movement, vesicle trafficking). In this work we describe how we have resolved these issues.

1P085 Amyloid beta oligomer studied by photon counting histogram(Protein:Function,The 48th Annual Meeting of the Biophysical Society of Japan)

1P085 Amyloid beta oligomer studied by photon counting histogram(Protein:Function,The 48th Annual Meeting of the Biophysical Society of Japan)

Caveolin-1 Boosts Clustering of Mu (μ) Opioid Receptors in the Plasma Membrane

Caveolin-1 (Cav1), is a structural protein component of many mammalian cell plasma membrane and is known to be involved in lipid and protein sorting, receptor desensitization, receptor trafficking, cell migration and many other cellular events. Here we determine if stable expression of Cav1 in cells alters the receptor organization prototype on the membrane. We use two different cell lines for this study: Fisher Rat Thyroid (FRTwt) cells that do not express detectable level of Cav1 and a sister line that is stably transfected with canine Cav1 protein (FRTcav). We express μ opioid receptors (MOR) tagged with either YFP (MOR-YFP) or CFP (MOR-CFP) in cells for different experiments. Forster resonance energy transfer (FRET) measurement between MOR-CFP and Gαi-YFP in FRTwt and FRTcav cells shows receptor sequestration in the presence of Cav1. We find that diffusion of MOR-YFP in plasma membrane of FRTcav cells is slower compared to FRTwt cells by scanning fluorescence correlation spectroscopy (scanning-FCS) experiments. Photon counting histogram (PCH) analyses provide higher average brightness for MOR-YFP in FRTcav cells. Taken together these data provide evidence for caveolin-assisted enhanced clustering of G-protein coupled receptors on the plasma membrane.

Nuclear Import and Assembly of Influenza A Virus RNA Polymerase Studied in Live Cells by Fluorescence Cross-Correlation Spectroscopy

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.

Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature.

Evidence for a Second, High Affinity Gβγ Binding Site on Gαi1(GDP) Subunits

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.

Photon Counting Histogram Analysis as a Tool for Studying the Nature of Intermolecular Interactions

Photon counting histogram (PCH) analysis is a statistical method that provides independent information about the relative brightness and the number of molecular entities in the system. This suggests that it could be very advantageous in the analysis of intermolecular interactions in multicomponent systems. In this paper we employ PCH to study the interaction of Rhodamine 6G (Rh-6G) with sodium dodecyl sulfate (SDS) micelles and show how the method can unveil the mechanism of quenching of Rh-6G by methyl viologen (MV).

Estimation of single-molecule blinking parameters using photon counting histogram

We have used computer simulations to explore the photon counting histogram (PCH) as a statistic for estimating kinetic parameters of a blinking immobilized fluorophore. Fluorescence trajectories made up of 500 on–off cycles allow recovery of the escape rates k on , k off with 20% error, when the bin size h is in an optimal range. The corresponding estimates of the intensities I on , I off improve with increasing resolution k on h , k off h of the on- and off-time, respectively. PCH analysis requires that the intensities I on , I off be well separated. The parameter estimates improve with increasing fluorescence trajectory length.

Amyloid Beta Oligomer Formation Analysis by Photon Counting Histogram

Amyloid beta (Ab) is 4 kDa peptide which is thought to form aggregates such as oligomers and fibrils, and to cause Alzheimer disease (AD). Recently, it has been suggested that soluble Ab oligomers are the causative agent of AD since such oligomers are more cytotoxic than fibrils. It was also suggested that Ab oligomers affect not only cell death but also early stage of cell dysfunction and cause memory loss.However, the mechanism how soluble oligomers are produced is still unknown. In this study, we analyzed formation of Ab oligomers in vitro at a single molecule level using photon counting histogram (PCH)(Chen, Y. et al. (1999) Biophys. J. 77, 553-67; Terada, N. et al. (2007) Biophys. J. 92, 2162-71.). Using PCH method, the number of protomers in oligomers and concentrations are obtained from histograms of photons from fluorescent molecules. Combination of a conforcal optics and a photon counting sensor enables us to catch the fluorescence from molecules diffusing through the conforcal volume at PCH. The concentration distribution of oligomers can be calculated from histograms.Fluorescent intensity of fluorescein-labeled Ab monomer (FL-Ab) was evaluated using PCH. After 30 min incubation of FL-Ab in buffer solution, dimer fraction was successfully observed with PCH, assuming that fluorescent intensity is in proportion to the number of protomers in oligomers. Detailed analysis of formation of Ab oligomers such as amyloid-beta derived diffusible ligands (ADDLs) using these techniques is in progress.

Investigation of Dnmt1-DNA Interaction using Fluorescence Fluctuation Spectroscopy

DNA methyltransferase 1 (dnmt1) is an important factor in the epigenetic process of DNA methylation. It is responsible for the regulation of tissue-specific patterns of methylated cytosine residues. Pathological changes in these methylation patterns are connected with various diseases, for example certain types of cancer. We investigated the functional nature of the interaction between dnmt1 and DNA. A construct was formed, consisting of a synthetic DNA strand, labeled with a synthetic fluorescent dye, and dnmt1, labeled with Green Fluorescent Protein (GFP). To determine whether the functional form of dnmt1 is monomeric, dimeric or consists of even larger complexes, we measured the ratio of GFP to synthetic dye molecules using Fluorescence Fluctuation methods such as Fluorescence Cross Correlation spectroscopy (FCCS), stoichiometry determination from a Burst Analysis experiment as well as Photon Counting Histograms (PCH) analysis.

Proteome-Wide Fluctuation Analysis Of S.cerevisiae

We measured over 40.000 single live yeast S. cerevisiae cells to determine the concentration and diffusion constants of more than 4100 proteins. These proteins account for more than 75% of the yeast proteome. We used Fluorescence Correlation Spectroscopy (FCS), Photon Counting Histograms (PCH), and Brightness & Number analysis (B&N) to analyze the intensity fluctuations of single molecules fused to GFP. The data was collected using a commercial FCS setup attached to a confocal microscope (ConfoCor3 and LSM 510 META, Carl Zeiss Jena GmbH, Germany) controlled by custom software.The cells were imaged in transmitted light. We acquired fluorescence images, using the avalanche photo diodes of the FCS setup. This allows us to determine the localization of proteins, the cell cycle as well as cell health.We developed a software package to automate the measurements and data analysis. We use the Open Microscopy Environment (OME) to organize our images, fluctuation measurements, and analysis results.We calculated the protein copy number per cell, and compare the noise in concentration levels between different proteins in respect to localization and biochemical pathway.We find that the diffusion coefficient for GFP is identical in nucleus and cytosol. But interestingly, most of the proteins localized in the nucleus diffuse slower than proteins localized in the cytoplasm.We will present our data and compare them to information gathered with different methods like flow-cytometry and mass-spectroscopy. We will discuss conclusions derived by complementing our data with information collected in public databases like Saccharomyces Geneome Database (www.yeastgenome.org), Yeast GFP Fusion Localization Database (yeastgfp.ucsf.edu), and the General Repository for Interaction Datasets (www.thebiogrid.org).

An Investigation Into the Membrane Diffusion and Organisation of Adenosine Receptor Homo-oligomers

Oligomerisation of G-protein coupled receptors (GPCRs) is now a widely accepted phenomenon, although its effects on receptor signalling, pharmacology and organisation are still unclear. Using a combination of bimolecular fluorescence complementation (BiFC, Hu, Mol. Cell, 9, 789, 2002) and fluorescence correlation spectroscopy (FCS), we have specifically monitored the diffusion of homo-oligomers of three members of the adenosine receptor family of GPCRs (the A1-, A2A- and A3-adenosine receptors (Ax-AR)) in microdomains of living cells. This approach has allowed us to directly investigate the membrane organisation of homo-oligomeric forms of these receptors.FCS measurements were carried out as previously described (Briddon, PNAS, 101, 4673, 2004).on the upper cell membrane of CHO-K1 cells transiently expressing C-terminal fusions of each AR subtype with either wtYFP (representing total receptor population) or C-YFP and N-YFP (representing oligomeric receptors).Homo-oligomers of all three subtypes were detected and showed a high degree of membrane localisation. For all three subtypes, receptors labelled with wtYFP (total receptor population) showed similar diffusion co-efficients (D=0.40, 0.51 and 0.43 μm2/s for A1-, A2A- and A3-AR, respectively). The oligomeric A3-AR (measured using BiFC) had a significantly faster diffusion co-efficient when compared to the A3-AR total population (D=0.60 vs. 0.43 μm2/s, P<0.05) suggesting that the homo-oligomeric A3-AR represents a faster diffusing fraction of the total receptor population. This was not the case for the A1- and A2A-ARs. Further investigation into the extent of receptor dimerisation for each ARs subtype among the total population and their membrane mobilities was carried out using photon counting histogram (PCH) analysis and fluorescence recovery after photobleaching (FRAP). These data indicate important differences in the molecular organisation of the monomeric vs oligomeric forms of the A3-AR, and also differences among receptor subtypes in their propensity for dimer formation.

Resolvability of PCH in Two Dimensional Systems

Although the ability of PCH analysis to resolve the components of mixtures of fluorescent molecules has been carefully studied in three-dimensional systems (Muller, Chen et al. 2000), it has not been investigated in two dimensions. We explored the characteristics of the reduced χ2 surface of two dimensional binary mixtures, specifically, the principal curvature at the χ2 minimum as a function of brightness and molecular concentration. Our results are in good agreement with the previously published results.A potential problem can arise from errors in focusing in two dimensional systems . When data acquisition time and therefore the data record is insufficient to to resolve species in a mixture, independent information about each species, e.g., measurements of brightness, can be used to extract more accurate results. Our experiments on Giant Unilamellar Vesicles (GUVs) labeled with a single lipid analog allow us to estimate brightness variations due to focusing. Combining this information and the PCH from mixtures yield reasonable estimations of the parameters of interestMuller, J. D., Y. Chen, et al. (2000). “Resolving heterogeneity on the single molecular level with the photon-counting histogram.” Biophys J 78(1): 474-86.

Coherent Anti-Stokes Raman Scattering Microscopy of Samples Probed with Gaussian Volumes

Coherent anti-Stokes Raman scattering (CARS) microscopy is becoming increasingly popular to characterize biochemical samples. Within this context, we show that theoretical analysis can still be accomplished under the simple assumption of Gaussian volumes instead of spatial shapes obtainable from diffraction necessary to describe the tight-focusing condition realized within the focus of microscopes with high numerical apertures. The assumption, common in other physical and chemical spectroscopic techniques based on microscopy (e.g., fluorescence correlation spectroscopy, photon counting histogram) and never applied to CARS, is here used to determine the expression of the anti-Stokes electric field. Contrary to the standard approach resorting to numerical methods, we find that either the field is analytical for certain shapes of the Raman scatterer or the numerical reconstruction is strongly limited. In addition, we examine tests against two typical problems found in the literature, namely, a description of CARS radiation patterns and CARS imaging. With regard to the latter, we remark that the loss of spatial symmetry, the treatment of which is onerous in standard CARS microscopy because of possible separations between the microscope focus and the Raman scatterer, can be handled with ease in the limit of Gaussian volumes. An example is considered for polystyrene beads that are usually employed as test model of a CARS response of relevant biochemical samples.

Money matters

<h3>Abstract</h3> Lifetimes of chemical species is typically estimated, on a pixel-by-pixel basis by either fitting time correlated single photon counting (TCSPC) histograms or, more recently, through phasor analysis from time-resolved photon arrivals. While both methods yield lifetimes in a computationally efficient manner, they have limitations. First, both TCSPC and phasor analysis set the number of chemical species by hand before lifetimes can be determined. Yet the number of species itself is encoded in photon arrival times and need not be set by hand <i>a priori</i>. Next, even to determine lifetimes under the assumption of a known number of species, both methods rely on heavy data post-processing of the signal thereby requiring large amounts of data to retrieve lifetimes. As a result, the sample is exposed to orders of magnitude more light than is required and the ability to resolve fast processes is compromised. Here we propose a direct photo-by-photon analysis of data drawn from pulsed excitation experiments to infer, simultaneously and self-consistently, the number of species and their associated lifetimes from as little as a few thousand photons for two species. We do so by leveraging new mathematical tools within the Bayesian nonparametric (BNP) paradigm that we have previously exploited in the analysis of single photon arrivals from single spot confocal microscopy. We benchmark our method on simulated as well as experimental data for one, two, three, and four species with data sets from both immobilized and freely diffusing molecules.