Photon Counting Histogram Research Articles

Non-contact elasticity contrast imaging using photon counting.

Tissues' biomechanical properties, such as elasticity, are related to tissue health. Optical coherence elastography produces images of tissues based on their elasticity, but its performance is constrained by the laser power used, working distance, and excitation methods. We develop a new method to reconstruct the elasticity contrast image over a long working distance, with only low-intensity illumination, and by non-contact acoustic wave excitation. We combine single-photon vibrometry and quantum parametric mode sorting (QPMS) to measure the oscillating backscattered signals at a single-photon level and derive the phantoms' relative elasticity. We test our system on tissue-mimicking phantoms consisting of contrast sections with different concentrations and thus stiffness. Our results show that as the driving acoustic frequency is swept, the phantoms' vibrational responses are mapped onto the photon-counting histograms from which their mechanical properties-including elasticity-can be derived. Through lateral and longitudinal laser scanning at a fixed frequency, a contrast image based on samples' elasticity can be reliably reconstructed upon photon level signals. We demonstrated the reliability of QPMS-based elasticity contrast imaging of agar phantoms in a long working distance, low-intensity environment. This technique has the potential for in-depth images of real biological tissue and provides a new approach to elastography research and applications.

Optimized Interference Suppression for TCSPC LiDAR

The increased use of light detection and ranging (LiDAR) systems for distance determination requires the investigation of mutual interference. In this article, we describe the conditions for the occurrence of LiDAR interference. We outline suppression methods for different LiDAR types identifying pulse-position modulation (PPM) as a solution for time-of-flight LiDAR with time-correlated single photon counting (TCSPC) histograms. Based on PPM, we present a suppression method, which randomly varies the laser pulse emission times. For optimal suppression, we switch on the suppression only when interference is present. To recognize the occurrence of LiDAR interference, we develop a multipulse detection algorithm that can also extract all pulse positions. Simulations show that the algorithm can be applied for a signal-to-noise ratio greater than 3. Determining the heights of all recognized pulse signatures, an appropriate suppression level can be chosen. We successfully show the optimized interference suppression for an example LiDAR measurement. For safe use by multiple systems, we suggest random numbers. We reuse the TCSPC histograms to generate random numbers, whose generation probability is calculated theoretically and confirmed by simulation and measurement data. For nearly all histogram distributions consisting of background- and laser-generated data, a sufficient amount of random numbers is produced.

PRINCIPAL COMPONENT ANALYSIS OF PHOTON COUNTING HISTOGRAMS IN FLUORESCENCE FLUCTUATION SPECTROSCOPY EXPERIMENTS

An integrated approach based on the use of data mining methods has been proposed to improve the efficiency of the analysis of photon counting histograms in the study of the molecular composition of a substance by the method of fluorescence fluctuation spectroscopy. The method of principal components is used to test the hypothesis about the cluster separability of multidimensional experimental data. The reason for the compression of a point cloud into a characteristic nonlinearity, or so-called arc-shaped cloud, in the space of first two principal components is investigated. Examples of simulated data sets on some selected molecular systems of various brightness and concentration are considered. Nonlinear effects complicate interpretation and subsequent quantitative analysis of data. It has been established that the arching of the data cloud is a consequence of the presence of a significant variation in one or more physical parameters. In particular, it is the result of a significant increase in the variation in the parameters of the brightness or concentration of molecules. These parameters can be as additional measure in assessing the quality of the experiments if only one type of molecule is studied, and also can be used for characterizing the system under study in the case of a mixture of molecules of different types. It is proposed to apply the locally weighted scatterplot smoothing normalization to eliminate the nonlinear effects in the space of principal components.

Poly(ethylene oxide) Is Positively Charged in Aqueous Solutions.

There have been controversies about the binding of cations to poly(ethylene oxide) (PEO) chains in aqueous solutions. In the current study, single molecular evidence of charging PEO chains by cation binding in aqueous solutions is provided. From the adoption of the photon-counting histogram method, it is discovered that the local pH value at the vicinity of the PEO chain is higher than the bulk solution, showing that the PEO chain is positively charged. Such a situation exists with and without the presence of salt (NaCl) in the solution, presumably due to the binding of cations, such as hydronium and sodium ions. Single molecular electrophoresis experiments using fluorescence correlation spectroscopy demonstrate that the PEO chains are weakly charged with a charging extent of ~5%. In comparison to the salt-free condition, the addition of external salt (NaCl) at moderate concentrations further charges the chain. The charging causes the PEO chains to expand and a further increase in the salt concentration causes the chain to shrink, exhibiting a polyelectrolyte-like behavior, demonstrated by the hydrodynamic radii of a single PEO chain. The effect of ion identity is discovered with alkali cations, with the order of the charging capacity of Li+ < Na+ < Cs+ < K+.

Shear-Induced Counterion Release of a Polyelectrolyte

For the purpose of understanding the molecular origins of the rheological properties of polyelectrolyte solutions, an in situ investigation into the distribution of counterions of a polyelectrolyte under shear is conducted. An apparatus named rheo-microspectrometer is constructed by integrating an advanced rheometer with multiple highly sensitive single-molecule fluorescence microscopic and spectroscopic techniques, including fluorescence correlation spectroscopy, photon counting histogram, single-molecule fluorescence imaging, and fluorescence emission spectroscopy. It has the capacity of measuring the directional and diffusive motions of single fluorescence-labeled polymer molecules as well as their fluorescence emission spectra when the samples are under shear. The shear-induced changes in counterion distribution of a model polyelectrolyte, sodium polystyrene sulfonate (NaPSS), is investigated at the single molecular level. Fluorescence resonance energy transfer between the fluorescence donor attached at the PSS– chain end and the cationic acceptor serving as the counterion probe in solution shows the increase of average counterion–chain distance. The emission spectra of the pH-responsive fluorophore labeled at the PSS– chain end show the dilution of hydronium ions near the chain by shear. The results indicate that the shear applied to the NaPSS solution releases the counterions from the PSS– chain. The mechanism of counterions’ redistribution is attributed to the shear-induced transient overlap of electric fields of multiple PSS– chains. The results indicate that the effective charge of a polyelectrolyte molecule increases with shear, in contrast to the conventional assumption of the invariable charges.

Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

Determination of the Partition Coefficient of Protein-Membrane Reversible Association By FCS and PCH

Protein-membrane reversible association is critical to many cell and physiological functions. In most cases, protein association to the membrane is not stoichiometric and is best described by the partition coefficient rather than the dissociation constant. Fluorescence Correlation Spectroscopy (FCS) is a suitable methodology for the determination of the partition coefficient (Kp) of proteins into membranes (1, 2). Using FCS, Kp is typically determined from the diffusion of lipid vesicles loaded with fluorescently-tagged proteins. However, the uneven distribution of protein onto vesicles and the presence of unbound fluorescently-tagged protein due to dissociation events or the coexistence of a non-membrane competent protein conformer biases the retrieved quantities. In this work we investigate the effects of unbound protein in Kp quantification by FCS using antibody-membrane association as a model. In addition, we discuss the likely distribution of proteins onto vesicles in the case of reversible protein-membrane association, when the conventionally used Poisson distribution does not fully describe association/dissociation events. We compare the experimentally determined Kp against the results obtained upon simulating the diffusion of vesicles loaded with fluorescently-tagged antibodies following different statistical distributions. Finally, we discuss the conditions in which the Photon Counting Histogram (PCH) can be used to quantify the distribution of proteins associated to the vesicles. (1.) Melo, A. M., M. Prieto, and A. Coutinho. 2011. The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy. Biochimica Et Biophysica Acta-Biomembranes 1808(10):2559-2568. (2.) Kruger, D., J. Ebenhan, S. Werner, and K. Bacia. 2017. Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy. Biophys. J. 113(6):1311-1320.

Exploring early time points of vimentin assembly in flow by fluorescence fluctuation spectroscopy.

Despite the importance for cellular processes, the dynamics of molecular assembly, especially on fast time scales, is not yet fully understood. To this end, we present a multi-layer microfluidic device and combine it with fluorescence fluctuation spectroscopy. We apply this innovative combination of methods to investigate the early steps in assembly of vimentin intermediate filaments (IFs). These filaments, together with actin filaments and microtubules, constitute the cytoskeleton of cells of mesenchymal origin and greatly influence their mechanical properties. We are able to directly follow the two-step assembly process of vimentin IFs and quantify the time scale of the first lateral step to tens of ms with a lag time of below 3 ms. Although demonstrated for a specific biomolecular system here, our method may potentially be employed for a wide range of fast molecular reactions in biological or, more generally, soft matter systems, as it allows for a precise quantification of the kinetics underlying the aggregation and assembly.

A Deep Subelectron Temporal Noise CMOS Image Sensor With Adjustable Sinc-Type Filter to Achieve Photon-Counting Capability

This letter introduces a Gm-cell-based CMOS image sensor (CIS) achieving deep subelectron noise performance. The CIS presents a new compensation block and low-noise current source to improve the performance of the Gm pixel. Furthermore, an optional first-order IIR filter is implemented to improve the output swing. The conversion gain, full well capacity, and dynamic range of the CIS can be easily adjusted by the charging time and the filter mode for different applications. The prototype chip is fabricated in a standard 180 nm CIS process and has a deep subelectron read noise of 0.31e-rms at minimum (of the noise distribution) and 0.42e-rms at peak (of the noise distribution). A smooth and clear photon-counting histogram is observed.

Studying the physics of charged macromolecules by single molecule fluorescence spectroscopy.

It is well documented that conventional methods such as dynamic light scattering have encountered difficulties in characterizing charged macromolecules and, therefore, it is desirable that new methods and techniques are introduced. With the ultra-high sensitivity, single molecule fluorescence spectroscopy has successfully lowered the detection limit considerably and enabled measurement under extreme dilution conditions-around the concentration of 10-9M-at which the effect of inter-chain electrostatic repulsion is suppressed. Furthermore, the excellent spatial and temporal resolution as well as the capacity of molecular recognition of these methods help in obtaining rich information of charged macromolecules. This paper summarizes the applications of single molecule fluorescence spectroscopy, especially fluorescence correlation spectroscopy and photon counting histogram, in the studies on charged macromolecules in aqueous solutions and plenty of new information has been revealed on the molecular conformation, counterion distribution, and a few important governing factors. The powerfulness and effectiveness of single molecule fluorescence spectroscopy make it promising in the investigations of charged macromolecules.

Complex Analysis of Fluorescence Intensity Fluctuations of Molecular Compounds

A method is proposed for the complex analysis of fluctuations in the fluorescence intensity of molecular compounds, which allows determining the structural composition of protein oligomers. The idea of the method is to analyze the photon counting histograms of experimental measurements using principal component analysis to assess the presence of oligomeric compounds, and to perform hierarchical cluster analysis, to determine the data classes corresponding to various molecular compounds, followed by selecting cluster medoids to determine the oligomeric composition of protein complexes. The efficiency of the analysis algorithms developed within the framework of the proposed method was confirmed on simulated and experimental photon counting histograms of the measured fluorescence intensity fluctuations of monomeric and dimeric forms of green-fluorescent protein (GFP).

Fluorescence Correlation Spectroscopy and Photon Counting Histograms in Finite, Bounded Domains

Analysis of fluctuations arising as fluorescent particles pass through a focused laser beam has enabled quantitative characterization of a broad range of molecular kinetic processes. Two key mathematical frameworks that have enabled these quantifications are fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis. Although these frameworks are effective and accurate when the focused laser beam is well approximated by an infinite Gaussian beam with a waist that is small compared to the size of the region over which the fluorescent particles can diffuse, they cannot be applied to situations in which this region is bounded at the nanoscale. We therefore derived general forms of the FCS and PCH frameworks for bounded systems. The finite-domain form of FCS differs from the classical form in its boundary and initial conditions and requires development of a new Fourier space solution for fitting data. Our finite-domain FCS predicts simulated data accurately and reduces to a previous model for the special case when the system is much larger than the Gaussian beam and can be considered to be infinite. We also derived the PCH form for the bounded systems. Our approach enables estimation of the concentration of diffusing fluorophores within a finite domain for the first time, to our knowledge. The method opens the possibility of quantification of kinetics in several systems for which this has never been possible.

Lensless compressive sensing with annulus-sector-shaped pixel geometry in the photon-starved environment

To achieve fast compressive object classification and imaging in photon-starved environment, we put forward a novel lensless sensing configuration based on annulus-sector-shaped pixels, where pixels located at different annulus correspond to different temporal profile of photon-counting histogram, thus mapping the spatial distribution features of target to the signatures at the temporal domain. A model is established based on the superposition of different temporal contributions, and solved by exploiting the rotational symmetry of each sector and dramatically reduced solution space. Object classification among 62 classes is conducted with a single-pixel single-photon detector, achieving the classification accuracy of 94.56% at the total operating time of 1 s, corresponding to ~0.82 photons per pixel and a compressive ratio as small as 0.98%. Our method is verified to have great potential in the high-speed photon-limited sensing.

Resolving fluorescent species by their brightness and diffusion using correlated photon-counting histograms.

Fluorescence fluctuation spectroscopy (FFS) refers to techniques that analyze fluctuations in the fluorescence emitted by fluorophores diffusing in a small volume and can be used to distinguish between populations of molecules that exhibit differences in brightness or diffusion. For example, fluorescence correlation spectroscopy (FCS) resolves species through their diffusion by analyzing correlations in the fluorescence over time; photon counting histograms (PCH) and related methods based on moment analysis resolve species through their brightness by analyzing fluctuations in the photon counts. Here we introduce correlated photon counting histograms (cPCH), which uses both types of information to simultaneously resolve fluorescent species by their brightness and diffusion. We define the cPCH distribution by the probability to detect both a particular number of photons at the current time and another number at a later time. FCS and moment analysis are special cases of the moments of the cPCH distribution, and PCH is obtained by summing over the photon counts in either channel. cPCH is inherently a dual channel technique, and the expressions we develop apply to the dual colour case. Using simulations, we demonstrate that two species differing in both their diffusion and brightness can be better resolved with cPCH than with either FCS or PCH. Further, we show that cPCH can be extended both to longer dwell times to improve the signal-to-noise and to the analysis of images. By better exploiting the information available in fluorescence fluctuation spectroscopy, cPCH will be an enabling methodology for quantitative biology.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

Molecular weight dependence of chain conformation of strong polyelectrolytes.

Using sodium polystyrene sulfonate (NaPSS) and quarternized poly 4-vinylpyridine (QP4VP) as model systems, the chain conformation of polyelectrolytes under finite salt concentrations is investigated at a single molecular level. By fluorescence correlation spectroscopy (FCS), the hydrodynamic radius (R h) of the samples with the molecular weight ranging more than one order of magnitude was measured. The variations of R h as a function of molecular weight reveal the molecular weight dependence: under moderate salt concentrations (such as 10-4 and 0.1M), the shorter chains of both NaPSS and QP4VP take the rod-like conformation, while the longer chains take the coiled conformation (random coil or swelled random coil conformation, respectively). At high enough salt levels, both the charged chains take the coiled conformations. Photon counting histogram (PCH) measurements of the local pH value at the vicinity of the NaPSS chain expose the higher extent of counterion adsorption for longer chains as well as higher salt concentrations, telling that the charge regularization process is the major governing factor.

Counterion Cloud Expansion of a Polyelectrolyte by Dilution

It has long been documented that the reduced viscosity of polyelectrolyte has an anomalous dependence on its concentration, i.e., the Fuoss law. To explore the molecular mechanism, the counterion distribution of sodium polystyrenesulfonate (NaPSS) as a function of concentration is investigated at the single-molecule level. By examination of the fluorescence resonance energy transfer (FRET) between a fluorescence donor on NaPSS chain and an acceptor in the counterions using single-molecule fluorescence spectroscopy, an increase of average counterion–chain distance is discovered upon dilution, indicating the expansion of counterion cloud. By photon counting histogram, an increase of effective charge of the NaPSS chain during dilution is exposed. The variation of these parameters agrees well with that of the reduced viscosity, helping to shed light on the molecular mechanism of the Fuoss law: the expansion of the counterion cloud increases hydrodynamic friction, and the increase of effective charges of NaPSS...

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures

The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (lo) and liquid-disordered (ld) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on lo-ld sequestration of integrins, thereby exhibiting overall ld preference in the absence of ligands and lo affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting lo and ld domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.