Single Molecule Concentration Research Articles

An Alternative Framework for Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a flexible and widely used tool routinely exploited in vivo. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high molecular concentrations, high signal to noise ratio and long time traces, typically in the minute range. This is in contrast to data acquisition which, in principle, provides information on microsecond timescales that has, thus far, evaded analysis. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics. Within this principled and generalizable framework, which starts from the direct analysis of photon counts, we achieve the same accuracy as FCS, but with much shorter traces, even at nearly single molecule concentrations. Our new analysis extends the capability of confocal microscopy based approaches, by (i) probing processes several orders of magnitude faster in time; (ii) reducing phototoxic effects on living samples induced by long periods of light exposure; (iii) tracking instantaneous molecules concentration; (iv) estimating the molecular and background photon emission rates; and (v) incorporating demanding illumination profiles, such as those arising in two-photon excitation and TIRF microscopy or even Airy patterns by changing the specified point spread function.

Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.

Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

The biomedical uses for the spectroscopy of scattered light by micro and nanoscale objects can broadly be classified into two areas. The first, often called light scattering spectroscopy (LSS), deals with light scattered by dielectric particles, such as cellular and sub-cellular organelles, and is employed to measure their size or other physical characteristics. Examples include the use of LSS to measure the size distributions of nuclei or mitochondria. The native contrast that is achieved with LSS can serve as a non-invasive diagnostic and scientific tool. The other area for the use of the spectroscopy of scattered light in biology and medicine involves using conducting metal nanoparticles to obtain either contrast or electric field enhancement through the effect of the surface plasmon resonance (SPR). Gold and silver metal nanoparticles are non-toxic, they do not photobleach, are relatively inexpensive, are wavelength-tunable, and can be labeled with antibodies. This makes them very promising candidates for spectrally encoded molecular imaging. Metal nanoparticles can also serve as electric field enhancers of Raman signals. Surface enhanced Raman spectroscopy (SERS) is a powerful method for detecting and identifying molecules down to single molecule concentrations. In this review, we will concentrate on the common physical principles, which allow one to understand these apparently different areas using similar physical and mathematical approaches. We will also describe the major advancements in each of these areas, as well as some of the exciting recent developments.

Threshold Particle Diameters in Miniemulsion Reversible-Deactivation Radical Polymerization

Various types of controlled/living radical polymerizations, or using the IUPAC recommended term, reversible-deactivation radical polymerization (RDRP), conducted inside nano-sized reaction loci are considered in a unified manner, based on the polymerization rate expression, Rp = kp[M]K[Interm]/[Trap]. Unique miniemulsion polymerization kinetics of RDRP are elucidated on the basis of the following two factors: (1) A high single molecule concentration in a nano-sized particle; and (2) a significant statistical concentration variation among particles. The characteristic particle diameters below which the polymerization rate start to deviate significantly (1) from the corresponding bulk polymerization, and (2) from the estimate using the average concentrations, can be estimated by using simple equations. For stable-radical-mediated polymerization (SRMP) and atom-transfer radical polymerization (ATRP), an acceleration window is predicted for the particle diameter range, . For reversible-addition-fragmentation chain-transfer polymerization (RAFT), degenerative-transfer radical polymerization (DTRP) and also for the conventional nonliving radical polymerization, a significant rate increase occurs for . On the other hand, for the polymerization rate is suppressed because of a large statistical variation of monomer concentration among particles.

Modeling Controlled/Living Radical Polymerization Kinetics: Bulk and Miniemulsion

AbstractA first‐principle model for the controlled/living radical polymerization (CLRP) is discussed. The polymerization rate of CLRP is conveniently represented by a simple relationship, Rp ∝ [Intermediate]/[Trapping Agent], which highlights the important characteristics of various types of CLRPs. In stable free radical polymerization and atom‐transfer radical polymerization, the relationship, [Trap] ≪ [Interm] holds, and the polymerization rate is controlled by [Trap]. When the polymerization is conducted in nanosized particles, even a single trapping agent in a particle may lead to a larger [Trap] than for bulk polymerization. This single‐molecule‐concentration (SMC) effect theory leads to determine the particle diameter, Dp,SMC below which Rp starts to decrease significantly compared with the corresponding bulk polymerization, and $R_{{\rm p}} \,{\propto} \,D_{{\rm p}}^{3} $ for Dp < Dp,SMC. For the particle sizes somewhat larger than Dp,SMC, the statistical variation in the number of trapping agents can make Rp larger. A simple equation to estimate the Dp,Fluct‐value, below which the acceleration due to the fluctuation effect is predicted to occur, is presented. In conjunction with the SMC effect, an acceleration window, in which Rp is larger than for bulk polymerization, may be observed for Dp,SMC < Dp < Dp,Fluct. On the other hand, many reversible‐addition‐fragmentation chain transfer polymerizations conform to the condition [Interm] ≪ [Trap], and Rp is controlled by [Interm]. If [Interm] in a particle under the zero‐one condition is larger than for bulk polymerization, Rp can be increased significantly by reducing the particle size due to the zero‐one intermediate molecule (ZIM) effect. The ZIM effect theory leads to determine the particle diameter, Dp,ZIM below which Rp increases significantly compared with the bulk polymerization, and $R_{{\rm p}} \,{\propto} \,D_{{\rm p}}^{{-} 3} $ for Dp < Dp,ZIM. magnified image

Characterizing Multiple Molecular States in Single-Molecule Multiparameter Fluorescence Detection by Probability Distribution Analysis

Probability distribution analysis (PDA) [M. Antonik et al., J. Phys. Chem. B 2006, 110, 6970] allows one to quantitatively analyze single-molecule (SM) data obtained in Forster resonance energy transfer (FRET) or fluorescence polarization experiments. By taking explicitly background and shot noise contributions into account, PDA accurately predicts the shape of one-dimensional histograms of various parameters, such as FRET efficiency or fluorescence anisotropy. In order to describe complex experimental SM-FRET or polarization data obtained for systems consisting of multiple non-interconverting fluorescent states, several extensions to the PDA theory are presented. Effects of brightness variations and multiple-molecule events are considered independently of the detection volume parameters by using only the overall experimental signal intensity distribution. The extended PDA theory can now be applied to analyze any mixture, by using any a priori model or a model-free deconvolution approach based on the maximum entropy method (MEM). The accuracy of the analysis and the number of free parameters are limited only by data quality. Correction of the PDA model function for the presence of multiple-molecule events allows one to measure at high SM concentrations to avoid artifacts due to a very long measurement time. Tools such as MEM and combined mean donor fluorescence lifetime analysis have been developed to distinguish whether extra broadening of PDA histograms could be attributed to structural heterogeneities or dye artifacts. In this way, an ultimate resolution in FRET experiments in the range of a few Angstrom is achieved which allows for molecular Angstrom optics distinguishing between a set of fixed distances and a distribution of distances. The extended theory is verified by analyzing simulations and experimental data.

High-Throughput Single Copy DNA Amplification and Cell Analysis in Engineered Nanoliter Droplets

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.

Kinetics of Stable Free Radical Mediated Polymerization inside Submicron Particles

AbstractControlled/living radical polymerization systems in which the active period is extremely small, ϕA ≪ 1, such as the cases of stable free radical mediated polymerization (or nitroxide mediated polymerization) and atom transfer radical polymerization, are considered theoretically. The polymerization rate, Rp, for such systems increases by lowering the trapping agent concentration [X]. When the polymerization is conducted inside small particles, Rp decreases with D below the diameter Dp,SMC at which a single molecule concentration (SMC) is equal to [X]bulk. On the other hand, when the average number of trapping agents in a particle $\bar n_X$ is smaller than about 10, the fluctuation of nX among particles is significant, which leads to a larger Rp than in the cases where all particles contain the same nX. Because of the effects of SMC and fluctuation, Rp may show an acceleration window, Dp,SMC < Dp < Dp,Fluct where Rp is slightly larger than that in bulk.magnified image

Single molecule FCS-based oxygen sensor (O 2-FCSensor): a new intrinsically calibrated oxygen sensor utilizing fluorescence correlation spectroscopy (FCS) with single fluorescent molecule detection sensitivity

We report about a new intrinsically calibrated sensor technology exemplified by means of an O 2-FCSensor. The O 2-FCSensor is particularly suited for the quantitative determination of molecular oxygen within biological gases and fluids. The technology is based on the determination of the highly O 2-dependent triplet transition rates of suitable fluorophores such as Rhodamine Green or Fluorescein. The method utilizes fluorescence correlation spectroscopy (FCS) with the ultimate detection sensitivity of a single fluorescent molecule. Calibration curves of the triplet fraction ( T) and triplet relaxation time ( τ T ) as a dependence of the oxygen concentration have been determined using a thin (μm) sensor layer, and can be well approximated by hyperbolic sensor characteristics with oxygen sensitivities in the order of 0.1/(%O 2). Interestingly, the oxygen sensitivity of triplet fractions of O 2-FCSensors are not influenced by changes in viscosity of the solution demonstrating that FCS-based O 2 measurements are not diffusion controlled (in contrast to the dynamic process of collisional fluorescence quenching by molecular oxygen). Due to the ultimate sensitivity of this technique it can be applied to nanomolar or even single molecule concentrations of fluorophores thus toxic or cancerogenic influences of the applied indicators are avoided or at least minimized. Furthermore, new fluorescent indicator classes with exceptional photophysical properties can be explored for FCS-based chemo- and biosensors enabling the measurement of other analytes such as, e.g. hydrogen ion activities and carbon dioxide. As a consequence, blood gas analysis (pH, pCO 2 and pO 2) may be realized on the basis of intrinsically calibrated FCS-Sensor technology comprising the potential of absolute measurement with unprecedented sensitivity.

Single molecule fluorescence spectroscopy of mutants of the Discosoma red fluorescent protein DsRed

We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity.