Fluorescence Correlation Spectroscopy

This paper describes the first experimental application of fluorescence correlation spectroscopy, a new method for determining chemical kinetic constants and diffusion coefficients. These quantities are measured by observing the time behaviour of the tiny concentration fluctuations which occur spontaneously in the reaction system even when it is in equilibrium. The equilibrium of the system is not disturbed during the experiment. The diffusion coefficients and chemical rate constants which determine the average time behaviour of these spontaneous fluctuations are the same as those sought by more conventional methods including temperature‐jump or other perturbation techniques. The experiment consists essentially in measuring the variation with time of the number of molecules of specified reactants in a defined open volume of solution. The concentration of a reactant is measured by its fluorescence; the sample volume is defined by a focused laser beam which excites the fluorescence. T...

Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally conta...

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipid...

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto-and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.

A new (to our knowledge) robust approach for the determination of lateral diffusion coefficients of weakly bound proteins is applied for the phosphatidylserine specific membrane interaction of bovine prothrombin. It is shown that z-scan fluorescence correlation spectroscopy in combination with pulsed interleaved dual excitation allows simultaneous monitoring of the lateral diffusion of labeled protein and phospholipids. Moreover, from the dependencies of the particle numbers on the axial sample positions at different protein concentrations phosphatidylserine-dependent equilibrium dissociation constants are derived confirming literature values. Increasing the amount of membrane-bound prothrombin retards the lateral protein and lipid diffusion, indicating coupling of both processes. The lateral diffusion coefficients of labeled lipids are considerably larger than the simultaneously determined lateral diffusion coefficients of prothrombin, which contradicts findings reported for the isolated N-terminus of prothrombin.

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.

The proteins of the Bcl-2 family are key regulators of apoptosis, but their molecular mechanisms remains controversial. Two important aspects that center the debate involve the interaction network between the pro-and antiapoptotic family members and the role of their translocation to the mitochondrial outer membrane (MOM) during apoptosis. We have used FCCS to examine quantitatively the dynamic interactions of Bid and tBid with Bcl-xLDCt in solution and in lipid membranes. We found that only the active form tBid binds to Bcl-xLDCt and that the membrane strongly promotes binding between them. Importantly for drug design, a BH3 peptide from Bid disrupts the tBid/Bcl-xL complex in solution but not in lipid bilayers. Our findings convincingly suggest that the most relevant interaction between tBid and Bcl-xL happens in the membrane and reveal its significance as an additional regulatory stage for MOM permeabilization.

The use of microscale thin polymer films is widespread in biomedical science and engineering, with applications in areas such as tissue engineering, drug delivery, microfluidic devices, bio-adhesion mediators, and bio-actuators. Much attention is devoted to the use of functional polymers that display stimuli-responsive behavior with the intention of providing "smart" coatings. One potential example is the use of thin thermoresponsive polymer films as drug eluting coatings on medical devices, where not only does the polymer function as a drug reservoir but it also acts as a biocompatibility modulator to improve device performance. Often these thin polymer coatings have to be applied to complex geometries, which can cause problems for in-situ analysis. Another important consideration is the fact that these films have large surface area to mass ratios and thus water uptake can be significant. This is serious because coating stability, device efficacy, and long-term storage are influenced by the physiochemical properties of the polymer which are modulated by water content. Thus, there is a need for a rapid, non-contact, non-destructive, analytical method capable of analyzing thermoresponsive polymers in solution, and in-situ of the solid-state on medical devices. Fluorescence spectroscopy based methods can deal with both sample types and provide additional benefits in terms of high sensitivity and low probe concentrations, which provide for minimal sample disruption. This article gives a brief overview of the application of various fluorescence methods for the physicochemical characterization of thermoresponsive polymers such as poly(Nisopropylacrylamide), PNIPAm.

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared ...

Nano-domains are sub-light-diffraction-sized heterogeneous areas in the plasma membrane of cells, which are involved in cell signalling and membrane trafficking. Throughout the last thirty years, these nano-domains have been researched extensively and have been the subject of multiple theories and models: the lipid raft theory, the fence model, and the protein oligomerization theory. Strong evidence exists for all of these, and consequently they were combined into a hierarchal model. Measurements of protein and lipid diffusion coefficients and patterns have been instrumental in plasma membrane research and by extension in nano-domain research. This has led to the development of multiple methodologies that can measure diffusion and confinement parameters including single particle tracking, fluorescence correlation spectroscopy, image correlation spectroscopy and fluorescence recovery after photobleaching. Here we review the performance and strengths of these methods in the context of...

The presented data show that the FCS technique can be used to detect the DNA condensation process induced with the cationic compound hexadecyltrimethylammonium bromide (HTAB). We have shown that HTAB induces plasmid condensation upon interaction with it. Condensation can be considered to be complete when the diffusion constant reaches its maximum. The HTAB induced increase in diffusion time does not correlate well with the changes observed when count rate and particle number are considered. This observation contradicts data published for another cationic agent, spermine. This apparent discrepancy proves that the mechanisms of interaction between these compounds and DNA are different. Consequently, the different characters of the plots of count rate, diffusion time, and particle number versus condensing agent concentration can be a source of additional information about the nature of cationic compound-DNA interaction.

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

MARTIN HOF, TERESA KRAL, MAREK LANGNER, NOPPADON ADJIMATERA and IAN S. BLAGBROUGH J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 3, CZ-18223 Prague, Czech Republic, Agricultural University, Department of Physics and Biophysics, Wrocław, Poland, Wrocław University of Technology, Wrocław, Poland, Academic Centre for Biotechnology of Lipids Aggregates, Wrocław, Poland, Department of Pharmacy and Pharmacology, University of Bath, Bath, U.K.

The presented data show that the FCS technique can be used to detect the DNA condensation process induced with the cationic compound hexadecyltrimethylammonium bromide (HTAB). We have shown that HTAB induces plasmid condensation upon interaction with it. Condensation can be considered to be complete when the diffusion constant reaches its maximum. The HTAB induced increase in diffusion time does not correlate well with the changes observed when count rate and particle number are considered. This observation contradicts data published for another cationic agent, spermine. This apparent discrepancy proves that the mechanisms of interaction between these compounds and DNA are different. Consequently, the different characters of the plots of count rate, diffusion time, and particle number versus condensing agent concentration can be a source of additional information about the nature of cationic compound-DNA interaction.

The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N 4 ,N 9 -dioleoylspermine and N 1 -cholesteryl spermine carbamate. Methods. Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen \ (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results. N 4 ,N 9 -Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 Â 10 j12 m 2 /s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0Y1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N 1 -Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5Y2.5) with D = 1.3 Â 10 j12 m 2 /s and PN value of 5.2. N 4 ,N 9 -Dioleoylspermine is a more efficient DNA-condensing agent than N 1 -cholesteryl spermine carbamate. Conclusions. FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto-and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.

Fesselin or avian synaptopodin 2 stimulates actin polymerization in a Ca 2þcalmodulin dependent manner. Fesselin binding to F-actin inhibits myosin S1 binding and yet fesselin binds with moderate affinity to smooth muscle myosin. These properties suggest that fesselin could tether actin and myosin together in an inactive complex as caldesmon does. This possibility was tested by observing the effect of fesselin on the rate of dissociation of actinmyosin by ATP in a stopped-flow device. Dissociation was measured by light scattering (a measure of particle size) and by pyrene actin fluorescence (a specific measure of actin-myosin binding). Fesselin reduced the multi-exponential rates of change of light scattering and pyrene fluorescence in concentration dependent manners. Each light scattering trace had a rapid initial transition that was not present in pyrene fluorescence traces. That rapid light scattering change was likely due to dissociation of filamentous myosin. The reduction in rate of that rapid process could mean that fesselin alters the structure and dissociation kinetics of smooth myosin filaments. We examined changes in smooth muscle myosin with electron microscopy. ATP caused dissociation of myosin filaments in both the presence and absence of fesselin. In the absence of ATP, fesselin increased the size of myosin filaments. Furthermore, the filaments were arranged in parallel arrays. These results indicate that fesselin cross-links myosin to actin and also organizes myosin filaments in solution.

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

The size of a synaptic vesicle (SV) is generally thought to be determined by the amount of lipid and membrane protein it contains. Once formed, it is thought to remain constant in size. Using fluorescence correlation spectroscopy and cryogenic electron microscopy, we show that glutamatergic vesicles reversibly increase their size upon filling with glutamate. The increase (~25% in diameter) corresponds to an increase in surface area of ~50% and in volume of ~100%. This large size increase implies a large structural change in the SV upon loading with neurotransmitters. Vesicles lacking SV protein 2A (SV2A) did not manifest a change in size after loading with glutamate, indicating that SV2A is required for this phenomenon.

Extracellular vesicles (EVs) and cell-derived vesicles (CDVs), generated by fragmenting cellular membranes, have both been explored as therapeutic delivery vehicles. Surface proteins on these vesicles are of great importance as they are characteristic to the cell of origin and modulate vesicle interactions with target cells. Here we introduced a high throughput fluorescence correlation spectroscopy (ht-FCS) approach capable of characterizing vesicle surface proteins across a large number of samples. We used automated screening and acquisition of FCS data to profile surface proteins of cell-derived vesicles with high fidelity based on changes in diffusion time upon antibody-vesicle interactions. We characterized vesicles generated from 4 cell types using antibodies for known exosome biomarkers. The ht-FCS technique presented here offers the capability to screen EVs or cell derived vesicles against a library of surface markers or to screen a library of cell-derived vesicles for a specific identifying marker at a high speed.

The American Physical Society Decorated Rods: Novel Self-Assembled DNA Nanoparticles for Gene Delivery JASON DEROUCHEY, Dept. of Physics, LMU-Muenchen, GREG WALKER, ERNST WAGNER, Dept. of Pharmaceutical Biology, LMU-Muenchen, JOACHIM R ÄDLER, Dept. of Physics, LMU-Muenchen -The complexation of linear DNA fragments with cationic diblock copolymers was studied as a model system for understanding "bottom-up" self-assembly of nanoscopic gene delivery systems. Fluorescence correlation spectroscopy (FCS) measurements were performed on monodisperse linear DNA fragments complexed with diblock copolymers consisting of a cationic charged moiety, branched polyethyleneimine (bPEI), of 2, 10 or 25kDa, and a neutral shielding moiety, poly(ethylene glycol) (PEG, 20kDa). For 10 and 25kDa bPEI-PEG diblocks, severe aggregation is observed despite the presence of the shielding PEG. By decreasing the bPEI length to 2 kDa, or conversely increasing the number of chains per DNA, controlled nanoparticle formation is observed. The resulting decorated particles are consistent with a core-shell particle consisting of a single DNA surrounded by a brush layer of densely packed PEG chains. Diffusion coefficients for both DNA and decorated DNA fragments were measured as a function of DNA length ranging from 75 to 1018 bp and are well described by a diffusing rod model. Decorated rod DNA nanoparticles showed high stability against both NaCl salt and bovine serum albumin and are of potential interest for gene delivery of short antisense DNA or siRNA.

Mechanisms that can alter nucleosome structure to enhance DNA accessibility are of great interest because of their potential involvement in genomic processes. One such mechanism is H2A/H2B release from nucleosomes; it occurs in vivo and is involved in the in vitro activities of several transcription-associated complexes. Using fluorescence approaches based on Fo ¨rster resonance energy transfer, we previously detected sequence-dependent structure/stability variations between 5S and two types of promoter nucleosomes (from yeast GAL10 or mouse mammary tumor virus promoters). Those variations included differing responses when nucleosomes were diluted to concentrations (sub-nM) known to produce H2A/H2B loss. Here, we show that treatment of these same three types of nucleosomes with the histone chaperone yNAP-1, which causes H2A/H2B release from nucleosomes in vitro, produces the same differential Fo ¨rster resonance energy transfer responses, again demonstrating sequence-dependent variations associated with conditions that produce H2A/H2B loss. Single-molecule population data indicate that DNA dynamics on the particles produced by diluting nucleosomes to sub-nM concentrations follow two-state behavior. Rate information (determined by fluorescence correlation spectroscopy) suggests that these dynamics are enhanced in MMTV-B or GAL10 compared to 5S particles. Taken together, the results indicate that H2A/H2B loss has differing effects on 5S compared to these two promoter nucleosomes and the differences reflect sequence-dependent structure/stability variations in the depleted particles.

Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal refl...

We observed single molecules of fluorescently labeled double-stranded (ds) DNA held in an anti-Brownian electrokinetic trap. From the measured density fluctuations we extract the density-density response function of the molecule over times >4:5 ms and distances >250 nm, i.e., how a perturbation in density in one part of the molecule propagates through the rest of the molecule. We find a nonmonotonic radial dependence of the relaxation time. In contrast with earlier measurements on freely diffusing dsDNA, we observe clear signs of internal hydrodynamic interactions.

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1–10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static str...

Fluorescence correlation spectroscopy (FCS), is a widely used tool routinely exploited for in vivo and in vitro applications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted from microseconds to seconds long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy approaches to probe processes several orders of magnitude faster and permits a reduction of photo-toxic effects on living samples induced by long periods of light e...

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic deri...