Fluorescence Correlation Spectroscopy Analysis Research Articles

Watching lanthanide nanoparticles one at a time: characterization of their photoluminescence dynamics at the single nanoparticle level.

Lanthanide nanoparticles (LnNPs) feature sharp emission lines together with millisecond emission lifetimes which make them promising luminescent probes for biosensing and bioimaging. Although LnNPs are attracting much interest, their photoluminescence properties at the single nanoparticle level remain largely unexplored. Here, we employ fluorescence correlation spectroscopy (FCS) and photoluminescence burst analysis to investigate the photodynamics of Sm- and Eu-based LnNPs with single nanoparticle sensitivity and microsecond resolution. By recording the photoluminescence intensity and the number of contributing LnNPs, we compute the photoluminescence brightness per individual nanoparticle and estimate the actual number of emitting centers per nanoparticle. Our approach overcomes the challenges associated with ensemble-averaged techniques and provides insights into LnNP photodynamics. Moreover, we demonstrate our microscope's ability to detect and analyze LnNPs at the single nanoparticle level, monitoring both photoluminescence brightness and burst duration. These findings expand our understanding of LnNPs and pave the way for advanced biosensing applications at the single nanoparticle level.

Dual-Wavelength On-Chip Integrated Metalens for Epi-Fluorescence Single-Molecule Sensing.

Single-molecule fluorescence spectroscopy offers unique capabilities for the low-concentration sensing and probing of molecular dynmics. However, employing such a methodology for versatile sensing and diagnostics under point-of-care demands device miniaturization to lab-on-a-chip size. In this study, we numerically design metalenses with high numerical aperture (NA = 1.1), which are composed of silicon nitride nanostructures deposited on a waveguide and can selectively focus guided light into an aqueous solution at two wavelengths of interest in the spectral range of 500-780 nm. Despite the severe chromatic focal shift in the lateral directions owing to the wavelength-dependent propagation constant in a waveguide, segmented on-chip metalenses provide perfectly overlapping focal volumes that meet the requirements for epi-fluorescence light collection. We demonstrate that the molecule detection efficiencies of metalenses designed for the excitation and emission wavelengths of ATTO 490LS, Alexa 555, and APC-Cy7 tandem fluorophores are sufficient to collect several thousand photons per second per molecule at modest excitation rate constants. Such sensitivity provides reliable diffusion fluorescence correlation spectroscopy analysis of single molecules on a chip to extract their concentration and diffusion properties in the nanomolar range. Achromatic on-chip metalenses open new avenues for developing ultra-compact and sensitive devices for precision medicine and environmental monitoring.

Critical aggregation concentration and reversibility of amyloid-β (1–40) oligomers

Amyloid-beta (Aβ) aggregation is a critical factor in the pathogenesis of Alzheimer's disease, with distinct aggregation behaviours observed between its isoforms Amyloid-β 1–40 (Aβ40) and 1–42 (Aβ42). In this study, we investigated the aggregation properties of Aβ40 using fluorescence correlation spectroscopy (FCS) and detailed data analysis. Our results reveal that Aβ40 undergoes a two-step cooperative aggregation process. The first step, characterized by a critical aggregation concentration (cac) of 0.5 ± 0.3 μM, results in the formation of metastable oligomers of 5–25 monomers and stable oligomers of 50–100 monomers, with less than 10 % of the total amyloid aggregated. The second step, with a cac of 19 ± 2 μM, leads to the formation of much larger aggregates, consistent with protofibrils, and approximately 50 % aggregated amyloid. Notably, the cac for Aβ40 is significantly higher, and the fraction of aggregated amyloid is much lower compared to Aβ42, indicating a lower propensity for aggregation. Additionally, our findings suggest that Aβ40 early oligomers are reversible upon dilution, albeit with a kinetic barrier to disaggregation. These insights into the aggregation mechanisms of Aβ40 enhance our understanding of its role in Alzheimer's disease and may inform therapeutic strategies targeting amyloid aggregation.

Unveiling the photoluminescence dynamics of gold nanoclusters with fluorescence correlation spectroscopy.

Gold nanoclusters (AuNCs) have captured significant interest for their photoluminescent properties; however, their rapid photodynamics remain elusive while probed by ensemble-averaging spectroscopy techniques. To address this challenge, we use fluorescence correlation spectroscopy (FCS) to uncover the photoluminescence dynamics of colloidal Au18(SG)14 nanoclusters. Our FCS analysis reveals the photoluminescence (PL) brightness per nanocluster, elucidating the impact of photoexcitation saturation and ligand interactions. Unlike DNA-encapsulated silver nanoclusters, their gold counterparts notably exhibit minimal blinking, with moderate amplitudes and 200 μs characteristic times. Our data also clearly reveal the occurrence of photon antibunching in the PL emission, showcasing the quantum nature of the PL process, with each AuNC acting as an individual quantum source. Using zero-mode waveguide nanoapertures, we achieve a 16-fold enhancement of the PL brightness of individual AuNCs. This constitutes an important enabling proof-of-concept for tailoring emission properties through nanophotonics. Overall, our study bridges the gap between ensemble-averaged techniques and single-molecule spectroscopy, offering new insights into AuNC photodynamics for biosensing and imaging applications.

Spectroscopic characterization of the interactions between poly(2-(trimethylamino)ethyl methacrylate) chloride and the xanthene dyes, 2′, 7′-difluorofluorescein and 2, 4, 5, 7-tetraiodofluorescein

Intermolecular interactions in buffered aqueous solution between the polycation, poly(2-(trimethylamino)ethyl methacrylate) chloride (pTMAEMC) and two anionic xanthene dyes, 2′, 7′-difluorofluorescein (Oregon Green 488) and 2, 4, 5, 7-tetraiodofluorescein (Erythrosin B), are characterized using multiple optical spectroscopic methods. Visible absorption spectroscopy indicates the formation of ground-state pTMAEMC-dye complexes. Benesi-Hildebrand binding isotherm analysis of visible absorption spectra for pTMAEMC-dye mixtures quantifies the strength of binding interactions producing the complexes. For both Oregon Green 488 (OG) and Erythrosin B (EB) in mixtures with pTMAEMC, the concentration of the solution’s sodium acetate buffer at a fixed pH alters the binding constants, Kb, suggesting that ionic strength plays a key role in determining the binding affinity of pTMAEMC for the dyes. Comparison of Kb, for the dyes indicates stronger binding of EB under all solution conditions. Steady-state fluorescence emission spectroscopy, fluorescence quenching, excited-state fluorescence lifetime measurements and fluorescence correlation spectroscopy provide complementary data for the interactions between pTMAEMC and the dyes. Mixtures of pTMAEMC with the dyes produce fluorescence enhancements and fluorescence quenching which exhibit a dependence on the buffer concentration used in the mixture. Excited-state lifetime analysis indicates that OG interacts with pTMAEMC through ground-state interactions while EB exhibits both ground-state and excited-state interactions with pTMAEMC. The spectroscopic measurements suggest that a polyelectrolyte effect for pTMAEMC due to ionic strength variation produced by the buffer concentration affects the dye binding profile of the polycation. This conclusion is supported by fluorescence correlation spectroscopy (FCS) analyses of the hydrodynamic diameter changes in pTMAEMC-OG binding in low buffer concentration (low ionic strength) solution. FCS analyses of pTMAEMC-OG mixtures also reveal diversity in the complexes formed in low ionic strength solution suggesting that other xanthene dyes will exhibit similar binding behaviors in mixtures with pTMAEMC as a function of solution ionic strength.

Image correlation spectroscopy approaches to probe diffusion in cell.

Among the methods for studying molecular dynamics through fluorescence, fluorescence correlation spectroscopy (FCS) is one of the most effective in vivo experimental techniques. It returns information about the diffusion of fluorescent molecule pools in a single well-defined small volume by means of auto/cross-correlating fluorescence intensity fluctuation. Since probing a single point may not be representative of a complex landscape, we can use the spatio-temporal image correlation spectroscopy (stICS) method. Using a sequence of images taken on the same functional biological area, such a method computes the auto/cross-correlation over the entire region. Since the diffusive parameters are extracted from the autocorrelation map as an average over a region of interest, stICS can be susceptible to spatial inhomogeneities of the fluorescent moiety in the same area. However, vesicles, membranes, confinements are sometimes poorly detectable from the sole fluorescence intensity. If different diffusion pools are present, some of them may be missed from the analysis of the global autocorrelation curve on account of the signal-to-noise ratio and/or their dynamic characteristics. This work explores different independent approaches to reveal the spatial feature of the molecular dynamics inside the cell. These approaches required a sequence of images to perform FCS analysis pixel by pixel. Each single-pixel autocorrelation evaluation avoids mixing different contributions due to average dynamics pools over a large field of view. Among the developed algorithm, the most promising is related to an analysis based on Maximum Entropy, which, for each point, gives a spectrum of Brownian Diffusion coefficients. This information can help link morphological and dynamics feature, also through correlative analysis with other spatial techniques, enabling new imaging schemes. These approaches are applied to recover membrane characteristics in cell's membrane such as PDL1, a significant player in an immune-checkpoint also involved in cancer progression.

Application of fluorescence correlation spectroscopy to investigate the dynamics of a ribosome-associated trigger factor in Escherichia coli.

Co-translational protein folding is one of the central topics in molecular biology. In Escherichia coli, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both in vitro and in vivo. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living E. coli cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.

In preprints: morphogens in motion.

In preprints: morphogens in motion.

Three in One: Stimuli-Responsive Fluorescence, Solid-State Emission, and Dual-Organelle Imaging Using a Pyrene-Benzophenone Derivative.

Small organic luminogens, owing to their contrasting stimuli-responsive fluorescence in solution along with strong emission in aggregated and solidstates, have been employed in optoelectronic devices, sensors, and bioimaging. Pyrene derivatives usually exhibit strong fluorescence and concentration-dependent excimer/aggregate emission in solution. However, the impacts of microenvironments on the monomer and aggregate emission bands and their relative intensities in solution, solid, and supramolecular aggregates are intriguing. The present study delineates a trade-off between the monomer and aggregate emissions of a pyrene-benzophenone derivative (ABzPy) in solution, in the solid-state, and in nanoaggregates through a combined spectroscopic and microscopic approach. The impact of external stimuli (viscosity, pH) on the aggregate emission was demonstrated using steady-state and time-resolved spectroscopy, including fluorescence correlation spectroscopy and fluorescence anisotropy decay analysis. The aggregate formation was noticed at a higher concentration (>10 μM) in solution, at 77 K (5 μM), and in the solid-state due to the π-π stacking interactions (3.6 Å) between two ABzPy molecules. In contrast, no aggregate formation was observed in the viscous medium as well as in a micellar environment even at a higher concentration of ABzPy (50 μM). The crystal structure analysis further shed light on the intermolecular hydrogen-bonding-assisted solid-state emission, which was found to be highly sensitive toward external stimuli like pH and mechanical forces. The broad emission band comprising both monomer and aggregate in the aqueous dispersion of nanoaggregates was used for the specific cellular imaging of lysosomes and lipid droplets, respectively.

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.

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid.

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer β-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of β-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (Kd) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.

One-photon excited photoluminescence of gold nanospheres and its application in prostate specific antigen detection via fluorescence correlation spectroscopy (FCS)

Gold nanoparticles are known to exhibit appealing intrinsic plasmon-modulated photoluminescence (PL) properties which can be explored in various fluorescence-based sensing applications. In this paper, we evaluate the PL of different-sized gold nanospheres (AuNSs) under one-photon excitation (1PE) and develop a sensitive homogeneous immunoassay for the detection of prostate specific antigen (PSA) in colloidal suspension via fluorescence correlation spectroscopy (FCS). The 1PE PL of AuNSs of three different sizes are evaluated in solution phase under excitation at 405 nm via steady-state fluorescence spectroscopy measurements, while FCS analysis emphasizes the feasibility of using 1PE PL properties to monitor their diffusion behavior. Fluorescence lifetime imaging microscopy (FLIM) assays coupled with PL spectral profile analysis performed on single-particles-like structures conform the plasmonic origin of the detected PL and validate their potential of synthesized AuNSs as fluorescent probes in bioimaging and bioassays. Finally, to the best of our knowledge, we provide the first demonstration of the successful use of the 1PE PL of the synthesized AuNSs as probes for the FCS-based one-step label-free sensitive optical detection of PSA biomarker. The approach consisting in monitoring the diffusion of the AuNSs-oligomers induced by the interaction of anti-PSA-conjugated AuNSs with PSA molecules is successfully validated for the detection of PSA levels as low as 4.4 ng/ml in solution. Considering that the development of rapid, efficient and label-free biosensing methods is of continuous interest nowadays, we are confident that our results may have a strong impact on medicine towards more efficient, sensitive and reliable diagnosis.

From Macro to Mesoporous ZnO Inverse Opals: Synthesis, Characterization and Tracer Diffusion Properties.

Oxide inverse opals (IOs) with their high surface area and open porosity are promising candidates for catalyst support applications. Supports with confined mesoporous domains are of added value to heterogeneous catalysis. However, the fabrication of IOs with mesoporous or sub-macroporous voids (<100 nm) continues to be a challenge, and the diffusion of tracers in quasi-mesoporous IOs is yet to be adequately studied. In order to address these two problems, we synthesized ZnO IOs films with tunable pore sizes using chemical bath deposition and template-based approach. By decreasing the size of polystyrene (PS) template particles towards the mesoporous range, ZnO IOs with 50 nm-sized pores and open porosity were synthesized. The effect of the template-removal method on the pore geometry (spherical vs. gyroidal) was studied. The infiltration depth in the template was determined, and the factors influencing infiltration were assessed. The crystallinity and photonic stop-band of the IOs were studied using X-Ray diffraction and UV-Vis, respectively. The infiltration of tracer molecules (Alexa Fluor 488) in multilayered quasi-mesoporous ZnO IOs was confirmed via confocal laser scanning microscopy, while fluorescence correlation spectroscopy analysis revealed two distinct diffusion times in IOs assigned to diffusion through the pores (fast) and adsorption on the pore walls (slow).

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.

Anomalous Subdiffusion in Living Cells: Bridging the Gap Between Experiments and Realistic Models Through Collaborative Challenges

The life of a cell is governed by highly dynamical microscopic processes. Two notable examples are the diffusion of membrane receptors and the kinetics of transcription factors governing the rates of gene expression. Different fluorescence imaging techniques have emerged to study molecular dynamics. Among them, fluorescence correlation spectroscopy (FCS) and single-particle tracking (SPT) have proven to be instrumental to our understanding of cell dynamics and function. The analysis of SPT and FCS is an ongoing effort, and despite decades of work, much progress remains to be done. In this paper, we give a quick overview of the existing techniques used to analyze anomalous diffusion in cells and propose a collaborative challenge to foster the development of state-of-the-art analysis algorithms. We propose to provide labelled (training) and unlabelled (evaluation) simulated data to competitors all over the world in an open and fair challenge. The goal is to offer unified data benchmarks based on biologically-relevant metrics in order to compare the diffusion analysis software available for the community.

Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis

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 the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

Estimation of parameters from time traces originating from an Ornstein-Uhlenbeck process.

In this article, we develop a Bayesian approach to estimate parameters from time traces that originate from an overdamped Brownian particle in a harmonic potential, or Ornstein-Uhlenbeck process (OU). We show that least-square fitting the autocorrelation function, which is often the standard way of analyzing such data, is significantly underestimating the confidence intervals of the fitted parameters. Here, we develop a rigorous maximum likelihood theory that properly captures the underlying statistics. From the analytic solution, we found that there exists an optimal measurement spacing (Δt=0.7968τ) that maximizes the statistical accuracy of the estimate for the decay-time τ of the process for a fixed number of samples N, which plays a similar role than the Nyquist-Shannon theorem for the OU process. To support our claims, we simulated time series with subsequent application of least-square and our maximum likelihood method. Our results suggest that it is quite dangerous to apply least-squares to autocorrelation functions both in terms of systematic deviations from the true parameter values and an order-of-magnitude underestimation of confidence intervals. To see whether our findings apply to other methods where autocorrelation functions are typically fitted by least-squares, we explored the analysis of membrane fluctuations and fluorescence correlation spectroscopy. In both cases, least-square fits exhibit systematic deviations from the true parameter values and significantly underestimate their confidence intervals. This fact emphasizes the need for the development of proper maximum likelihood approaches for such methods. In summary, our results have strong implications for parameter estimation for processes that result in a single exponential decay in the autocorrelation function. Our analysis can directly be applied to single-component dynamic light scattering experiments or optical trap calibration experiments.

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.

The assembly of protein-templated gold nanoclusters for enhanced fluorescence emission and multifunctional applications

Protein-templated gold nanoclusters have attracted attention in fluorescence imaging due to their simple synthesis and good biocompatibility. However, limitations still exist such as poor colloid stability and undesirable fluorescence intensity. Here we describe the self-assembly of keratin-templated gold nanoclusters via a simple and mild preparation process, including keratin-templated synthesis of gold nanoclusters (AuNCs@Keratin), silver ions modification of AuNCs@Keratin (AuNCs-Ag@Keratin), and gadolinium ions-induced aggregation of AuNCs-Ag@Keratin (AuNCs-Ag@Keratin-Gd). It was demonstrated that the AuNCs-Ag@Keratin-Gd obtained an enhanced fluorescence intensity (6.5 times that of AuNCs@Keratin), high colloid stability for more than 4 months, and good biocompatibility. Moreover, the AuNCs-Ag@Keratin-Gd holds promise in multifunctional applications such as near-infrared (NIR) fluorescence imaging, magnetic resonance (MR) imaging, and redox-responsive drug delivery, extending the applicability of fluorescent gold nanoclusters, especially in biomedical fields. Statement of significanceAssembly-induced fluorescence enhancement has been rarely reported on as it relates to the protein-templated gold nanoclusters (AuNCs). In this work, self-assembly of protein-templated AuNCs was developed for enhanced fluorescence intensity and multifunctional applications, including bioimaging and responsive drug delivery. A cysteine-rich protein, keratin, was utilized as the template to synthesize AuNCs, which underwent silver ion modification and gadolinium ion-induced aggregation. The silver modification of the keratin-templated AuNCs facilitated the formation of a dense aggregate after gadolinium ion-induced assembly, thus generating an enhanced fluorescence intensity. Such a mechanism was confirmed by fluorescence correlation spectroscopy analysis. We believe that this work will extend the applicability of the fluorescent gold nanoclusters, especially in biomedical fields, and provided an effective approach for the mechanism analysis of the assembly-induced fluorescence enhancement via fluorescence correlation spectroscopy.

Computational design and interpretation of single-RNA translation experiments.

Advances in fluorescence microscopy have introduced new assays to quantify live-cell translation dynamics at single-RNA resolution. We introduce a detailed, yet efficient sequence-based stochastic model that generates realistic synthetic data for several such assays, including Fluorescence Correlation Spectroscopy (FCS), ribosome Run-Off Assays (ROA) after Harringtonine application, and Fluorescence Recovery After Photobleaching (FRAP). We simulate these experiments under multiple imaging conditions and for thousands of human genes, and we evaluate through simulations which experiments are most likely to provide accurate estimates of elongation kinetics. Finding that FCS analyses are optimal for both short and long length genes, we integrate our model with experimental FCS data to capture the nascent protein statistics and temporal dynamics for three human genes: KDM5B, β-actin, and H2B. Finally, we introduce a new open-source software package, RNA Sequence to NAscent Protein Simulator (rSNAPsim), to easily simulate the single-molecule translation dynamics of any gene sequence for any of these assays and for different assumptions regarding synonymous codon usage, tRNA level modifications, or ribosome pauses. rSNAPsim is implemented in Python and is available at: https://github.com/MunskyGroup/rSNAPsim.git.