Fluorescence Fluctuation Research Articles

Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.

Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

Quantitative models for transcriptional dynamics monitored using an MS2-GFP system

Gene expression begins with transcription, in which the information in the DNA sequence of a gene is copied into mRNA. The process of transcription can be divided into three distinct steps: initiation, elongation, and termination. In this review, we discuss how the time rates of these three steps can be obtained by analyzing fluorescence signals from the transcription sites of mRNA labeled with the MS2-GFP system. Particularly, we focus on the analysis methods using (1) the auto-correlation of fluorescence fluctuation and (2) the fluorescence change upon inhibition of transcription initiation. These analysis methods can be also applied to the translational dynamics of a protein labeled using the SunTag system. We provide the full mathematical derivation of the models and relate the theoretical predictions to experimental data to facilitate broader applications of the analysis methods.

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.

Molecular simulation, characteristics and mechanism of thermal-responsive acetylated amylose V-type helical complexes.

To explore the thermal-responsive characteristics of acetylated amylose-guest V-type helical complexes (AAGHCs) and their potential use as thermal-responsive drug carriers, different types of AAGHCs were built, in which acetylated amylose was used as a host, and iodine, propofol, or hexane was utilized as the guest molecule. Their thermal-responsive characteristics were investigated through molecular dynamic (MD) simulation and corresponding experiments. MD simulation showed that the thermal-responsive helix-unfolding and guest-release behavior in AAGHCs, and the complete unfolding of AAGHC could be divided into brewing, triggering and collapsing periods. Energy analysis revealed that the Lana-Jones potential is an important binding energy that bridges host and guest molecules and enhances the stability of the helix. The various types or number of guests showed different binding energies. The stronger the binding energy, higher is the temperature required to trigger the unfolding of the helix and the releasing of guests. FT-IR and X-ray diffraction analyses confirmed the structures of AAGHCs. The change in hydrated size, and UV-VIS absorption of AAGHCs at high temperatures both confirmed the thermal-responsiveness of AAGHCs. The fluorescence fluctuation of loaded 7-hydroxycoumarin reflected the same thermal-responsive process and mechanism as MD simulation. This study provides meaningful theoretical guidance for the design of thermal-responsive drug carriers based on acetylated amylose-guest V-type helical complexes.

Target specific aptamer-induced self-assembly of fluorescent graphene quantum dots on palladium nanoparticles for sensitive detection of tetracycline in raw milk

The excessive use of tetracyclines (TCs), a bacteriostaticantibiotic, in food products, has led to the accumulation of TCs residues in the human body, affecting human health seriously. Therefore, the development of a highly sensitive method to detect TCs in food is of utmost importance. This study reports a novel sensing strategy using aptamer-induced fluorescence fluctuation of graphene quantum dots (GQDs) and palladium nanoparticles (Pd NPs) for the rapid and label-free detection of tetracycline with a limit of detection of 45 ng.mL−1. A novel single-step synthesis of positively charged Pd NPs and one-step green synthesis of GQDs directly from graphite has been developed. The proposed strategy provides an efficient way to detect low traces of TCs and a new technique for the development of aptamer-based sensors.

Fluorescent Visualization of Nucleolar G-Quadruplex RNA and Dynamics of Cytoplasm and Intranuclear Viscosity

The nucleolus, the locus of ribosome biogenesis, was found to be the predominant intracellular target of a new fluorescent probe, V-P1. In solution, the probe demonstrated both a selectivity to RNA...

FCSlib: an open-source tool for fluorescence fluctuation spectroscopy analysis for mobility, number and molecular brightness in R.

FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. It encompasses techniques such as Fluorescence Correlation Spectroscopy, Number and Brightness, Pair Correlation Function and Pair Correlation of Molecular Brightness. Source code available at https://cran.r-project.org/web/packages/FCSlib/ for Linux, Windows and macOS platforms. Sample data as well as a user's guide are available at https://github.com/FCSlib/FCSlib. Supplementary data are available at Bioinformatics online.

Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

Quantification of fluctuations from fluorescence correlation spectroscopy experiments in reaction-diffusion systems.

Fluorescence correlation spectroscopy (FCS) is commonly used to estimate diffusion and reaction rates. In FCS the fluorescence coming from a small volume is recorded and the autocorrelation function (ACF) of the fluorescence fluctuations is computed. Scaling out the fluctuations due to the emission process, this ACF can be related to the ACF of the fluctuations in the number of observed fluorescent molecules. In this paper the ACF of the molecule number fluctuations is studied theoretically, with no approximations, for a reaction-diffusion system in which the fluorescence changes with binding and unbinding. Theoretical ACFs are usually derived assuming that fluctuations in the number of molecules of one species are instantaneously uncorrelated to those of the others and obey Poisson statistics. Under these assumptions, the ACF derived in this paper is characterized only by the diffusive timescale of the fluorescent species and its total weight is the inverse of the mean number of observed fluorescent molecules. The theory is then scrutinized in view of previous experimental results which, for a similar system, gave a different total weight and correct estimates of other diffusive timescales. The total weight mismatch is corrected by assuming that the variance of the number of fluorescent molecules depends on the variance of the particle numbers of the other species, as in the variance decomposition formula. Including the finite acquisition time in its computation, it is shown that the ACF depends on various timescales of the system and that its total weight coincides with the one obtained with the variance decomposition formula. This calculation implies that diffusion coefficients of nonobservable species can be estimated with FCS experiments performed in reaction-diffusion systems. Ways to proceed in future experiments are also discussed.

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Green synthesis of gold nanoparticles using cinnamon bark extract, characterization, and fluorescence activity in Au/eosin Y assemblies

This study reports the synthesis of gold nanoparticles (AuNPs) using an unconventional, eco-friendly method that utilizes cinnamon bark extract as a reducing agent and stabilizer. The synthesized AuNPs were characterized using UV–Vis spectroscopy and transmission electron microscopy (TEM). UV–Vis spectrum shows the surface plasmon resonance (SPR) peak around 535 nm. TEM shows the spherical shape of AuNPs and the particle size distribution at around 35 nm. The ability of the synthesized AuNPs as a quencher of eosin Y dye was monitored using a fluorometer. It was found that in the presence of AuNPs, the fluorescence peak of eosin is quenched. The fluorescence of the mixture of eosin and AuNPs is enhanced in the presence of bovine serum albumin (BSA) protein. TEM shows that the average size of the AuNPs is reduced from 35 to 5 nm with the addition of eosin, and the size of AuNPs then increased to 26 nm upon adding BSA to the mixture of ANPs and eosin. These fluorescence fluctuations may be used for trace identification and biosensing.

SOFISM: Super-resolution optical fluctuation image scanning microscopy

Super-resolution optical microscopy is a rapidly evolving scientific field dedicated to imaging sub-wavelength-sized objects, leaving its mark in multiple branches of biology and technology. While several super-resolution optical microscopy methods have become a common tool in life science imaging, new methods, supported by cutting-edge technology, continue to emerge. One rather recent addition to the super-resolution toolbox, image scanning microscopy (ISM), achieves up to twofold lateral resolution enhancement in a robust and straightforward manner. To further enhance ISM’s resolution in all three dimensions, we present and experimentally demonstrate here super-resolution optical fluctuation ISM (SOFISM). Measuring the fluorescence fluctuation contrast in an ISM architecture, we obtain images with a × 2.5 lateral resolution beyond the diffraction limit along with an enhanced axial resolution for a fixed cell sample labeled with commercially available quantum dots. The inherent temporal averaging of the ISM technique enables image acquisition of the fluctuation correlation contrast within millisecond-scale pixel dwell times. SOFISM can therefore offer a robust path to achieve high-resolution images within a slightly modified confocal microscope, using standard fluorescent labels and reasonable acquisition times.

Coastal geomorphology and oceanographic features shape subtidal benthic communities in management areas of central Chile

Abstract The spatial variation in community structure among 30 areas for the management and exploitation of benthic resources (AMEBRs) in central Chile was assessed from surveys conducted during the same spring–summer season. One hundred taxa identified in the subtidal surveys were grouped into 10 functional groups. The influence and relative importance of external factors over benthic associations were assessed through nonmetric multidimensional scaling (NMDS) and path analysis. Three main categories of external factors were considered: ‘terrain traits’ (e.g. coastline exposure), ‘surface‐ocean conditions’ (e.g. sea surface temperature, SST), and ‘fishery‐related variables’ (e.g. cumulative landings). The NMDS revealed a strong association between benthic community structure and both terrain and surface‐ocean conditions (42% of variance explained by permutational multivariate analysis of variance), but not with fishery‐related variables. Assemblages typical of rocky substrates were associated with larger seasonal fluctuations in SST and surface chlorophyll a. The presence of soft‐bottom functional groups was associated with higher levels of surface fluorescence and larger seasonal fluctuations in a satellite‐derived proxy for river‐plume influence (nLw645). A path analysis model for hard‐bottom communities reached a 49% goodness of fit (GOF), with the presence of epifaunal filter‐feeders, grazers, predators, and scavengers being significantly correlated with leathery and corticated foliose algae, which in turn depend on the fraction of rocky substrate in the AMEBR; the fraction of rocky substrate is positively correlated with coastline exposure. The model for soft‐bottom communities reached a 52% GOF with the presence of infaunal filter‐feeders and epifaunal detritivores depending directly on SST and surface chlorophyll a. The fishery‐related variables considered in the model had no apparent effect on the structure of either type of subtidal community. These results provide a hierarchy of physical–biological associations determining the functional composition of subtidal benthic communities in management areas along central Chile. This knowledge may improve future assessments of ecosystem response to changes in environmental conditions and management strategies.

Proposal for simultaneous analysis of fluorescence intensity fluctuations and resonance energy transfer (IFRET) measurements

Resonance energy transfer (RET) and fluorescence fluctuation spectroscopies (FFS) are powerful fluorescence-based techniques for quantifying the self-association of membrane receptors within oligomeric complexes in living cells. However, RET spectrometry’s ability to extract information on the detailed quaternary structure of oligomers sometimes rests on assumptions regarding the relative abundances of oligomers of different sizes, while FFS techniques may provide oligomer size information but not quaternary structure details, as they lack a probe for inter-molecular distances. In this report, we introduce a method which we termed ‘intensity fluctuations and resonance energy transfer’ (IFRET), which combines analysis of donor and acceptor intensity fluctuations with RET efficiency determination. Because the three measured quantities each have a unique dependence on the acceptor mole fraction (XA), simultaneous global fitting of all three dramatically reduces ambiguity in the data fitting and choice of the most appropriate fitting model. We demonstrate the effectiveness of the method on simulated brightness and RET efficiency data incorporating mixtures of monomers, dimers, and tetramers and show that IFRET analysis provides a major improvement in both identifying the correct quaternary structure model and extracting the relative abundances of the monomers, dimers, and tetramers. Conceivably, the enhanced resolution of IFRET could potentially provide insight into the functional significance of receptor oligomerization in the presence and absence of cognate ligands.

Correlated Exciton Fluctuations in a Two-Dimensional Semiconductor on a Metal.

Excitons in nanoscale materials can exhibit fluorescence fluctuations. Intermittency is pervasive in zero-dimensional emitters such as single molecules and quantum dots. In contrast, two-dimensional semiconductors are generally regarded as stable light sources. Noise contains, however, valuable information about a material. Here, we demonstrate fluorescence fluctuations in a monolayer semiconductor due to sensitivity to its nanoscopic environment focusing on the case of a metal film. The fluctuations are spatially correlated over tens of micrometers and follow power-law statistics, with simultaneous changes in emission intensity and lifetime. At low temperatures, an additional spectral contribution from interface trap states emerges with fluctuations that are correlated with neutral excitons and anticorrelated with trions. Mastering exciton fluctuations has implications for light-emitting devices such as single-photon sources and could lead to novel excitonic sensors. The quantification of fluorescence fluctuations, including imaging, unlocks a set of promising tools to characterize and exploit two-dimensional semiconductors and their interfaces.

Spectral cross-cumulants for multicolor super-resolved SOFI imaging

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

Efficient super-resolution volumetric imaging by radial fluctuation Bayesian analysis light-sheet microscopy.

Various computational super-resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super-resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light-sheet fluorescence microscopy, to achieve super-resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700-nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high-contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super-resolved 3D image at 4-times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2-orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual-color imaging of cell nucleus in live Drosophila brain. The spatial co-localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.

Measuring protein surface density on glass substrate using fluorescence fluctuation spectroscopy

Surface functionalization implies to know the surface density of proteins, which, for cellular biology, impacts on cells behavior (motility, spreading, and fate). Contradictory, the quantification of this parameter is lacking. We studied the surface density of deposited proteins on glass using the Image Correlation Spectroscopy (ICS) technique as part of the Fluorescence Fluctuation Microscopy (FFM) methods. As the confocal microscope scans the surface, the proteins deposited on it contribute to the image through the confocal point spread function (PSF). The width of the image autocorrelation is equal to the width of the PSF; its amplitude provides the average number of proteins within the PSF, proportional to the surface density. Nevertheless, the assessment of the surface density can be underestimated if the surface coverage is not uniform or overestimated if a background contributes to the fluorescence signal. Hence, we combine the ICS and photobleaching (pICS) to detect these artifacts and estimate the actual surface density. The pICS method makes it possible to have a better control of the estimation of the surface density. We noticed that the surface density of proteins increased with their initial concentration in solutions. Overall, FFM is a quantitative technique to infer the surface density of protein.

Temperature sensitivity based on Er3+ fluorescence fluctuation in Gd2Ti2O7: Er3+-Yb3+ porous nanofibers

Abstract (Gd1−x−yErxYby)2Ti2O7 (GTOEY) fibers were synthesized by electrospinning technique and heat-treatment processes, in which nano-scale fibers vested with homogeneous porous morphology, high crystallinity and elevated temperature heat-resistance. Under 977 nm laser excitation, GTOEY nanofibers exhibit effective frequency upconversion (UC) fluorescence, therefore, the violent green and dimmed red UC emissions are identified as two-photon absorption processes, attributing to 2H11/2/4S3/2 → 4I15/2 and 4F9/2 → 4I15/2 radiation transitions of Er3+, respectively. Moreover, the temperature sensitivity based on UC emission fluctuation of Er3+ over a wide temperature range of 303–633 K has been derived. Thereby, having used a 980 nm laser and achieved green UC luminescence of GTOEY fibers centered at 525 and 545 nm, the maximum absolute and relative sensitivity values are obtained to be 0.382% K−1 and 1.12% K−1, respectively. These significant results demonstrate that the GTOEY nanofibers with distinct porous structure and admirable photothermal sensitivities can be regarded as a promising temperature sensing material used for overheating alarm and safety protection equipment.

Deriving a Proxy for Iron Limitation From Chlorophyll Fluorescence on Buoyancy Gliders

Chlorophyll fluorescence, primarily used to derive phytoplankton biomass, has long been an underutilized source of information on phytoplankton physiology. Diel fluctuations in chlorophyll fluorescence are affected by both photosynthetic efficiency and non-photochemical quenching (NPQ), where NPQ is a decrease in fluorescence through the dissipation of excess energy as heat. NPQ variability is linked to iron and light availability, and has the potential to provide important diagnostic information on phytoplankton physiology. Here we establish a relationship between NPQsv (Stern-Volmer NPQ) and indices of iron limitation from nutrient addition experiments in the sub-Antarctic zone (SAZ) of the Atlantic Southern Ocean, through the derivation of NPQmax (the maximum NPQsv value) and αNPQ (the light limited slope of NPQsv). Significant differences were found for both Fv/Fm and αNPQ for iron versus control treatments, with no significant differences for NPQmax. Similar results from CTDs indicated that changes in NPQ were driven by increasing light availability from late July to December, but by both iron and light from January to February. We propose here that variability in αNPQ, which has removed the effect of light availability, can potentially be used as a proxy for iron limitation (as shown here for the Atlantic SAZ), with higher values being associated with greater iron stress. This approach was transferred to data from a buoyancy glider deployment at the same location by utilising the degree of fluorescence quenching as a proxy for NPQGlider, which was plotted against in situ light to determine αNPQ. Seasonal increases in αNPQ are consistent with increased light availability, shoaling of the mixed layer depth (MLD) and anticipated seasonal iron limitation. The transition from winter to summer, when positive net heat flux dominates stratification, was coincident with a 24% increase in αNPQ variability and a switch in the dominant driver from incident PAR to MLD. The dominant scales of αNPQ variability are consistent with fine scale variability in MLD and a significant positive relationship was observed between these two at a ~10 day window. The results emphasise the important role of fine scale dynamics in driving iron supply, particularly in summer when this micronutrient is limiting.