Fluorescence Correlation Spectroscopy Measurements Research Articles

Evidence for Nanostructures of at Least 20 nm in a Phosphonium Ionic Liquid at Room Temperature Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) measurements are performed on the ionic liquid (IL), tetradecyl(trihexyl) phosphonium chloride, [P66614+][Cl-], using fluorescent probes of varying sizes: ATTO 532, ∼2 nm; and 20- and 40 nm fluorescent beads. The fluorescence correlation function, G(t), is analyzed in terms of a distribution of diffusion coefficients using a maximum entropy method (MEM). For ATTO 532 and the 20 nm beads, the fit to G(t) yields two well-defined distributions; for the 40 nm beads, however, only one is obtained. These results are consistent with the existence of two nanodomains whose size is greater than or equal to 20 nm and less than 40 nm. The origin of such nanodomains is attributed to a liquid-liquid phase transition. Other groups have observed liquid-liquid phase transitions experimentally in a number of systems, including [P66614+][Cl-]. We suggest that because large regions (i.e., greater than 1-2 nm) resulting from the liquid-liquid phase transition would be expected to have different properties, such as viscosity, and because their presence would necessarily increase the number of interfaces in the IL, these regions may provide an explanation for the exceptional behavior of ILs in various separation systems.

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

Photoisomerization of heptamethine cyanine (Cy7) dyes: A theoretical study

In this study, density functional theory (DFT) combined with time-dependent (TD) DFT calculations were employed to investigate the photoisomerization reaction kinetics of two near infrared (NIR) heptamethine cyanine (Cy7-NH3 and Cy7-SO3) dyes in the ground singlet state and the first excited singlet state. We found that the photoisomerization of the ground state all-trans Cy7 molecules results in at least one mono-cis and one all-cis species that demonstrate redshifted emission, in agreement with recently published transient state excitation modulation spectroscopy and fluorescence correlation spectroscopy measurements. The transition states were estimated for a whole photoisomerization pathway for both the ground singlet and first excited singlet state potential energy surfaces. We have found that all-cis isomers of the studied Cy7 dyes can be achieved through a sequential two-step photoisomerization within the excited singlet state potential energy surface, along the double CC bond adjacent to edge group (leading to mono-cis isomer 1) and along the double CC bond adjacent to the central-chain group (leading to mono-cis isomer 2). Computations show that all-trans→ mono-cis isomer 1→all-cis kinetics is limited by the first trans→ mono-cis isomer 1 stage, while the all-trans→ mono-cis isomer 2→all-cis pathway is limited by the second mono-cis isomer 2→all-cis stage. Accounting for the fact that mono-cis isomer 2 demonstrates red-shifted emission compared to the all-trans form and that this mono-cis isomer 2 is reachable through the energetically favorable all-trans→ mono-cis isomer 2 stage, we concluded that the experimentally observed red-shifted emission by Cy7-NH3 and Cy7-SO3 should be assigned to the formation of mono-cis isomer 2 species. If the all-cis isomer is populated through the double-step photoisomerization it can also be considered as a source of red-shifted emission. However, as follows from our simulations, the all-cis isomer is kinetically intricate to achieve compared to the mono-cis isomer 2.

Effect of molecular crowders on ligand binding kinetics with G-quadruplex DNA probed by fluorescence correlation spectroscopy

Guanine-rich single-stranded DNA folds into G-quadruplex DNA (GqDNA) structures, which play crucial roles in various biological processes. These structures are also promising targets for ligands, potentially inducing antitumor effects. While thermodynamic parameters of ligand/DNA interactions are well-studied, the kinetics of ligand interaction with GqDNA, particularly in cell-like crowded environments, remain less explored. In this study, we investigate the impact of molecular crowding agents (glucose, sucrose, and ficoll 70) at physiologically relevant concentrations (20% w/v) on the association and dissociation rates of the benzophenoxazine-core based ligand, cresyl violet (CV), with human telomeric antiparallel-GqDNA. We utilized fluorescence correlation spectroscopy (FCS) along with other techniques. Our findings reveal that crowding agents decrease the binding affinity of CV to GqDNA, with the most significant effect—a nearly three-fold decrease—observed with ficoll 70. FCS measurements indicate that this decrease is primarily due to a viscosity-induced slowdown of ligand association in the crowded environment. Interestingly, dissociation rates remain largely unaffected by smaller crowders, with only small effect observed in presence of ficoll 70 due to direct but weak interaction between the ligand and ficoll. These results along with previously reported data provide valuable insights into ligand/GqDNA interactions in cellular contexts, suggesting a conserved mechanism of saccharide crowder influence, regardless of variations in GqDNA structure and ligand binding mode. This underscores the importance of considering crowding effects in the design and development of GqDNA-targeted drugs for potential cancer treatment.

Thickness of Nanoscale Poly(Dimethylsiloxane) Layers Determines the Motion of Sliding Water Drops.

Layers of nanometer thick polydimethylsiloxane (PDMS) are applied as hydrophobic coatings because of their environmentally friendly and chemically inert properties. In applications such as heat exchangers or fog harvesting, low water drop friction on surfaces is required. While the onset of motion (static friction) has been studied, the knowledge of dynamic friction needs to be improved. To minimize drop friction, it is essential to understand which processes lead to energy dissipation and cause dynamic friction? Here, the dynamic friction of drops on PDMS brushes of different thicknesses is measured, covering the whole available velocity regime. The brush thickness L turns out to be a predictor for drop friction. 4-5nm thick PDMS brush shows the lowest dynamic friction. A certain minimal thickness is necessary to form homogeneous surfaces and reduce the attractive van der Waals interaction between water and the substrate. The increase in dynamic friction above L = 5nm is also attributed to the increasing viscoelastic dissipation of the capillary ridge formed at the contact line. The height of the ridge is related to the brush thickness. Fluorescence correlation spectroscopy and atomic force measurements support this interpretation. Sum-frequency generation further indicates a maximum order at the PDMS-water interface at intermediate thickness.

Diffusion behavior of rhodamine 6G in single octadecylsilyl-functionalized silica particle revealed by fluorescence correlation spectroscopy.

We investigated the diffusion behavior of rhodamine 6G (Rh6G) within single octadecylsilyl-functionalized (ODS) silica particle in an acetonitrile (ACN)/water system using fluorescence correlation spectroscopy (FCS). FCS measurements were conducted at the center of the particle to exclusively determine the intraparticle diffusion coefficient (D). The obtained D values were analyzed based on a pore and surface diffusion model, the results of which indicate that surface diffusion primarily governs the intraparticle diffusion of Rh6G. Furthermore, an increase in the concentration of ACN (CACN) resulted in a corresponding increase in the surface diffusion coefficient (Ds), whereas the addition of NaCl did not significantly affect the Ds values. We attributed this dependence of Ds to the dielectric constant change in the interfacial liquid phase formed on the ODS layer. Specially, Ds values of (4.0 ± 0.5) × 10-7, (7.7 ± 1.1) × 10-7, (1.0 ± 0.3) × 10-6, and (1.1 ± 0.2) × 10-6cm2s-1 were obtained for CACN = 20, 30, 40, and 50vol%, respectively. We anticipate that this approach will contribute to advancing research on intraparticle mass transfer mechanisms.

Fluorescence correlation spectroscopy measurements of DNA unwinding/bending fluctuations with DNA backbone-incorporated dyes

Fluorescence correlation spectroscopy measurements of DNA unwinding/bending fluctuations with DNA backbone-incorporated dyes

Rotational and translational diffusion of biomolecules in complex liquids and HeLa cells.

Diffusive motion accompanies many physical and biological processes. The Stokes-Sutherland-Einstein relation for the translational diffusion coefficient, DT, agrees with experiments done in simple fluids but fails for complex fluids. Moreover, the interdependence between DT and rotational diffusion coefficient, DR, also deviates in complex fluids from the classical relation of DT/DR = 4r2/3 known in simple fluids. Makuch et al. Soft Matter, 2020, 16, 114-124 presented a generalization of the classical translational and rotational diffusion theory for complex fluids. In this work, we empirically verify this model based on simultaneous translational and rotational diffusion measurements. We use fluorescently stained cowpea chlorotic mottle virus (CCMV) particles as monodisperse probes and aqueous polyethylene glycol (PEG) solutions as a model complex fluid. The theory and experimental data obtained from fluorescence correlation spectroscopy (FCS) measurements agreed. Finally, we used the same model and analyzed the diffusion of Yo-Pro-1 stained large ribosomal subunits (LSU) in the cytoplasm and nucleus of living HeLa cells.

Deep learning reduces data requirements and allows real-time measurements in imaging FCS

Imaging fluorescence correlation spectroscopy (FCS) is a powerful tool to extract information on molecular mobilities, actions, and interactions in live cells, tissues, and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1-ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Third, as FCS evaluation is model dependent, data evaluation is significantly slowed unless analytic models are available. Here, we introduce two convolutional neural networks—FCSNet and ImFCSNet—for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both convolutional neural networks are trained on simulated data, are model agnostic, and allow autonomous, real-time evaluation of imaging FCS measurements.

Fluorescence correlation spectroscopy measurements on amyloid fibril reveal at least two binding modes for fluorescent sensors

Interaction of fluorophore with amyloid structure is central to the design and development of fluorescent sensors. In literature ensemble spectroscopy results reveal at least two distinct binding modes for most of the torsional probes like Thioflavin-T, SYPRO Orange, etc. though single molecule localization microscopy and spectroscopy results consider only one binding mode. To address this discrepancy we employed fluorescence correlation spectroscopy (FCS) to measure the binding kinetics on immobilized fibril structures with single molecule sensitivity. Our results indicate that there are at least two possible biding configurations with differing relaxation time constants indicating possible weaker and stronger binding configurations depending on the local environment on the fibril structure. Critical evaluation of on-time distributions from the recorded wide-field image stack for single molecule localization microscopy image, further highlight the presence of two burst times. Such bi-modal binding behavior is attributed to the differing binding configurations on fibril architecture.

Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications.

The Parkinson's disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked β-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS87 enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release. Here, we show how one can study these effects by synthesizing αS constructs containing authentic PTMs and labels for single molecule fluorescence correlation spectroscopy measurements. We synthesize αS-gS87 and αS-pS87 by combining native chemical ligation with genetic code expansion approaches. We introduce the fluorophore by a click reaction with a non-canonical amino acid. Beyond the specific problem of PTM effects on αS, our studies highlight the value of this combination of methods for multiply modifying proteins.

Spectroscopic and Chemical Properties of Ionic Liquids: Computational Study.

A brief account is given of highlights of our computational efforts - often in collaboration with experimental groups - to understand spectroscopic and chemical properties of ionic liquids (ILs). Molecular dynamics, including their inhomogeneous character, responsible for key spectral features observed in dielectric absorption, infra-red (IR) and fluorescence correlation spectroscopy (FCS) measurements are elucidated. Mechanisms of chemical processes involving imidazolium-based ILs are illustrated for CO2 capture and related reactions, transesterification of cellulose, and Au nanocluster-catalyzed Suzuki cross-coupling reaction with attention paid to differing roles of IL ions. A comparison with experiments is also made.

Achieving High Temporal Resolution in Single‐Molecule Fluorescence Techniques Using Plasmonic Nanoantennas

AbstractSingle‐molecule fluorescence techniques are essential for investigating the molecular mechanisms in biological processes. However, achieving sub‐millisecond temporal resolution to monitor fast molecular dynamics remains a significant challenge. The fluorescence brightness is the key parameter that generally defines the temporal resolution for these techniques. Conventional microscopes and standard fluorescent emitters fall short in achieving the high brightness required for sub‐millisecond monitoring. Plasmonic nanoantennas are proposed as a solution, but despite huge fluorescence enhancement having been obtained with these structures, the brightness generally remains below 1 million photons/s/molecule. Therefore, the improvement of temporal resolution is overlooked. This article presents a method for achieving high temporal resolution in single‐molecule fluorescence techniques using plasmonic nanoantennas, specifically optical horn antennas. This work demonstrates about 90% collection efficiency of the total emitted light, reaching a high fluorescence brightness of 2 million photons/s/molecule in the saturation regime. This enables observations of single molecules with microsecond binning time and fast fluorescence correlation spectroscopy measurements. This work expands the applications of plasmonic antennas and zero‐mode waveguides in the fluorescence saturation regime toward brighter single‐molecule signal, faster temporal resolutions, and improved detection rates to advance fluorescence sensing, DNA sequencing, and dynamic studies of molecular interactions.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy

BackgroundSkeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy.MethodsTo identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse.ResultsIn this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest.ConclusionsOur findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Measuring the fluidity of the membrane of Bacillus subtilis using TIR-FCS.

Bacteria need to constantly maintain the fluidity of their membrane in response to external stimuli such as changes in temperature, growth medium, etc. So far, bacterial membrane remodeling could only be assessed indirectly via biochemical methods like fatty acid analysis or in live bacteria using environment-sensitive dyes. Fluorescence correlation spectroscopy (FCS), a gold-standard technique in membrane dynamics investigations, was however barely used to study bacterial membranes. This is likely due to an increased technical complexity, caused by the small size of bacteria and mounting difficulties, which complicates FCS measurements on confocal microscopes as they are usually performed. Here, we show that imaging FCS on a TIRF microscope (TIR-FCS) can remediate this thanks to an increased axial resolution with respect to confocal and ability to correct for sample drift using image registration. To facilitate the analysis of the large amount of data generated by TIR-FCS, we developed a simple method to automatically exclude artefactual FCS curves. As a result, we could perform high-throughput measurements of the diffusion coefficients of lipophilic dyes like Nile Red or TopFluor-sphingomyelin in the membrane of B. subtilis. Using this assay, we observed the effects of membrane remodeling in response to known stimuli like a cold shock. We also looked at the effects of the widely used membrane-perturbing drug benzyl alcohol, revealing an unexpectedly temperature-dependent fluidization effect. Finally, we used TIR-FCS to explore membrane fluidity adaptation during the sporulation process. We believe that this method has significant potential in microbiology and can easily be applied in the future to different problems, to study membrane proteins diffusion or to understand the effects of selected drugs on membrane viscosity.

Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy.

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated "stealth" liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.

Molecular crowders modulate ligand binding affinity to G-quadruplex DNA by decelerating ligand association

The study of kinetics of ligand binding to G-quadruplex DNA (GqDNA) structures is of paramount importance for comprehending (possible) ligand-induced anticancer activity involving GqDNA within cells. However, cellular environment is crowded with variety of small- and macro-molecules that occupy ∼30–40 % of cell-volume. While only few earlier studies dealt with deciphering the kinetics of ligands’ interactions with GqDNA in dilute solution, such studies in cell-like crowded milieu are absent. Here we investigate the effect of small and macro molecular crowders, glucose, sucrose and ficoll 70, on the kinetic steps of association and dissociation of a benzophenoxazine-ligand (Cresyl Violet: CV) with human telometic (hybrid) GqDNA structure using fluorescence correlation spectroscopy (FCS), aided by other methods. We find that the binding constants of the ligand to GqDNA change appreciably with nearly fivefold decrease in the presence of ficoll 70, compared to that in pure buffer solution. FCS measurements unfold that this decrease of binding constants is mainly modulated by the viscosity-induced deceleration of the association of the ligand to GqDNA in the crowded solution; however, the rate-determining dissociation rates remain nearly unchanged in the presence of all three crowders. These results have important implications in the context of ligand/GqDNA interactions within cellular environment, which indicate that even if the binding affinity of a ligand to GqDNA structures may be influenced by cellular crowders, they may not influence the unbinding rate of the ligand from a stable ligand/GqDNA complex formed by strong π-π stacking interactions.

Measurement of Protein and Nucleic Acid Diffusion Coefficients Within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation of protein and RNA complexes into biomolecular condensates has emerged as a ubiquitous phenomenon in living systems. These protein-RNA condensates are thought to be involved in many biological functions in all forms of life. One of the most sought-after properties of these condensates is their dynamical properties, as they are a major determinant of condensate physiological function and disease processes. Measurement of the diffusion dynamics of individual components in a multicomponent biomolecular condensate is therefore routinely performed. Here, we outline the experimental procedure for performing in-droplet fluorescence correlation spectroscopy (FCS) measurements to extract the diffusion coefficient of individual molecules within a biomolecular condensate in vitro. Unlike more common experiments such as fluorescence recovery after photobleaching (FRAP), where data interpretation is not straightforward and strictly model dependent, FCS offers a robust and more accurate way to quantify biomolecular diffusion rates in the dense phase. The small observation volume allows FCS experiments to report on the local diffusion coefficient within a spatial resolution of <1μm, making it ideal for probing spatial inhomogeneities within condensates as well as variable dynamics within subcompartments of multiphasic condensates.