Z-scan Fluorescence Correlation Spectroscopy Research Articles

Investigation of nano- and microdomains formed by ceramide 1 phosphate in lipid bilayers

Biological membranes are renowned for their intricate complexity, with the formation of membrane domains being pivotal to the successful execution of numerous cellular processes. However, due to their nanoscale characteristics, these domains are often understudied, as the experimental techniques required for quantitative investigation present significant challenges. In this study we employ spot-variation z-scan fluorescence correlation spectroscopy (svzFCS) tailored for artificial lipid vesicles of varying composition and combine this approach with high-resolution imaging. This method has been harnessed to examine the lipid-segregation behavior of distinct types of ceramide-1-phosphate (C1P), a crucial class of signaling molecules, within these membranes. Moreover, we provide a quantitative portrayal of the lipid membranes studied and the domains induced by C1P at both nano and microscales. Given the lack of definitive conclusions from the experimental data obtained, it was supplemented with comprehensive in silico studies—including the analysis of diffusion coefficient via molecular dynamics and domain populations via Monte Carlo simulations. This approach enhanced our insight into the dynamic behavior of these molecules within model lipid membranes, confirming that nano- and microdomains can co-exist in lipid vesicles.

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.

From LUVs to GUVs─How to Cover Micrometer-Sized Pores with Membranes.

Pore-spanning membranes (PSMs) are a versatile tool to investigate membrane-confined processes in a bottom-up approach. Pore sizes in the micrometer range are most suited to visualize PSMs using fluorescence microscopy. However, the preparation of these PSMs relies on the spreading of giant unilamellar vesicles (GUVs). GUV production faces several limitations. Thus, alternative ways to generate PSMs starting from large or small unilamellar vesicles that are more reproducibly prepared are highly desirable. Here we describe a method to produce PSMs obtained from large unilamellar vesicles, making use of droplet-stabilized GUVs generated in a microfluidic device. We analyzed the lipid diffusion in the free-standing and supported parts of the PSMs using z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching experiments in combination with finite element simulations. Employing atomic force indentation experiments, we also investigated the mechanical properties of the PSMs. Both lipid diffusion constants and lateral membrane tension were compared to those obtained on PSMs derived from electroformed GUVs, which are known to be solvent- and detergent-free, under otherwise identical conditions. Our results demonstrate that the lipid diffusion, as well as the mechanical properties of the resulting PSMs, is almost unaffected by the GUV formation procedure but depends on the chosen substrate functionalization. With the new method in hand, we were able to reconstitute the syntaxin-1A transmembrane domain in microfluidic GUVs and PSMs, which was visualized by fluorescence microscopy.

One-Photon Lithography for High-Quality Lipid Bilayer Micropatterns.

A relevant question in cell biology with broad implications in biomedicine is how the organization and dynamics of interacting membranes modulate signaling cascades that involve cell-cell contact. The functionalization of surfaces with supported lipid bilayers containing tethered proteins is a particularly useful method to present ligands with membrane-like mobility to cells. Here, we present a method to generate micrometer-sized patches of lipid bilayers decorated with proteins. The method uses an economic microcontact printing technique based on one-photon lithography that can be easily implemented in a commercial laser scanning microscope. We verified that both proteins and lipids freely diffuse within the patterned bilayer, as assessed by z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. These results suggest that the supported lipid bilayer patterns constitute an optimal system to explore processes involving direct interactions between cells. We also illustrate possible applications of this method by exploring the interaction of cells expressing the Fas receptor and patterns of lipid bilayers containing an agonist antibody against Fas.

Agonist mobility on supported lipid bilayers affects Fas mediated death response

Extrinsic apoptosis is initiated by recognition and clustering of the single-pass transmembrane proteins Fas ligand and Fas expressed at the surface of closely apposed lymphocytes and target cells, respectively. Since Fas-mediated death response was mainly studied with soluble antibodies, the mobility constraints for receptor activation by a membrane embedded agonist is not well understood. We explored this influence by stimulating apoptosis on functionalized supported lipid bilayers, where we quantified agonist mobility by z-scan fluorescence correlation spectroscopy. Using different lipid compositions, we show that the apoptotic response correlates with increased lateral mobility of the agonist in the lipid bilayer.

Comparison of Actin- and Glass-Supported Phospholipid Bilayer Diffusion Coefficients

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20–44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes.

Molecular Organization and Dynamics in Polymersome Membranes: A Lateral Diffusion Study

Amphiphilic block copolymers self-assemble into artificial membranes of enhanced strength and stability compared to lipid membranes and are still able to incorporate biological membrane proteins. Membrane fluidity is a key parameter for retaining function of incorporated proteins. In this study, lateral diffusion properties of membranes of diblock and triblock copolymers based on poly(2-methyl-2-oxazoline) and poly(dimethylsiloxane), with thicknesses between 6 and 21 nm, were systematically investigated. Z-scan fluorescence correlation spectroscopy was used to obtain highly accurate diffusion coefficients. The lateral diffusion coefficients (D) scale with the molecular weight of the hydrophobic block (Mh) for both diblock and triblock configurations as D ∝ Mh–1.25. A significant diffusion increase of diblocks compared to triblocks revealed that diffusion is primarily related to the different structural conformation of the macromolecules assembled in the membrane. Moreover, hindered diffusion for higher mo...

On multivalent receptor activity of GM1 in cholesterol containing membranes

Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Phospholipid diffusion coefficients of cushioned model membranes determined via z-scan fluorescence correlation spectroscopy.

Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir-Schaefer method on a hydrogel layer is potentially an effective mimic of the cross section of a biological membrane and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and co-workers revealed that phospholipid diffusion changes from raftlike to free diffusion as the temperature is increased-an insight into the dynamic behavior of hydrogel supported membranes not previously reported.

Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.

Low axial drift stage and temperature controlled liquid cell for z-scan fluorescence correlation spectroscopy in an inverted confocal geometry

A recent iteration of fluorescence correlation spectroscopy (FCS), z-scan FCS, has drawn attention for its elegant solution to the problem of quantitative sample positioning when investigating two-dimensional systems while simultaneously providing an excellent method for extracting calibration-free diffusion coefficients. Unfortunately, the measurement of planar systems using (FCS and) z-scan FCS still requires extremely mechanically stable sample positioning, relative to a microscope objective. As axial sample position serves as the inherent length calibration, instabilities in sample position will affect measured diffusion coefficients. Here, we detail the design and function of a highly stable and mechanically simple inverted microscope stage that includes a temperature controlled liquid cell. The stage and sample cell are ideally suited to planar membrane investigations, but generally amenable to any quantitative microscopy that requires low drift and excellent axial and lateral stability. In the present work we evaluate the performance of our custom stage system and compare it with the stock microscope stage and typical sample sealing and holding methods.

Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS

Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.

Cholesterol Depletion Mimics the Effect of Cytoskeletal Destabilization on Membrane Dynamics of the Serotonin1A Receptor: A zFCS Study

Single-point fluorescence correlation spectroscopy (FCS) of membrane-bound molecules suffers from a number of limitations leading to inaccurate estimation of diffusion parameters. To overcome such problems and with the overall goal of addressing membrane heterogeneities, we performed z-scan FCS (zFCS) of the serotonin1A receptor. We analyzed the results according to FCS diffusion laws that provide information on the organization of the diffusing species. Analysis of our results shows that the diffusion coefficients of the receptor and a fluorescently labeled phospholipid are similar when probed at length scales ∼210 nm. We discuss the significance of the spatiotemporal evolution of dynamics of membrane-bound molecules in the overall context of membrane domains and heterogeneity. Importantly, our results show that the serotonin1A receptor exhibits confinement in cell membranes, possibly due to interaction with the actin cytoskeleton. Surprisingly, depletion of membrane cholesterol appears to reduce receptor confinement in a manner similar to that observed in the case of cytoskeletal destabilization, implying possible changes in the actin cytoskeleton induced upon cholesterol depletion. These results constitute the first report on G-protein-coupled receptor dynamics utilizing a combination of zFCS and the FCS diffusion laws, and present a convenient approach to explore cell membrane heterogeneity at the submicron level.

Real-time monitoring of melittin-induced pore and tubule formation from supported lipid bilayers and its physiological relevance

The temporal evolution of effects of antimicrobial peptide melittin on supported phospholipid bilayers (SPBs) containing negatively charged phospholipids was monitored by ellipsometry and laser scanning microscopy together with measurements of lipid mobility by Z-scan fluorescence correlation spectroscopy. Under all conditions used in our study, we observed reproducibly two effects. The first one is formation of pores in the SPB, which occupy approximately 40% of the bilayer. The formation of pores was accompanied by a decrease in lateral diffusion coefficient of the lipids to approximately 60% of its initial value. The second, simultaneous, effect is the formation of tubules of approximately 30nm radius and length of the order of 10mum. Flushing of the sample with excess of buffer removes most of the tubules, but it does not affect the pores. Further experiments performed under various conditions demonstrated reproducibility of both phenomena.

Simultaneous Characterization of Lateral Lipid and Prothrombin Diffusion Coefficients by Z-Scan Fluorescence Correlation Spectroscopy

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.

The Effect of Shear Stress on the Fluidity of Supported Lipid Bilayers

Previous studies show that fluid shear stress on cell membranes causes metabolic changes in cells. One question that arises is what physical effects shear stress has on the fluidity of artificial lipid bilayers. As demonstrated by Haidekker et al, shear stress can change membrane fluidity of endothelial cells within 5 seconds, and membrane fluidity recovered completely after flow stopped. Due to the speed and reversibility of the change in membrane fluidity caused by shear stress, we hypothesized that the change in fluidity may be caused by the direct effect of shear stress on the packing of the lipid bilayer itself, rather than an effect mediated through an intracellular signaling cascade. This research studied the effect of shear stress on a protein-free membrane by inducing shear stress on a supported lipid bilayer in a micro-volume flow chamber. The effect of changing fluid flow in step increments on the membrane fluidity was measured using z-scan fluorescence correlation spectroscopy (FCS).

On the use of Z-scan fluorescence correlation experiments on giant unilamellar vesicles

Z-scan fluorescence correlation spectroscopy (FCS) uses multiple focus planes to determine diffusion coefficients in supported phospholipid bilayers (SPBs) without the need for extrinsic calibration. The approach can be used to discriminate diffusion modes. In this work, Z-scan FCS was applied to study the diffusion of the fluorescent lipid analogue DiI–C18(5) in the top membrane of giant unilamellar vesicles (GUVs) in the electroformation chamber. In contrast to SPBs, the intrinsic estimate of the radial waist might differ from the one obtained by extrinsic calibration. We describe a pragmatic approach to verify the validity of the Z-scan approach under these conditions.

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.

New concepts for fluorescence correlation spectroscopy on membranes

Fluorescence correlation spectroscopy (FCS) is a powerful tool to measure useful physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in model and cell membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we assess the potential and limitations of standard confocal FCS on lipid membranes and present recent developments which facilitate accurate and quantitative measurements on such systems. In particular, we discuss calibration-free diffusion and concentration measurements using z-scan FCS and two focus FCS and present several approaches using scanning FCS to accurately measure slow dynamics. We also show how surface confined FCS enables the study of membrane dynamics even in presence of a strong cytosolic background and how FCS with a variable detection area can reveal submicroscopic heterogeneities in cell membranes.

Combination of ellipsometry, laser scanning microscopy and Z‐scan fluorescence correlation spectroscopy elucidating interaction of cryptdin‐4 with supported phospholipid bilayers

The present study has two main objectives. The first is to characterize antimicrobial peptide (AMP) cryptdin-4 (Crp-4) interactions with biological membranes and to compare those interactions with those of magainin 2. The second is to combine the complementary experimental approaches of laser scanning microscopy (LSM), ellipsometry, and Z-scan fluorescence correlation spectroscopy (FCS) to acquire comprehensive information on mechanisms of AMP interactions with supported phospholipid bilayers (SPBs)-a popular model of biological membranes. LSM shows appearance of inhomogeneities in spatial distribution of lipids in the bilayer after treatment with Crp-4. Ellipsometric measurements show that binding of Crp-4 does not significantly change the lipid structure of the bilayer (increase in adsorbed mass without a change in thickness of adsorbed layer). Furthermore, Crp-4 slows the lateral diffusion of lipids within the membrane as shown by Z-scan FCS. All changes of the bilayer induced by Crp-4 can be partially reversed by flushing the sample with excess of buffer. Bilayer interactions of magainin 2 are significantly different, causing large loss of lipids and extensive damage to the bilayer. It seems likely that differences in peptide mode of action, readily distinguished using these combined experimental methods, are related to the distinctive beta-sheet and alpha-helical structures of the respective peptides.