Martin Štefl

Molecular rheometry: Direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Applications of phasors to in vitro time-resolved fluorescence measurements.

The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented.

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.

Applications of Phasor Plots to in Vitro Protein Studies

In a recent article, we described the application of phasor analysis to fluorescence intensity decay data on in vitro samples. As detailed in that article, this method provides researchers with a simple graphical method for viewing lifetime data that can be used to quantify individual components of a mixture as well as to identify excited state reactions. In the current article, we extend the use of in vitro phasor analysis to intrinsic protein fluorescence. We show how alterations in the excited state properties of tryptophan residues are easily visualized using the phasor method. Specifically, we demonstrate that protein-ligand and protein-protein interactions can result in unique shifts in the location of phasor points, indicative of protein conformational changes. Application of the method to a rapid kinetic experiment is also shown. Finally, we show that the unfolding of lysozyme with either urea or guanidine hydrochloride results in different phasor trajectories, indicative of unique denaturation pathways.

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.

Accurate Determination of the Orientational Distribution of a Fluorescent Molecule in a Phospholipid Membrane

Orientation of lipophilic dye molecules within a biological membrane can report on the molecular environment, i.e., the physical and chemical properties of the surrounding membrane. This fact, however, remains under-utilized, largely because of our limited quantitative knowledge of molecular orientational distributions and the fact that robust techniques allowing experimental observation of molecular orientations of dyes in biological membranes are only being developed. In order to begin filling this lack of knowledge and to develop appropriate tools, we have investigated the membrane orientational distribution of the 3-hydroxyflavone-based membrane dye F2N12S. Results of our single- and two-photon polarization microscopy observations of linear dichroism of F2N12S-labeled giant unilamellar vesicles are consistent with a Gaussian-like orientational distribution of the transition dipole moment of the dye, with a mean tilt angle of 53.2 ± 0.1° with respect to the bilayer normal and a standard deviation of 13.3 ± 0.6°. Independently, by combining quantum chemical calculations and molecular dynamics simulations, we obtained very similar values; a mean tilt angle of 48 ± 4° and a standard deviation of 13 ± 2°. The good agreement between the experimentally and computationally obtained values cross-validates both approaches and gives confidence to the results obtained. The results open a door to robust quantitative determinations of orientational distributions of fluorescent molecules (ranging from simple synthetic dyes to fluorescent proteins attached to membrane proteins) associated with lipid membranes. Such determinations enable rational development of a novel class of sensitive fluorescent optical probes, reporting on cellular events through changes in linear dichroism.

Comprehensive portrait of cholesterol containing oxidized membrane.

Biological membranes are under significant oxidative stress caused by reactive oxygen species mostly originating during cellular respiration. Double bonds of the unsaturated lipids are most prone to oxidation, which might lead to shortening of the oxidized chain and inserting of terminal either aldehyde or carboxylic group. Structural rearrangement of oxidized lipids, addressed already, is mainly associated with looping back of the hydrophilic terminal group. This contribution utilizing dual-focus fluorescence correlation spectroscopy and electron paramagnetic resonance as well as atomistic molecular dynamics simulations focuses on the overall changes of the membrane structural and dynamical properties once it becomes oxidized. Particularly, attention is paid to cholesterol rearrangement in the oxidized membrane revealing its preferable interaction with carbonyls of the oxidized chains. In this view cholesterol seems to have a tendency to repair, rather than condense, the bilayer.

The fast polarization modulation based dual-focus fluorescence correlation spectroscopy

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

Phospholipid lateral diffusion in phosphatidylcholine-sphingomyelin-cholesterol monolayers; Effects of oxidatively truncated phosphatidylcholines

Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally containing oxidatively truncated phosphatidylcholines, using fluorescence correlation spectroscopy (FCS). To our knowledge, these are the first FCS results on miscibility transition in ternary lipid monolayers, confirming previous results obtained using Brewster angle microscopy on such lipid monolayers. Wide-field fluorescence microscopy was additionally employed to verify the transition, i.e. the loss and reformation of SM/Chol domains.

Z-Scan Fluorescence Correlation Spectroscopy: A Powerful Tool for Determination of Lateral Diffusion in Biological Systems

The characterization of the dynamics of biological membranes is a topic which currently grasps a high level of attention. Biological membranes are extremely important as they are required for both protection and communication of eukaryotic cells. They also play a key role for transportation of nutrients into and out of the cell. Recent studies have proved that biological membranes are not homogeneous but are instead composed of microdomains, which complicate the precise determination of lateral diffusion coefficients. Z-scan fluorescence correlation spectroscopy (Z-scan FCS), one of the fluorescence fluctuation methods, is a technique which can be employed to determine lateral diffusion coefficients of membrane lipids and also membrane-associated molecules. Moreover, when Z-scan FCS is used in combination with Wawrezinieck diffusion law, lipid rafts in heterogeneous membranes can be monitored. This review is focused firstly on the theory of lateral diffusion in biological systems and secondly on FCS, especially Z-scan FCS as a very useful approach for determination of lateral diffusion coefficients in planar systems.