Biomedical subjects

Martin Hof

Publications and source records attributed to Martin Hof.

At least 19 recordsLinked to original sources

PubMed2006

Fluorescence lifetime correlation spectroscopy.

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

Journal Article↗

PubMed2006

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research.

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.

Lipid Bilayers↗

PubMed2006

Surface sticking and lateral diffusion of lipids in supported bilayers.

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes.

Diffusion↗

PubMed2006

Lipid diffusion in giant unilamellar vesicles is more than 2 times faster than in supported phospholipid bilayers under identical conditions.

The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D=7.8+/-0.8 microm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D=3.1+/-0.3 microm2 s-1).

Diffusion↗

PubMed2006

Headgroup hydration and mobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study.

The biophysical properties of liposome surfaces are critical for interactions between lipid aggregates and macromolecules. Liposomes formed from cationic lipids, commonly used to deliver genes into cells in vitro and in vivo, are an example of such a system. We apply the fluorescence solvent relaxation technique to study the structure and dynamics of fully hydrated liquid crystalline lipid bilayers composed of mixtures of cationic dioleoyltrimethylammoniumpropane (DOTAP) and neutral dioleoylphosphatidylcholine (DOPC). Using three different naphthalene derivatives as fluorescent dyes (Patman, Laurdan and Prodan) allowed different parts of the headgroup region to be probed. Wavelength-dependent parallax quenching measurements resulted in the precise determination of Laurdan and Patman locations within the DOPC bilayer. Acrylamide quenching experiments were used to examine DOTAP-induced dye relocalization. The nonmonotonic dependence of dipolar relaxation kinetics (occurring exclusively on the nanosecond time scale) on DOTAP content in the membrane was found to exhibit a maximum mean solvent relaxation time at 30 mol % of DOTAP. Up to 30 mol %, addition of DOTAP does not influence the amount of bound water at the level of the sn(1) carbonyls, but leads to an increased packing of phospholipid headgroups. Above this concentration, elevated lipid bilayer water penetration was observed.

Acrylamide↗

PubMed2006

Lipopolyamine-mediated single nanoparticle formation of calf thymus DNA analyzed by fluorescence correlation spectroscopy.

PURPOSE: The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N4,N9-dioleoylspermine and N1-cholesteryl spermine carbamate. METHODS: Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. RESULTS: N4,N9-dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 x 10(-12) m2/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio = 1.0-1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N1-cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5-2.5) with D = 1.3 x 10(-12) m2/s and PN value of 5.2. N4,N9-dioleoylspermine is a more efficient DNA-condensing agent than N1-cholesteryl spermine carbamate. CONCLUSIONS: FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.

Carbamates↗

PubMed2006

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy.

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.

Biological Transport↗

PubMed2006

DNA-spermine and DNA-lipid aggregate formation visualized by fluorescence correlation spectroscopy.

BACKGROUND: Fluorescence correlation spectroscopy (FCS) can be used for the determination of diffusion coefficients of single molecules. Since diffusion coefficients are correlated with size and shape of the labeled species, FCS provides information on conformational changes in plasmids aggregates. METHODS: A 10-kbp plasmid stained with PicoGreen was condensed by spermine or liposomes formulated from cationic lipid and egg phosphatidylcholine. RESULTS: The diffusion coefficient of DNA increases from 1.0 x 10(-12) m2/s to 3.2 x 10(-12) m2/s by the addition of spermine, whereas the addition of cationic liposomes leads to complexes characterized by diffusion coefficients with values ranging from 1.7 to 1.9 x 10(-12) m2/s. CONCLUSIONS: FCS experiments allow determining the diffusion coefficients of DNA-containing aggregates which provide information regarding the topology and homogeneity of the aggregate.

DNA↗

PubMed2005

Solvent relaxation study of pH-dependent hydration of poly(oxyethylene) shells in polystyrene-block-poly(2-vinylpyridine)-block-poly(oxyethylene) micelles in aqueous solutions.

The hydration of the poly(oxyethylene) shell in polystyrene-block-poly(2-vinylpyridine)-block-poly(oxyethylene) micelles was investigated by monitoring the solvent relaxation response of a solvent-sensitive fluorophore (patman). It has been found that the relaxation occurs on the nanosecond time scale. Results for triblock copolymer micelles have been compared with those obtained for polystyrene-block-poly(2-vinylpyridine) micelles in order to evaluate the effect of the outer polyoxyethylene layer. Considerable pH-dependent changes in the hydration of poly(oxyethylene) units at the poly(2-vinylpyridine)/polyoxyethylene interface were observed. Additionally, the paper shows that the solvent relaxation technique is a suitable tool for studying polymeric nanoparticles and that the measurement of time-dependent half-width of the emission spectrum allows for estimation of the extent of relaxation process observed by a given experimental setup.

Journal Article↗

PubMed2005

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy.

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93. The lipid was fluorescently labelled sphingomyelin (Bodipy FL-C5 SM). The protein was the myelin oligodendrocyte glycoprotein (MOG). In order to monitor the lateral diffusion of MOG, OLN-93 cells were transfected with a MOG-EGFP (enhanced green fluorescent protein) fusion plasmid. The measurements were performed at room temperature. FCS data were analyzed for two-dimensional (2D) diffusion according to three models which all included a triplet fraction: (a) 2D 1 component (2D1C), (b) 2D anomalous diffusion (2D1Calpha), and (c) 2D 2 components (2D2C). Preliminary results indicate that for the lipid case, the best fits are obtained with 2D2C. In the case of MOG-EGFP, 2D2C and 2D1Calpha give fits of similar quality. The parameter estimates obtained with 2D1Calpha, however, have a lower standard deviation. The anomaly parameter for MOG-EGFP is 0.59+/-0.01.

Animals↗

PubMed2005

Influence of the curvature on the water structure in the headgroup region of phospholipid bilayer studied by the solvent relaxation technique.

Solvent relaxation (SR) in 1,2-dioleoyl-palmitoyl-sn-glycero-3-phosphocholine (DOPC) unilamellar vesicles of different size was probed by 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (Patman), 6-propionyl-2-dimethylaminonaphthalene (Prodan) and 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)-coumarin (DTMAC). Patman probes the amount and mobility of the bound water molecules located at the carbonyl region of the bilayer. Membrane curvature significantly accelerates the solvent relaxation process, but does not influence the total Stokes shift, showing that membrane curvature increases the mobility, without affecting the amount of water molecules present in the headgroup region. This pattern was also verified for other phosphatidylcholines. Prodan is located in the phosphate region of the bilayer and probes a more polar, mobile and heterogeneous environment than Patman. The influence of membrane curvature on SR probed by Prodan is similar, however, less pronounced compared to Patman. DTMAC (first time used in SR) shows a broad distribution of locations along the z-axis. A substantial amount of the coumarin chromophores face bulk water. No effect of curvature on SR probed by DTMAC is detectable.

Lipid Bilayers↗

PubMed2005

Solvent relaxation in phospholipid bilayers: principles and recent applications.

Although there exist a number of methods, such as NMR, X-ray, e.g., which explore the hydration of phospholipid bilayers, the solvent relaxation (SR) method has the advantage of simple instrumentation, easy data treatment and possibility of measuring fully hydrated samples. The main information gained from SR by the analysis of recorded "time-resolved emission spectra" (TRES) is micro-viscosity and micro-polarity of the dye microenvironment. Based on these parameters, one can draw conclusions about water structure in the bilayer. In this review, we focus on physical background of this method, on all the procedures that are needed in order to obtain relevant parameters, and on the requirements on the fluorescence dyes. Furthermore, a few recent applications (the effect of curvature, binding of antibacterial peptides and phase transition) illustrating the versatility of this method are mentioned. Moreover, limitations and potential problems are discussed.

Cell Membrane↗

PubMed2005

Propidium iodide and PicoGreen as dyes for the DNA fluorescence correlation spectroscopy measurements.

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe-nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

DNA↗

PubMed2004

Surface-dependent transitions during self-assembly of phospholipid membranes on mica, silica, and glass.

Formation of supported membranes by exposure of solid surfaces to phospholipid vesicles is a much-used technique in membrane research. Freshly cleaved mica, because of its superior flatness, is a preferred support, and we used ellipsometry to study membrane formation kinetics on mica. Neutral dioleoyl-phosphatidylcholine (DOPC) and negatively charged dioleoyl-phosphatidylserine/dioleoyl-phosphatidylcholine (20% DOPS/80% DOPC) vesicles were prepared by sonication. Results were compared with membrane formation on silica and glass, and the influence of stirring, buffer, and calcium was assessed. Without calcium, DOPC vesicles had a low affinity (Kd approximately 30 microM) for mica, and DOPS/DOPC vesicles hardly adsorbed. Addition of calcium promptly caused condensation of the adhering vesicles, with either loss of excess lipid or rapid additional lipid adsorption up to full surface coverage. Vesicle-mica interactions dominate the adsorption process, but vesicle-vesicle interactions also seem to be required for the condensation process. Membranes on mica proved unstable in Tris-HCl buffer. For glass, transport-limited adsorption of DOPC and DOPS/DOPC vesicles with immediate condensation into bilayers was observed, with and without calcium. For silica, vesicle adsorption was also rapid, even in the absence of calcium, but the transition to condensed layers required a critical surface coverage of about 50% of bilayer mass, indicating vesicle-vesicle interaction. For all three surfaces, additional adsorption of DOPC (but not DOPS/DOPC) vesicles to condensed membranes was observed. DOPC membranes on mica were rapidly degraded by phospholipase A2 (PLA2), which pleads against the role of membrane defects as initial PLA2 targets. During degradation, layer thickness remained unchanged while layer density decreased, in accordance with recent atomic force microscopy measurements of gel-phase phospholipid degradation by PLA2.

Adsorption↗

PubMed2003

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.

Antimicrobial Cationic Peptides↗

PubMed2003

Associating oligonucleotides with positively charged liposomes.

Oligonucleotides (ODNs) are short (up to 30 bases) fragments of single-stranded nucleic acids that are used as sequence specific regulators of gene expression and anti-sense based therapeutics. ODNs are frequently aggregated with particulates in order to improve their pharmacological characteristics. Complexes of ODN and lipid aggregates are among the most commonly mentioned in the literature. In order to control the formation and final properties of such aggregates, a detailed description of how ODN interacts with the lipid surface is needed. In this paper, we present the results of fluorescence measurements regarded an association of 20 base ODN, labelled with fluorescein, and a lipid surface containing various amount of positive charge. Unilamellar lipid vesicles were formed from egg phosphatidylcholine (PC) and various amounts of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). It was found that about 20 mol% of DOTAP in the lipid bilayer suffices to obtain complete ODN association. This result was further confirmed via measurements performed by fluorescence correlation spectroscopy (FCS). These in turn showed that the diffusion time of labelled ODN in the presence of cationic liposomes decreases. Also, the particle number and count rate were reduced, concurring with conclusions derived from steady-state fluorescence spectroscopy results.

Base Sequence↗

PubMed2002

The application of fluorescence correlation spectroscopy in detecting DNA condensation.

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).

Algorithms↗

PubMed2002

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy.

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHbetaAPr-1-neo (10 kbp; contour length 3.4 microm) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 microM (spermine/DNAphosphate=0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.0x10(-12) m2/s to 6.0x10(-12) m2/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dye's dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

DNA↗