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P Schwille

Publications and source records attributed to P Schwille.

15 recordsLinked to original sources
PubMed1999

Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy.

We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1, 1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C(20)) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3 -phosphoc holine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D(T), of the DiI-C(20) was measured by FCS. D(T) values at approximately 25 degrees C ranged from approximately 3 x 10(-8) cm(2)/s in the fluid phase, to approximately 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to approximately 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of D(T) in a region of two-phase coexistence on a single vesicle.

Carbocyanines↗
PubMed1999

Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.

Animals↗
PubMed1999

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.

Animals↗
PubMed1998

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 microseconds at pH 7 to 45 microseconds at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 microseconds and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Fluorescence↗
PubMed1998

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy.

A method for sensitively monitoring enzyme kinetics and activities by using dual-color fluorescence cross-correlation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two fluorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and fluorophores at each 5' end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored on-line at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a KM of 14 +/- 1 nM and a kcat of 4.6 +/- 0.2 min-1. In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color fluorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.

Biochemistry↗
PubMed1997

Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies.

This review demonstrates the large analytical and diagnostic potential of fluorescence correlation spectroscopy applied to freely diffusing biomolecules in solution. All applications discussed here in detail are based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. However, the principle of the measurement can be extended to many different reactions with characteristic association times between several minutes up to several hours. If the reaction significantly affects the diffusion constants of at least one partner, single-color auto-correlation analysis is sufficient to extract kinetic parameters. If the observed binding process has only a moderate effect on diffusion coefficients, the detection selectivity and sensitivity can be improved by dual-color cross-correlation analysis. Finally, we show that diffusional analysis on the single-molecule level even opens up diagnostic applications, such as the detection of minute amounts of infectious agents like HIV-1 viruses in blood.

DNA↗
PubMed1997

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.

DNA↗
PubMed1996

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR.

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe's average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 micrograms unspecific DNA without post-PCR probe manipulations could be achieved with different primer/ probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Automation↗
PubMed1996

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy.

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1-1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)-this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis.

Binding kinetics in solution of six N,N,N',N'-tetramethyl-5-carboxyrhodamine-labeled oligodeoxyribonucleotide probes to a 101mer target RNA comprising the primer binding site for HIV-1 reverse transcriptase were characterized using fluorescence correlation spectroscopy (FCS). FCS allows a sensitive, non-radioactive real time observation of hybridization of probes to the RNA target in the buffer of choice without separation of free and bound probe. The binding process could directly be monitored by the change in translational diffusion time of the 17mer to 37mer DNA probe upon specific hybridization with the larger RNA target. The characteristic diffusion time through a laser-illuminated open volume element with 0.5 micron in diameter increased from 0.13-0.2 ms (free) to 0.37-0.50 ms (bound), depending on the probe. Hybridization was approximated by biphasic irreversible second-order reaction kinetics, yielding first-phase association rate constants between 3 x 10(4) and 1.5 x 10(6) M-1 s-1 for the different probes. These varying initial rates reflected the secondary structures of probes and target sites, being consistent with a hypothetical binding pathway starting from loop-loop interactions in a kissing complex, and completion of hybridization requiring an additional interaction involving single-stranded regions of both probe and target. FCS thus permits rapid screening for suitable antisense nucleic acids directed against an important target like HIV-1 RNA with low consumption of probes and target.

Animals↗
PubMed1976

Fasting uric acid and phosphate in urine and plasma of renal calcium-stone formers.

Clearance experiments in calcium-stone patients (n = 60) and controls (n = 60) demonstrated significantly higher urinary uric acid (UA) in younger (less 40 years) stone patients than controls (median: 480 vs. 351 mug/min) but not in older (greater than 40 years) patients. Serum UA and urinary oxypurines were comparable in health and stone disease. Conversely urinary phosphate was significantly lower in younger patients than matched controls (males: 224 vs. 304 mug) and presumably is responsible for the more alkaline pH. It is suggested that calcium-stone formation in humans is represented by two different populations.

Adult↗
PubMed1976

Ultrafiltrable uric acid in serum of healthy humans and patients with renal calcium lithiasis.

Ultrafiltrable serum uric acid (u.a.) was determined by ultrafiltration under in vivo conditions in humans using a reliable technique described in detail. It could be demonstrated that u.a. binding to macromolecules occurs in healthy humans (controls) and in patients with renal calcium stones. The percentage of free u.a. in controls (n=60) averages 86.2 +/- 0.9 SEM. With increasing age, bound u.a. rises slightly. On the other hand, younger (less than 40 years) stone patients have significantly more bound u.a. than matched controls (80.7 +/- 1.0 SEM; p less than 0.001), whereas this is not found in elderly patients. The degree of binding is not related to concentration of plasma proteins but inversely related to free fatty acid concentration in healthy controls (r= -0.52; p less than 0.01). It is suggested that no augmentation of tubular u.a. filtered by the glomeruli could have occurred. The origin of fasting hyperuricosuria shown earlier to be a prominent feature of young renal calcium stone formers is yet unknown.

Adolescent↗