The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.

• MeSH
• Fluorescence MeSH
• Fluorescent Dyes * MeSH
• Spectrometry, Fluorescence MeSH
• Luminescent Proteins genetics   MeSH
• Green Fluorescent Proteins genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluorescent Dyes * MeSH
• Luminescent Proteins MeSH
• Green Fluorescent Proteins MeSH

The plasma membrane is a complex system, consisting of two layers of lipids and proteins compartmentalized into small structures called nanodomains. Despite the asymmetric composition of both leaflets, coupling between the layers is surprisingly strong. This can be evidenced, for example, by recent experimental studies performed on phospholipid giant unilamellar vesicles showing that nanodomains formed in the outer layer are perfectly registered with those in the inner leaflet. Similarly, microscopic phase separation in one leaflet can induce phase separation in the opposing leaflet that would otherwise be homogeneous. In this review, we summarize the current theoretical and experimental knowledge that led to the current view that domains are - irrespective of their size - commonly registered across the bilayer. Mechanisms inducing registration of nanodomains suggested by theory and calculations are discussed. Furthermore, domain coupling is evidenced by experimental studies based on the sparse number of methods that can resolve registered from independent nanodomains. Finally, implications that those findings using model membrane studies might have for cellular membranes are discussed.

• Keywords
• biomembranes, domain registration, interleaflet coupling, membrane asymmetry, nanodomains, phase separation, plasma membranes,
• Publication type
• Journal Article MeSH
• Review MeSH

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

• MeSH
• Diffusion MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence * MeSH
• Photons MeSH
• Liposomes chemistry   MeSH
• Models, Theoretical MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Liposomes MeSH

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.

• MeSH
• Time Factors MeSH
• Chemistry Techniques, Analytical methods   MeSH
• Diffusion MeSH
• Fluorescence * MeSH
• Fluorescent Dyes chemistry   MeSH
• Phosphatidylcholines chemistry   MeSH
• Phosphatidylethanolamines chemistry   MeSH
• Phosphatidylserines chemistry   MeSH
• Phospholipids chemistry   MeSH
• Kinetics MeSH
• Lipid Bilayers chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1,2-dielaidoylphosphatidylethanolamine MeSH Browser
• 1,2-dioleoylphosphatidylserine MeSH Browser
• 1,2-oleoylphosphatidylcholine MeSH Browser
• Fluorescent Dyes MeSH
• Phosphatidylcholines MeSH
• Phosphatidylethanolamines MeSH
• Phosphatidylserines MeSH
• Phospholipids MeSH
• Lipid Bilayers MeSH

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

• Keywords
• biomembranes, confocal microscopy, fluorescence fluctuation spectroscopy, giant unilamellar vesicles, lateral diffusion, supported lipid bilayers,
• MeSH
• Diffusion MeSH
• Spectrometry, Fluorescence * MeSH
• Microscopy, Confocal MeSH
• Lipid Bilayers chemistry   MeSH
• Membrane Lipids chemistry   MeSH
• Unilamellar Liposomes chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Lipids MeSH
• Unilamellar Liposomes MeSH

The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Thus, it is in the scope of research interest. It was shown, that spermine-induced compaction of large DNA molecules occurs in a discrete "all-or-non" regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (approximately 10 kbp), so far, it was stated that the mechanism of folding is continuous. Here, we show, that neither a standard benchmark technique-dynamic light scattering, nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach-fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen during the titration with spermine and based on that, it reveals the discrete mechanism of the process. Furthermore, we show that it allows for observation of the equilibrium state transition dynamics.

• MeSH
• Algorithms MeSH
• Spectrometry, Fluorescence MeSH
• Nucleic Acid Conformation * MeSH
• DNA, Circular chemistry   MeSH
• Models, Molecular MeSH
• Organic Chemicals chemistry   MeSH
• Plasmids chemistry   MeSH
• Scattering, Radiation MeSH
• Spermine chemistry   MeSH
• Light MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Circular MeSH
• Organic Chemicals MeSH
• PicoGreen MeSH Browser
• Spermine MeSH