Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Membrane aging during cell growth ascertained by laurdan generalized polarization

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.     

Two-photon polarization microscopy reveals protein structure and function

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.     

Two-photon excitation in fluorescence polarization receptor-ligand binding assay

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.     

Imaging domains in model membranes with atomic force microscopy

Lateral segregation in biomembranes can lead to the formation of biologically functional domains. This paper reviews atomic force microscopy studies on domain formation in model membranes, with special emphasis on transbilayer asymmetry, and on lateral domains induced by lipid-lipid interactions or by peptide-lipid interactions.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.     

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.     

Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors

Transport parameters determine the access of drugs to tumors. However, technical difficulties preclude the measurement of these parameters deep inside living tissues. To this end, we adapted and further optimized two-photon fluorescence correlation microscopy (TPFCM) for in vivo measurement of transport parameters in tumors. TPFCM extends the detectable range of diffusion coefficients in tumors by one order of magnitude, and reveals both a fast and a slow component of diffusion. The ratio of these two components depends on molecular size and can be altered in vivo with hyaluronidase and collagenase. These studies indicate that TPFCM is a promising tool to dissect the barriers to drug delivery in tumors.     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

An epifluorescence microscopy method for generalized polarization imaging

Generalized polarization (GP) microscopy represents an excellent tool to study lipid–lipid and lipid–protein interactions in situ and in vitro. Here, we present an efficient and cost effective method to perform GP microscopy using a standard light-emitting diode (LED) epifluorescence microscope equipped with a digital color camera.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Identification of sterol-containing domains in vacuolar membranes by confocal microscopy

Sterol-containing domains in the tonoplast of beet root cells were identified by confocal microscopy with a membrane probe filipin. The presence of sterol-containing domains in the tonoplast was confirmed using methyl-β-cyclodextrin. Sterol-containing domains with separate small intensely fluorescing sterol-enriched sites were found in the vacuolar membrane; these sites can be attributed to raft structures. It was shown that sterol content in the isolated rafts can be assessed by confocal microscopy. β-Sitosterol, stigmasterol, α-tocopherol, and cholesterol were identified among the sterols of the isolated rafts.     

Laser-driven microsecond temperature cycles analyzed by fluorescence polarization microscopy

We demonstrate a novel technique to achieve fast thermal cycles of a small sample (a few femtoliters). Modulating a continuous near-infrared laser focused on a metal film, we can drive the local temperature from 130 to 300 K and back, within a few microseconds. By fluorescence microscopy of dyes in a thin glycerol film, we record images of the hot spot, calibrate its temperature, and follow its variations in real time. The temperature dependence of fluorescence anisotropy, due to photophysics and rotational diffusion, gives a steady-state temperature calibration between 200 and 350 K. From 200 to 220 K, we monitor temperature more accurately by fluorescence autocorrelation, a probe for rotational diffusion. Time-resolved measurements of fluorescence anisotropy give heating and cooling times of a few microseconds, short enough to supercool pure water. We designed our method to repeatedly cycle a single (bio)molecule between ambient and cryostat temperatures with microsecond time resolution. Successive measurements of a structurally relevant variable will decompose a dynamical process into structural snapshots. Such temperature-cycle experiments, which combine a high time resolution with long observation times, can thus be expected to yield new insights into complex processes such as protein folding.     

Interaction of retinol and intestinal microvillus membranes studied by fluorescence polarization

The interaction of retinol and microvillus membranes prepared from the duodenum, jejunum and ileum of the rat can be studied readily by fluorescence and fluorescence polarization. A characteristic pattern of increased retinol anisotropy in membranes from more distal segments is demonstrated. Studies with trypsin indicate that the membrane proteins influence retinol fluorescence intensity and anisotropy.