Taking another look with fluorescence microscopy: Image processing techniques in Langmuir monolayers for the twenty-first century

Fluorescence microscopy has become a powerful and standard complementary technique in the study of amphiphilic films at the air-water interface. For nearly three decades the coupling of traditional thermodynamic measurements with direct visualization has provided a better understanding of self-assembled Langmuir monolayers and their application in the study of the physical properties of membranes and interfaces. As an introduction we provide a brief overview of this established technique and demonstrate its continued utility in the recent observation of novel phase behavior in monolayers of 25-hydroxycholesterol (25-OH) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). We then focus our review on new analysis techniques which take advantage of the ability to store, process, and analyze large sets of images. We pay particular attention to efforts measuring the line tension between coexisting two dimensional fluid phases in the Langmuir monolayer. Using non-perturbative methods, we can measure fundamental mechanical properties of these two dimensional systems. Finally, we highlight the use of Model Convolution Microscopy as a new tool to provide insight on the experimental limits in these studies.     

Highly cohesive monolayers of lipid derivatives of colchicine: a dynamics study

Monolayers of lipid derivatives of colchicine spread at the air--water interface reach the thermodynamic equilibrium over an abnormally long period of time. Dynamics of this equilibration and the behavior of the film during compression--decompression cycles are observed by fluorescence microscopy. The thermodynamically disfavored structures observed are unrelated to previously described unusual shapes in the liquid expanded-gas coexistence regions. The relation between the cholesterol-like effect of the colchicinoid moiety, its propensity to dimerize, and the high viscosity of the monolayer are discussed.     

The phase behavior of lipid monolayers containing pulmonary surfactant protein C studied by fluorescence light microscopy

Three compounds of the pulmonary surfactant – dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant associated protein C (SP-C) – were spread at the air-water interface of a Langmuir trough as a model system to mimic the properties of natural surfactant. Fluorescence microscopical images of the film formed at the interface were obtained during compression using a fluorescence dye bound covalently either to phosphatidylcholine or to SP-C. The images were quantified using statistical methods in respect to relative areas and relative fluorescence intensities of the domains found. In the early stage of compression, film pressure rose slightly and was accompanied by a phase separation which could be recognized in the images by the formation of bright and dark domains. On further compression, after a steep increase of film pressure, a plateau region of constant film pressure started abruptly. During compression in the plateau region, fluorescence intensity of the bright domain formed in the early stage of compression increased. The increasing fluorescence intensity, the non-Gaussian intensity distribution of the bright domain, and the small mean molecular area of the film in the plateau region gave rise to the assumption that multilayer structures were formed in the late stage of compression. The formation of the multilayer structures was fully reversible in repeated compression-expansion cycles including the plateau region of the phase diagram. The ability of lipid/SP-C mixtures to form reversible multilayer structures during compression may be relevant to stability in lungs during expiration and inhalation. Received: 13 February 1997 / Accepted: 22 May 1997     

A scanning force- and fluorescence light microscopy study of the structure and function of a model pulmonary surfactant

The structure of an artificial pulmonary surfactant was studied by scanning force- and fluorescence light microscopy (SFM, and FLM, respectively). The surfactant – a mixture of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and recombinant surfactant-associated protein C (SP-C) – was prepared at the air-water interface of a Langmuir film balance and imaged by FLM under various states of compression. In order to visualize their topography by SFM, the films were transferred onto a solid mica support by the Langmuir-Blodgett (LB) technique. We found that a region of high film compressibility of the spread monolayer close to its equilibrium surface pressure (π=50 mN/m) was due to the exclusion of layered protrusions with each layer 5.5 to 6.5 nm thick. They remained associated with the monolayer and readily reinserted upon expansion of the film. Comparison with the FLM showed that the protrusions contained the protein in high concentration. The more the film was compressed, the larger was the number of layers on top of each other. The protrusions arose from regions of the monolayer with a distinct microstructure that may have been responsible for their formation. The molecular architecture of the microstructure remains to be elucidated, although some of it can be inferred from spectroscopic data in combination with the SFM topographical images. We illustrate our current understanding of the film structure with a molecular model. Received: 20 September 1996 / Accepted: 22 May 1997     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

A comparison of the packing behavior of egg phosphatidylcholine with cholesterol and biogenically related sterols in Langmuir monolayer films

Cholesterol and selected derivatives were studied as mixed Langmuir monolayers with egg phosphatidylcholine (PC). As an extension of our earlier work, which employed binary sterol/PC mixtures, here we examined ternary mixed monolayers containing cholesterol along with an alternate sterol and PC in different molar ratios, using pressure-area isotherms. The ternary systems behaved similarly to the binary sterol/PC systems reported previously, with similar condensation noted for the sterol/PC films. To better understand how variations in sterol structure affect sterol packing in such membrane monolayers, binary mixtures containing cholestenone, cholestanol, and lanosterol with PC were also studied. Cholestanol behaved similarly to cholesterol when incorporated with PC, while cholestenone and lanosterol did not cause as much film condensation. The observed differences in molecular packing, and attributed sterol structural differences, are considered within the context of sterol/phospholipid mixtures in biological membranes.     

Condensed complexes of cholesterol and phospholipids

There is overwhelming evidence that lipid bilayer regions of animal cell membranes are in a liquid state. Quantitative models of these bilayer regions must then be models of liquids. These liquids are highly non-ideal. For example, it has been known for more than 75 years that mixtures of cholesterol and certain phospholipids undergo an area contraction or condensation in lipid monolayers at the air-water interface. In the past 3 years, a thermodynamic model of “condensed complexes” has been proposed to account for this non-ideal behavior. Here we give an overview of the model, its relation to other models, and to modern views of the properties of animal cell membranes.     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

Cathodoluminescence-emitting lipid droplets in rat testis: a study by analytical color fluorescence electron microscopy

Cathodoluminescence (CL) from lipid droplets (LDs) in the rat testis was examined by analytical color fluorescence electron microscopy. The results show that (1) the Cl at wavelengths of 320 nm (CL320) and 450 nm (CL450) is derived from cholesterol esters and a mixture of lipids including vitamin A esters, respectively; (2) CL320 in the LDs of Leydig cells sharply decreases on postnatal day 21, while CL320 and CL450 in the LDs of Sertoli cells begin to be detectable; (3) the CL450-emitting LDs in seminiferous tubules, whose distributional patterns display cyclic changes during the spermatogenic cycle, are involved in spermatogenesis; and (4) the intensity of CL as well as the distributional patterns of CL-emitting LDs in testicular cells change after hypophysectomy, vitamin-A deficiency, and treatment with ethylene dimethane sulfonate and testosterone propionate. This study demonstrates that analytical color fluorescence electron microscopy is a useful tool for in-vivo observation of some specific compounds which cannot be visualized by other methods.     

Find your coat: Using correlative light and electron microscopy to study intracellular protein coats

Protein coats, important for vesicular trafficking in eukaryotic cells, help shape membranes and package cargo. But their dynamic construction cannot be fully understood until the distinct steps of their assembly in their native intracellular context at molecular resolution can be visualized. For this, correlative light and electron microscopy (CLEM) is an essential tool. Here, we discuss how emerging CLEM techniques have been used to study the assembly of protein coats inside cells. We review how current and developing CLEM technologies are poised to answer fundamental questions of protein coat architecture at the nanoscale.     

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F ₀- and the F _m-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 μm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.     

Spatial variation in the molecular tilt orientational order within the solid domains of phase-separated, mixed dialkylphosphatidylcholine monolayers

The miscibility of the solid-phase-forming distearoylphosphatidylcholine (DSPC) and the fluid-phase-forming dilauroylphosphatidylcholine (DLPC) at the air/water interface was investigated by the Langmuir film balance. Surface pressure-area isotherms suggest that mixtures containing 25.0-62.5-mol% DLPC (range of composition investigated) are phase-separated. The lateral structure of the DSPC/DLPC monolayers was imaged by Brewster angle microscopy (BAM) as a function of the surface pressure. Quasi-circular condensed domains appeared at pressures between 0 and 0.5mN m(-1), and these structures were already fully developed at approximately 1mN m(-1). Further compression of the monolayers above 1mN m(-1) merely brought the domains closer together. The mixed monolayers consisted of solid domains of DSPC, approximately 3-20 micro in size, in a fluid matrix of DLPC. BAM and the phase contrast mode of intermittent-contact atomic force microscopy (AFM) revealed that the quasi-circular DSPC domains are divided into segments of different reflectivities (BAM) or phase shift (AFM) that arise from abrupt changes in the long-range orientational order of the tilted hydrocarbon chains. The DSPC domains in DSPC/DLPC internally exhibited star and cardioid textures that were heretofore only reported for single-component lipid monolayers in the phase coexistence region.     

Surface film pressure of actin: interactions with lipids in mixed monolayers

The interactions of actin with neutral lipid films made from DLPC, and with positively charged films built from DLPC and stearylamine (SA), have been characterized by the monolayer technique. Injection of actin underneath an expanded lipid film produces an increase in the surface pressure that is consistent with a penetration of the lipid molecules by actin. This adsorption of actin to the lipid is more pronounced either with positively charged films or with Mg(2+) present in the sub-phase, suggesting that the mechanism involves an electrostatic attraction. During compression, the actin molecules are squeezed out into the sub-phase, carrying along some lipid molecules; this suggests a strong affinity of the lipids for actin. An analysis of the dilational modulus shows that when actin is found as monomers at the interface, the mixed actin-lipid film undergoes three phase changes upon compression. On the other hand, when actin is polymerized at the interface, the actin and the lipid form a rigid film for which the compressibility is mostly dominated by actin.     

Membrane localization and dynamics of Nile Red: Effect of cholesterol

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.     

Thematic Review Series: Lipids and Lipid Metabolism in the Eye: The ins and outs of cholesterol in the vertebrate retina

The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterified form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that significantly compromises the structure and function of the retina. Although the relative contributions of de novo synthesis versus extraretinal uptake are not yet known, herein we review what is known about these processes and the dynamics of cholesterol in the vertebrate retina and indicate some future avenues of research in this area.     

Observation of the Yarrowia lipolytica peroxisome-vacuole dynamics by fluorescence microscopy with a single filter set

We constructed the fusion of peroxisomal acyl-CoA oxidase 3 and the enhanced yellow fluorescent protein (EYFP) for fluorescent labeling of Yarrowia lipolytica peroxisomes. Using the spectral overlap between EYFP and FM4-64, we developed a procedure for simultaneous observation of Y. lipolytica peroxisomes and vacuoles with the single fluorescein isothiocyanate filter set. Using this procedure we were able to follow the Y. lipolytica peroxisome-vacuole dynamics under pexophagy conditions and show that Y. lipolytica peroxisomes are degraded in the vacuoles by a macropexophagic mechanism.     

Influence of the chain length of ubiquinones on their interaction with DPPC in mixed monolayers

The thermodynamic behavior of representative short (UQ2), middle (UQ4 and UQ6) and long-chain (UQ10) ubiquinones (UQ) mixed with dipalmitoyl-phosphatidylcholine (DPPC) was studied in monolayers at the air-water interface. The influence of isoprenoid chain-length of UQ on miscibility of both lipids was investigated by analysis of surface pressure-area isotherms and using fluorescence microscopy. Analysis of excess areas (A_ex) and free energies of mixing (ΔG_m), calculated from compression isotherms in the full range of ubiquinones concentrations, has given evidences for UQ-rich constant-size (UQ6, UQ10) or less growth limited (UQ2, UQ4) microdomains formation within mixed films. Fluorescence microscopy observation revealed that ubiquinones are preferentially soluble in the expanded phase. When lateral pressure increased, concomitant evolutions of A_ex and ΔG_m parameters, and composition dependence of collapse surface pressures, argue for an evolution towards a total segregation, never reached due to expulsion of ubiquinones from the film. The possible significance of these observations is discussed in relation to ubiquinones organization and similar chain length effects in membranes.     

Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.     

Quantification and calibration of images in fluorescence microscopy

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue “phantoms” that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (?40 and ?4 °C).