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. 2005 Dec 16;90(6):2170–2178. doi: 10.1529/biophysj.105.073510

Correlated Fluorescence-Atomic Force Microscopy of Membrane Domains: Structure of Fluorescence Probes Determines Lipid Localization

James E Shaw *,‡, Raquel F Epand §, Richard M Epand §, Zaiguo Li , Robert Bittman , Christopher M Yip *,†,‡
PMCID: PMC1386795  PMID: 16361347

Abstract

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.

INTRODUCTION

Lipid microdomains, or rafts, formed as a result of specific lipid-lipid or lipid-protein interactions in the cell membrane, are thought to play an important role in regulating signal transduction, cellular transport, and lipid sorting (1–8). Applying fluorescence microscopy or spectroscopy to study these structures and their dynamics in vivo is a challenge because of the lack of adequate imaging techniques with sufficient spatial and/or temporal resolution (9) as well as the limited number of specific lipid antibodies or lipid-binding protein domains that are available to serve as probes (10,11). Incorporating an artificial chromophore such as NBD, BODIPY, anthracene, pyrene, diphenylhexatriene, or a polyene chain into one of the fatty acid side chains is a common strategy to create fluorescent lipids to study metabolism, cellular transport, and distribution of lipids (12). The majority of these probes were introduced before the importance of lipid phase separation was recognized. Recently it has been demonstrated, using fluorescence quenching assays with model membranes (12–15), that chemical modification of a raft-associated lipid with a fluorophore can alter its ability to remain associated with (or associate to) these domains (15–17). In addition, correlated atomic force-fluorescence microscopic methods have recently shown promise in visualizing microdomains and fluorescence probe distribution simultaneously (18–21), including the effect of extrinsic markers on the morphology of lipid monolayers (22). This study was motivated by the realization that atomic force microscopy (AFM) can detect membrane microdomains using the differences in height between lipids existing in different phase regimes (20,23–26), including rafts or liquid-ordered (lo) phases reported to be ∼0.5–1 nm above the surrounding liquid-disordered (ld) phase in model membranes composed of DOPC/sphingomyelin (SM)/cholesterol (Chl) (27–31). Herein we describe the use of simultaneous coupled AFM-confocal imaging to investigate the ability of the various lipids such as ganglioside GM1, phosphatidylcholine, SM, and Chl labeled with NBD or BODIPY, to partition into the lo or ld domains of phase-segregated planar lipid bilayers. Our findings demonstrate that correlated AFM/fluorescence microscopy is a simple approach for identifying the preferential localization of fluorescent lipid probes. The attachment of a fluorophore such as NBD or BODIPY on the acyl chain of a saturated lipid or of Chl generally decreases the affinity of the lipid or sterol for the more ordered domains.

MATERIALS AND METHODS

Materials

N-stearoyl-D-erythro-sphingosylphosphorylcholine (synSM, 18:0), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC, 18:1), brain bovine GM1 ammonium salt (brain GM1), 1-palmitoyl-2-(6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-phosphocholine (NBD-PC) (Fig. 1 A), 25-(N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl] amino]-27-norcholesterol (NBD-Cholesterol) (Fig. 1 B), and N-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino)dodecanoyl]sphingosine-1-phosphocholine (NBD-SM) (Fig. 1 C) were purchased from Avanti Polar Lipids (Alabaster, AL). Chl was purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). BODIPY FL C5-ganglioside GM1 (BODIPY-GM1) (Fig. 1 D) and Alexa Fluor 555 cholera toxin subunit B (Alexa555-CTX-B) were purchased from Invitrogen Canada (Burlington, ON, Canada). BODIPY-Chl analogs (1, 2, and 3) (Fig. 1, E1, E2, and F) were synthesized as described (Z. Li, E. Mintzer, and R. Bittman, unpublished). BODIPY-SM (Fig. 1 G) was synthesized by N-acylation of sphingosylphosphorylcholine with a p-nitrophenyl ester of a BODIPY-fatty acid conjugate.

FIGURE 1.

FIGURE 1

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Chemical structures of (A) NBD-PC, (B) NBD-Chl, (C) NBD-SM, (D) BODIPY-GM1, (E1 and E2) BODIPY-Chl 1 and 2, (F) BODIPY-Chl 3, and (G) BODIPY-SM.

Liposome preparation

The liposomes were prepared by dissolving the required amounts of the various lipids in chloroform or chloroform/methanol (2:1), removing the solvent by rotary evaporation and keeping the films under vacuum for 3–4 h to remove occluded solvent. The lipid films were hydrated with 10 mM HEPES buffer containing 150 mM NaCl at pH 7.4 to a final lipid concentration of ∼1 mM. Unilamellar vesicles were formed by sonication in a heated ultrasonic cleaner (∼60°C) until the suspension became clear or only slightly hazy.

Supported planar bilayer preparation

Supported planar bilayers (SPBs) were formed by in situ fusion of 100 μl of 1 mM liposome suspension containing the desired lipid composition and 400 μl of 10 mM HEPES buffer, pH 7.4, containing 4 mM CaCl2, 150 mM NaCl, onto a freshly cleaved mica disc (∼25 mm in diameter and ∼0.17 mm in thickness) held in a custom-made open fluid cell. The liposomes were first allowed to fuse at ambient temperature for 3 min, heated in place to 70°C for 3 min, and then allowed to cool to room temperature. This heating-cooling step facilitated the nucleation and growth of micron-size gel-phase domains, as has been reported by others (32). This preparation step was performed in place on the AFM-confocal-total internal reflection fluorescence (TIRF) instrument, as described in the following section.

Coaxial confocal-AFM imaging

All imaging was performed using an integrated AFM-confocal-TIRF imaging system. In this system, a confocal microscope (FluoView 500, Olympus, Melville, NY) was combined into a single imaging platform with an atomic force microscope (Nanoscope IIIa Bioscope, Digital Instruments/Veeco, Santa Barbara, CA) through an Olympus IX70 inverted microscope base. All confocal images were acquired using an Olympus 60× TIRF objective (NA ∼ 1.45). SPBs were formed in situ as described earlier. The fluid cell was washed sequentially with pH 7.4 buffers containing 10 mM HEPES, 4 mM CaCl2, 150 mM NaCl, followed by 10 mM HEPES, 4 mM EDTA, 150 mM NaCl, and finally 10 mM HEPES, 150 mM NaCl to remove excess solution liposomes before confocal-AFM imaging. All procedures were performed in the dark to avoid photobleaching of the fluorophores. Once the formation of the SPB was confirmed by AFM and confocal imaging, for some experiments a small volume of concentrated Alexa555 CTX-B stock solution (∼1 mg/ml) was injected into the custom-designed open fluid cell, allowing it to distribute evenly by diffusion. From the total quantity of the toxin injected and the total cell volume immediately after injection, the final concentration of CTX-B was estimated to be ∼1 μg/ml. All BODIPY- and NBD-containing compounds were excited by an Ar-ion 488 nm laser, and the emission was collected using a photomultiplier tube (PMT) fitted with a 510–525 nm band-pass filter. A 543 nm HeNe laser was used to excite the Alexa555 fluorophores, and the Alexa555 fluorescence was captured using another PMT equipped with a 560-nm high-pass filter. Confocal pinholes for both PMTs were set at 800 μm to maximize fluorescence counts emitted by the fluorophores. The BODIPY/NBD and Alexa555 fluorophores were sequentially excited to minimize possible cross talk between the BODIPY/NBD and Alexa555 PMT channels.

Although the fluorescence and AFM images are collected simultaneously, there are differences in the timescale of imaging and the actual area being imaged by the two techniques. The AFM data were collected as 512 × 512 pixel 16-bit images at a typical line scan rate of 1.2 Hz, whereas the 1024 × 1024 pixel 12-bit confocal images were acquired at a line scan rate of ∼105 Hz. It is also important to note that the region imaged by the AFM is a subset of the confocal microscope's field of view. This is a result of the limited (∼125 μm × ∼125 μm) scan window afforded the AFM scanner compared with the much larger field of view provided by the 60× objective used by the confocal microscope.

Image analysis

AFM image analysis was conducted using the Digital Instruments Nanoscope software (version 4.42r9). Height images were flattened and plane-fit in the x-scan direction. Quantitative height measurements were determined by section analysis and shown as an average of 50 measurements. Contrast adjustments for the fluorescent images were made using ImagePro Plus (version 4.5, Silver Spring, MD) or the public domain program ImageJ (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij). AFM-confocal image correlation was accomplished using Adobe Photoshop or custom in-house developed Java software.

RESULTS AND DISCUSSION

Binding of cholera toxin to GM1

Simultaneous AFM-confocal imaging at ambient temperature (∼23°C) revealed that SPBs containing 1:1 DOPC/SM/30 mol % Chl/2 mol % brain GM1 formed phase-segregated high (lo) and low (ld) domains with an average height difference of ∼1 nm, regardless of the type of exogenous SM (synSM, 18:0 or 16:0, or brain SM) or fluorescently labeled component. Not unexpectedly, we were unable to resolve the individual GM1 molecules by AFM. This is likely a consequence of the flexibility of the GM1 headgroup. Accordingly, to visualize the GM1-containing domains, the B-subunit of CTX-B, labeled with Alexa555, was introduced into the AFM fluid cell.

It is well known that CTX-B binds to the GM1 headgroup to form, depending on the local GM1 concentration, well-ordered two-dimensional crystalline arrays extending ∼2.5 nm in height above GM1-containing domains (18,19,33). Although our correlated AFM/confocal imaging confirmed that Alexa555-CTX-B was associated with the higher domains, we were unable to resolve the individual CTX-B molecules by topography imaging, nor was the expected increase in height attributable to association of the labeled CTX-B to the GM1 molecules in the lo domains detected. This is likely a consequence of the low ∼2 mol % brain GM1 in the lo domains and incomplete coverage by CTX-B as a densely packed two-dimensional monolayer. However, we were able to resolve a direct spatial correspondence between fluorescence intensity and the lo domains, as identified by in situ AFM (see yellow arrows in Fig. 2 A).

FIGURE 2.

FIGURE 2

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Simultaneous AFM-confocal imaging of reconstituted rafts in model SPBs composed of 34 mol % DOPC/34 mol % synSM (C18)/30 mol % Chl/2 mol % brain GM1 with (A) 0.2% NBD-SM or (B) 0.2 mol % BODIPY-SM. Alexa555-CTX-B was added to locate the ganglioside receptor GM1, a raft-associated lipid. (Inset) Corresponding section analysis along A-A′ and B-B′.

BODIPY-SM and NBD-SM preferentially partition into the ld domains

A consequence of its long alkyl chains and corresponding high melting temperatures, SM is expected to be colocalized with the taller GM1-containing high domains, a model that has been supported by x-ray diffraction studies (34). Surprisingly, our correlated AFM/confocal imaging revealed that BODIPY and NBD analogs of SM in fact preferentially localize in the lower (ld) domains (Fig. 2). In the BODIPY and NBD-labeled lipids, one of the fatty acyl chains was replaced with an NBD- or BODIPY-labeled acyl chain. A recent study using a headgroup-labeled BODIPY-GM1 revealed that it failed to enter a more ordered gel domain, whereas a headgroup labeled Alexa488-GM1 preferentially partitioned into the gel domain (19). These data strongly suggest that phase partitioning of fluorophores between domains is a complex phenomenon that depends not only on the site of fluorophore attachment but also on the physical properties of the fluorophore.

BODIPY-GM1 and NBD-PC preferentially partition into the ld domains

Simultaneous AFM-confocal imaging at ambient temperature (∼23°C) revealed that SPBs containing 1:1 DOPC/synSM, 18:0/30 mol % Chl/2 mol % brain GM1/0.2 mol % BODIPY-GM1 and 0.2 mol % of NBD-PC, phase-segregated into high (lo) and low (ld) domains with height difference ranging from ∼0.5 to 1.5 nm, in agreement with previous AFM and x-ray diffraction studies (28–30,33). NBD-PC is known to partition into ld phase (32,34–36), whereas CTX-B is known to strongly associate with the ganglioside GM1 (34). Close inspection revealed that the green fluorescence arising from the BODIPY and NBD labels was predominately localized to the low domains (ld), whereas the red fluorescence associated with Alexa555 labeled CTX-B remained mainly associated with the tall (lo) domains (Fig. 3). Our data suggest that brain GM1 preferentially segregates to the tall (lo) domains, whereas BODIPY-GM1 localizes preferentially in the nonraft domains. One would expect that there would be some binding of CTX-B to the low domain because of the presence of the BODIPY GM1; however, the relative fluorescence intensity of CTX-B would be much higher in the high domain because of the 10-fold higher content of brain GM1 (the molar ratio of brain GM1/BODIPY GM1 is 10:1, in this case). Although CTX-B is known to induce clustering of GM1 (37–39), it is not likely that the introduction of CTX-B would result in the translocation of BODIPY-GM1 from the low to high domains. Thus, this Alexa555-CTX-B labeling experiment demonstrates that the unmodified GM1 sequesters into the high domains as expected; however, it also clearly reveals that BODIPY-GM1 unexpectedly associates with the lower, nonraft, domains (Fig. 3).

FIGURE 3.

FIGURE 3

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Simultaneous AFM-confocal imaging of reconstituted rafts in model SPBs composed of 34 mol % DOPC/34 mol % SynSM (C18)/30 mol % Chl/2 mol % brain GM1 with (A) 0.2 mol % BODIPY-GM1 or (B) 0.2 mol % NBD-PC. Alexa555-CTX-B was added to locate the ganglioside receptor GM1, a raft-associated lipid. (Inset) Corresponding section analysis along A-A′ and B-B′.

Domain partitioning of fluorescently labeled cholesterol

A major constituent of eukaryotic membranes, Chl plays a critical role in defining membrane structure and function (40), including regulating lipid trafficking and sorting (41). Our correlated AFM-fluorescence data clearly reveal that NBD-Chl and BODIPY-Chl 1 and 3 do not distribute preferentially in the lo phase (Fig. 4), which is enriched in Chl and sphingolipids (8). The side chain of Chl resides in the bilayer interior (40,42); however, the NBD group, which is generally considered to reside deep in the bilayer (43), was reduced when a water-soluble reducing agent (dithionite) was added to NBD-Chl-containing bilayers (44), indicating that the NBD moiety at C25 of Chl can loop back toward the lipid/water interface. Our study agrees with a recent finding that NBD-Chl (45) does not mimic Chl (44,46).

FIGURE 4.

FIGURE 4

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Simultaneous AFM-confocal imaging of reconstituted rafts in model SPBs composed of 34 mol % DOPC/34 mol % SynSM (C18)/28 mol % Chl/2 mol % brain GM1 with (A) 2 mol % NBD-Chl, (B) 34 mol % DOPC/34 mol % SynSM (C18)/30 mol % Chl/2 mol % brain GM1 with 0.2 mol % BODIPY-Chl 1, or (C) 0.2 mol % BODIPY-Chl 3. Alexa555-CTX-B was added to locate the ganglioside receptor GM1, a raft-associated lipid. (Inset) Corresponding section analysis along A-A′, B-B′, and C-C′ in the AFM topography images.

BODIPY-Chl 3 is an analog of coprostanol. Although structurally related to Chl, this analog was designed not to mimic Chl, but to serve as a nonraft probe. Since the sterol nucleus is not flat in BODIPY-Chl 3, this compound would differ from natural Chl with respect to lipid-lipid interactions, consistent with the results from this study.

BODIPY-Chl 2 is a more faithful mimic of Chl since it retains the planar conformation of the Chl ring system and does not contain any polar atoms linking BODIPY to the sterol ring. This is in agreement with our findings that BODIPY-Chl 2 partitions into a Triton X-100 low density insoluble fraction, whereas BODIPY-Chl 3 does not (47). Nevertheless, the localization of BODIPY-Chl 2 within the bilayer is dependent on the type of SM used to form the SPBs. For instance, BODIPY-Chl 2 distributes uniformly across the higher (lo) and the lower (ld) domains in bilayers containing synSM 18:0. As shown in Fig. 5 A, binding of Alexa555-CTX-B induced quenching of BODIPY fluorescence at the higher (ld) domains. Sequestering of BODIPY dyes or resonance energy transfer between BODIPY and Alexa555 are possible mechanisms that can result in localized quenching of BODIPY fluorescence. The exact mechanisms remain unclear. In contrast, BODIPY-Chl 2 preferentially partitioned into the lower domains in the bilayers containing synSM 16:0 (Fig. 5 B) and preferentially localized in the higher (lo) domains in bilayers containing brain SM (Fig. 6).

FIGURE 5.

FIGURE 5

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Simultaneous AFM-confocal imaging of reconstituted rafts in model SPBs composed of (A–B) 34 mol % SynSM (C18)/34 mol % DOPC/30 mol % Chl/2 mol % brain GM1 with 0.2 mol % BODIPY-Chl 2 or (C–D) 34 mol % SynSM (C16)/34 mol % DOPC/30 mol % Chl/2 mol % brain GM1 with 0.2 mol % BODIPY-Chl 2. Alexa555-CTX-B was added to locate the ganglioside receptor GM1, a raft-associated lipid. (Inset) Corresponding section analysis along A-A′, B-B′, C-C′, and D-D′ in the AFM topography images.

FIGURE 6.

FIGURE 6

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Simultaneous AFM-confocal imaging of reconstituted rafts in model SPBs composed of 35 mol % DOPC/35 mol % brain SM/28 mol % Chl/2 mol % BODIPY-Chl 2. (Inset) Corresponding section analysis along A-A′.

The higher hydrophobicity of BODIPY relative to NBD allows for better insertion into the bilayer (17,48). However, this does not guarantee that a BODIPY-bearing lipid would preferentially partition into the more ordered (lo) phase, as shown by the behavior of BODIPY-GM1 and BODIPY-SM. Different linkers that connect the BODIPY and the sterol side chain result in differences among BODIPY-Chl analogs. The linker coupling BODIPY to the sterol in BODIPY-Chl 1 contains an ester functionality, whereas in BODIPY-Chl 2 a short linker devoid of oxygen atoms was employed. Interestingly, a relatively small change in structure from BODIPY-Chl-1 to BODIPY-Chl-2 can change the location of the sterol. The sensitivity of the domain partitioning of BODIPY-Chl 2 to the nature of the surrounding lipid is illustrated by the change caused by the nature of the SM (Figs. 5 and 6).

IMPLICATIONS AND CONSIDERATIONS

Although our confocal images show significant contrast associated with the segregation of the dyes into different spatial domains, it should be noted that the fluorescence intensity in the two domains depends not only on the concentration of the fluorophore in that domain, but also on its quantum yield in that particular domain. We have not quantitatively determined the partition coefficient of the dyes examined in our studies, but rather used the correlated AFM approach to assign and assess the phases that the dyes are in favor of staining. Using planar supported lipid bilayers to study membrane or protein-membrane dynamics and interactions allows us to systematically vary lipid composition and to employ the correlated AFM-confocal approach with very little ambiguity in the results. Planar supported lipid bilayers lack membrane curvatures that are present in the spherical model membranes such as giant unilamellar vesicles (GUVs) or cell membranes. However, it has been demonstrated that GUVs partition into liquid/liquid immiscible domains in the same way that planar supported lipid bilayers do, and the lipids in both leaflets of the supported bilayers are freely mobile (49).

CONCLUSIONS

Our studies have several important implications. Upon conjugation with NBD or BODIPY, the lipids examined in this study with the exception of BODIPY-Chl 2, and that had been shown to associate with raft domains in the absence of the fluorophore no longer preferentially segregated to the taller raft domains. This is consistent with previous reports (12,15). Topographical AFM can provide details of the physical structure of the domains where the fluorescent probes are preferentially located. This correlated approach is an excellent means of investigating the partitioning of fluorescent probes in a coexisting raft/nonraft system. In addition, this study illustrates the possible adverse effects arising from the use of NBD- and BODIPY-labels to identify raft domains.

Acknowledgments

This work was supported by the Canadian Institutes of Health Research (R.M.E.: MA-7654; C.M.Y.: MT:14769) and the Natural Sciences and Engineering Research Council of Canada (C.M.Y.: 194435). C.M.Y. acknowledges support from the Canada Research Chairs program, the Canadian Foundation for Innovation, the Ontario Innovation Trust, and the Ontario Research and Development Challenge Fund.

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