Sphingomyelin distribution in lipid rafts ofartificial monolayer membranes visualized by
Raman microscopy
Jun Andoa,b,c,d, Masanao Kinoshitae,f,1, Jin Cuie,g, Hiroyuki Yamakoshia,d, Kosuke Dodoa,b,d, Katsumasa Fujitaa,b,c,Michio Muratae,g,2, and Mikiko Sodeokaa,b,d,2
aSodeoka Live Cell Chemistry Project, Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology, Saitama 351-0198, Japan;
Core Research for Evolutionary Science and Technology (CREST), JST, Saitama 351-0198, Japan; cDepartment of Applied Physics, Osaka University,
Osaka 565-0871, Japan; dSynthetic Organic Chemistry Laboratory, RIKEN, Saitama 351-0198, Japan; and eLipid Active Structure Project, JST, Exploratory
Research for Advanced Technology, fProject Research Center for Fundamental Science, and gDepartment of Chemistry, Graduate School of Science,
Osaka University, Osaka 560-0043, Japanb

Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved March 10, 2015 (received for review September 19, 2014)

Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell
membranes, called lipid rafts, are thought to have importantbiological functions related to membrane signaling and protein
trafficking. To visualize the distribution of SM in lipid rafts bymeans of Raman microscopy, we designed and synthesized an SM
analog tagged with a Raman-active diyne moiety (diyne-SM).
Diyne-SM showed a strong peak in a Raman silent region that is
free of interference from intrinsic vibrational modes of lipids anddid not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize
the distribution of diyne-SM in raft-mimicking domains formed in
SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman
images visualized a heterogeneous distribution of diyne-SM,
which showed marked variation, even within a single ordereddomain. Specifically, diyne-SM was enriched in the central area ofraft domains compared with the peripheral area. These results
seem incompatible with the generally accepted raft model, in
which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SMconcentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique ofhyperspectral Raman imaging of a single lipid monolayer opens
the door to quantitative analysis of lipid membranes by providing
both chemical information and spatial distribution with high(diffraction-limited) spatial resolution.

|

lipid raft Raman imaging
sphingomyelin

direct observation is essential to investigate the lipid distribution in intact lipid preparations.
Fluorescence microscopy has been widely used to observe
phase separation in lipid membranes (7), and fluorescently labeled lipids or lipophilic dyes have generally been used as imaging agents (8). However, these fluorescent lipids are frequently
excluded from the raft-like ordered domains, probably because
the large fluorophores perturb the lipid packing (8, 9). So far,there is no lipid probe that can directly visualize the distributionof intrinsic SM molecules in multicomponent membranes, althoughsome peptide-based large probes that recognize raft domains havebeen developed (10, 11). Consequently, direct observation of SM
distribution in lipid rafts has not proved feasible with fluorescencebased imaging modalities.
Here, we focused on spontaneous Raman scattering microscopy to directly and chemoselectively visualize the distribution ofa lipid constituent in lipid rafts using a ternary lipid monolayer
system as a model. Raman spectroscopy has chemical specificity,
because it detects characteristic molecular vibration frequencies.
The molecular distribution in a sample can be determined by
Raman microscopy, and the image has quantitative chemical
contrast, because the scattering intensity is proportional to thenumber of molecules in the detection volume. A molecule-specificfull spectrum is obtained for each pixel, allowing us to analyze the
Significance| alkyne tag | supported monolayer |Phase separation in lipid rafts has been observed with fluorescently labeled lipids, but they are often excluded from theordered domain because of the steric effect of the bulky fluorophore on lipid packing, making it difficult to analyze the interior of the raft domain. Here, we synthesized an analog ofsphingomyelin tagged with a small Raman active diyne moiety,which provides high chemical selectivity without affecting themembrane properties. Raman microscopy successfully visualized,
at single lipid-layer sensitivity, a heterogeneous spatial distribution of this probe within raft-like ordered domains, which wasdifferent from the generally accepted raft model. This approachprovides both chemical selectivity and quantitative imaging capability and is useful for functional studies of lipid rafts.

S

pecific membrane microdomains, called lipid rafts, are thought
to have important biological functions in cells (1, 2). The rafts
are frequently defined as detergent-resistant membrane domains
and enriched in sphingomyelin (SM) and cholesterol (chol). Certain
membrane proteins, such as glycosylphosphatidylinositol-anchoredproteins and acylated cytosolic proteins, are considered to show
preferential association with those raft lipids, which thereby, facilitate various biological functions, including membrane trafficking
and signal transduction (3). Therefore, an understanding of the
distribution of SMs within lipid rafts is expected to provide basic
information about site-specific cellular functions.
The basic features of lipid rafts have often been examinedusing SM/phosphatidylcholine (PC)/chol mixtures as model membrane systems, because these ternary mixtures undergo phase separation into raft-like ordered domains and fluid disordered domains(4). In addition, the membrane properties of these ordered domains, such as detergent insolubility, are similar to those of lipid
rafts in biomembranes (5). However, there are few reports on thelipid distribution in the raft mixtures, because detergent extraction provokes reorganization of the lipids and consequently,
obscures information about the inherent domains (6). Thus,

4558–4563 | PNAS | April 14, 2015 | vol. 112 | no. 15

Author contributions: J.A., M.K., K.D., M.M., and M.S. designed research; J.A., M.K., J.C.,
and H.Y. performed research; K.F. contributed new reagents/analytic tools; J.A., M.K., andK.F. analyzed data; and J.A., M.K., K.F., M.M., and M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1

Present address: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka
812-8581, Japan.

2

To whom correspondence may be addressed. Email: murata@chem.sci.osaka-u.ac.jp or
sodeoka@riken.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1418088112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1418088112

Results
The Raman spectra in Fig. 2 are those of supported monolayer
membranes consisting of SM (Fig. 2A), diyne-SM (Fig. 2B),
dioleoyl-PC (DOPC) (Fig. 2C), and chol (Fig. 2D). As shownin Fig. 2B, the diyne-SM monolayer showed a strong peak at
2,263 cm−1, which is attributed to the stretching vibrational modeof the diyne moiety (18). Because the peak appears in the silent
region of membrane lipids, it would be possible to unambiguously
identify diyne-SM in multicomponent membranes by means ofRaman spectroscopy without any interference from intrinsic vibrational modes of other membrane constituents. For this measurement, 532-nm laser light was focused onto the membrane by using
an objective lens to excite Raman scattering from the lipid. It wasFig. 1.

Ando et al.

Chemical structures of SM, diyne-SM, DOPC, and chol.

Fig. 2. Raman spectra of supported lipid monolayers of (A) SM, (B) diyneSM, (C) DOPC, and (D) chol on a quartz substrate. The Raman peak of diyne
at 2,263 cm−1 is marked by a red arrow. The supported sample was preparedat 12 mN/m and 25 °C using the LB technique. Raman measurement was
performed 15 times at different positions in each membrane, with an exposure time of 6 s. Averaged Raman spectra are shown. Raman peaks of O2
at ∼1,555 cm−1 and N2 at ∼2,330 cm−1 have been deleted so that Ramanpeaks from lipid molecules can be clearly seen, which is explained in Fig. S1.

confirmed that our microscope was able to acquire a Raman
spectrum from a small, diffraction-limited area within a lipid
monolayer, with a signal-to-noise ratio sufficient to detect the
Raman peak of the diyne moiety.
Next, we observed a diyne-SM/DOPC/chol ternary monolayer(1:1:1 molar ratio) by laser Raman microscopy in a pointscanning mode. SM-based lipid monolayer and bilayer are known
to have similar phase segregation properties (4, 19); moreover,by using a monolayer, we can exclude possible asymmetry of lipid
composition between the two layers that would weaken thecontrast of Raman images. The Raman image, reconstructedfrom the peak intensity of diyne at 2,264 cm−1, visualized aheterogeneous distribution of diyne-SM (Fig. 3A), which wasconcentrated in micrometer-sized round domains. When an image was constructed using the peak bottom intensity at 2,222 cm−1,
it showed no contrast or distribution, ruling out the possibility that
artifacts affected the imaging (Fig. 3B). We also performed Raman
imaging of lipid membrane (Fig. 4 A–C) with three different composition ratios of diyne-SM/DOPC/chol (molar ratios of 1:1:1, 3:7:3,
and 1:0:0). Phase segregation occurred at the 1:1:1 ratio, whereas
uniform distributions were observed for 3:7:3 and 1:0:0 ratios. The
difference between Fig. 4 A and B can be accounted for by thenotion that the increased content of chol leads to recruitment of SM
from the disordered phase and increases the concentration of SM in
the ordered domains (20). Judging from the image contrast, thedensity of diyne-SM is higher in ordered domains at a 1:1:1 ratio(Fig. 4A) than at a 3:7:3 ratio (Fig. 4B) but lower than that at a 1:0:0
ratio (Fig. 4C). The averaged diyne peak intensity of the ordereddomains in Fig. 4A was ∼2.3 times higher than that of the disordered region in Fig. 4A when the domains were separated based on
the mean value of the entire Raman image (Fig. S2 and Table S1).
On the same basis, the diyne-SM density in the ordered domains
in Fig. 4A was 52.2% of the diyne-SM density in Fig. 4C (Fig. S2and Table S1). Note that spatial variations of the signal in Fig.
4C (Fig. S2E) are mainly caused by the low signal-to-noise ratio
in the detection as summarized in Table S1.
Hyperspectral Raman imaging, as performed in this experiment, allows us to examine spatially resolved Raman spectra atdifferent phases in the diyne-SM/DOPC/chol ternary monolayer(Fig. 4 D and E and Fig. S3). In particular, the intensity profile oflipid CH2/CH3 stretching at 2,800–3,000 cm−1 was different between the raft-like ordered domains and the disordered domain(Fig. 4D). The peak profile of CH2/CH3 stretching (such as sharp
peaks at 2,850 and 2,883 cm−1) in the raft-like ordered domains was
PNAS | April 14, 2015 | vol. 112 | no. 15 | 4559

CHEMISTRY

molecular composition and state of the lipid mixture at eachposition, with the help of accumulated knowledge on Raman
spectra of lipid membranes obtained over the last few decades(12). Because Raman microscopy uses an optical detection
scheme, minimally invasive noncontact observation of a sample
can be achieved under atmospheric pressure.
Despite these advantages, spontaneous Raman microscopy
has not yet been used for imaging of lipid rafts to our knowledge.
It is partly because Raman imaging of a single lipid membrane
has long been thought to be infeasible from a sensitivity point ofview owing to the extremely weak scattering signal from thelimited number of lipid molecules in the detection volume (13).
Spatial resolution is also restricted by the imaging time, becausea weak scattering signal tends to require a long exposure time,
resulting in limited pixel numbers for imaging within a realistic
observation time. However, recent developments in spontaneous
Raman microscopy have allowed us to overcome this problem; efficient optical systems and a parallel Raman excitation/detection
configuration (14–16) improve imaging speed with both high sensitivity and high diffraction-limited spatial resolution.
As a target lipid component for observation by Raman microscopy in a raft-containing membrane, we focused on SM, andnewly synthesized an SM analog with a Raman-active alkyne
moiety. Alkyne is a promising tag in terms of both its small
chemical structure and its high Raman scattering intensity in thesilent region of biomolecules, allowing selective detection ofalkyne-tagged molecules (17, 18). We first attempted Raman
imaging using a propargyl-SM analog with a single alkyne group,
but the intensity was insufficient for imaging. Because a conjugated diyne group shows a stronger Raman band (18), we next
aimed to introduce a 6-hydroxy-hexa-2,4-diynyl group at theammonium moiety of the head group; the 6-hydroxyl group was
expected to increase the hydrophilicity of this relatively hydrophobic Raman tag (Fig. 1). We found that diyne-SM was effective for spontaneous Raman imaging of SM in a raft-mimicking
ternary monolayer system.

Fig. 3. Raman images of a diyne-SM/DOPC/chol ternary monolayer (1:1:1
molar ratio) on a quartz substrate. The images were reconstructed based on(A) the intensity of the diyne peak at 2,264 cm−1 and (B) that of the peakbottom at 2,222 cm−1. The images consist of 54 × 28 pixels. (Scale bar: 10 μm.)

similar to that of the diyne-SM monolayer (Fig. 4D and Fig. S4). A
characteristic peak of chol at 2,936 cm−1 was also observed in theordered domains (Fig. S4), suggesting a higher content of chol aswell as diyne-SM. However, the peak profile at the disordereddomain (Fig. 4D) was similar to that of the DOPC monolayer (Fig.
S4). The spatial distribution of Raman spectra along the yellow line
in Fig. 4A was reconstructed, and the position-dependent changeof the peak profile of CH2/CH3 stretching corresponded well tothe intensity change of the diyne peak (Fig. 4E).
High-resolution Raman imaging was performed to visualize
the distribution of diyne-SM inside a raft-like ordered domain of
the ternary monolayer (Fig. 5A). We used slit-scanning Raman
microscopy, which used a line-shaped laser focus for parallel
Raman spectroscopic detection (16). Because the imaging speedof the slit-scanning configuration is more than 100 times fasterthan that of the point-scanning configuration, we could increase
the image pixel number to observe fine structure in the sample.
The Raman image in Fig. 5A shows a heterogeneous distributionof diyne-SM within the round-shaped ordered domains; diyneSM was enriched in the central area of the domain comparedwith the peripheral area. We also performed Raman imaging ofpure diyne-SM monolayer by using slit-scanning Raman microscopy and confirmed the uniform intensity distribution of theimage (Fig. S5). The fluorescence image of the ternary monolayer containing 0.2 mol% Bodipy-PC was also obtained (Fig. 5B,
Lower, and Fig. S6A), in which the ordered domains appeared as
dark areas. A Raman image of the diyne peak at 2,264 cm−1 atthe same area of the same sample was also obtained (Fig. 5B,
Upper, and Fig. S6B) to compare the two imaging modalities.
Diyne-SM was observed as round-shaped domains showing
inverted contrast with respect to the fluorescence image. The
dark regions in the fluorescence image overlap well with thebright regions in the Raman image (Fig. S6C), which is in accordance with the previous finding that Bodipy-PC is preferentially localized in the disordered domain (7, 8). This result also
supports the idea that diyne-SM is preferentially incorporated
into the ordered domain. The line profiles of Raman and fluorescence images were taken (Fig. 5C). The fluorescence image
shows a dip at ordered domains, and larger domains have a flatbottom, indicating that fluorescent probe cannot reach the center of the domains. However, the Raman image shows protrusions rather than trapezoidal peaks, which should represent
the distribution of diyne-SM in the ordered domain; the resultsin Fig. 5 confirm that the alkyne tag directly reflected the contents of a specific lipid constituent in the raft-like ordered domains, which is usually impossible with fluorescence probes.
The influence of substitution of SM with the diyne moiety
should be carefully considered, because it may potentially alter
the membrane properties. Among various parameters, the interactions between SM-SM and SM-chol are thought to be apivotal driving force for phase segregation and ordered domain
formation (21, 22). We, therefore, investigated the interaction
between SM and chol or diyne-SM and chol using differential
scanning calorimetry (DSC) and surface pressure-area isotherm(π-A isotherm) measurements. DSC of pure SM and diyne-SMbilayers revealed values of the main transition temperature(Tm) of 44.5 °C and 39.5 °C, respectively (arrows in Fig. S7); asimilar drop in Tm values was reported for perdeuterated PC
4560 | www.pnas.org/cgi/doi/10.1073/pnas.1418088112(d62-dipalmitoyl-PC), which was previously used as a Ramanprobe (23). The π-A isotherms of diyne-SM/chol and SM/cholbinary monolayers are shown in Fig. 6 A and B. Pure diyne-SMand pure SM monolayers showed phase transition from liquidexpanded (LE) to liquid-condensed phase at 20–30 and 10–20
mN/m, respectively (black lines in Fig. 6 A and B). To evaluate
the influence of chol on the molecular packing of the SMs, themean molecular areas (Amean) were plotted as a function of themolar fraction of chol (xchol) at 5 mN/m (Fig. 6 C and D), wherepure diyne-SM and SM monolayers form homogeneous LEphase. The lateral molecular area of SM at 5 mN/m (61 Å2) isclose to that in the fluid-phase bilayer (57 Å2), which was calculated from the molecular volume (1,225 Å3) and bilayer thickness(43 Å) (24). In both the diyne-SM/chol and the SM/chol mixtures,
the Amean values were smaller than the additive functions over allof the experimental xchol range, suggesting the existence of similarintermolecular condensation in the diyne-SM/chol and SM/chol
monolayers. For additional analysis, we calculated the partial
molecular area (PMA) of chol APMA by fitting the data to two
linear functions according to a previous report (25). Similar APMA
values were obtained for diyne-SM/chol (−7 ± 5 Å2) and SM/chol(−3 ± 5 Å2) monolayers at lower concentrations of chol (Fig. 6E).
Assuming that the lateral area of rigid chol is constant at 38 ±1 Å2, irrespective of composition, these results suggest that thechol-induced condensation of diyne-SM is similar to that of SM
at lower concentrations of chol. The APMA values increased to∼40 Å2 at similar compositions: at xchol = 0.37 for diyne-SM/choland xchol = 0.35 for SM/chol. These xchol values, called breakpoints, are likely to correspond to the composition at which allmonolayer domains transform completely into the orderedphase; the same analysis and interpretation have been applied to
the Ld/Lo phase transition in SM/chol bilayer systems (26). Because the APMA value above the break point is consistent with thelateral occupied area of pure chol (∼38 ± 1 Å2), no condensationof SM occurs above the break point, and thus, the capacity forchol should be similar between diyne-SM and SM. Direct evidence for the chol-induced condensation of SM could be obtainedby membrane rigidity and areal compressional modulus Cs−1
analysis. For both the diyne-SM/chol and the SM/chol monolayers,
the Cs−1 values of the diyne-SM/chol and SM/chol monolayers
were in line with the theoretical curves (27) below each breakpoint (solid lines in Fig. 6 F and G). However, additional increase

Fig. 4. Raman images of diyne-SM/DOPC/chol ternary monolayers withcomposition ratios of (A) 1:1:1, (B) 3:7:3, and (C) 1:0:0 reconstructed using
the intensity of the diyne peak at 2,263 cm−1. (D) Averaged Raman spectrafrom rectangular areas marked i–iv in A–C. Area i corresponds to an ordereddomain, whereas area ii corresponds to a disordered region. Each rectangular area includes 9 (3 × 3) pixels. The Raman peak of N2 at 2,330 cm−1 wasremoved so that the diyne peak could be clearly seen. (E) Spatial distributionof Raman spectra of the diyne-SM/DOPC/chol ternary monolayer at a 1:1:1
ratio acquired along the yellow line marked in A. Wavenumber region includes the diyne peak and CH2/CH3 stretching vibrational mode. The images
consist of 36 × 24 pixels. (Scale bar: 10 μm.)

Ando et al.

Fig. 5. (A) High-resolution Raman imaging of a 1:1:1 diyne-SM/DOPC/chol
ternary monolayer taken with slit-scanning Raman microscopy. The image
was reconstructed using the diyne peak intensity at 2,262 cm−1. Exposure
time and laser power were 100 s per line and 10.5 mW/μm2. The image isshown in a 16-color display. The images consist of 412 × 400 pixels. (Scale bar:
10 μm.) (B) Raman and fluorescence images of a 1:1:1 diyne-SM/DOPC/chol
ternary monolayer containing 0.2 mol% Bodipy-PC. Raman and fluorescence
images were obtained in the same imaging area of the same sample. The
Raman image was reconstructed using the diyne peak intensity at 2,264 cm−1.
The fluorescence image was reconstructed using the average fluorescenceintensity at 542–603 nm. Fluorescence background during Raman imaging was
suppressed by photobleaching of Bodipy-PC under 532-nm laser exposure.
Exposure time and laser power for Raman imaging were 60 s per line and 14.1
mW/μm2. Exposure time and laser power for fluorescence imaging were 0.5 s
per line and 0.3 mW/μm2. Each image consists of 387 × 250 pixels. (Scale bar:
10 μm.) (C) Line profiles of lipid rafts calculated along the dotted lines of theRaman and fluorescence images in B (red and gray, respectively). The line
profile from the Raman image was smoothed using the moving average.

in chol concentration led to positive deviation of Cs−1 from theideal function. On the basis of the results in Fig. 6 F and G, themolecular compressional modulus C−1
mol of diyne-SM and SM
turned out to be similar in the chol-absent LE and the cholpresent ordered phases (Table 1). These results clearly indicatethat the interaction properties of diyne-SM and SM with chol
are closely similar.
Moreover, we compared the mobility and orientation of acyl
chains of diyne-SM and SM in SM/DOPC/chol (1:1:1 mol:mol:

Discussion
The heterogeneous distribution of diyne-SM in ordered domains,
as seen in Fig. 5A, implies the existence of intermediate regions
between SM-rich ordered and SM-poor disordered domains. This
gradual change in the concentration of diyne-SM from the core to
the peripheral areas of the SM-rich domain implies a slightly
different view from that of the common raft model, in which theraft and nonraft phases show a relatively clear biphasic separation.
According to previous atomic force microscope observations, themonolayer thickness is larger for the ordered domain than for thedisordered domain because of the extension of acyl chains in
the former domains (29). Considering that SM-induced intermolecular hydrogen bonding facilitates the ordering of lipid
packing and increases membrane thickness, the intermediate region may possibly work to mitigate the thickness gap between the
SM-rich and -poor domains, avoiding direct contact of these tworegions. Because Raman imaging required relatively long acquisition times, we prepared the membrane on a quartz substrateusing the LB technique. According to previous literature, thedistribution of domains and the compositions of phases wereusually preserved on the sample transfer from the water surface toa relatively hydrophilic substrate and also, unchanged for longtime (30). Therefore, our Raman images are likely to be snapshotsof the dynamic LB membrane on the water surface. Although theresults in this study cannot be directly applied to biologicalmembranes, the images indicate that the distribution of SM is nothomogeneous, even in a single raft domain, which may have

Fig. 6. Surface pressure vs. molecular area isotherms of (A) diyne-SM/chol and (B) SM/cholbinary monolayers. Reported data for SM/chol
mixtures in B, D, and G were redrawn for comparison (24). The molar fraction of chol xchol isdirectly shown. The plots show mean moleculararea Amean vs. composition of (C) diyne-SM/choland (D) SM/chol binary monolayers at 5 mN/m.
Each result can be fitted to two lines as indicated by dashed lines. Theoretical mean molecular areas (additivity functions) are indicatedby solid lines. (E) The PMAs APMA of chol in(blue) diyne-SM/chol and (red) SM/chol monolayers at 5 mN/m were estimated from C and D,
respectively (27). The cross-sections between
dashed lines and xchol = 1 in C and D show APMA
values, which are sums of the lateral areas occupied by chol and the chol-induced lateral expansion of neighboring lipids. (F and G) The−1of themolecular compressional modulus Cmol
diyne-SM and SM in the ordered phase was estimated by fitting the data to the theoretical
function in the region of xchol ≥ 0.5 (dashed lines
in F and G), in which all monolayer domains
form the ordered phase. The cross-sections between xchol = 0 and the dashed lines correspond−1(24). The Cs−1 vs. composito the values of Cmoltion plots are shown for (F) diyne-SM/chol and(G) SM/chol binary monolayers at 5 mN/m (27).
The solid lines indicate the theoretical Cs−1 values of SM/chol mixtures, and the dashed lines were obtained by fitting the data to the theoretical equation in
the region of xchol ≥ 0.5. Analysis is in SI Materials and Methods.

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PNAS | April 14, 2015 | vol. 112 | no. 15 | 4561

CHEMISTRYmol) bilayers by means of 2H NMR analysis (Fig. 7) of partly
deuterated analogs (Fig. 7 A and B). The SM mixture gave twopairs of Pake doublets at Δν = 51.9 kHz and Δν = 36.3 kHz. The
outer and inner Pake doublets correspond to the Lo and Ld domains, respectively (Fig. 7C) (28). The diyne-SM mixture gave apair of Pake doublets at Δν = 48.1 kHz, which is close to the outer
Pake obtained from the SM mixture (Δν = 51.9 kHz) (Fig. 7D); theinner doublet of the diyne-SM membrane was not clearly observed,
probably because of a smaller content of Ld phase in the liposome
preparation. Thus, the Lo phase developed in the diyne-SM ternary mixture in bilayer form showed similar mobility to that of SM.

−1
Table 1. Molecular compressional modulus of SM Cmolin thediyne-SM/chol and SM/chol monolayers

Diyne-SMSM

xchol = 0 (LE phase;mN/m)xchol ≧ 0.5 (orderedphase; mN/m)

33 ± 1047 ± 10120 ± 20115 ± 20

−1
The Cmolvalues were calculated from Fig. 6 F and G; the cross-sectional−1in thevalues between the dashed curves and xchol = 0 correspond to Cmol
ordered phase (24).

implications for the dynamic properties of lipid rafts with diverse
lipid compositions in biomembranes. To elucidate the dynamic
behavior of SM molecules in a biological membrane, additional
improvement of the performance of Raman microscopy in termsof imaging speed and sensitivity will be an important next step.
We substituted a Raman tag at the head group of lipid toobserve SM molecules bearing intrinsic acyl chains in the raft
mixture. Perdeuterated acyl-chained lipids, such as those of d62dipalmitoyl-PC, have been often used as Raman-tagged lipid
probes, because C–D bonds exhibit peaks in the silent region of
biomolecules, and relatively strong Raman scattering occurs
owing to the copious C–D bonds (23). This strategy is useful to
examine head group effects on the phase behavior but is not
ideal for investigating the interactions between acyl chains;deuterium labeling is known to slightly but significantly alter
thermodynamic parameters, such as transition enthalpy, andalso, influence phase behavior in single and multicomponent
systems (23, 31). For the lipid head group, the number of sites
available for introducing a Raman tag is relatively limited. Alkyne, particularly conjugated diyne, is a promising Raman tag forthe head group because of its strong Raman intensity (18). We
incorporated a conjugated diyne moiety into the ammonium
group at the polar head and successfully recorded its image in an
artificial monolayer with a raft-mimicking composition. It is
important to examine the influence of modification of the headgroup with diyne on the membrane properties of SM. We
quantitatively evaluated the membrane properties of diyne-SMand naturally occurring SM in the presence and absence of cholby using DSC, π-A isotherm measurements, and solid-state
NMR. DSC revealed that the Tm of pure diyne-SM bilayer was
5 °C lower than that of SM, which is the same as the difference
between PC and perdeuterated-PC (23). This correspondence
implies that introduction of the diyne group into the SM headgroup perturbs lipid–lipid interactions to a similar extent as thecase of perdeuteration of the acyl chains of PC. Areal and compressional modulus analysis further showed that the interactions
between diyne-SM and chol are similar to those between SM andchol (Fig. 6E and Table 1). The NMR spectra of ternary mixtures
showed that the mobility of the hydrocarbon chains of the d2diyne-SM (Fig. 7B) in the ordered phase is similar to that of d2SM (Fig. 7A) in the SM/DOPC/chol mixture (1:1:1 molar ratio)(Fig. 7). In addition, both of the fluorescence images of diyneSM/DOPC/chol and SM/DOPC/chol monolayers in the presenceof Bodipy-PC showed that the ordered domains with circularshape are separated from the disordered matrix (Fig. S8). Considering that the domain shape is sensitive to the environmental
conditions, such as the membrane properties of the coexisting
phases, phase equilibrium, and electrostatic force between lipid
constituents (32), these results suggested that the diyne moiety
hardly modulates the membrane properties and phase behavior.
As an imaging modality for lipid membranes, IR absorption
spectroscopy shows high chemoselectivity, detecting molecularvibrations with higher sensitivity than Raman spectroscopy.
However, the spatial resolution of IR absorption microscopy is
restricted to micrometer scale owing to the long wavelength ofthe probe light (33). Raman microscopy, however, provides higher
resolution with visible or near-IR excitation. Recently developed
coherent Raman microscopy allows fast molecular imaging (34, 35).
4562 | www.pnas.org/cgi/doi/10.1073/pnas.1418088112

Lipid bilayer in lipid vesicles or supported membranes has been
observed with coherent anti-Stokes Raman scattering microscopy(36, 37). Imaging of lipid monolayer, however, has not beenachieved with this type of microscopy to our knowledge. Although
it requires long imaging times, spontaneous Raman microscopy
may have an advantage for observing low-concentration molecules, because image contrast of coherent Raman microscopy canbe affected by signals that are not related to molecular vibration, such as nonresonant background or spurious backgroundcaused by cross-phase modulation. Secondary ion mass spectrometry (SIMS) is an emerging technique, in which lipid molecules on the substrate are bombarded with an ion beam, andlipid-specific fragment ions are analyzed by time-of-flight MS
without labeling of target molecules (30, 38). Although thistechnique requires destructive imaging conditions, including ultrahigh vacuum, and suffers from a matrix effect that prevents
quantitative analysis, it has been applied to molecule-specificimaging of raft mixtures (38). Quantitative analysis with SIMS
was recently achieved by using a magnetic sector detection system (39, 40). This imaging modality achieves chemical specificity
by using isotopic labeling of target molecules and affords highspatial resolution up to 50 nm. Chemoselective and quantitative
imaging modalities based on MS are also expected to bring a new
dimension to lipid raft research (41).
One of the disadvantages of Raman microscopy is its low
sensitivity. Nevertheless, it has unique capabilities, including theobservation of living organisms in aqueous and physiologicalenvironments (14, 15). In addition, intrinsic vibrational modes ofmembrane lipids in Raman spectra can provide a great deal ofmolecular information about the composition, structure, andphase state of lipid molecules (12). Furthermore, fluorescenceimaging and Raman observation can be performed with the same
sample in the same microscopic setup (Fig. 5 and Fig. S6),
providing complementary information. In addition, Raman microscopy, in principle, can perform label-free imaging of lipid
membranes by observing their intrinsic vibrational modes. As
shown in Fig. 2, the peak profile of intrinsic CH2/CH3 stretchingvibrations at 2,800–3,000 cm−1 varied depending on the lipid
species. Detailed analysis using spectral unmixing (42) may enable
us to discriminate the distributions of plural lipid components,
potentially enabling label-free Raman observation of lipid rafts.
In conclusion, Raman microscopic observation of a raftmimicking SM/DOPC/chol ternary monolayer was performed.
We could specifically detect the distribution of SM tagged with adiyne group from the complicated Raman spectra of the lipid
mixture. The membrane properties of diyne-SM were similar to
those of naturally occurring SM, which was confirmed by DSC,
π-A isotherm, and solid-state NMR. Raman intensity distribution, reconstructed from the Raman peak height of the diyne,
clearly visualized segregated diyne-SM microdomains in themonolayer membrane, whereas such domains are usually observed as uniformly dark areas in fluorescence microscopy. We

Fig. 7. Chemical structures of (A) d2-SM and (B) d2-diyne-SM and the NMR
spectra of (C) d2-SM/DOPC/chol and (D) d2-diyne-SM/DOPC/chol (molar ratioof 1:1:1) ternary bilayers at 30 °C. The d2-SM/DOPC/chol mixtures gave two
Pake doublets at Δν = 51.9 kHz (Lo) and Δν = 36.3 kHz (Ld). The d2-diyneSM/DOPC/chol mixtures gave a single Pake doublet at Δν = 48.1 kHz (Lo).

Ando et al.

period of 10 min for evaporation of the organic solvent, the monolayers
were compressed at a rate of 20 mm2/s. The subphase and ambient temperatures were controlled at 25.0 °C ± 0.1 °C and 25 °C ± 2 °C, respectively. A
thin quartz plate was dipped horizontally into the water followed by compression of the sample at 20 mm2/s to reach π = 12 mN/m. After compression,
the quartz substrate was extracted from the water at a rate of 0.1 mm/s, andquartz-supported monolayers were formed.

Materials and Methods

Raman/Fluorescence Microscopy. Raman scattering and fluorescence images
and spectra in Figs. 2–4 were obtained using laser-scanning Raman microscopy (Raman-11; Nanophoton) with a 532-nm excitation laser. The laser
beam was focused to a point at the sample and scanned in 2D over a sample
to acquire the Raman image. Raman scattering and fluorescence images inFig. 5 were obtained using a home-built slit-scanning Raman microscopewith a 532-nm excitation laser. The laser beam was focused to a line at the
sample and scanned in 1D over a sample to acquire the Raman image.
Details of the experimental procedures for Raman/fluorescence imaging andspectroscopy, DSC, π-A isotherm measurements, 2H measurements, and synthesis
and characterization of diyne-SM are described in SI Materials and Methods.

Materials. Porcine brain SM and DOPC were purchased from Avanti Polar
Lipids, and chol was purchased from Sigma Aldrich. SM with a saturated 18:0
acyl chain was extracted from brain SM and purified by HPLC. The purity of the
SM was checked by MS and TLC. These lipids were separately dissolved inchloroform/methanol (4:1 vol/vol) at a concentration of 1 mg/mL and stored at−20 °C until use. A fluorescent probe, Bodipy-PC, was brought from Molecular
Probes. This probe was dissolved in chloroform/methanol (4:1 vol/vol) at aconcentration of 50 μg/mL and stored in the dark at −20 °C. Raman-tagged
SM (diyne-SM) was newly synthesized (details in SI Materials and Methods
and Figs. S9 and S10); d2-diyne-SM was synthesized similarly to diyne-SM, andd2-SM was prepared (details in Figs. S11 and S12), as reported (28). Otherchemicals were purchased from Wako Pure Chemical Industries, Ltd.
Supported Monolayer Preparation. Monolayers of lipid mixtures were preparedon a computer-controlled Langmuir film balance (USI System) calibrated using
stearic acid (Sigma Aldrich). The subphase, which consisted of distilled, freshly
deionized water, was obtained using a Milli-Q System. The sample solution was
prepared by mixing the appropriate amount of each lipid dissolved in chloroform/methanol (4:1 vol/vol) in a microvial. A total of 30 μL lipid solution(1 mg/mL) was spread onto the aqueous subphase (100 × 290 mm2) using a
glass micropipette (Drummond Scientific Company). After an initial delay

ACKNOWLEDGMENTS. We thank Dr. Fuminori Sato, Associate Prof. Shigeru
Matsuoka, and Assistant Prof. Yuichi Umegawa [Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology Lipid
Active Structure Project, Osaka University] and Mr. Naoya Inazumi (School of
Science, Osaka University) for their support for NMR measurements. We also
thank Dr. Almar Palonpon (JST Exploratory Research for Advanced Technology
Sodeoka Live Cell Chemistry Project, Osaka University) for his support for the
analysis of Raman imaging data. We thank Dr. Taisuke Ota (Nanophoton) for
technical support for the Raman microscope system. This work was partially
supported by RIKEN, JST, Osaka University Photonics Advanced Research Center,
and Japan Society for the Promotion of Science KAKENHI (Grant-in-Aid for
Scientific Research) Grants 23710276 (to H.Y.) and 25242073 (to M.M.).

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Ando et al.

PNAS | April 14, 2015 | vol. 112 | no. 15 | 4563

CHEMISTRY

found that diyne-SM shows a heterogeneous distribution eveninside raft-like ordered domains; it was enriched in the central
area compared with peripheral areas. Our proposed alkyne-tag
Raman imaging, performed in this study at single lipid-layer
sensitivity, should be a useful tool for research on lipid membranes,
especially lipid rafts, by providing both quantitative and moleculespecific chemical contrast with hyperspectral imaging capability.

Supporting Information
Ando et al. 10.1073/pnas.1418088112
SI Materials and Methods
Experimental Procedures for Raman/Fluorescence Imaging andSpectroscopy.
Experimental procedures used for Fig. 2. Supported monolayer mem-branes of single lipid composition, including SM, diyne-SM, DOPC,
and chol, were prepared on a quartz substrate (0.17-mm thickness;
Starbar Japan), fixed in a metal chamber (Attofluor; Life Technologies), and placed on the sample stage of the microscope(Raman-11; Nanophoton). For Raman spectroscopic measurement, the 532-nm laser was focused at a point on the membranewith a water immersion objective lens (CFI Plan Apo IR 60XWI;
Nikon). The laser intensity at the sample plane was 340 mW.
Exposure time was 6 s. Slit width for the spectrophotometer was set
as 50 μm. Raman measurement was performed 15 times at different positions on the same membrane. Averaged Raman spectraof each membrane are shown in Fig. 2. The Raman peak of diyneSM appeared at 2,263 cm−1.
Experimental procedures used for Fig. 3. Supported monolayer membrane of diyne-SM/DOPC/chol ternary monolayer with the composition ratio 1:1:1 was prepared on a quartz substrate (0.17-mm
thickness; Starbar Japan). The membrane was fixed in a metalchamber (Attofluor; Life Technologies) and placed on the sample
stage of the microscope (Raman-11; Nanophoton). For Raman
spectroscopic measurement, the 532-nm laser was focused at apoint on the membrane with a water immersion objective lens
(CFI Plan Apo IR 60XWI; Nikon). Focus drift was suppressed
during imaging by using a real-time feedback system (PFS; Nikon).
The laser intensity at the sample plane was 360 mW. Exposure timewas 6 s/pixel. Slit width for the spectrophotometer was set as 50 μm.
Each Raman spectrum was smoothed using a moving averagemethod. Raman images were reconstructed using peak intensityof diyne at 2,264 cm−1 and peak bottom intensity at 2,222 cm−1. The
Raman image of diyne was obtained after subtraction of the signal
intensity between 2,264 and 2,222 cm−1, whereas the Raman imageof the peak bottom (2,222 cm−1) was obtained after subtraction of
the signal intensity between 2,222 and 2,180 cm−1.
Experimental procedures used for Fig. 4. Supported monolayer membranes of diyne-SM/DOPC/chol ternary monolayer with composition ratios of 1:1:1, 3:7:3, and 1:0:0 were prepared on a quartz
substrate (0.17-mm thickness; Starbar Japan). Each membrane on
the substrate was fixed in a metal chamber (Attofluor; Life
Technologies) and placed on the sample stage of the microscope(Raman-11; Nanophoton). For Raman spectroscopic measurement, a 532-nm laser was focused as a point on the membranewith a water immersion objective lens (UPLSAPO 60XW;
Olympus). The laser intensity at the sample plane was 370 mW.
Exposure time was 6 s/pixel. Slit width for the spectrophotometer
was set as 50 μm. Smoothing of Raman data was done using amoving average. Raman images were reconstructed using peakintensity of diyne at 2,263 cm−1 after subtraction of peak bottom intensity.
Experimental procedures used for Fig. 5. The experimental setup was
the same as used previously (1). Briefly, a frequency-doubled
Nd:YVO4 laser (Verdi; Coherent Inc.) was used as the excitation
laser source. A line-shaped laser beam formed by a cylindricallens was focused on the sample by a water immersion objective
lens (CFI Plan Apo IR 60XWI; Nikon). Scattering light from thesample was collected by the same objective lens, chromaticallyseparated by an edge filter (LP03-532RU-25; Semrock) to transmit Raman scattering light, and focused on the spectrophotometer
slit (MK-300; Bunko Keiki) for detection with a cooled CCD
camera (Pixis 400B; Princeton Instruments). Laser scanning was
Ando et al. www.pnas.org/cgi/content/short/1418088112

performed with a single-axis galvanometer mirror (710-745825,
000-3014016; GSI Lumonics).
For Raman imaging in Fig. 5A, a supported 1:1:1 ternarymonolayer membrane of diyne-SM/DOPC/chol was prepared ona quartz substrate (0.17-mm thickness; Starbar Japan). The membrane on the substrate was fixed in a metal chamber (Attofluor; Life
Technologies) and placed on the sample stage of the home-built
slit-scanning Raman microscope. For Raman spectroscopic measurement, a 532-nm laser was focused as a line on the membranewith a water immersion objective lens (CFI Plan Apo IR 60XWI;
Nikon). Focus drift was suppressed during imaging by using a realtime feedback system (PFS; Nikon). The laser intensity at the
sample plane was 10.5 mW/μm2. Exposure time was 100 s per line.
Slit width for the spectrophotometer was set as 50 μm. The scanning
number was 240 lines for Raman imaging, with an image size of
240 × 400 pixels. For noise reduction, the obtained Raman data
were subjected to singular value decomposition (SVD) (1). We
used a spectral region (1,799–2,301 cm−1) in the calculation
procedure of SVD. We chose four loading vectors that significantly contributed to the image contrast. After SVD processing,Raman images were reconstructed using the peak intensity of
diyne at 2,262 cm−1 based on the averaged peak top intensity
between 2,259 and 2,265 cm−1 after subtraction of the averaged
peak bottom intensity between 2,223 and 2,229 cm−1. To compensate for aspect ratio, the image in the scanning (horizontal)
direction was extended 1.72 times by linear interpolation. The
final image consists of 412 × 400 pixels.
For Raman and fluorescence imaging in Fig. 5B, a supported
1:1:1 ternary monolayer membrane of diyne-SM/DOPC/cholcontaining 0.2 mol% Bodipy-PC was prepared on a quartz substrate (0.17-mm thickness; Starbar Japan). The membrane on
the substrate was fixed in a metal chamber (Attofluor; Life
Technologies) and placed on the sample stage of the home-built
slit-scanning Raman microscope. For both Raman and fluorescence imaging, a 532-nm laser was focused as a line on themembrane with a water immersion objective lens (CFI Plan Apo
IR 60XWI; Nikon). First, fluorescence imaging was performed,
and second, Raman imaging was performed at the same area ofthe same sample. Fluorescence background was suppressed
during Raman imaging by photobleaching of Bodipy-PC under
532-nm laser exposure. Focus drift was suppressed during imaging by using a real-time feedback system (PFS; Nikon). The
laser intensity at the sample plane was 14.1 mW/μm2, and theexposure time was 60 s per line for Raman imaging. The laser
intensity for fluorescence imaging was 0.3 mW/μm2, and theexposure time was 0.5 s per line. Slit width of the spectrophotometer was set as 50 μm. The scanning number was 225 lines forboth Raman and fluorescence imaging, with the image size of
225 × 250 pixels. For noise reduction, the obtained Raman data were
subjected to SVD. We used a spectral region (2,000–2,314 cm−1)in the calculation procedure of SVD. We chose four loading vectorsthat significantly contributed to the image contrast. After SVD
processing, the Raman image was reconstructed using the peakintensity of diyne at 2,264 cm−1 based on the averaged peak top
intensity between 2,261 and 2,267 cm−1 after subtraction of theaveraged peak bottom intensity between 2,225 and 2,231 cm−1.
Fluorescence images were reconstructed using average intensity at
542–603 nm. To compensate for aspect ratio, the image in thescanning (horizontal) direction was extended 1.72 times by linear
interpolation. The final image consists of 387 × 250 pixels.
Line profiles in Fig. 5C were calculated from the average intensity of 4 pixels along the vertical direction, which is indicated
1 of 11

by a red dotted line in the Raman image in Fig. 5B, Upper and agray dotted line in the fluorescence image in Fig. 5B, Lower. The
profile from the Raman image was smoothed by use of themoving average.
Experimental procedures used for Fig. S5. In Fig. S5A, the experimental setup was the same as used in Fig. 5. For Raman imaging,a supported membrane of diyne-SM monolayer was preparedon a quartz substrate (0.17-mm thickness; Starbar Japan). The
membrane on the substrate was fixed in a metal chamber (Attofluor; Life Technologies) and placed on the sample stage ofthe home-built slit-scanning Raman microscope. For Raman
spectroscopic measurement, a 532-nm laser was focused as a lineon the membrane with a water immersion objective lens (CFI
Plan Apo IR 60XWI; Nikon). Focus drift was suppressed during
imaging by using a real-time feedback system (PFS; Nikon). The
laser intensity at the sample plane was 13.9 mW/μm2. Exposure
time was 60 s per line. Slit width for the spectrophotometer was
set as 50 μm. The scanning number was 200 lines for Raman
imaging, with an image size of 200 × 190 pixels. For noise reduction, the obtained Raman data were subjected to SVD (1).
We used a spectral region (1,800–2,310 cm−1) in the calculation
procedure of SVD. We chose four loading vectors that significantly contributed to the image contrast. After SVD processing,Raman images were reconstructed using the peak intensity of
diyne at 2,263 cm−1 based on the averaged peak top intensity
between 2,260 and 2,266 cm−1 after subtraction of the averaged
peak bottom intensity between 2,212 and 2,218 cm−1. To compensate for aspect ratio, the image in the scanning (horizontal)
direction was extended 1.72 times by linear interpolation. The
final image consists of 344 × 190 pixels. In Fig. S5B, the image
was extracted from lower left portion in Fig. 5A, which consist of
344 × 190 pixels. In Fig. S5C, line profiles were calculated fromthe average intensity of 7 pixels along the vertical direction,
which is indicated by a dotted lines.
Experimental procedures used for Fig. S6. A supported monolayer
membrane of 1:1:1 diyne-SM/DOPC/chol ternary monolayer
containing 0.2 mol% Bodipy-PC was prepared on a quartz substrate (0.17-mm thickness; Starbar Japan). The membrane on thesubstrate was fixed in a metal chamber (Attofluor; Life Technologies) and placed on the sample stage of the microscope(Raman-11; Nanophoton). For both fluorescence and Raman
spectroscopic measurement, a 532-nm laser was focused as apoint on the membrane with a water immersion objective lens
(CFI Plan Apo IR 60XWI; Nikon). Focus drift was suppressed
during imaging by using a real-time feedback system (PFS;
Nikon). For fluorescence imaging, the laser intensity at the
sample plane was 0.5 mW, and the exposure time was 1 s/pixel.
For Raman imaging, the laser intensity at the sample was 360 mW,
and the exposure time was 6 s/pixel. First, fluorescence imaging was
performed, and second, Raman imaging was performed at thesame area of the same sample. Slit width for the spectrophotometer
was set as 50 μm. For reconstruction of fluorescence images, average intensity between 555 and 605 nm was used. Smoothing ofRaman data was done using a moving average. Raman images
were reconstructed using peak intensity of diyne at 2,264 cm−1
after subtraction of peak bottom intensity.
DSC Thermograms of Diyne-SM Bilayers. The thermal-phase behavior of SM bilayers was examined with a nanodifferential
scanning calorimeter (Calorimetry Science Corp.). Bilayer samples were prepared by a conventional method. Briefly, SM dissolved in chloroform/methanol (4:1) was dried under a flowof nitrogen and then, reduced pressure for at least 24 h. The
resulting lipid film was dispersed into distilled and deionizedwater (Simplicity UV; Merck Millipore) and incubated for∼30 min at 60 °C with intermittent vortexing. The final concentration was 2.47 mM. Then, 330 μL sample was placed in
the DSC immediately before measurement. A scanning rate ofAndo et al. www.pnas.org/cgi/content/short/1418088112

0.5 °C/min was used. The main transition temperatures of diyneSM and SM bilayers were found to be 39.5 °C and 44.5 °C, respectively, as shown by the arrows in Fig. S7.
π-A Isotherm Measurements and Supported Monolayer Preparation.

Monolayers of lipid mixtures were prepared on a computercontrolled Langmuir film balance (USI System) calibrated using
stearic acid (Sigma Aldrich). The subphase, which consisted of
distilled, freshly deionized water, was obtained using a Milli-Q
System. The sample solution was prepared by mixing the appropriate amount of each lipid solution in a microvial. A total of
30 μL lipid dissolved in chloroform/methanol (4:1 vol/vol; 1 mg/mL)
was spread onto the aqueous subphase (100 × 290 mm2) using a
glass micropipette (Drummond Scientific Company). After an
initial delay period of 10 min for evaporation of the organic
solvent, the monolayers were compressed at a rate of 20 mm2/s.
The subphase and ambient temperatures were controlled at
25.0 °C ± 0.1 °C and 25 °C ± 2 °C, respectively. The π-A isotherm
measurements were repeated three to five times under the same
conditions. These measurements provided the molecular area ata corresponding pressure within an error of ∼ ±1 Å2. The influence of oxidation on the unsaturated chains at the air–waterinterface was checked by intentionally exposing pure SM andpure DOPC monolayers to air for 10–30 min before compression. The change in the isotherm after prolonged exposure of SM
or DOPC monolayer to air was within the error described above.
Analysis. In Fig. 6, we evaluated the intermolecular interaction inlipid binary mixtures at the surface pressure of 5 mN/m on thebasis of the deviations of experimentally obtained mean molecular areas (Amean) from those of ideal mixtures (A12):A12 = A1 ð1 − xÞ + A2 x,
where A1 and A2 are the molecular areas of pure components 1
and 2 (e.g., SM and chol), respectively, and x is the molar fraction of component 2. Thus, the value of A12 corresponds to themean molecular area in the mixture constituted of noninteractive or completely immiscible molecules. According to previous
literature (2), PMAs of components 1 (A1PMA) and 2 (A2PMA) canbe defined as!
∂NA1mean
1
APMA =∂N1and
A2PMA =


∂NA2mean,∂N2

where N, N1, and N2 are the total amounts of all constituents andcomponents 1 and 2, respectively. On the basis of the additivity
rule, the Amean also can be expressed as
Amean ðxÞ = ð1 − xÞA1PMA ðxÞ + xA2PMA ðxÞ.
Here, denoting derivatives with respect to x by prime yields, thefollowing equations are obtained:
A1PMA ðxÞ = Amean ðxÞ − xA′mean ðxÞ
and
A2PMA ðxÞ = Amean ðxÞ + ð1 − xÞA′mean ðxÞ.
2 of 11

Areal compressibility (Cs) at the surface pressure of 5 mN/m
was calculated from the π-A isotherm using
1∂AmeanCs = −.
∂πAmean
πThe compressibility in ideal mixtures (C12) is calculated according to Ali et al. (3):


1
C12 =
fðCs1 A1 Þð1 − xÞ + ðCs2 A2 Þxg ,A12
where Cs1 and Cs2 are the areal compressibilities of the purecomponents 1 and 2, respectively. They suggested that C12 isadditive with respect to the product of Csi and Ai rather thanCsi for either ideal or completely nonideal mixing. Areal compressibility (Cs) was expressed in term of areal compressional
modulus (Cs−1) for easy comparison with previous data.
Fluorescence Observation of Ordered Domains in Diyne-SM/DOPC/
Chol and SM/DOPC/Chol Ternary Monolayers. Fig. S8 shows fluo-

rescence images of diyne-SM/DOPC/chol (1:1:1 mol/mol/mol)(Fig. S8A) and SM/DOPC/chol (1:1:1 mol/mol/mol) (Fig. S8B)
quartz-supported monolayers in the presence of 0.2 mol% BodipyPC at 12 mN/m and 25 °C. Fluorescence observations were conducted using a confocal laser-scanning microscope (FV1000-D
IX81; Olympus) with an air objective lens with a long working
distance (LUCPLFLN 60X; Olympus). A wavelength of 488 nm
was used for excitation of Bodipy. A laser-scanning rate of 4.0 or
8.0 μs/pixel was used for acquisition of confocal images (1,024 ×
1,024 pixels).
2
H NMR Measurements. A mixture of lipids comprising 10.0 μmol
d2-SM or d2-diyne-SM (Fig. 7), 10.0 μmol chol, and 10.0 μmol
DOPC was dissolved in chloroform/methanol (1:1 vol/vol). Afterremoving the solvent in vacuo for 20 h, the dried membrane film
was hydrated with 1 mL distilled water and vigorously vortexed
at 65 °C to make multilamellar vesicles. The sample was frozen andthawed three times, lyophilized, and rehydrated with deuteriumdepleted water to make 50% (wt/wt) water. Then, the mixture
was again frozen and thawed 10 times. The sample was transferred into a glass tube (5 × 26 mm), which was sealed with epoxy
glue. 2H NMR measurements were recorded on a 300-MHz
CMX300 Spectrometer (Chemagnetics; Varian) with a 5-mm 2H
static probe (Otsuka Electronics) using a quadrupolar echo sequence (4). The 90° pulse width was 2 μs, interpulse delay was
30 μs, and repetition rate was 0.5 s. The sweep width was 200 kHz,and the number of scans was around 100,000.

Synthetic Procedures for Diyne-SM and d2-Diyne-SM.
(2S,3R,E)-(2-stearoylamino-3-hydroxyoctadec-4-en-1-yl)-{2-[(6-hydroxyhexa2,4-diyn-1-yl)dimethylammonio]ethyl}phosphate (diyne-SM). To a solutionof propargyl alcohol S1 (300 mg, 5.35 mmol) in acetone (15 mL),
N-bromosuccinimide (1.02 g, 5.78 mmol) and silver nitrate (91 mg,
0.54 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 2 h and then, concentrated. The residue was extracted two times with diethyl ether. The
combined organic layers were dried with Na2SO4, filtered, andconcentrated to afford S2 (645 mg, 90% yield) as a pale yellow oil,which was used directly in the next step. 1H NMR (500 MHz,CDCl3): δ 1.63 (brs, 1H), 4.30 (s, 2H). CuCl (0.3 mg, 3.1 μmol),i-PrNH2 (5.1 μL, 0.06 mmol), and NH2OH·HCl (1.0 mg, 14.5 μmol)
were added to MeOH (1 mL) at room temperature under argon.
The mixture was cooled to 0 °C, and then, S3 (5) (compound S3
was the intermediate for the synthesis of SM head group analogs;
40 mg, 0.05 mmol) was added, forming a yellow acetylide suspension. A solution of S2 (21 mg, 0.16 mmol) in MeOH (0.2 mL) was
added immediately. The reaction mixture was stirred at the same
temperature for 30 min, concentrated, and extracted with CHCl3.
The combined organic layers were dried with Na2SO4, filtered, andconcentrated. The mixture was passed through a short bed of silica
gel eluting with CHCl3/MeOH (5:1). The crude product was purified on a Cosmosil 5C18-AR-II column (10 × 150 mm; Nacalai
Tesque) with MeOH as the eluent to give diyne-SM (5.1 mg, 12%)as a white solid. TLC: Rf = 0.18 (CH2Cl2/MeOH/NH4OH 70:30:3);
1
H NMR (500 MHz, CD3OD): δ 0.88 (t, J = 7.5 Hz, 6H); 1.24–1.42(m, 50H); 1.53–1.63 (m, 2H); 2.01 (dt, J = 7.0, 7.0 Hz, 2H); 2.13–
2.20 (m, 2H); 3.24 (s, 6H); 3.70 (t, J = 5.5 Hz, 2H); 3.87–3.98(m, 2H); 4.03 (dd, J = 8.0, 8.0 Hz, 1H); 4.06–4.13 (m, 1H); 4.27
(s, 2H); 4.25–4.30 (m, 2H); 4.56 (s, 2H); 5.43 (ddt, J = 15.5, 8.0,
1.5 Hz, 1H); 5.69 (dtd, J = 15.5, 7.0, 1.0 Hz, 1H); 7.90 (d, J = 9.0 Hz,
1H); 13C NMR (125 MHz, CD3OD): δ 13.11, 22.41, 25.85, 29.15,
29.18, 29.37, 29.44, 29.74, 31.75, 32.15, 36.05, 49.55, 50.86, 53.93,
55.79, 58.90, 64.22, 64.61, 65.64, 66.54, 71.22, 75.42, 81.27, 129.89,
133.82, 174.58; HRMS (electrospray ionization) calculated for
C46H86N2O7P [M + H]+ 809.6167, found 809.6184. 1H and 13C
NMR spectra of diyne-SM are shown in Figs. S9 and S10. The
synthetic scheme is summarized in Scheme S1.
(2S,3R,E)-[2-(10,10-dideuteriumstearoylamino)-3-hydroxyoctadec-4-en-1-yl]-{2[(6-hydroxyhexa-2,4-diyn-1-yl)dimethylammonio]ethyl}phosphate (d2-diyne-SM).

General Information for the Synthesis of Diyne-SM. Chemicals andsolvents were purchased from Nacalai Tesque, Aldrich, TCI, or
Kanto Chemicals Inc. and used without additional purification
unless otherwise noted. TLC was done on Merck Precoated Silica
Gel 60 F-254 Plates. Spots on TLC plates were stained withphosphomolybdic acid. NMR spectra were collected on a JEOLECA 500 (500 MHz) using deuterated solvent as the lock.
Chemical shift is given in parts per million (δ), and coupling
constant (J) is in hertz. The following terms are used to designate
multiplicity: singlet (s), doublet (d), triplet (t), quartet (q), quintuplet (quint), multiplet (m), and broad (b). High-resolution mass
spectra (HRMS) were recorded on an LTQ-Orbitrap XL.

This compound was prepared from S4 (a full account of thesynthesis of compound S4 will be reported elsewhere) in 14%
yield as a white solid by following the same procedure as described for S3. TLC: Rf = 0.18 (CH2Cl2/MeOH/NH4OH 70:30:3);
1
H NMR (500 MHz, CD3OD): δ 0.88 (t, J = 7.5 Hz, 6H); 1.22–
1.43 (m, 48H); 1.50–1.63 (m, 2H); 2.01 (dt, J = 7.0, 7.0 Hz, 2H);
2.13–2.20 (m, 2H); 3.25 (s, 6H); 3.71 (t, J = 5.0 Hz, 2H); 3.88–3.99(m, 2H); 4.02 (dd, J = 8.0, 8.0 Hz, 1H); 4.06–4.12 (m, 1H); 4.27
(s, 2H); 4.21–4.34 (m, 2H); 4.56 (s, 2H); 5.43 (ddt, J = 15.0, 8.0,
1.2 Hz, 1H); 5.69 (dtd, J = 15.0, 7.0, 0.6 Hz, 1H); 7.90 (d, J = 9.0
Hz, 1H); 13C NMR (125 MHz, CD3OD): δ 13.16, 22.43, 25.87,
29.18, 29.21, 29.33, 29.47, 29.52, 29.55, 29.58, 31.77, 31.79, 32.18,
36.06, 49.56, 50.87, 53.87, 53.93, 55.80, 58.90, 59.94, 64.22, 64.28,
64.61, 64.65, 65.67, 66.56, 71.21, 75.43, 81.29, 129.93, 133.79,
174.54; HRMS (electrospray ionization) calculated for
C46H83D2N2O7PNa [M + Na]+ 833.6112, found 833.6124. 1H
and 13C NMR spectra of d2-diyne-SM are shown in Figs. S11
and S12. Synthetic scheme is summarized in Scheme S2.

1. Palonpon AF, et al. (2013) Raman and SERS microscopy for molecular imaging of live
cells. Nat Protoc 8(4):677–692.
2. Edholm O, Nagle JF (2005) Areas of molecules in membranes consisting of mixtures.
Biophys J 89(3):1827–1832.
3. Ali S, Smaby JM, Brockman HL, Brown RE (1994) Cholesterol’s interfacial interactions
with galactosylceramides. Biochemistry 33(10):2900–2906.

4. Davis JH, Jeffrey KR, Bloom M, Valic MI, Higgs TP (1976) Quadrupolar echo deuteronmagnetic resonance spectroscopy in ordered hydrocarbon chains. Chem Phys Lett
42(2):390–394.
5. Goretta SA, Kinoshita M, Mori S, Tsuchikawa H, Matsumori N, Murata M (2012) Effects
of chemical modification of sphingomyelin ammonium group on formation of liquidordered phase. Bioorg Med Chem 20(13):4012–4019.

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Fig. S1. Raman spectrum of the diyne-SM monolayer supported on a quartz substrate shown in Fig. 2A. Raman peaks of N2, O2, and diyne are indicated by red
arrows. Lower is a five times enlarged view of Upper in terms of intensity (vertical axis). Strong Raman scattering by N2 and O2 in air was always detectedduring Raman measurement of lipid membranes. For ease of picking out the important Raman peaks, such as diyne, the two peaks of N2 and O2 have been
removed in Figs. 2 and 4 and Figs. S3 and S4.

Fig. S2. (A) Raman image of the diyne-SM/DOPC/chol ternary monolayer with a 1:1:1 ratio reconstructed using the intensity of the diyne peak at 2,263 cm−1.
The image is the same as Fig. 4A. Mean value of the image is 5.64. The two phases were separated by taking pixel regions (B) below the mean value as
disordered domains and (C) above the mean value as ordered domains. Raman images of diyne-SM/DOPC/chol ternary monolayers with (D) a 3:7:3 ratio and(E) a 1:0:0 ratio. The images are the same as Fig. 4 B and C, respectively. (Scale bar: 10 μm.)

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Fig. S3. Raman spectra obtained along the red lines in the Raman images of the diyne-SM/DOPC/chol ternary monolayer with 1:1:1, 3:7:3, and 1:0:0 ratios. The
images are the same as in Fig. 4 A–C, with 36 × 24 pixels. Raman peak of N2 at ∼2,330 cm−1 was removed so that it would be easier to see Raman peaks fromlipid molecules. Pix, pixel. (Scale bar: 10 μm.)

Fig. S4. Comparison between Raman spectra of supported lipid monolayers with single-component composition (diyne-SM, DOPC, or chol) and Raman spectraof diyne-SM/DOPC/chol ternary monolayer at ordered and disordered domains. The spectra of the ternary monolayer were obtained from five representative
areas (3 × 3 pixels) calculated using the Raman image in Fig. 4A. Characteristic Raman peaks are displayed in the image, including diyne stretching vibration(2,263 cm−1), CH2 symmetric/asymmetric stretching vibration (2,850/2,883 cm−1), and CH3 symmetric stretching vibration (2,936 cm−1). Raman peak of N2 at∼2,330 cm−1 was removed so that it would be easier to see Raman peaks from lipid molecules.

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Fig. S5. (A) Raman image of a pure diyne-SM monolayer taken with slit-scanning Raman microscopy. The image was reconstructed using the diyne peakintensity at 2,263 cm−1. Exposure time and laser power were 60 s per line and 13.9 mW/μm2, respectively. The image consists of 344 × 190 pixels. (Scale bar:
10 μm.) (B) Raman image of a 1:1:1 diyne-SM/DOPC/chol ternary monolayer extracted from the lower left portion in Fig. 5A. The image consists of 344 × 190
pixels. (Scale bar: 10 μm.) (C) Line profiles of lipid membranes calculated along the dotted lines of the images in A and B.

Fig. S6. (A) Fluorescence and (B) Raman images of a 1:1:1 diyne-SM/DOPC/chol ternary monolayer containing 0.2 mol% Bodipy-PC. The fluorescence image
was reconstructed using the average fluorescence intensity at 555–605 nm (blue). The Raman image was reconstructed using the diyne peak intensity at
2,264 cm−1 (red). Both images were obtained in the same imaging area of the same sample. Fluorescence background during Raman imaging was suppressedby photobleaching of Bodipy-PC under 532-nm laser exposure. (C) Superimposed image of fluorescence and Raman images obtained in Fig. S5 A and B. The
images consist of 54 × 28 pixels. (Scale bar: 20 μm.)

Fig. S7.

DSC heating thermograms of (Upper) diyne-SM and (Lower) SM bilayers immediately after preparation. The main transitions are indicated by arrows.

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Fig. S8. Fluorescence images of (A) diyne-SM/DOPC/chol and (B) SM/DOPC/chol quartz-supported monolayers in the presence of 0.2 mol% Bodipy-PC at
12 mN/m and 25 °C. (Scale bar: 50 μm.)

Fig. S9.

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H NMR spectrum of diyne-SM.

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Fig. S10.

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C NMR spectrum of diyne-SM.

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Fig. S11.

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H NMR spectrum of d2-diyne-SM.

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Fig. S12.

13

C NMR spectrum of d2-diyne-SM.

Scheme S1. Synthesis of diyne-SM.

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Scheme S2.

Synthesis of d2-diyne-SM.

Table S1. Mean value of Raman intensity of the diyne peak indiyne-SM/DOPC/chol ternary monolayer calculated from theimages shown in Fig. S2
Diyne-SM/DOPC/chol

Pixel no.

Mean (± SD)

Relative, %1:1:1 (ordered)
1:1:1 (disordered)
3:7:3
1:0:0

406
458
864
864

8.01 (± 1.60)
3.54 (± 1.23)
5.29 (± 1.60)
15.35 (± 1.48)

52.2
23.1
34.5
100

Relative value was calculated using diyne peak intensity of the membranewith a 1:0:0 ratio as 100%.

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