Lp

RAa

b

6

a

ARRAA

KFRNNML

1

ciipapap

s11p[vpT

h0

Chemistry and Physics of Lipids 183 (2014) 1–8

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids

jou rn al hom epage : www.elsev ier .com/ locate /chemphys l ip

ocation, dynamics and solvent relaxation of a nile red-basedhase-sensitive fluorescent membrane probe

oopali Saxenaa,1, Sandeep Shrivastavaa,1, Sourav Haldara, Andrey S. Klymchenkob,mitabha Chattopadhyaya,∗

CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, IndiaLaboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74, Route du Rhin,7401 Illkirch Cedex, France

r t i c l e i n f o

rticle history:eceived 18 March 2014eceived in revised form 22 April 2014ccepted 27 April 2014vailable online 4 May 2014

eywords:

a b s t r a c t

Fluorescent membrane probes offer the advantage of high sensitivity, suitable time resolution, and mul-tiplicity of measurable parameters, and provide useful information on model and cell membranes. Inthis paper, we have explored the location, dynamics, and solvent relaxation characteristics of a novelNile Red-based phase-sensitive probe (NR12S). Unlike Nile Red, NR12S enjoys unique orientation andlocation in the membrane, and is localized exclusively in the outer leaflet of the membrane bilayer. Byanalysis of membrane depth using the parallax approach, we show that the fluorescent group in NR12S is

luorescent membrane probeEESile RedR12Sembrane penetration depth

iquid-ordered phase

localized at the membrane interface, a region characterized by slow solvent relaxation. Our results showthat NR12S exhibits REES (red edge excitation shift), consistent with its interfacial localization. Moreinterestingly, REES of NR12S displays sensitivity to the membrane phase. In addition, fluorescence emis-sion maximum, anisotropy, and lifetime of NR12S are dependent on the membrane phase. We envisionthat NR12S may prove to be a useful probe in future studies of complex natural membranes.

. Introduction

Biological membranes are complex two-dimensional, non-ovalent assemblies of a diverse variety of lipids and proteins. Theympart an identity to the cell and its organelles, and represent andeal milieu for the proper function of a diverse set of membraneroteins. The eukaryotic cell is composed of diverse lipids (van Meernd de Kroon, 2011) and tracking lipids in a crowded cellular milieu

oses considerable challenge. In this scenario, use of lipid probesssumes significance (Chattopadhyay, 2002). Various types of lipidrobes have proved to be useful in membrane biology due to their

Abbreviations: 2-AS, 2-(9-anthroyloxy)stearic acid; 12-AS, 12-(9-anthroyloxy)-tearic acid; 5-PC, 1-palmitoyl-2-(5-doxyl)stearoyl-sn-glycero-3-phosphocholine;2-PC, 1-palmitoyl-2-(12-doxyl)stearoyl-sn-glycero-3-phosphocholine; DMPC,,2-dimyristoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-hosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ET(30),2,6-diphenyl-4-(2,4,6-triphenyl-N-pyridino)phenoxide]; LUV, large unilamellaresicle; Nile Red, 9-diethylamino-5H-benzo[�]phenoxazine-5-one; POPC, 1-almitoyl-2-oleoyl-sn-glycero-3-phosphocholine; REES, red edge excitation shift;empo-PC, 1,2-dioleoyl-sn-glycero-3-phosphotempocholine.∗ Corresponding author. Tel.: +91 40 2719 2578; fax: +91 40 2716 0311.

E-mail address: amit@ccmb.res.in (A. Chattopadhyay).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.chemphyslip.2014.04.007009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.

© 2014 Elsevier Ireland Ltd. All rights reserved.

ability to monitor lipid molecules by a variety of physicochemicalapproaches at increasing spatiotemporal resolution (Eggeling et al.,2009). In particular, application of spectroscopic and microscopictechniques using fluorescent lipid analogs represents a convenientapproach for monitoring membrane lipid organization and dynam-ics. Fluorescence-based approaches are preferred due to their highsensitivity, suitable time resolution, and multiplicity of measur-able parameters. Lipids covalently linked to extrinsic fluorophoreswith suitable fluorescence properties are generally used for suchstudies. The advantage of using fluorescently labeled lipids is thechoice available for the fluorescent label. Probes with appropriatecharacteristics can therefore be designed for specific applications.

A major criterion of a fluorescent membrane probe is its sensitiv-ity to environmental factors. Nile Red, an uncharged phenoxazonedye (see Fig. 1), is such a probe whose fluorescence propertiesare altered by the polarity of its immediate environment due toa large change in its dipole moment upon excitation (Greenspanand Fowler, 1985; Golini et al., 1998). This large change in dipolemoment has been attributed to charge separation between thediethylamino group which acts as the electron donor and the

quinoid part of the molecule which serves as the electron accep-tor. Nile Red has been used as a fluorescent probe for monitoringhydrophobic surfaces in proteins (Sackett et al., 1990), and as alipid stain in membranes (Gao et al., 2006). It has also been used for

2 R. Saxena et al. / Chemistry and Phy

O

O

S

O

OO–

N+O

N

N

Fig. 1. Chemical structure of NR12S. The part of NR12S responsible for its membranea(r

mia

calbttpbtfpi(abtsiegdpsicm(s1bwnac

2

2

C

nchoring property is shown in red and the fluorophore (Nile Red) is shown in blue.For interpretation of the references to color in this figure legend, the reader iseferred to the web version of this article.)

onitoring organization and heterogeneity induced by cholesteroln model (Krishnamoorthy and Ira, 2001; Mukherjee et al., 2007b)nd natural (Mukherjee et al., 2007a) membranes.

The chemical structure and design of a membrane probe arerucial in terms of its usefulness as a reporter molecule. It is desir-ble that the probe should be able to intercalate with membraneipids with the fluorophore part suitably embedded in the mem-rane. Hydrophobic molecules (such as Nile Red) partition intohe membrane, but do not get oriented in a specific conforma-ion due to lack of anchoring. As a result, these probes essentiallyrovide a weighted average information, depending on the num-er of locations they occupy, and their fluorescence properties inhese locations. The information obtained from such probes there-ore lacks specificity. This can be avoided by covalently linking therobe with a fatty acyl chain (which helps in alignment of the probe

n the membrane) and an anchoring group which is often chargedShynkar et al., 2007). This strategy was recently utilized to gener-te NR12S (see Fig. 1 for chemical structure), a fluorescent probeased on Nile Red, which has unique orientation and location inhe membrane (Kucherak et al., 2010). Besides being environment-ensitive, NR12S has the advantage of being localized exclusivelyn the outer leaflet of the membrane (Chiantia et al., 2012; Darwicht al., 2012). Nonetheless, the exact location of the fluorescentroup in NR12S in membranes is not known. In this paper, weetermined the exact depth of the NR12S fluorophore using thearallax approach (Chattopadhyay and London, 1987). Our resultshow that the fluorophore in NR12S is localized at the membranenterface, a region characterized by unique motional and dielectricharacteristics different from both the bulk aqueous phase, and theore isotropic hydrocarbon-like deeper regions of the membrane

Haldar et al., 2011). This specific region of the membrane exhibitslow rates of solvent relaxation (Chattopadhyay and Mukherjee,993; Jurkiewicz et al., 2006; Das et al., 2008), a feature that haseen associated with red edge excitation shift (REES). Consistentith this, we report here that NR12S exhibits REES and the mag-itude of REES displays sensitivity to membrane phase. Our resultsre useful for future studies of NR12S in natural membranes withomplex behavior.

. Materials and methods

.1. Materials

DMPC, cholesterol and ET(30) dye were obtained from Sigmahemical Co. (St. Louis, MO). DOPC, DPPC, POPC, Tempo-PC, 5-PC

sics of Lipids 183 (2014) 1–8

and 12-PC were obtained from Avanti Polar Lipids (Alabaster, AL).2-AS and 12-AS were obtained from Molecular Probes (Eugene, OR).Lipids were checked for purity by thin layer chromatography onsilica gel precoated plates (Sigma) in chloroform/methanol/water(65:35:5, v/v/v) and were found to give only one spot in allcases with a phosphate-sensitive spray and on subsequent char-ring (Dittmer and Lester, 1964). Concentrations of phospholipidswere determined by phosphate assay subsequent to total oxidationby perchloric acid (McClare, 1971). DMPC was used as an inter-nal standard to assess lipid digestion. NR12S was synthesized asdescribed previously (Kucherak et al., 2010). The concentration ofa stock solution of NR12S prepared in DMSO was estimated usingits molar extinction coefficient (ε) of 45,000 M−1 cm−1 at 550 nmin ethanol. All other chemicals used were of the highest purityavailable. Solvents used were of spectroscopic grade. Purity of sol-vents was further confirmed by the ET(30) procedure (Mukherjeeet al., 1994). Water was purified through a Millipore (Bedford, MA)Milli-Q system and used throughout.

2.2. ET(30) procedure

The ET(30) dye is a solvatochromic dye which undergoes one ofthe largest known solvent-induced shifts in absorption maximum.The extremely large solvent-induced shift has been used to intro-duce an empirical parameter of solvent polarity, called the ET(30)value. The ET(30) value for a solvent is defined as the transitionenergy of the dissolved ET(30) dye measured in kcal/mol accordingto the following equation:

ET = hc �NA = 2.859 × 10−3� (1)

where h is Planck’s constant, c is the velocity of light, � is thewavenumber of the photon in cm−1 which produces the electronictransition, and NA is Avogadro’s number. Due to extremely largesolvatochromism of this dye, the ET(30) values serve as sensitiveindicators of solvent polarity and are sensitive to any impuritypresent in trace amounts. ET(30) values have been previously deter-mined for a large number of solvents. A few grains of the ET(30) dyewere dissolved in a given solvent, and its absorption maximum wasmonitored. From this absorption maximum, the ET(30) value wascalculated using Eq. (1). The ET(30) values so obtained were com-pared with the literature values (Reichardt, 1988). The ET(30) valuesobtained showed a maximum deviation of <0.5% from the reportedvalues.

2.3. Sample preparation

All experiments were performed using large unilamellar vesi-cles (LUVs) of 100 nm diameter of POPC, DPPC, or POPC/40 mol%cholesterol. While POPC and POPC with 40 mol% cholesterol sam-ples contained 1 mol% NR12S, DPPC samples contained 0.5 mol%NR12S. In general, 640 nmol of total lipid and 6.4 (or 3.2 in case ofDPPC) nmol of NR12S were mixed well and dried under a stream ofnitrogen while being warmed gently (∼40 ◦C). After further dry-ing under a high vacuum for at least 3 h, the lipid mixture washydrated (swelled) by addition of 1.5 ml of buffer A (10 mM sodiumphosphate, 150 mM sodium chloride, pH 7.4), and each sample wasvortexed for 3 min to uniformly disperse the lipids and form homo-geneous multilamellar vesicles. The buffer was always maintainedat a temperature well above the phase transition temperature ofthe phospholipid used as the vesicles were made. Lipids weretherefore swelled at a temperature of 40 ◦C for POPC and 60 ◦Cfor DPPC samples. LUVs of 100 nm diameter were prepared by the

extrusion technique using an Avestin Liposofast Extruder (Ottawa,Ontario, Canada) as previously described (MacDonald et al., 1991;Mukherjee and Chattopadhyay, 2005). Briefly, the multilamellarvesicles were freeze–thawed five times using liquid nitrogen to

d Phy

eao(1setwatAp1u

2

pqds(1(nidewb1p(gflflfl

2

alossvoI

rie

r

wavest

e

R. Saxena et al. / Chemistry an

nsure solute equilibration between trapped and bulk solutionsnd then extruded through polycarbonate filters (pore diameterf 100 nm) mounted in an extruder fitted with Hamilton syringesHamilton Company, Reno, NV). The samples were subjected to1 passes through the polycarbonate filters to give the final LUVuspension. Background samples were prepared in the same wayxcept that NR12S was not added to them. The optical density ofhe samples measured at 530 nm was less than 0.15 in all cases,hich rules out any possibility of inner filter effect or scattering

rtifacts. Samples were incubated in dark for 12 h at room tempera-ure (∼23 ◦C) for equilibration prior to fluorescence measurements.ll experiments were performed with at least three sets of sam-les at room temperature (∼23 ◦C). For experiments using solvents,

nmol of NR12S in DMSO was mixed with 2 ml of a given solventsed in the study.

.4. Depth measurements using the parallax method

The actual spin (nitroxide) contents of the spin-labeled phos-holipids (Tempo-, 5- and 12-PC) were assayed using fluorescenceuenching of anthroyloxy-labeled fatty acids (2- and 12-AS) asescribed previously (Abrams and London, 1993). For depth mea-urements, liposomes were made by the ethanol injection methodKremer et al., 1977). These samples were made by codrying60 nmol of DOPC containing 10 mol% spin-labeled phospholipidTempo-, 5- or 12-PC) and 1 mol% NR12S under a steady stream ofitrogen with gentle warming (∼35 ◦C), followed by further dry-

ng under a high vacuum for at least 3 h. The dried lipid film wasissolved in ethanol to give a final concentration of 40 mM. Thethanolic lipid solution was then injected into 1.5 ml of buffer A,hile vortexing to give a final concentration of 0.11 mM DOPC in

uffer. The lipid composition of these samples was 90% DOPC and0% spin-labeled PC (Tempo-, 5- or 12-PC). Duplicate samples wererepared in each case except for samples lacking the quencherTempo-, 5- or 12-PC), for which triplicates were prepared. Back-round samples lacking NR12S were prepared in all cases, and theiruorescence intensity was subtracted from the respective sampleuorescence intensity. Samples were kept in dark for 12 h beforeuorescence measurements.

.5. Steady state fluorescence measurements

Steady state fluorescence measurements were performed with Hitachi F-4010 spectrofluorometer (Tokyo, Japan) using 1 cm pathength quartz cuvettes. Excitation and emission slits with bandpassf 5 nm were used for all measurements. Background intensities ofamples in which NR12S was omitted were subtracted from eachample spectrum to cancel out any contribution due to the sol-ent Raman peak and other scattering artifacts. The spectral shiftsbtained with different sets of samples were identical in most cases.n other cases, the values were within ±1 nm of those reported.

Fluorescence anisotropy measurements were performed atoom temperature (∼23 ◦C) using a Hitachi Glan-Thompson polar-zation accessory. Anisotropy values were calculated from thequation (Lakowicz, 2006):

= IVV − GIVH

IVV + 2GIVH(2)

here IVV and IVH are the fluorescence intensities (after appropri-te background subtraction) with the excitation polarizer orientedertically and the emission polarizer vertically and horizontally ori-nted, respectively. G is the ratio of the efficiencies of the detection

ystem for vertically and horizontally polarized light and is equalo IHV/IHH.

For depth measurements, samples were excited at 530 nm, andmission was collected at 600 nm. Excitation and emission slits

sics of Lipids 183 (2014) 1–8 3

with bandpass of 5 nm were used. Fluorescence was measured atroom temperature (∼23 ◦C) and averaged over two 5 s readings.Intensities were found to be stable over time. In all cases, theintensity from background samples without NR12S was subtracted.Membrane penetration depths were calculated using Eq. (5) (seeSection 3).

2.6. Time-resolved fluorescence measurements

Fluorescence lifetimes were calculated from time-resolved flu-orescence intensity decays using IBH 5000F NanoLED equipment(Horiba Jobin Yvon, Edison, NJ) with DataStation software in thetime-correlated single photon counting (TCSPC) mode. A pulsedlight-emitting diode (LED) (NanoLED-01) was used as an excita-tion source. This LED generates optical pulses at 490 nm with pulseduration of 1.2 ns and is run at 1 MHz repetition rate. The LED pro-file (instrument response function) was measured at the excitationwavelength using Ludox (colloidal silica) as the scatterer. To opti-mize the signal-to-noise ratio, 10,000 photon counts were collectedin the peak channel. All experiments were performed using emis-sion slits with bandpass of 4 or 8 nm. The sample and the scattererwere alternated after every 5% acquisition to ensure compensationfor shape and timing drifts occurring during the period of data col-lection. This arrangement also prevents any prolonged exposure ofthe sample to the excitation beam, thereby avoiding any possiblephotodamage of the fluorophore. Data were stored and analyzedusing DAS 6.2 software (Horiba Jobin Yvon, Edison, NJ). Fluores-cence intensity decay curves so obtained were deconvoluted withthe instrument response function and analyzed as a sum of expo-nential terms

F(t) =∑

i˛i exp

(−t

�i

)(3)

where F(t) is the fluorescence intensity at time t and ˛i is a pre-exponential factor representing the fractional contribution to thetime-resolved decay of the component with a lifetime �i such that�i˛i = 1. The decay parameters were recovered using a nonlinearleast squares iterative fitting procedure based on the Marquardtalgorithm (Bevington, 1969). The program also includes statisticaland plotting subroutine packages (O’Connor and Phillips, 1984).The goodness of fit of a given set of observed data and the cho-sen function was evaluated by the �2 ratio, the weighted residuals(Lampert et al., 1983), and the autocorrelation function of theweighted residuals (Grinvald and Steinberg, 1974). A fit was con-sidered acceptable when plots of the weighted residuals and theautocorrelation function showed random deviation about zero witha minimum �2 value not more than 1.5. Intensity-averaged meanlifetimes 〈�〉 for biexponential decays of fluorescence were calcu-lated from the decay times and pre-exponential factors using thefollowing equation (Lakowicz, 2006):

〈�〉 = ˛1�21 + ˛2�2

2˛1�1 + ˛2�2

(4)

3. Results

3.1. Membrane penetration depth of NR12S

Knowledge of the exact location of the fluorescent group in amembrane probe is crucial since it allows to correlate the fluo-rescence parameters of the probe with a defined location in themembrane. This is important since some probes (such as probes

labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group)tend to loop up in the membrane (Chattopadhyay, 1990; Haldarand Chattopadhyay, 2013). Knowledge of the precise depth of amembrane-embedded group or molecule often helps define the

4 R. Saxena et al. / Chemistry and Physics of Lipids 183 (2014) 1–8

-------------------------------------------------------

30

20

10

0Center of the bilay er

Dis

tanc

e fr

om t

he c

ente

r of

the

bila

yer

(Å)

Flb

c(ptrdMmlu

z

wttfdtbwoPit(affltam2ia

3v

sppflaspt

NO

RM

ALI

ZED

FLU

ORES

CEN

CE

INTE

NSIT

Y (A

RBIT

RARY

UN

ITS)

EMISSION WAVELENGTH (nm)

560 580 60 0 620 64 0

0.2

0.4

0.6

0.8

1.0

Fig. 3. Effect of membrane phase on fluorescence emission spectra of NR12S. Rep-resentative fluorescence emission spectra of NR12S in membranes of POPC ( ),DPPC (—) and POPC/cholesterol (—) are shown. Spectra are intensity-normalized atthe respective emission maximum. Measurements were carried out at room tem-perature (∼23 ◦C). The excitation wavelength used was 530 nm for POPC and DPPCmembranes, and 525 nm for membranes containing POPC/40 mol% cholesterol. The

2005; Demchenko, 2008). An attractive aspect of REES is thatit allows to monitor the mobility parameters of the environ-ment itself (represented by the relaxing solvent molecules) using

2 We have used the term maximum of fluorescence emission in a somewhat wider

ig. 2. A schematic representation of one-half of the membrane bilayer showing theocalization of NR12S in phosphatidylcholine membranes. The horizontal line at theottom indicates the center of the bilayer.

onformation and topology of membrane probes and proteinsChattopadhyay, 1992; London and Ladokhin, 2002). Interestingly,roperties such as polarity, fluidity, segmental motion, abilityo form hydrogen bonds, extent of solvent penetration, and theesultant environmental heterogeneity are known to vary in aepth-dependent manner in the membrane (Chattopadhyay andukherjee, 1999a; Chattopadhyay, 2003; Haldar et al., 2012). Theembrane penetration depth of the NR12S fluorophore was calcu-

ated by the parallax method (Chattopadhyay and London, 1987)sing the equation:

cF = Lc1 +[

(−1/�C) ln(F1/F2) − L221

2L21

](5)

here zcF is the distance of the fluorophore from the center ofhe bilayer, Lc1 is the distance of the center of the bilayer fromhe shallow quencher (Tempo-PC in this case), L21 is the dif-erence in depth between the two quenchers (i.e., the verticalistance between the shallow and deep quenchers), and C is thewo dimensional quencher concentration in the plane of the mem-rane (molecules/A2). Here, F1/F2 is the ratio of F1/Fo and F2/Fo, inhich F1 and F2 are the fluorescence intensities in the presence

f the shallow quencher (Tempo-PC) and the deep quencher (5-C), respectively, both at the same quencher concentration C; Fo

s the fluorescence intensity in the absence of any quencher. Allhe bilayer parameters used were the same as described previouslyChattopadhyay and London, 1987). Our results show that the aver-ge depth of penetration of the fluorescent group of NR12S is ∼18 Arom the center of the bilayer (see Fig. 2). This suggests that theuorescent group in NR12S is localized at the interfacial region ofhe membrane. This region of the membrane typically exhibits rel-tively slow solvent relaxation and therefore is suitable for REESeasurements (see below) (Haldar et al., 2011; Chattopadhyay,

003; Chattopadhyay and Haldar, 2014). It should be noted that thiss a time-averaged value of depth and does not take into accountny distribution in probe depth.

.2. Fluorescence characteristics of NR12S in membranes ofarying phase

The fluorescence properties of NR12S have been reported to beensitive to membrane phase (Kucherak et al., 2010). Membranehase represents a crucial determinant of membrane physicalroperties (Van Meer et al., 2008). We therefore monitored theuorescence properties of NR12S in LUVs made of POPC, DPPC

nd POPC with 40 mol% cholesterol, as these vesicles repre-ent liquid-disordered (fluid), gel (ordered), and liquid-orderedhase membranes, respectively (Brown and London, 1998). Whilehe lipid acyl chains are ordered and extended in all trans

ratio of NR12S to total lipid was 1:100 (mol/mol) in case of membranes of POPCand POPC/cholesterol, and 1:200 (mol/mol) for DPPC membranes. The total lipidconcentration was 0.43 mM in all cases. See Section 2 for other details.

conformation in the gel phase, they are fluid and disordered inthe liquid-disordered phase. The liquid-ordered phase representsan interesting phase, and is characterized by acyl chains that areextended and ordered (such as in the gel phase), but display highlateral mobility similar to the liquid-disordered phase (Mouritsen,2010). The liquid-ordered phase exists above a threshold level ofcholesterol for binary lipid mixtures (Mouritsen, 2010). The fluores-cence emission spectra of NR12S in these membranes are shownin Fig. 3. The emission maximum2 of NR12S in POPC (fluid) andDPPC (gel) vesicles was found to be 594 and 592 nm, respectively,when excited at 530 nm. In POPC vesicles with 40 mol% choles-terol (the liquid-ordered phase), the emission maximum displayeda blue shift toward lower wavelength and was found to be 583 nmwhen excited at 525 nm. Such blue shift in emission maximumof NR12S in membranes of liquid-ordered phase has previouslybeen reported, and assigned to decreased membrane hydrationand defined vertical orientation of the fluorophore in this phase(Kucherak et al., 2010).

3.3. REES in membranes of varying phase

REES is defined as the shift in the wavelength of maximum flu-orescence emission toward higher wavelengths, caused by a shiftin the excitation wavelength toward the red edge of the absorp-tion band. This effect assumes relevance for polar fluorophoresin motionally restricted environment where the dipolar relax-ation time for the solvent shell around a fluorophore becomescomparable to or longer than its fluorescence lifetime (Haldaret al., 2011; Chattopadhyay, 2003; Chattopadhyay and Haldar,2014; Mukherjee and Chattopadhyay, 1995; Raghuraman et al.,

sense here. In every case, we have monitored the wavelength corresponding tomaximum fluorescence intensity, as well as the center of mass of the fluorescenceemission, in the symmetric part of the spectrum. In most cases, both these methodsyielded the same wavelength. In cases where minor discrepancies were found, thecenter of mass of emission has been reported as the fluorescence maximum.

R. Saxena et al. / Chemistry and Physics of Lipids 183 (2014) 1–8 5

(A)

(B)

POPC POPC + Chol

20

16

12

8

4

REE

S (

nm)

585570555540525

605

600

595

590

585

580

EXCITATION WAVE LEN GTH (nm)

EMIS

SIO

N M

AXIM

UM

(nm

)

Fig. 4. (A) Effect of changing excitation wavelength on the wavelength of maximumemission for NR12S in membranes of POPC (�), and POPC/40 mol% cholesterol (�).The lines joining the data points are provided merely as viewing guides. (B) Com-parison of the magnitude of REES of NR12S in these membranes. The magnitudeof REES corresponds to the total shift in emission maximum when the excitationwS

ttbCSMC

vwcespP∼cettef(s

FLU

ORES

CEN

CE

AN

ISO

TRO

PY

0.14

0.16

0.18

0.20

0.22

DPPCPOPC POPC + Chol

Fig. 5. Fluorescence anisotropy of NR12S in membranes of varying phase. The exci-

of fit is shown in Fig. 6. The lifetimes of NR12S in membranesof varying phase are shown in Table 1. All fluorescence decayscould be fitted well to a biexponential function. We chose to usethe intensity-averaged mean fluorescence lifetime as an important

Table 1Representative fluorescence lifetimes of NR12S in membranes of varying phase.a

Condition ˛1 �1 (ns) ˛2 �2 (ns) 〈�〉 (ns)

POPC 0.12 1.05 0.88 3.96 3.86DPPC 0.30 0.76 0.70 3.72 3.48

avelength is changed from 525 to 580 nm. All other conditions are as in Fig. 3. Seeection 2 for other details.

he fluorophore merely as a reporter group. REES has provedo be a useful tool to monitor probe environment in mem-ranes and membrane-mimetic environments (Mukherjee andhattopadhyay, 2005; Chattopadhyay and Mukherjee, 1999a;hrivastava et al., 2009; Rawat and Chattopadhyay, 1999;ukherjee et al., 2004; Raghuraman et al., 2004; Kelkar and

hattopadhyay, 2004).The shifts in the maxima of fluorescence emission of NR12S in

esicles of POPC and POPC/cholesterol as a function of excitationavelength are shown in Fig. 4A. As the excitation wavelength is

hanged from 525 to 580 nm, the emission maximum of NR12Sxhibits a shift toward longer wavelengths in both cases. The emis-ion maximum is shifted from 594 to 603 nm in liquid-disorderedhase POPC vesicles and from 583 to 602 nm in liquid-orderedOPC/cholesterol membranes. These shifts correspond to REES of9 and 19 nm for liquid-disordered and liquid-ordered phase vesi-

les, respectively, and are shown in Fig. 4B. Such dependence ofmission spectrum on excitation wavelength is characteristic ofhe red edge effect. Observation of REES in both cases suggests thathe fluorescent moiety of NR12S experiences motionally restrictednvironment in these membranes. This is consistent with the inter-

acial localization of the fluorescent group of NR12S in membranessee Fig. 2), since the membrane interface exhibits characteristiclow solvent relaxation (Haldar et al., 2011; Chattopadhyay, 2003;

tation wavelength used was 530 nm and emission was monitored at 596 nm in allcases. Data shown are means ± S.E. of at least three independent measurements. Allother conditions are as in Fig. 3. See Section 2 for other details.

Chattopadhyay and Haldar, 2014). Importantly, although REES hasbeen earlier reported in liquid-disordered and gel phase mem-branes (Chattopadhyay and Mukherjee, 1999b; Raghuraman et al.,2007), our present results of REES of NR12S in POPC/cholesterolmembranes constitute one of the early reports of REES for probesin liquid-ordered phase membranes.

3.4. Fluorescence anisotropy and lifetime in membranes ofvarying phase

Fluorescence anisotropy is extensively used to monitor therotational diffusion rate of membrane embedded probes, whichis sensitive to the packing of lipid fatty acyl chains (Lakowicz,2006; Jameson and Ross, 2010). This is due to the fact that fluo-rescence anisotropy depends on the extent to which the probe isable to reorient after excitation, and probe reorientation is depend-ent on local lipid packing. Fig. 5 shows the steady state anisotropyof NR12S in membranes of varying phase. As apparent from thefigure, the anisotropy is lowest in the liquid-disordered phase(POPC). This is due to the relatively loose packing in the liquid-disordered phase. Interestingly, the anisotropy of NR12S in theliquid-ordered (POPC/cholesterol) phase, although higher than thatin liquid-disordered phase, appears to be much lower than the cor-responding value in the gel (DPPC) phase. This is surprising since,as mentioned above, the acyl chains in the liquid-ordered phaseare reported to be extended and ordered, similar to the packingarrangement in the gel phase (but see later).

Fluorescence lifetime serves as a reliable indicator of thelocal environment in which a given fluorophore is localized(Prendergast, 1991). A typical decay profile of NR12S in liquid-ordered phase vesicles of POPC/cholesterol with its biexponentialfitting and the statistical parameters used to check the goodness

POPC/cholesterol 0.11 1.39 0.89 4.36 4.25

a The excitation wavelength was 490 nm and emission was monitored at 596 nm.All other conditions are as in Fig. 3. Mean fluorescence lifetimes were calculatedusing Eq. (4). See Section 2 for other details.

6 R. Saxena et al. / Chemistry and Physics of Lipids 183 (2014) 1–8

Fig. 6. Time-resolved fluorescence intensity decay of NR12S in POPC membranes containing 40 mol% cholesterol. The excitation wavelength used was 490 nm correspondingto pulsed diode light source and emission was monitored at 596 nm. The sharp peak on the left corresponds to the profile of the pulsed light emitting diode (LED). Ther tion.

o r deta

pNndvEcDl(∼vllriTpldlepfl

elatively broad peak on the right is the decay profile, fitted to a biexponential funcf the weighted residuals. All other conditions are as in Fig. 3. See Section 2 for othe

arameter for describing the behavior of membrane embeddedR12S because it is independent of the method of analysis and theumber of exponentials used to fit the time-resolved fluorescenceecay. The mean fluorescence lifetimes of NR12S in membranes ofarying phase were calculated from data shown in Table 1 usingq. (4), and are shown in Table 1 and Fig. 7A. The mean fluores-ence lifetime of NR12S was found to be minimum in gel phasePPC membranes with a value of ∼3.5 ns. The mean fluorescence

ifetimes of NR12S in liquid-disordered (POPC) and liquid-orderedPOPC/cholesterol) phase membranes were longer, with values of3.9 and 4.2 ns, respectively. In order to explore the effect of sol-ent polarity on the fluorescence lifetime of NR12S, we measuredifetime of NR12S in solvents of varying polarity. The fluorescenceifetimes of NR12S in different solvents are shown in Fig. S1. Ouresults show that fluorescence lifetime of NR12S is relatively highn solvents of low polarity, and low in solvents of high polarity.his suggests that fluorescence lifetime of NR12S is sensitive toolarity of the surrounding medium. The longer mean fluorescence

ifetime of NR12S in POPC-cholesterol vesicles could therefore beue to the relatively tight packing of the lipid fatty acyl chains in the

iquid-ordered phase, which results in less water penetration. Inter-

stingly, the relatively low fluorescence lifetime of NR12S in gelhase DPPC vesicles, characterized by compact packing of the lipidatty acyl chains, merits comment. It is possible that the interfacialocation of NR12S observed in liquid-disordered phase membranes

The two lower plots show the weighted residuals and the autocorrelation functionils.

is perturbed in gel phase DPPC vesicles due to extreme tight packingof the lipid fatty acyl chains. As a result, the probe is pushed towardthe membrane surface, thereby experiencing more polar environ-ment which results in a shorter lifetime. This is in accordance withthe observed red shifted emission in gel phase compared to theliquid ordered phase (Fig. 3) (Kucherak et al., 2010). Fluorescencelifetime of NR12S in POPC vesicles was found to be intermediatebetween gel and liquid-ordered phases (Fig. 7A).

In order to ensure that the anisotropy values measured forNR12S (Fig. 5) are not influenced by lifetime-induced artifacts, theapparent (average) rotational correlation times were calculatedusing Perrin’s equation (Lakowicz, 2006):

�c = 〈�〉 r

r◦ − r(6)

where ro is the limiting (fundamental) anisotropy of the fluorescentgroup in NR12S (in the absence of any other depolarizing pro-cesses such as rotational diffusion), r is the steady state anisotropy(Fig. 5), and 〈�〉 is the mean fluorescence lifetime from Table 1.Although Perrin’s equation is not strictly applicable to this system,it is assumed that this equation will apply to a first approximation,

especially because we used mean fluorescence lifetimes for theanalysis of multiple component lifetimes. The values of the appar-ent rotational correlation times, calculated using Eq. (6) with a ro

value of 0.34 (Ferrer and del Monte, 2005), are shown in Fig. 7B.

R. Saxena et al. / Chemistry and Phy

APP

AREN

T RO

TATI

ON

AL

CO

RREL

ATIO

N T

IME

(ns)

DPPCPOPC POPC + Chol

3

4

5

MEA

N F

LUO

RES

CEN

CE

LIFE

TIM

E(n

s)

(A)

(B)

2.5

3.0

3.5

4.0

4.5

DPPCPOPC POPC + Chol

Fig. 7. (A) Mean fluorescence lifetimes of NR12S in membranes of varying phase.Mean fluorescence lifetimes were calculated using Eq. (4). The excitation wave-length used was 490 nm and emission was monitored at 596 nm. Data shown aremeans ± S.E. of at least three independent measurements. All other conditions areas in Fig. 3. See Section 2 for other details. (B) Apparent rotational correlation timesof NR12S in membranes of varying phase. Apparent rotational correlation timeswfld

AtiiTiwufw

4

eiMtmwrupof

ere calculated from fluorescence anisotropy values of NR12S from Fig. 5 and meanuorescence lifetimes from panel (A) of this figure using Eq. (6). See text for otheretails.

s expected, the apparent rotational correlation time was foundo be minimum in the liquid-disordered (POPC) phase. Interest-ngly, the apparent rotational correlation times are almost samen the liquid-ordered (POPC/cholesterol) and gel (DPPC) phases.his is consistent with similar compact packing of lipid acyl chainsn both these phases. The apparent disagreement of these results

ith anisotropies shown in Fig. 5 reveals that the anisotropy val-es were indeed influenced by fluorescence lifetimes. The resultsrom anisotropy measurements therefore should be interpretedith caution.

. Discussion

It is becoming increasingly evident that the eukaryotic cellularnvironment is characterized by multiple membranes with vary-ng phase, differing in physical dimensions such as thickness (Van

eer et al., 2008; Sharpe et al., 2010). These membranes providehe varied and dynamic backdrop for carrying out cellular signaling

odulated by a range of slow solvent relaxation dynamics. In thisork, we have monitored the organization, dynamics and solvent

elaxation characteristics of NR12S in membranes of varying phase

tilizing fluorescence-based approaches including REES and thearallax approach for depth analysis. NR12S is a recently devel-ped membrane probe based on Nile Red with several specialeatures. These include environmental sensitivity, phase-sensitive

sics of Lipids 183 (2014) 1–8 7

fluorescence, and exclusive localization in the outer leaflet(Chiantia et al., 2012; Darwich et al., 2012). Although Nile Red isfrequently used as a lipid stain and in exploring membrane organi-zation (Gao et al., 2006; Krishnamoorthy and Ira, 2001; Mukherjeeet al., 2007a,b), NR12S enjoys certain advantages over Nile Red.Unlike Nile Red, NR12S specifically labels the outer leaflet of plasmamembranes in cells due to its very slow flip-flop rate. In this work,we analyzed the membrane penetration depth of the fluorescentgroup in NR12S using the parallax method. We show here that thefluorescent group in NR12S is localized at the membrane interfa-cial region, characterized by an average depth of penetration of∼18 A from the center of the bilayer (see Fig. 2). The interfacialregion of the membrane is uniquely characterized by relativelyslow solvent relaxation and offers an appropriate environment forREES measurements (Haldar et al., 2011; Chattopadhyay, 2003;Chattopadhyay and Haldar, 2014). As a consequence, our resultsshow that NR12S displays phase-sensitive REES in membranes(Fig. 4). These results constitute the first report on membrane pen-etration depth and solvent relaxation characteristics of this novelprobe in membranes. In addition, we show that the fluorescenceemission maximum, anisotropy, and lifetime of NR12S are depend-ent on the phase of the membrane.

The phase-dependent solvent relaxation properties of NR12Scould be potentially useful since cellular membranes display com-plex solvent relaxation patterns, depending on the membranephase. Taken together, our results show that NR12S can distin-guish different membrane phases through a variety of fluorescenceparameters such as emission maximum, REES, anisotropy and life-time. We conclude that NR12S appears to be a promising probeto explore membrane organization in model and biological mem-branes.

Conflict of interest

The authors declare that there are no conflict of interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgments

This work was supported by the Council of Scientific and Indus-trial Research (CSIR, India), and Centre National de la RechercheScientifique (CNRS, France). R.S. and S.H. thank the Council of Sci-entific and Industrial Research for the award of Senior ResearchFellowships. A.C. is an Adjunct Professor at the Special Centre forMolecular Medicine of Jawaharlal Nehru University (New Delhi,India) and Indian Institute of Science Education and Research(Mohali, India), and Honorary Professor at the Jawaharlal NehruCentre for Advanced Scientific Research (Bangalore, India). A.C.gratefully acknowledges J.C. Bose Fellowship (Department of Sci-ence and Technology, Govt. of India). We thank Arunima Chaudhuriand G. Aditya Kumar for help during the preparation of themanuscript, and members of A.C.’s research group for criticallyreading the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.chemphyslip.2014.04.007.

8 d Phy

R

A

B

B

C

C

C

C

C

C

C

C

C

C

D

D

D

D

E

F

G

G

G

G

H

H

H

J

J

K

R. Saxena et al. / Chemistry an

eferences

brams, F.S., London, E., 1993. Extension of the parallax analysis of membrane pen-etration depth to the polar region of model membranes: use of fluorescencequenching by a spin-label attached to the phospholipid polar headgroup. Bio-chemistry 32, 10826–10831.

evington, P.R., 1969. Data Reduction and Error Analysis for the Physical Sciences.McGraw-Hill, New York.

rown, D.A., London, E., 1998. Structure and origin of ordered lipid domains inbiological membranes. J. Membr. Biol. 164, 103–114.

hattopadhyay, A., 1990. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)-labeled lipids: fluorescence probes of biological and model membranes.Chem. Phys. Lipids 53, 1–15.

hattopadhyay, A., 1992. In: Gaber, B.P., Easwaran, K.R.K. (Eds.), Biomembrane Struc-ture and Function: The State of the Art. Adenine Press, Schenectady, NY, pp.153–163.

hattopadhyay, A., 2002. Lipid probes in membrane biology. Chem. Phys Lipids 116,1–188.

hattopadhyay, A., 2003. Exploring membrane organization and dynamics by thewavelength-selective fluorescence approach. Chem. Phys. Lipids 122, 3–17.

hattopadhyay, A., Haldar, S., 2014. Dynamic insight into protein structure utilizingred edge excitation shift. Acc. Chem. Res. 47, 12–19.

hattopadhyay, A., London, E., 1987. Parallax method for direct measurement ofmembrane penetration depth utilizing fluorescence quenching by spin-labeledphospholipids. Biochemistry 26, 39–45.

hattopadhyay, A., Mukherjee, S., 1993. Fluorophore environments inmembrane-bound probes: a red edge excitation shift study. Biochemistry32, 3804–3811.

hattopadhyay, A., Mukherjee, S., 1999a. Depth-dependent solvent relaxation inmembranes: wavelength-selective fluorescence as a membrane dipstick. Lang-muir 15, 2142–2148.

hattopadhyay, A., Mukherjee, S., 1999b. Red edge excitation shift of a deeplyembedded membrane probe: implications in water penetration in the bilayer.J. Phys. Chem. B 103, 8180–8185.

hiantia, S., Klymchenko, A.S., London, E., 2012. A novel leaflet-selective fluorescencelabeling technique reveals differences between inner and outer leaflets at highbilayer curvature. Biochim. Biophys. Acta 1818, 1284–1290.

arwich, Z., Klymchenko, A.S., Kucherak, O.A., Richert, L., Mély, Y., 2012. Detection ofapoptosis through the lipid order of the outer plasma membrane leaflet. Biochim.Biophys. Acta 1818, 3048–3054.

as, R., Klymchenko, A.S., Duportail, G., Mély, Y., 2008. Excited state proton transferand solvent relaxation of a 3-hydroxyflavone probe in lipid bilayers. J. Phys.Chem. B 112, 11929–11935.

emchenko, A.P., 2008. Site-selective red-edge effects. Methods Enzymol. 450,59–78.

ittmer, J.C., Lester, R.L., 1964. A simple, specific spray for the detection of phospho-lipids on thin-layer chromatograms. J. Lipid. Res. 5, 126–127.

ggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova,S., Belov, V.N., Hein, B., von Middendorff, C., Schönle, A., Hell, S.W., 2009. Directobservation of the nanoscale dynamics of membrane lipids in a living cell. Nature457, 1159–1163.

errer, M.L., del Monte, F., 2005. Enhanced emission of nile red fluorescent nanopar-ticles embedded in hybrid sol–gel glasses. J. Phys. Chem. B 109, 80–86.

ao, F., Mei, E., Lim, M., Hochstrasser, R.M., 2006. Probing lipid vesicles by bimolec-ular association and dissociation trajectories of single molecules. J. Am. Chem.Soc. 128, 4814–4822.

olini, C.M., Williams, B.W., Foresman, J.B., 1998. Further solvatochromic, ther-mochromic, and theoretical studies on Nile Red. J. Fluoresc. 8, 395–404.

reenspan, P., Fowler, S.D., 1985. Spectrofluorometric studies of the lipid probe, nilered. J. Lipid Res. 26, 781–789.

rinvald, A., Steinberg, I.Z., 1974. On the analysis of fluorescence decay kinetics bythe method of least-squares. Anal. Biochem. 59, 583–598.

aldar, S., Chattopadhyay, A., 2013. In: Mely, Y., Duportail, G. (Eds.), FluorescentMethods to Study Biological Membranes. Springer, Heidelberg, pp. 37–50.

aldar, S., Chaudhuri, A., Chattopadhyay, A., 2011. Organization and dynamics ofmembrane probes and proteins utilizing the red edge excitation shift. J. Phys.Chem. B 115, 5693–5706.

aldar, S., Kombrabail, M., Krishnamoorthy, G., Chattopadhyay, A., 2012.Depth-dependent heterogeneity in membranes by fluorescence lifetime distri-bution analysis. J. Phys. Chem. Lett. 3, 2676–2681.

ameson, D.M., Ross, J.A., 2010. Fluorescence polarization/anisotropy in diagnosticsand imaging. Chem. Rev. 110, 2685–2708.

urkiewicz, P., Olzynska, A., Langner, M., Hof, M., 2006. Headgroup hydration andmobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study. Lang-muir 22, 8741–8749.

elkar, D.A., Chattopadhyay, A., 2004. Depth-dependent solvent relaxation inreverse micelles: a fluorescence approach. J. Phys. Chem. B 108, 12151–12158.

sics of Lipids 183 (2014) 1–8

Kremer, J.M.H., van der Esker, M.W., Pathmamanoharan, C., Wiersema, P.H., 1977.Vesicles of variable diameter prepared by a modified injection method. Bio-chemistry 16, 3932–3935.

Krishnamoorthy, G., Ira, 2001. Fluorescence lifetime distribution in characterizingmembrane heterogeneity. J. Fluoresc. 11, 247–253.

Kucherak, O.A., Oncul, S., Darwich, Z., Yushchenko, D.A., Arntz, Y., Didier, P., Mély,Y., Klymchenko, A.S., 2010. Switchable nile red-based probe for cholesteroland lipid order at the outer leaflet of biomembranes. J. Am. Chem. Soc. 132,4907–4916.

Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy, 3rd ed. Springer, NewYork.

Lampert, R.A., Chewter, L.A., Phillips, D., O’Connor, D.V., Roberts, A.J., Meech, S.R.,1983. Standards for nanosecond fluorescence decay time measurements. Anal.Chem. 55, 68–73.

London, E., Ladokhin, A.S., 2002. In: Benos, D., Simon, S. (Eds.), Current Topics inMembranes. Elsevier, New York, pp. 89–115.

MacDonald, R.C., MacDonald, R.I., Menco, B.Ph.M., Takeshita, K., Subbarao, N.K., Hu,L.R., 1991. Small-volume extrusion apparatus for preparation of large, unilamel-lar vesicles. Biochim. Biophys. Acta 1061, 297–303.

McClare, C.W.F., 1971. An accurate and convenient organic phosphorus assay. Anal.Biochem. 39, 527–530.

Mouritsen, O.G., 2010. The liquid-ordered state comes of age. Biochim. Biophys. Acta1798, 1286–1288.

Mukherjee, S., Chattopadhyay, A., 1995. Wavelength-selective fluorescence as anovel tool to study organization and dynamics in complex biological systems. J.Fluoresc. 5, 237–246.

Mukherjee, S., Chattopadhyay, A., 2005. Influence of ester and ether linkage inphospholipids on the environment and dynamics of the membrane interface:a wavelength-selective fluorescence approach. Langmuir 21, 287–293.

Mukherjee, S., Chattopadhyay, A., Samanta, A., Soujanya, T., 1994. Dipole momentchange of NBD group upon excitation studied using solvatochromic and quan-tum chemical approaches: implications in membrane research. J. Phys Chem.98, 2809–2812.

Mukherjee, S., Kombrabail, M., Krishnamoorthy, G., Chattopadhyay, A., 2007a.Dynamics and heterogeneity of bovine hippocampal membranes: role of choles-terol and proteins. Biochim. Biophys. Acta 1768, 2130–2144.

Mukherjee, S., Raghuraman, H., Chattopadhyay, A., 2007b. Membrane localizationand dynamics of Nile Red: effect of cholesterol. Biochim. Biophys. Acta 1768,59–66.

Mukherjee, S., Raghuraman, H., Dasgupta, S., Chattopadhyay, A., 2004. Organiza-tion and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: afluorescence approach. Chem. Phys. Lipids 127, 91–101.

O’Connor, D.V., Phillips, D., 1984. Time-Correlated Single Photon Counting. AcademicPress, London, pp. 180–189.

Prendergast, F.G., 1991. Time-resolved fluorescence techniques: methods and appli-cations in biology. Curr. Opin. Struct. Biol. 1, 1054–1059.

Raghuraman, H., Kelkar, D.A., Chattopadhyay, A., 2005. Novel insights into proteinstructure and dynamics utilizing the red edge excitation shift approach. In: Ged-des, C.D., Lakowicz, J.R. (Eds.), Reviews in Fluorescence. Springer, New York, pp.199–222.

Raghuraman, H., Pradhan, S.K., Chattopadhyay, A., 2004. Effect of urea on the organi-zation and dynamics of Triton X-100 micelles: a fluorescence approach. J. Phys.Chem. B 108, 2489–2496.

Raghuraman, H., Shrivastava, S., Chattopadhyay, A., 2007. Monitoring the loopingup of acyl chain labeled NBD lipids in membranes as a function of membranephase state. Biochim. Biophys. Acta 1768, 1258–1267.

Rawat, S.S., Chattopadhyay, A., 1999. Structural transition in the micellar assembly:a fluorescence study. J. Fluoresc. 9, 233–244.

Reichardt, C., 1988. Solvents and Solvent Effects in Organic Chemistry. VCH Publish-ers, Weinheim, Germany.

Sackett, D.L., Knutson, J.R., Wolff, J., 1990. Hydrophobic surfaces of tubulin probedby time-resolved and steady-state fluorescence of Nile Red. J. Biol. Chem. 265,14899–14906.

Sharpe, H.J., Stevens, T.J., Munro, S.A., 2010. Comprehensive comparison oftransmembrane domains reveals organelle-specific properties. Cell 142,158–169.

Shrivastava, S., Haldar, S., Gimpl, G., Chattopadhyay, A., 2009. Orientation anddynamics of a novel fluorescent cholesterol analogue in membranes of varyingphase. J. Phys. Chem. B 113, 4475–4481.

Shynkar, V.V., Klymchenko, A.S., Kunzelmann, C., Duportail, G., Muller, C.D.,Demchenko, A.P., Freyssinet, J.M., Mély, Y., 2007. Fluorescent biomembraneprobe for ratiometric detection of apoptosis. J. Am. Chem. Soc. 129, 2187–

2193.

van Meer, G., de Kroon, A.I.P.M., 2011. Lipid map of the mammalian cell. J. Cell Sci.124, 5–8.

Van Meer, G., Voelker, D.R., Feigenson, G.W., 2008. Membrane lipids: where they areand how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124.