Analytical Biochemistry 440 (2013) 123–129

Contents lists available at SciVerse ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Fluorescence spectra decomposition by asymmetric functions: Laurdanspectrum revisited

0003-2697/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ab.2013.05.031

⇑ Corresponding author at: Department of Neurological, Neuropsychological,Morphological, and Movement Sciences, Section of Anatomy and Histology,University of Verona, Verona 37134, Italy. Fax: +39 045 8027163.

E-mail address: mihai.radu@univr.it (M. Radu).1 Abbreviations used: LN, log-normal; FWHM, full width at half-maximum; GP,

generalized polarization; LUV, large unilamellar vesicle; PBS, phosphate-bufferedsaline; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; MLV, multilamellarvesicle.

Mihaela Bacalum a,b,c, Bogdan Zorila a, Mihai Radu a,d,⇑a Department of Life and Environmental Physics, Horia Hulubei National Institute of Physics and Nuclear Engineering, 077125 Magurele, Romaniab Biomedical Research Institute, Hasselt University, B-3590 Diepenbeek, Belgiumc Department of Electricity, Solid State and Biophysics, Faculty of Physics, University of Bucharest, 077125 Magurele–Ilfov, Romaniad Department of Neurological, Neuropsychological, Morphological, and Movement Sciences, Section of Anatomy and Histology, University of Verona, Verona 37134, Italy

a r t i c l e i n f o

Article history:Received 8 April 2013Received in revised form 17 May 2013Accepted 22 May 2013Available online 6 June 2013

Keywords:Complex emission spectrumLog-normal functionLarge unilamellar vesiclesLaurdanGeneralized polarizationBilayer hydration

a b s t r a c t

Due to their asymmetric nature, complex fluorescence spectra of molecules can be analyzed much betterby log-normal distributions than by Gaussian ones. So far, the log-normal function has been used fordeconvolution of emission spectra of different fluorescent molecules, such as Tryptophan and Prodan,but to our knowledge it is far less used for Laurdan (2-dimethylamino-6-lauroylnaphthalene). In this arti-cle, we present the decomposition of Laurdan emission spectra in large unilamellar vesicles using a pro-cedure that relies on the log-normal asymmetric function. The procedure was calibrated using Laurdanspectra in homogeneous solutions of various solvents. Comparing our results with the ones obtained froma Gaussian fit, we show that (i) the position of the elementary peaks (�440 and 490 nm) is preserved in alarge range of temperatures that include the main phase transition of lipid bilayer and (ii) the bilayerhydration, as reported by Laurdan, increases approximately 8 times from the gel phase to the liquid crys-talline one, a result that fits with other reports, providing a more realistic description. In addition, we pro-pose a new parameter to globally evaluate Laurdan emission spectra with the prospect of acquiring alarger range of values than the classical ‘‘generalized polarization’’.

� 2013 Elsevier Inc. All rights reserved.

The complex emission spectrum decomposition is a very impor- function to describe band shape in absorption spectra [2]. Burstein

tant issue in analytical fluorescence spectroscopy. The structure of aspectrum is produced by the presence of either several differentfluorophores or different excited states of the same fluorophore inthe investigated solution. Most fluorophores produce asymmetricemission spectra even in a homogeneous solution where only oneemitting state is presumed to be present. The main reason is givenby the differences in vibronic level distribution between groundand excited states [1]. The solution for analysis of such complexspectra is the decomposition in elementary emission peaks. Thisis not a trivial task, with a valid mathematical model for an analyt-ical description of the elementary peak shape being needed. Veryoften, the first choice is oriented to the popular Gaussian andLorentzian functions in spite of their symmetry. Few alternativesfor complex spectrum decomposition have been proposed in the lit-erature, from which one requires the use of asymmetric functions.Siano and Metzler proposed a four-parameter log-normal (LN)1

and coworkers developed a procedure based on the mirror symmetricform of LN to analyze the emission spectra of a larger class of fluoro-phores [3]. In particular, they developed and validated a robust algo-rithm for analysis of the tryptophan emission spectrum in proteins[4]. The procedure was extended by the same group for Prodan andAcrylodan [5]. Kalauzi and coworkers presented a comparison amongtheir own asymmetric model and the LN and Gaussian models, con-cluding that LN and their new asymmetric function provide very sim-ilar results with a better fit than Gaussian-based models [6]. LN hasbeen successfully used as the base of an algorithm to analyze thespectra of a class of fluorophores presenting two coupled groundand excited states (3-hydroxychromone derivatives) [7].

Another sensitive issue of the complex spectra decomposition isthe meaning of the parameters resulting from a fit procedure, par-ticularly the parameters related to the shape function (e.g., fullwidth at half-maximum, FWHM). If only v2 is considered as a un-ique criterion to establish the optimal parameter values, it is pos-sible that some of these values are far from a physical meaning. Inthe particular case of complex spectrum produced by several pop-ulations of the same fluorophore, Burstein and coworkers proposeda procedure to calibrate the fitting algorithm, imposing someempirically derived constraints on the LN shape parameters [3].

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a deriva-tive of Prodan (6-propionyl-2-(dimethy-1-amino)-naphthalene)

124 M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129

[8], and its structure consists of a fluorescent naphthalene moietylinked to a 12-carbon atom chain. With this special design, Laurdanwas proposed from the beginning to be used in studies on cellmembranes and lipid bilayers [9,10]. Its aliphatic tail ensures agood anchorage into the lipid hydrophobic core of both cell mem-branes and artificial lipid bilayers. The fluorescence moiety is lo-cated at the level of sn-1 carbonyl of the phospholipids insidethe bilayer [11], being able to ‘‘sense’’ changes in the interface re-gion between the hydrophobic core and the external hydrophilicbulk. The transition dipole of Laurdan (4.4–5.0 D) [12] enables alarge bathochromic shift of the emission spectrum in solvents withdifferent polarity (from �400 nm in cyclohexane to �500 nm inmethanol). This sensitivity to environment polarity makes Laurdanvery useful in evaluating the hydration of lipid bilayer interfaces,and for this reason during past decades it has become one of themost used fluorescent dyes for characterizing lipid bilayer proper-ties (reviewed in Ref. [13]).

Even though there is an ongoing debate concerning the solvato-chromic effect in Laurdan emission spectrum, one mechanism thatis generally accepted takes into account the solvent dipole relaxa-tion around the large dipole of Laurdan in the excited state [14]. Ina heterogeneous medium such as lipid bilayer, some Laurdan mol-ecules have access to water and others do not. In this situation, thecomplex steady-state emission spectrum appears as a superposi-tion of the elementary emissions from the two excited states (non-relaxed emitting states–blue channel and relaxed emitting states–green channel) [15].

The spectral changes of Laurdan emission in lipid bilayers havebeen previously analyzed by a global parameter named ‘‘general-ized polarization’’ (GP) [15]:

GP ¼ IB � IG

IB þ IGð1Þ

where IB and IG are the intensities of emission in blue and greenchannels, respectively. Its simplicity and other interesting charac-teristics (for details, see Refs. [15,16]) have made GP a very popularway to present the data obtained by Laurdan spectroscopy on mod-el and biological membranes. GP values range from 1 to �1 if theblue channel is set at the dodecane peak position and the greenone is set at the methanol peak position [16]. In the lipid bilayer,GP is calculated setting the blue and green channels at 440 and490 nm, respectively. These values represent the peak positionsreached in the two pure lipid phases: 440 nm for the gel phaseand 490 nm for the liquid crystalline phase. Because of the spectraloverlapping of the relaxed and nonrelaxed state emissions, GP actu-ally cannot reach its extreme values in the lipid bilayer. Neverthe-less, if the number of emitting molecules in each state would beused instead of IB and IG, a more reliable parameter could be de-fined. The decomposition of Laurdan spectrum in a superpositionof elementary peaks can provide such information.

Until now, an elaborate procedure for evaluation of Laurdanspectrum recorded on a suspension of large unilamellar vesicles(LUVs) has been performed using decomposition by two Gaussianfunctions [17,18]. Instead of GP, the relative area of the elementarypeaks was used as a quantitative global parameter characterizingchanges in Laurdan spectrum as a function of temperature. Toour knowledge, only one report has used the LN decompositionfor analyzing the Laurdan emission spectrum, but in a completelydifferent environment (reverse micelles of aerosol OT [AOT]–waterin isooctane) [19].

In this article, we propose a new method to analyze the Laurdancomplex spectrum in model lipid membranes (particularly LUVs)by decomposition of the spectra using LN functions. The first stepis to evaluate the emission spectra of Laurdan in different solvents.These data are further used to calibrate the algorithm developedfor the complex spectrum decomposition. The results are discussed

by comparison with Gaussian decomposition, and the lipid bilayerhydration in gel and liquid crystalline phases is analyzed.

Materials and methods

Materials

All solvents used were spectroscopic grade. Na2HPO4�2H2O,KH2PO4 anhydrous, and NaCl were purchased from Sigma–Aldrichand used to prepare the phosphate-buffered saline (PBS, 10 mM,pH 7.4). 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC)was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Laur-dan was purchased from Invitrogen/Molecular Probes (Eugene, OR,USA).

LUV preparation

DMPC LUVs with a final lipid concentration of 50 lM were pre-pared using the extrusion method according to the Avanti pub-lished protocol. Briefly, the appropriate amount of lipids from thestock solutions prepared in chloroform was dried under nitrogenflow to remove the solvent. The lipids were then hydrated withPBS heated above the transition temperature (Tm) of the used lipidsand vigorously vortexed to form a suspension of multilamellar ves-icles (MLVs). The MLV suspension was repeatedly freeze–thawed(5 cycles) and extruded (25 times) through a 200-lm filter usinga standard extruder (Avanti Polar Lipids). The extrusion was per-formed at a temperature above the transition temperature of thelipids used for liposome preparation, resulting in a suspension ofLUVs. After liposome preparation, Laurdan was added into thesamples to a final lipid/probe ratio of 500:1 (the Laurdan final con-centration was 0.1 lM).

Fluorescence spectroscopy measurements

Steady-state fluorescence measurements were performed usinga FluoroMax 3 spectrofluorimeter (Horiba Jobin Yvon, Edison, NJ,USA) equipped with a Peltier thermostated cell holder. Fluores-cence spectra of the Laurdan dissolved in solvents were recordedat room temperature.

The emission spectra of the Laurdan inserted into the mem-brane of the LUVs were recorded in the range from 5 to 55 �C.The excitation wavelength was set at 378 nm, and the spectra wererecorded in the range from 400 to 600 nm, with excitation andemission slits being set at 3 nm. The spectra recorded were firstcorrected for the spectral sensitivity of the emission channel ofthe spectrofluorimeter. A second correction for Raman and scatter-ing artifacts was done by subtracting from the spectra the contri-bution of liposome suspension without Laurdan. All of theemission recordings have been done at a suspension absorptionsmaller than 0.05; consequently, no correction for the inner effectwas needed [20].

Before fitting, all recorded spectra were converted in the wave-number scale using the relation I = Ik*k2, where k is the wavelengthand Ik is the intensity in wavelength scale [1].

Data processing

Spectra of Laurdan in pure solventsAs it was first shown by Siano and Metzler, a four-parameter LN

function better describes the shape of peaks in absorption spectraof hydroxypyridine derivatives as compared with the Gaussian one[2]. Here, we used the LN mirror symmetric form (in wavenumberscale; see Fig. 1) proposed to describe the emission spectra of or-ganic fluorophores [3]:

M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129 125

I ¼ Imexp � ln2ln2ðqÞ

ln2 a�ta�tm

� �h iif t < a

I ¼ 0 if t � að2Þ

(

where I is the emission intensity, Im is the maximum of intensity, tis the wavenumber, tm is the position of the peak, q ¼ tm�tmin

tmax�tmis the

asymmetry of the function (tmax and tmin are the wavenumber val-ues at half-intensity), and a is the limiting wave number:

a ¼ tm þ ðtmax�tminÞqðq2�1Þ .

The spectra of Laurdan in homogeneous solution have been fit-ted by Eq. (2) using the nonlinear fitting tools of the Origin 8.0 soft-ware package (OriginLab, Northampton, MA, USA). The values oftm, tmax, and tmin have been derived for each solvent.

Spectra of Laurdan in LUVsUsing the LN function, Burstein and coworkers showed for sev-

eral fluorophores that for a probe found in different environmentsthere is a linear relationship between the tmin and tm (similarly fortmax). Using such constraints, the LN function parameters can bereduced to only two: Im and tm.

Spectra recorded for Laurdan inserted into the lipid bilayer ofDMPC LUVs were fitted with a superposition of two LN functions(Eq. (2)) adjusted with the constraining relationships of tmax andtmin against tm, inferred from fitting of spectra recorded in the sol-vents (for details, see Results and Discussion). The script used torun the LN fitting of LUV spectra and to infer the intensity and po-sition of each LN elementary peak was written using MatLab 9bsoftware (MathWorks, Natick, MA, USA). For comparison, the com-plex spectra in LUVs have been decomposed by two Gaussian func-tions using the Origin 8.0 software package.

GP values were calculated according to Eq. (1) using Origin 8.0.

Results and discussion

Solvent measurements and shape parameter relationships

The main working hypothesis in our approach is based on thefact that only one species of emitting Laurdan molecules is presentin a homogeneous solution. A similar consideration was previouslyused in characterizing Laurdan or Prodan emission spectra in puresolvents [21,22]. However, there are reports in the literature sup-porting the idea that even in pure organic solvents Laurdan has acomposite spectrum, but in most of these reports the recordingsare made in very viscous media (glycerol [23] or Primol and Marcoloils [24]) or at very low temperature (ethanol at �108 to 25 �C[19]). Indeed, ethanol high viscosity at very low temperature slowsthe reorientation of solvent molecules around the Laurdan dipole

Fig.1. Log-normal function with shape parameters.

in the excited state, and an apparent heterogeneity of the emissionspectrum is revealed. This heterogeneity is given by superposingthe contributions of Laurdan molecules that emit from nonrelaxedand relaxed states. At room temperature, the relaxation process isso fast that in steady-state recordings only the relaxed state emis-sion is observed [19].

To find a correlation among parameters restraining the possibleshapes of peak emitted by Laurdan, we first recorded the emissionspectrum of Laurdan in homogeneous solvents ranging from non-polar to polar protic ones (Table 1).

A few examples of spectra recorded in homogeneous solutionscovering all ranges of solvent polarity (nonpolar, polar aprotic,and polar protic) are presented in Fig. 2A. Two main aspects canbe emphasized: (i) the well-conserved asymmetric shape of thepeak and (ii) the large red shift of emission spectrum when thepolarity of solvent increases.

The shape of Laurdan spectra is clearly asymmetric, and theasymmetry (q) has a slow trend to decrease with the increase ofpolarity (Table 1). Spectra have been fitted very well by the LNfunction (Fig. 2B–D), with the residuals being under 2% for all ofthe solvents (Table 1).

The red shift of the spectrum with respect to the solvent polar-ity is a feature of Laurdan as a solvatochromic fluorescent probe.The peak position (tm) is related to polarity [expressed as ET(30)]by a linear relationship (Fig. 3A). Only few studies from the litera-ture have analyzed the dependence of peak position with respectto a polarity scale in the case of Laurdan [21,25], and our resultsare in agreement with these reports. This linearity proves the pos-sibility to interpret the change of Laurdan spectrum directly interms of the environment polarity.

In Fig. 3B, the values of the positions of the two half-maximalamplitude limits (tmin and tmax) are presented against the peak po-sition. The dependences are linear, and the relationships providedby the fit can be used to restrain the shape of elementary peaks.Analyzing this graph in detail, we found a small difference amongthe parameters of linear functions fitting the polar range of sol-vents and corresponding parameters for the nonpolar solvents.For this reason, the constraining functions were implemented con-sidering specific linear relationships for each range of solvents. Thewavenumber limit separating the range of polar solvents from thenonpolar ones was set to 22,300 cm�1 (between the acetone andchloroform peak positions). Linear relationships resulting from fitof tmax and tmin against tm dependences are:

tmin ¼ �958:4þ 0:966 � tm

tmax ¼ 1688:8þ 0:986 � tm for

tm < 22;300 cm�1 ðpolar solventsÞ ð3Þ

and

t�

min ¼ 1150:7þ 0:877 � t�

m

t�

max ¼ �99:3þ 1:058 � t�

m for

t�

m � 22;300 cm�1 ðnon-polar solventsÞ: ð4Þ

As stated before, LN function is controlled by four parameters:the amplitude (Im) and the shape parameters (tm, tmin, and tmax).Considering the linear relationships for tmin and tmax against tm

as constraints, the number of independent parameters has been re-duced to two (intensity and peak position). This fact provides amore robust fit and at the same time forces the selection of thepeak shape according to the empirical calibration provided bythe linear constraints. Thus, the elementary peak shape will alwaysbe correlated with the observed shape spectrum emitted in anappropriate polarity solvent.

Table 1Spectral characteristics of Laurdan emission in homogeneous solutions of solvents with different polarization.

Solvent tm

(cm�1)tmin

(cm�1)tmax

(cm�1)FWHMa (cm�1) q R2b

NonpolarCyclohexane 24,814 23,048 26,349 3301 1.391 0.9978Hexane 24,752 23,025 26,416 3390 1.393 0.9982Toluene 23,753 21,935 25,031 3096 1.375 0.9996Benzene 23,641 21,884 24,948 3064 1.374 0.9996Cloroformc 22,523 20,659 23,809 3150 1.249 0.9999

Polar aproticEthyl acetate 23,095 21,238 24,392 3154 1.286 0.9999Tetrahydrofuran 23,041 21,237 24,342 3105 1.289 0.9998Acetone 22,272 20,517 23,644 3127 1.232 0.9999Dimethylformamide 21,786 20,038 23,117 3078 1.226 0.9999Acetonitrile 21,692 20,024 23,130 3106 1.200 0.9999Dimethyl sulfoxide 21,413 19,684 22,744 3060 1.215 0.9999

Polar proticIsopropanol 20,704 19,101 22,082 2981 1.183 0.9993Propanol 20,661 19,098 22,073 2976 1.183 0.9993Butanol 20,492 18,836 21,843 3007 1.178 0.9993Ethanol 20,161 18,541 21,588 3047 1.180 0.9996Methanol 19,763 18,121 21,164 3044 1.167 0.9996

a Full width at half-maximum of fitted spectrum.b R2 coefficient characterizing the quality of fit.c Chloroform is classified either as nonpolar, based on its very low miscibility with water (<1%), or as polar, based on the asymmetry of its charge distribution (it has a

dipole moment of 1.5 D comparable to that of polar aprotic solvents). For this reason, its peak position is actually more left-shifted than that of the two polar aprotic solvents(ethyl acetate and tetrahydrofuran).

Fig.2. (A) Laurdan emission spectra in several solvents. (B–D) Three examples of Laurdan spectra fitted by LN function.

126 M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129

Fig.3. (A) Peak position against polarity of solvents in ET(30) scale. (B) Shape-parameters (tmin and tmax) against peak position for all the considered solvents.

Fig.4. (A) Complex spectra of Laurdan emitted from heterogeneous medium ofDMPC LUV lipid bilayer for range of temperatures from 5 to 55 �C (normalizationwas done at the maximum intensity of emission at 5 �C). (B) Comparison betweendecomposition of complex spectrum (at 15 �C) by LN functions and by Gaussianfunctions. For an simpler view of the graphs, some of the values of raw data havebeen omitted.

M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129 127

Decomposition of emission spectra of Laurdan

The complex spectrum of Laurdan was analyzed as a superposi-tion of two LN functions, one for each of the two excited states ofLaurdan molecules: nonrelaxed and relaxed states.

The procedure was applied to the emission spectra of Laurdaninserted into LUVs prepared from DMPC. A comparison with the re-sults provided by GP evaluation and the Gaussian decompositionmade on the same spectra is presented below. An example of thespectra recorded for an LUV suspension in a range of temperaturescovering the lipid transition from gel to liquid crystalline phase ispresented in Fig. 4A.

The spectra in Fig. 4A show the typical pattern of Laurdan emis-sion from LUVs with a high emission peak located at approxi-mately 440 nm at low temperature and a smaller one located atapproximately 490 nm at high temperature (see Refs. [18,26] fora comparison with other similar results). The spectra are highlyasymmetric, with an important emission in blue and green chan-nels even in the extreme values of temperature where the lipid bi-layer is presumed to be in one phase (gel or liquid crystalline). Anelaborate analysis of Laurdan emission from liposomes, conductedusing the multivariate frequency domain fluorescence techniqueproved that Laurdan in DMPC LUVs emits from three states: locallyexcited state (�415 nm), charge transfer state (�435 nm), and sol-vent relaxed state (�490 nm) [14]. In our steady-state recordings,we found only two emission bands (one at �440 nm and the sec-ond at �490 nm) corresponding to the charge transfer and solventrelaxed states reported by Rowe and Neal [14]. According to thesame reference, the molecules in the locally excited state areswitching very fast to the charge transfer state, resulting in thesteady-state spectrum with almost zero intensity of the locally ex-

cited state. This explains why in our recordings the locally excitedstate is not visible. Beside the LN decomposition, performed as de-scribed above, decomposition with two Gaussians was performedusing the OriginPro software package tools. Comparing the twodecomposition procedures we used, the fit was good in both cases,but the one case using LN functions was better (R2 = 0.999 vs. 0.998for Gaussian decomposition). Nevertheless, the results of decom-position are completely different from one procedure to the other.A comparison between the decomposition of complex spectrum byLN functions and by Gaussian functions is exemplified in Fig. 4B fora spectrum recorded at 15 �C. The LN decomposition provides twopeaks located at approximately 440 nm and 490 nm. Because atthis low temperature the bilayer is mostly in the gel phase (theDMPC transition temperature is 23 �C) and the majority of theLaurdan molecules emit from the nonrelaxed state, a higher areaof the peak located at approximately 440 nm is expected. In con-trast with this, the Gaussian decomposition provides a peak inthe blue channel at a bit lower wavelength (�435 nm) and the sec-ond one in the green channel at a significantly lower value(�465 nm) as compared with the positions provided by LN decom-position. Comparing the FWHM of the Gaussian peaks with the val-ues recorded on Laurdan emission in homogeneous solution, aclear discrepancy can be observed for the FWHM of the blue peak(�2357 cm�1), which is significantly smaller than the usual valueof approximately 3000 to 3300 cm�1 in organic solvents (see Ta-ble 1). The reason is given by the symmetry of the Gaussian func-tion. The blue peak especially fits the sharp shape of the complexspectrum at low wavelengths (<440 nm) and consequently isforced to be narrow.

128 M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129

The difference is deeper when comparing the areas of the peaks.Completely unexpected, the Gaussian decomposition provides alower peak area in the blue channel, suggesting that even at lowtemperature (when the bilayer is in the gel phase) most of theLaurdan molecules emit from the relaxed state. According to theGaussian decomposition results, the bilayer has a sufficiently highlevel of hydration even in the gel phase. An explanation can befound again based on the symmetry of the Gaussian function.The blue symmetric peak, being very narrow, covers only a smallerarea of the complex spectrum, forcing the presence of a secondpeak at approximately 460 nm that needs to cover the rest of thespectrum. Consequently, it results in this unexpected distributionof the complex spectrum area between the peaks. In the case ofthe LN function, the larger blue peak covers nearly all of the spec-trum area, and only a small peak in the green channel is necessaryfor a good fit.

Similar results can be found in other reports using the Gaussiandecomposition for analyzing the Laurdan complex spectrum inLUVs [17,18,26]. A complete analysis of the records from Fig. 4Ais given in Fig. 5. The LN decomposition provides stable positionsof the peaks, whereas the Gaussian one predicts a gradual shiftof the high wavelength peak from approximately 460 to 490 nmfor the entire range of temperatures (Fig. 5A). These results suggestdifferent scenarios for the molecular processes produced duringthe lipid bilayer phase transition. According to LN decomposition,

Fig.5. (A,B) Elementary peak positions (A) and relative areas (B) resulting fromfitting procedures. (C) A comparison among different parameters can be used toglobally describe the spectral changes in Laurdan emission (GP, DSr differencebetween relative areas of elementary LN peaks, and ratio of LN peak areas). Theresults are means ± standard deviations on three repetitions of the recordings.

the transition starts in some localized sites where the water pene-trates up to the Laurdan fluorescence moiety and saturates therelaxation process (these Laurdan molecules switch from the non-relaxed state to the relaxed state). The number of these sites in-creases with temperature. Such a scenario is close to thecoexisting phase domains of the main phase transition as describedfor dipalmitoylphosphocholine (DPPC) LUVs by fluorescence spec-troscopy [27] and by Monte Carlo simulations [28]. As reported bythe Gaussian decomposition, there is an enhanced number of watermolecules that reorient around the Laurdan dipole in the nonre-laxed state. As the penetration of water in the lipid bilayer isincreasing with temperature, the gradual loss of energy results ina continuous shift of peak position toward 490 nm, where the sol-vent relaxation process is saturated.

Analyzing the evolution of elementary peak areas (Fig. 5B), thescenario resulting from LN decomposition became more reliable.For this type of decomposition, the relative area of the peak inthe blue channel, emission from the nonrelaxed state (SrB LN), isnearly 1 at low temperature, suggesting that the water moleculespenetrate with great difficulty in the lipid bilayer because the bi-layer is in the gel phase. Increasing the temperature, SrB LN de-creases (the decrease is faster in a range around the phasetransition temperature) up to approximately 0.25 in the liquidcrystalline phase. The peak area for the emission from the relaxedstate (SrG LN) has an opposite variation. A completely different pic-ture arises from the Gaussian decomposition (Fig. 5B). The relativearea of the peak in the blue channel (SrB Gauss) starts from low val-ues (�0.35) and decreases more with the temperature up to nearlyzero. Moreover, the peak in the green channel (SrG Gauss) startsfrom high values (�0.65) and increases more with temperature.Based on this model, the degree of hydration of the lipid bilayer(as reported by Laurdan) found in the gel phase is quite high(>50%), a result that is difficult to match with the usual pictureof the lipid bilayer in the gel phase. The evaluation of the hydrationlevel of the bilayer by means of another fluorescent probe (F2N8)revealed that hydration in the liquid crystalline phase is 2 timeshigher compared with gel phase [29]. However, the F2N8 is locatedbetween the sn-1 carbonyls and phosphorus planes, whereas theLaurdan fluorescent moiety is located at approximately 10 Å fromthe middle plane of the lipid bilayer, along the normal direction[30], close to the sn-1 carbonyl plane [11] as proved by parallaxmethod measurements. Consequently, there is a smaller probabil-ity for Laurdan than for F2N8 to interact with water molecules, anda smaller hydration level can be reported by Laurdan. A moleculardynamics simulation of the phosphatidylcholine bilayer hydrationsuggests a double number of bond water molecules in the liquidcrystalline phase compared with the gel phase [31], and the predic-tion is confirmed by the experimental findings based on F2N8 fluo-rescence. In the same report, the water density at the location ofLaurdan is approximately 8 times higher in the liquid crystallinephase compared with the gel phase, in good agreement with ourresults (SrG LN is increasing �8 times over the phase transition;see Fig. 5B).

The relative areas of the elementary peaks can be used to definea parameter similar to GP, the difference of relative areas: DSr = SrB

LN – SrG LN. This parameter depends, due to the peak areas, on thefractions of emitting molecules in each state and, consequently,will actually range from �1 (emission from relaxed state only) to1 (emission from nonrelaxed state only). DSr and GP for the record-ings on DMPC LUVs are plotted in Fig. 5C. Indeed, in our recordingsthe DSr start from high values (�0.85) and drop to approximately�0.6, covering a significantly larger range than GP and becomingmore sensitive than GP. Another parameter proposed in the litera-ture [26] to characterize the changes in Laurdan spectrum is the ra-tio of elementary peak areas (SG/SB), and it is depicted in Fig. 5C forour recordings on DMPC LUVs analyzed by LN decomposition. This

M. Bacalum et al. / Anal. Biochem. 440 (2013) 123–129 129

parameter has a sharp increase starting close to the phase transi-tion temperature.

Conclusion

In this article, we have proposed a new method to evaluate thecomplex spectrum of Laurdan inserted into the membrane of LUVs.This method is based on a decomposition algorithm that uses theasymmetric LN function as a validated analytical model for theshape of elementary peaks. An empirical calibration of the decom-position algorithm is generated that relies on the relationshipsamong the shape parameters of the LN function as derived fromthe analysis of Laurdan emission in homogeneous solutions of sol-vents with various polarities. This procedure allows for a bettercharacterization of the hydration level at the Laurdan location, ingood agreement with the literature data. The new parameter pro-posed to evaluate Laurdan emission spectra (DSr) proved to be amore sensitive parameter than GP.

Although in this study we analyzed only the data recorded onDMPC LUVs, the method described here can be extended to vesicleswith different lipid compositions. Further studies are directed to-ward the analysis of more complex lipid systems.

Acknowledgments

M.B. is preparing a PhD under a bilateral agreement betweenthe University of Bucharest (Romania) and Hasselt University (Bel-gium). This work was supported by Grant POSDRU/88/1.5/S/56668and by national grant PNII-123/2012 from the Romanian Ministryof Research.

References

[1] B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley–VCH,Weinheim, Germany, 2002.

[2] B.D. Siano, D.E. Metzler, Band shapes of the electronic spectra of complexmolecules, J. Chem. Phys. 51 (1969) 1856–1861.

[3] E.A. Burstein, V.I. Emelyanenko, Log-normal description of fluorescence spectraof organic fluorophores, Photochem. Photobiol. 64 (1996) 316–320.

[4] E.A. Burstein, S.M. Abornev, Y.K. Reshetnyak, Decomposition of proteintryptophan fluorescence spectra into log-normal components: I.Decomposition algorithms, Biophys. J. 81 (2001) 1699–1709.

[5] V.I. Emel’ianenko, I. Reshetniak, O.A. Andreev, E.A. Burstein, Analysis of log-normal components of fluorescence spectra of Prodan and Acrylodan bound toproteins, Biofizika 45 (2000) 207–219.

[6] A. Kalauzi, D. Mutavdzic, D. Djikanovic, K. Radotic, M. Jeremic, Application ofasymmetric model in analysis of fluorescence spectra of biologically importantmolecules, J. Fluoresc. 17 (2007) 319–329.

[7] W. Caarls, C.M. Soledad, A.P. Demchenko, T.M. Jovin, Characterization ofcoupled ground state and excited state equilibria by fluorescence spectraldeconvolution, J. Fluoresc. 20 (2010) 181–190.

[8] G. Weber, F.J. Farris, Synthesis and spectral properties of a hydrophobicfluorescent probe: 6-Propionyl-2-(dimethylamino)naphthalene, Biochemistry18 (1979) 3075–3078.

[9] J.R. Lakowicz, J.R. Sheppard, Fluorescence spectroscopic studies of Huntingtonfibroblast membranes, Am. J. Hum. Genet. 33 (1981) 155–165.

[10] T. Parasassi, F. Conti, E. Gratton, Time-resolved fluorescence emission spectraof Laurdan in phospholipid vesicles by multifrequency phase and modulationfluorometry, Cell Mol. Biol. 32 (1986) 103–108.

[11] P. Jurkiewicz, A. Olzynska, M. Langner, M. Hof, Headgroup hydration andmobility of DOTAP/DOPC bilayers: a fluorescence solvent relaxation study,Langmuir 22 (2006) 8741–8749.

[12] A. Samanta, R.W. Fessenden, Excited state dipole moment of PRODAN asdetermined from transient dielectric loss measurements, J. Phys. Chem. A 104(2000) 8972–8975.

[13] S.A. Sanchez, M.A. Tricerri, G. Gunther, E. Gratton, Laurdan generalizedpolarization: from cuvette to microscope, in: A. Méndez-Vilas, J. Diaz (Eds.),Modern Research and Educational Topics in Microscopy, FORMATEX, Badajoz,Spain, 2007, pp. 1007–1014.

[14] B.A. Rowe, S.L. Neal, Photokinetic analysis of PRODAN and LAURDAN in largeunilamellar vesicles from multivariate frequency-domain fluorescence, J. Phys.Chem. B 110 (2006) 15021–15028.

[15] T. Parasassi, G. De Stasio, A. d’Ubaldo, E. Gratton, Phase fluctuation inphospholipid membranes revealed by Laurdan fluorescence, Biophys. J. 57(1990) 1179–1186.

[16] T. Parasassi, G. De Stasio, G. Ravagnan, R.M. Rusch, E. Gratton, Quantitation oflipid phases in phospholipid vesicles by the generalized polarization ofLaurdan fluorescence, Biophys. J. 60 (1991) 179–189.

[17] C.C. De Vequi-Suplicy, C.R. Benatti, M.T. Lamy, Laurdan in fluid bilayers:position and structural sensitivity, J. Fluoresc. 16 (2006) 431–439.

[18] A.D. Lucio, C.C. Vequi-Suplicy, R.M. Fernandez, M.T. Lamy, Laurdan spectrumdecomposition as a tool for the analysis of surface bilayer structure andpolarity: a study with DMPG, peptides, and cholesterol, J. Fluoresc. 20 (2010)473–482.

[19] M. Vincent, B. de Foresta, J. Gallay, Nanosecond dynamics of a mimickedmembrane–water interface observed by time-resolved Stokes shift ofLAURDAN, Biophys. J. 88 (2005) 4337–4350.

[20] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York,2006.

[21] A. Kawski, B. Kuklinski, P. Bojarski, H. Diehl, Ground and excited state dipolemoments of LAURDAN determined from solvatochromic and thermochromicshifts of absorption and fluorescence spectra, Z. Naturforsch. A 55 (2000) 817–822.

[22] F. Moyano, M.A. Biasutti, J.J. Silber, N.M. Correa, New insights on the behaviorof PRODAN in homogeneous media and in large unilamellar vesicles, J. Phys.Chem. B 110 (2006) 11838–11846.

[23] K.A. Kozyra, J.R. Heldt, J. Heldt, M. Engelke, H.A. Diehl, Concentration andtemperature dependence of Laurdan fluorescence in glycerol, Zeitschrift fürNaturforschung A 58 (2003) 581–588.

[24] M. Viard, J. Gallay, M. Vincent, O. Meyer, B. Robert, M. Paternostre, Laurdansolvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction, Biophys. J. 73 (1997) 2221–2234.

[25] Y.P. Morozova, O.M. Zharkova, T.Y. Balakina, V.Y. Artyukhov, B.V. Korolev,Conformal transitions of the Laurdan molecule in the absorption andfluorescence spectra, Russ. Phys. J. 54 (2011) 594–600.

[26] D. Ionescu, C. Ganea, A study of quercetin effects on phospholipid membranescontaining cholesterol using Laurdan fluorescence, Eur. Biophys. J 41 (2012)307–318.

[27] A.J. Metso, A. Jutila, J.P. Mattila, J.M. Holopainen, P.K.J. Kinnunen, Nature of themain transition of dipalmitoylphosphocholine bilayers inferred fromfluorescence spectroscopy, J. Phys. Chem. B 107 (2003) 1251–1257.

[28] J.H. Ipsen, K. Jorgensen, O.G. Mouritsen, Density fluctuations in saturatedphospholipid bilayers increase as the acyl-chain length decreases, Biophys. J.58 (1990) 1099–1107.

[29] G. M’Baye, Y. Mely, G. Duportail, A.S. Klymchenko, Liquid ordered and gelphases of lipid bilayers: fluorescent probes reveal close fluidity but differenthydration, Biophys. J. 95 (2008) 1217–1225.

[30] S.S. Antollini, F.J. Barrantes, Disclosure of discrete sites for phospholipid andsterols at the protein–lipid interface in native acetylcholine receptor-richmembrane, Biochemistry 37 (1998) 16653–16662.

[31] M. Stepniewski, A. Bunker, M. Pasenkiewicz-Gierula, M. Karttunen, T. Rog,Effects of the lipid bilayer phase state on the water membrane interface, J.Phys. Chem. B 114 (2010) 11784–11792.