NONLINEAR OPTICAL PROPERTIES OF POTENTIAL

SENSITIVE STYRYL DYESJUNG YAW HUANG, AARON LEWIS, AND LESLIE LOEW*Department ofApplied Physics, Cornell University, Ithaca, New York 14853; and *Department OfPhysiology, University of Connecticut Health Center, School of Medicine, Farmington, Connecticut06032

ABSTRACT The nonlinear optical properties of dyes that alter their optical characteristics rapidly with membranepotential are described. The second harmonic signals from these dyes characterized in this paper are among the largestthat have been detected to date. Structural conclusions are drawn from the second harmonic signals generated by theLangmuir Blodgett monolayers used in these measurements. Our results indicate that with appropriate instrumentationsecond harmonic signals could readily be detected from living cells stained with these dyes.

INTRODUCTION

Recently, in cell biology, there has been great progress insynthesizing molecules that have rapid alterations inoptical properties in response to electrical fields across cellmembranes (1-5). In one class of molecules, the essentialaspect that allows them to respond rapidly to such mem-brane potential variations appears to be a large alterationin the charge distribution within the chromophore inresponse to optical excitation (6). Thus, the effect of anelectrical field is to perturb this light-induced chargedistribution which causes a rapid and readily detectableeffect on the absorption spectrum of the dye in question. Inthis paper the nonlinear optical properties of these dyes arestudied, and it is demonstrated that they exhibit some ofthe largest second order nonlinear hyperpolarizabilitiesthat have been measured thus far.The dyes considered in this investigation were designed

by the use of molecular orbital calculations that allowedthe prediction of the molecular structures that would givethe largest light-induced dipole alterations (7). As a resultof these calculations molecules were synthesized that hadnegative charges that were covalently linked to a positivelycharged pyridinium styryl structure (8). The moleculeschosen for this study are shown in Fig. 1.To study the nonlinear optical properties of such mole-

cules, we generated monolayer films of the pyridiniumstyryl dyes on a Langmuir trough (9). The structures of thefilms were investigated by observing their nonlinear opticalproperties (10, 1 1). The results of this study are consistentwith the notion that these molecules have unique electro-optical characteristics.

Address reprint requests to Aaron Lewis, Division of Applied Physics,Bergmann Bldg., The Hebrew University of Jerusalem, Jerusalem,Israel.

EXPERIMENTAL PROCEDURES

A Q-switched frequency doubled Nd:YAG laser with a 10-Hz repetitionrate and 10-ns pulsewidth was used. The Nd:YAG laser was adjusted togive a green 532-nm beam of <20 mW, which was focused onto a 3-mmdiameter spot on the sample surface after passing through a half-waveplate, a Glan-Thompson laser prism polarizer, and a KG-5 color glassfilter. The reflected second harmonic signal at a wavelength of 266 nmwas passed through a UG-5 color glass filter which blocked the funda-mental at 532 nm. The UG-5 filter was followed by a UV Glan-Taylorpolarizer and a Schoeffel 0.2-m double monochromator. The SH signalwas detected by a cooled RCA C3 1034 photomultiplier and averaged by aStanford Research Model SR250 boxcar integrator.The dyes were all synthesized by the aldol condensation or Pd-

catalyzed coupling procedures as detailed by Hassner et al. (12). Thespreading solutions were prepared by adding a specific amount of dyemolecules into spectrophotometry grade chloroform (Fisher ScientificCo., Pittsburgh, PA) according to the published extinction coefficients(8). The molecules were spread on the surface of deionized distilled purewater subphase at pH 6.4.The Langmuir trough was made out of Teflon. A movable barrier

controlled the surface density of molecules and a #40 filter paper withdimension 0.7 x 1.0 cm was attached to a UC-3 force transducer (GouldInc., Santa Clara, CA) to measure the surface pressure (13). Duringdetection of the second harmonic signal, the movable barrier wascontrolled by a stepping motor such that the surface pressure of the filmwas kept at constant value.

Molecular orientation was determined by using the Heinz nullingtechnique (14). The incident pump beam was arranged to satisfy thefollowing condition:

[el (w)/e±Q(W)]2 = 2 (1)

where e(Z) is the product of the polarization vector and the Fresnel factorfor the pump field in the monolayer. The subscripts denote the compo-nents of e(w) on or perpendicular to the layer plane. This condition can bemet by adjusting the pump beam to have an angle of incidence of 450 anda polarization of 35.30 away from the incident plane. The molecularorientation was determined by the second harmonic extinction direction,as indicated by the UV polarizer.Our detection sensitivity was calibrated against the second harmonic

intensity from an x-cut quartz plate, observed under the same experimen-tal conditions. The quartz plate was rotated 28.750 about the y-axis of the

BIOPHYS. J. © Biophysical Society - 0006-3495/88/04/665/06 $2.00Volume 53 May 1988 665-670

665

SO \ H2n+1

(ml dI-m-ASPPS

so- - 73

-

(b) di-4-ANEPPS

FIGURE 1 The chemical structures of (a) the di-n-ASPPS and (b) thedi-4-ANEPPS dye molecules.

crystal to obtain one of the maxima of the Maker's fringes. Thepolarizations of the fundamental and second harmonic beams werechosen to be p-polarized relative to the plane of incidence. In this case, thetwo beams are the extraordinary waves of the quartz crystal. Using theknown nonlinear second order susceptibility d,, = 2.4 x 10 esu (15) andthe index of refraction (16) of quartz at 532 and 266 nm, we obtained aratio of (17)

Ip_p (2w)/ [I4(W)]2 = 3.96 x 10-26 cm2 erg- 1, (2)

where the first index in the subscript indicates the polarization of thefundamental beam and the second index for that of the second harmonicsignal. The p (or s) denotes that the polarization vector is parallel (orperpendicular) to the plane of incidence. For measurements of dispersionof the dye molecular hyperpolarizabilities, a fundamental beam from adye laser which was pumped by the Nd:YAG pulse laser was used. Thesame thin x-cut quartz plate was used as the calibration standard byassuming that the quartz plate has negligible dispersion for its second-order susceptibility.

mation in the monolayer may break this mirror symme-try.

Monolayer Molecular OrientationThere are two approaches to obtaining the angular

orientation of the styryl dyes in the monolayer. The firstmethod involves measuring and calculating the intensity ofthe SH signal under different polarization conditions, s, p,and 45°. A second method is to extinguish the SH signal byrotating a polarizer in the output beam relative to a fixedinput polarization.

For the first approach, the SH intensities of Is_p andIp_p for di-4-ANEPPS and di-6-ASPPS monolayers atvarious surface pressures are compared in Fig. 2, a and b.In di-4-ANEPPS, the ratio of Ip_p/I.,p was found todecrease from 0.4 to 0.1 with the increase of the surface

v

--

Qc.11

-o

c

Isp

di-6-ASPPS

pp

0.0 5.0 10.0 15.0 20.0

surface pressure

25.0 30.0 35.0 40.0

(dyn/cm)

RESULTS AND DISCUSSION

Monolayer Molecular Symmetry PropertiesThe s-polarized second harmonic (SH) intensity for theexcitation of a p-polarized fundamental beam, Ip,s, wasmeasured to characterize the symmetry properties of themonolayer structure of the pyridinium styryl dyes thatwere the focus of this study. For the di-4-ANEPPS dyemonolayer at 16 dynes/cm of surface presssure, the Ip-, is-10% of the I_p. The Ip_, SH intensity is proportional tothe absolute square of the xyz component of the surfacesusceptibility tensor, x(2) . This component vanishes if theLangmuir film possesses a mirror plane containing thetrough surface normal. However, molecules without mirrorplanes cannot be arranged in such a way as to give thewhole ensemble mirror symmetry. Thus when there ismirror symmetry, a mirror plane which contains themolecular long axis must exist in the molecule as well (1 8).The high value of Ip4 (10% of 1,p) that we observeindicates that although the chromophores have a mirrorplane which contains the molecular long axis, the confor-

0OC)0

2:1 ojn7 IZc (::)

c

IEU)

-o0

t4 0

0E0c

0IIsp

di-4-ANEPPS

o

0

0q0

o.C

-pp

5.0 10.0 15.0 20.0 25.0surfoce pressure (dyn/cm)

30.0 35.0 40.0

FIGURE 2 The second-harmonic (SH) intensities of styryl dye mono-layers, (a) di-6-ASPPS and (b) di-4-ANEPPS, at different surfacepressures. The p-polarized SH intensity with an s-polarized fundamentalbeam, I,-p, is shown in open circles. Solid circles are for I,-, SH intensitygenerated by a p-polarized fundamental beam. The laser beam was set atan incident angle of 450 in all experiments.

BIOPHYSICAL JOURNAL VOLUME 53 1988

00

0Iq

-0

D

2.,in- I.D

Co00

000

00

I

0

666

pressure for the monolayer. For di-6-ASPPS monolayer,this ratio was found to decrease from 0.12 at 0 dynes/cm ofsurface pressure to 0.05 at high surface pressure. Tounderstand the difference of this variation in the Ip_p toIs_p ratio between the two monolayers, calculations wereperformed using previously derived equations (19), and theresults are shown in Fig. 3 where the intensities of threedifferent polarized SH beams, IS-P. Ip_p, and I45-p, areplotted at various incident angles. To calculate theseintensities as a function of the incident laser angle, threequantities are needed. These quantities are the componentsof the hyperpolarizability tensor, ,B, the orientationalparameter, i, which is the angle between the molecularplane and the water surface and the molecular inclinationangle, 0, which is the polar angle of the molecule relative tothe normal surface. The two in-plane components of themolecular hyperpolarizability tensor, fBzzz and ozxx, aredominant for these types of molecules as reported by Dirket al. (20). The values estimated by Dirk et al. are lzzz =6 x 10-28 esu and Izxx = -3.6 x 10-28 esu. For thecalculation, the orientational parameter, it, was assumed tobe either randomly distributed or equal to 00. For therandom distribution assumption the result for Is-p is shownas a solid line in Fig. 3 and the ' = 00 assumption is shownas a curve with boxes. In the above calculations themolecular inclination angle, 0, was chosen to be 550 with awidth of ± 100. The ratio of Ip-pII,-p for the case of 4' = 00is found to be 0.47 at the incident angle of 450 but itbecomes 0.08 for the case of 4 being randomly distributed.

The measured value of Ip-p/I,-p = 0.4 for di-4-ANEPPSmonolayer with an incident angle of 450 and under lowsurface pressure indicates that the di-4-ANEPPS dyemolecule due to its larger ir-electron system and planarring structure tends to contact the water surface with apreferred geometry of ' = 00. For the di-6-ASPPS mono-layer, the geometry with the randomly distributed 4' angleis chosen since the measured ratio of Ip_p to Is_p is found tobe close to the values graphically represented in Fig. 3 forrandom systems. In Fig. 2, a and b, the small but finitevalue of Ip_p at an incident angle of 450 for the di-6-ASPPS and di-4-ANEPPS monolayers at all surfacepressures indicates that the out-of-plane transition moment

0

_7 O

I C)

-r

Lnn

0 L80.0

'4

22.3

di-6-ASPPS

.0 /

o1/..7I/,

90.0 100.0 110.0 120.0 1 30.0 140.0 1 50.0 1 60.0SH extinction angle (degrees)

90

rl

.Oc

- (

.-

(1)

c o

-r

0.0

0

oo

o~~~~~o o

22.5 45.0Incident Angle (Degrees)

67.5

FIGURE 3 The calculated intensities of three different polarized second-harmonic beams, I,sp, Ip_p, and 145_p, are plotted at various incidentangles. The two most significant in-plane components of the modelmolecular hyperpolarizability tensor were chosen to be ,,,,,= 6 x 10-28esu and i,,, =- 3.6 x 10-28 esu, respectively. The molecular inclinationangle, 0, has a central value of 550 with a width of ± 100. The orientationalparameter, st, which is related to the angle between the molecular planeand the water surface, was assumed to be either randomly distributed(solid line, dashed line, and dashed-dotted line) or equal to 0° (shown inthe curves with open boxes, open circles, and open triangles).

di-4-ANE PPS3

90.0 _I

180.0 90.0 100.0 110.0 120.0 130.0SH extinction angle (degrees)

140.0 150.0 160.0

FIGURE 4 The extinction angle measurements of the second-harmonic(SH) beams for the (a) di-6-ASPPS monolayer at surface pressures of 3,14, 22, and 32 dynes/cm and (b) di-4-ANEPPS monolayer at surfacepressures of 3, 16, and 26 dynes/cm, respectively. The x-axis indicates therotational angle of the SH beam polarization analyzer, where 900corresponds to the p-polarized direction. The polarization of the incidentpump beam is adjusted to be 35.50 from the plane of incidence. The linesin this figure are generated by a cubic spline fit to the experimental data(symbols) to guide the reader.

HUANG ET AL. Nonlinear Properties ofStyryl Dyes

0

0

Co

ISP V =0-C3

0

0

ur,0

0

U?

0

c

-r

Vl)

0

0

667

is small and that there is no single dominant component ofthe hyperpolarizability tensor.As noted above an alternate approach to measure the

molecular inclination angle, 0, in the monolayer is tomeasure the SH extinction angle. In Fig. 4 a, the extinctioncurves for di-6-ASPPS monolayer at surface pressures of3, 14, 22, and 32 dynes/cm are shown. The angle of thepolarization at which the minimum SH intensity wasobserved gradually increases with the surface pressure.The typical angle for this monolayer is -1200 when thesurface pressure is -22 dynes/cm. The same measure-ments were also made for the di-4-ANEPPS monolayer.Three extinction curves at surface pressures of 3, 16, and26 dynes/cm are shown in Fig. 4 b. An extinction angle of110° was observed for the monolayer when the surfacepressure is above 16 dynes/cm. By choosing the molecularhyperpolarizability component zzz = 9 x 10-28 esu and a

q

co01

04

o

*to \\\\.\ /'/I ~~~~~~~~~~~~~~~~~~~~~~~~601-'

C14'~~ ~ ~ ~ ~ ~ /

I /

o

0.0 45.0 90.0 135.0

Rotation Angle of SH Analyzer (Degrees)

0oo

0

(0

0

oX~ o.N a

Ea

>0'i0

LIr

06

\

\ \ \\

\-N

\.

0.0 45.0 90.0 135.0Rotation Angle of SH Analyzer (Degrees)

/

randomly distributed orientational parameter, 1, threecalculated SH extinction curves are shown in Fig. 5 for themolecular inclination angle 0 = 500 (solid line), 550(dashed line), and 600 (dashed-dotted line). The measuredSH extinction data of di-6-ASPPS monolayer at a surfacepressure of 3 dynes/cm (see Fig. 4 a) compares very wellwith the calculated curve for the molecular inclinationangle of 550. To fit satisfactorily the measured curves atsurface pressures of 14, 22 and 32 dynes/cm, the molecularinclination angle had to be decreased to 51, 49, and 470.Similar calculations were also made for the monolayer ofthe di-4-ANEPPS molecule with the molecular hyperpo-larizability 3zzz = 6 x 1028 esu and a randomly distrib-uted orientational parameter i. Three curves with themolecular inclination angles of 55, 65, and 750 are shownin Fig. 5 b. Compared with the measured SH extinctiondata for the di-4-ANEPPS monolayer, the calculatedcurve with the inclination angle of 550 simulates quite wellthe measured SH extinction data at the surface pressuresof 16 and 26 dynes/cm. The correlations of the measuredand calculated SH extinction curves have revealed areasonable picture that at low surface pressures the di-4-ANEPPS molecule with the more extended ir-electronsystem tends to lie flatter on the water surface than thedi-6-ASPPS molecule does. However, at high surfacepressures both molecules exhibit approximately similarinclination angles.The preceding discussion of the extinction angle and

monolayer structure did not consider the observation ofmore than one contributing hyperpolarizability compo-nent. When molecules such as styryl dyes are considered,where there is not one dominant hyperpolarizability com-ponent (20), the effect of other components has to beconsidered especially when there are nonrandom distribu-

00to

ai,IU) o_

180.0 90

0.0

FIGURE 5 The calculated extinction curves of the SH beams from (a)di-6-ASPPS and (b) di-4-ANEPPS monolayers. The geometries of thepump and SH beams are the same as those described in Fig. 4. In thiscalculation, the orientational parameter, 4,, was assumed to be randomlydistributed. The molecular inclination angle, 6, is indicated near thecurves.

1' = 0.

03

0000 0 0 0a0

~~~~~~ /000

.1- 0 - 0

- ,_F '~~ '' <'~~ 0 11a~~~"I

I- &0

0.3

45.0 90.0 135.0Rotation Angle of SH Analyzer (degrees)

180.0

FIGURE 6 The calculated extinction curves of the SH beams underdifferent values of f,,,. In this calculation, the chosen parameters are 0 =550, A/ = 00, and f,. = 6 x 10-28 esu. The second hyperpolarizabilitycomponent, fl,., was determined by a parameter r with the definition r =f3,,/1. and this parameter was varied from -0.3 to +0.3.

BIOPHYSICAL JOURNAL VOLUME 53 1988

C?I

S)10

6O

O [oo

I

r-i0N4

668

0o6

0u-i

0

10'o

I)0

o

sn O

,io

r-i

q0

0o

t /e °f=6x/0-28 esu

9=65'

0.0 45.0 90.0 135.0

Rotation Angle of SH Anolyzer (Degrees)

E

180.0

FIGURE 7 The same calculation as Fig. 5 b except that the molecularinclination angle, 6, was assumed to be 650 and with a spread of ± 100(solid line) and ± 10 (solid circles), respectively.

tions of the molecular plane relative to the surface. Suchconditions are exemplified by ,6 = 00. The results ofcalculating the extinction curves under the condition of A1 =00 where the effect of the fzxx component has to beconsidered is shown in Fig. 6 for a defined inclination angle

== 55° + 100. In this figure, the second hyperpolarizabilitycomponent #zx was determined by a parameter r with thedefinition r = fzxx/fzzz and this parameter was variedfrom -0.3 to +0.3. The direction of the SH polarizationvector at which the minimum SH intensity is detecteddecreases from 152 to 900 as a function of r. The results inFig. 6 indicate that for di-4-ANEPPS at low surfacepressure where A, = 00 the inclination angle, 0, would beunderestimated if Ozxx is not taken into account.Our results yield little information on the width of the

molecular inclination angle, 0. This is seen in Fig. 7 wheretwo curves with widths of ±10 and ± 10 are compared in amodel calculation. No significant shifting and broadeningof the SH extinction curve is observed indicating the lack

cn *\ . ._ 30

r11

a-~~~~~~~~~~~~~~~~i4) O.- \ \

O o~~~~~N\00

0

0.0 15.0 30.0 45.0 60.0 75.0 90.0 105.0 120.0

Surface Area (A2/molecule)

FIGURE 8 The surface pressure-surface area (7r-A) isotherms of thedi-4-ASPPS (dashed line), di-4-ANEPPS (dashed-dotted line), di-6-ASPPS (solid circles), and the di-10-ASPPS (solid line) for Lang-muir-Blodgett monolayers on a 0.5 mM CdC12 water subphase (pH = 7),respectively.

of sensitivity of these measurements to the variation ininclination angle.

Wavelength DependenceDispersions for these dye molecular hyperpolarizabili-

ties are listed in Table I. The SH measurements wereperformed at surface pressures of 14, 24, 24, and 17dynes/cm for the di-4-ASPPS, di-6-ASPPS, di-10-ASPPS, and di-4-ANEPPS monolayers, respectively. Atthese surface pressures, the monolayers have a surfacedensity of 2 x IO'4 cm-2 (see Fig. 8 for the ur-A isotherms).Resonance is observed as the SH photon energyapproaches the absorption band of the dye molecules whichwere found to be -480 nm for dye monolayers on glasssubstrates. For these molecules, the contribution from thewater surface to the surface susceptibility could beneglected (9). Due to the lack of a near infrared laser,

TABLE IDISPERSIONS OF THE SURFACE SUSCEPTIBILITIES OF THE PYRIDINIUM STYRYL DYE MONOLAYERS

AT SURFACE DENSITY OF NS = 2 x 1014 MOLECULES/cm2

Di-4-ASPPS Di-6-ASPPS Di-10-ASPPS Di-4-ANEPPS(NS = 2 x 1014) (Ns= 2 x 1014) (Ns= 2 x 1014) (NS = 2 x 1014)

2XZX XZ2 2

Xzxx Xxx Xxx XzxxWavelength x 10-14 X 10-14 X 10-14 X 10-4

nm532 1.2 1.2 1.2 0.8546 0.4 0.4 0.4 0.4560 0.4 0.4 0.4 0.5584 0.7 0.7 0.7 0.8604 1.0 1.0 0.9 1.0624 0.8 0.8 0.8640 1.3 1.3 1.3 1.4660 2.5 2.4 2.4 2.51064 1.7 1.7 1.9 2.1

HUANG ET AL. Nonlinear Properties ofStyryl Dyes 669

dispersions above 680 nm were not measured except for themeasurement at 1.06 ,tm generated by the fundamentalbeam of the Nd:YAG laser. The replacement of the phenylring of di-n-ASPPS molecules by the naphthalene ofdi-4-ANEPPS has little effect on the SHG properties ofthese dye molecules.

CONCLUSION

The second-order nonlinear optical responses of four typesof pyridinium styryl dye molecules with different lengths ofir-electron systems and hydrocarbon side chains weremeasured in this study. The correlations of the measuredand calculated SH extinction curves have revealed that thedi-4-ANEPPS molecule tends to lie flatter on the watersurface than the di-6-ASPPS molecule. The experimentsalso indicate that the tilt angle between the plane of thedi-4-ANEPPS molecule and the water surface is notrandomly distributed at low surface pressure and thisallows for interactions between the water surface and thenonpyridinium nitrogen of the dye that cannot take placeat high surface pressure.

Based on these second harmonic measurements we arenow in a position to estimate whether such signals could bedetected in live cells stained with such dyes. For themeasurement of cell membrane potential with these dyesusing fluorescence detection, a concentration of -I1% rela-tive to membrane lipid is typically attained. This wouldyield a surface susceptibility of 2 x 10-16 esu for di-4-ANEPPS at 1.06 ,im and such a signal can be detectedeven in our low repetition rate system. If a mode-lockedNd:YAG laser system with a 10 ps pulsewidth and 10MHz repetition rate is used, the signal intensity couldincrease by several orders (21). The infrared nature of thelight source should be specifically important in preventingdamage mechanisms induced by the laser and thus allow-ing for extended illumination without bleaching. In addi-tion, the molecules endocytotically internalized into thecell will not contribute to the observed signal. Therefore,simply those molecules in the membrane that areresponding to the membrane potential will be responsiblefor the signal without any background from internalizeddye molecules. Thus, for all of these reasons the study ofalterations in the surface susceptibility as a function ofchanges in cell membrane potential should be most inter-esting. Furthermore, because the second harmonic signal isgenerated instantaneously the only limitation to the kineticdetection of membrane potential with this technique is theresponse time of the dye. For the dyes we have investigated,this response time is thought to be nanoseconds or better.

This research was supported by a grant from the United States Air ForceGrant #AFOSR-87-0381 to A.L. L.M. is pleased to acknowledge supportfrom the United States Public Health through National Institutes ofHealth Grant GM 35063.

Receivedfor publication 18 August 1987 and in finalform 3 December1987.

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670 BIOPHYSICAL JOURNAL VOLUME 53 1988