FromVesicle Size Distributions to Bilayer Elasticity via

Cryo-Transmission and Freeze-Fracture Electron

Microscopy
B. Col dren, R. van Zanten, M. J. Mackel , and J. A. Zasadzi nski *
Department of Chemical Engineering and Materials Research Laboratory,
University of California, Santa Barbara, California 93106-5080
Hee-Tae Jung
Department of Chemical and Biochemical Engineering, Korea Advanced I nstituteof Science&
Technology, 373-1 Guseong-dong, Yuseong-gu, Daejon 305-701, Korea
Received February 21, 2003. I n Final Form: April 18, 2003
Three methods of eval uati ng vesi cl e mean radi i and pol ydi spersi ty, quasi -el asti c l i ght scatteri ng (QLS),
freeze-fracture el ectron mi croscopy (FF-TEM), and cryo-transmi ssi on el ectron mi croscopy (cryo-TEM),
were used to determi ne the si ze di stri buti ons of spontaneous vesi cl es made from mi xtures of cetyl tri -
methyl ammoni um tosyl ate (CTAT) and sodi um dodecyl benzene sul fonate (SDBS). Whi l e QLS i s probabl y
the most commonl y used method tosi ze vesi cl es, i t i s l i mi ted tomeasures of the mean hydrodynami c radi us
and an esti mate of the pol ydi spersi ty, both of whi ch are heavi l y wei ghted toward the l argest structures
i n the sol uti on. Cryo-TEM can provi de the enti re si ze di stri buti on of the outer di ameters of spheri cal
vesi cl es, from whi ch the sum of the Hel fri ch bi l ayer el asti c parameters, K ) + j/2 and the spontaneous
curvature radi us, R0, can be determi ned. FF-TEM can provi de the number-average mean di ameter and
pol ydi spersi ty once the i nfl uence of the fracture pl ane has been factored i nto the di stri buti on, thereby
confi rmi ng the cryo-TEM si ze di stri buti on. For 7:3 wt CTAT/SDBS at 1% total surfactant i n water, K )
+ j/2 ) 0.15 ( 0.03 kBT and R0 ) 55 nm ( 10 nm. For CTAT/SDBS, w/w, at 2% total surfactant, K )
0.54 kT ( 0.05 kBT and R0 ) 36 nm ( 1 nm. We fi nd that surfactant mi xi ng i s l i kel y the ori gi n of the l ow
bi l ayer el asti ci ty i n catani oni c vesi cl es. However, the l ower val ue of K i n the CTAT-ri ch sampl e i s l i kel y
due to the hydrophobi c tosyl ate counteri on i ncreasi ng the area per headgroup.
Introduction
Parti cl e si ze di stri buti on, mean di ameter, and pol y-
di spersi ty are i mportant experi mental parameters de-
scri bi ng a col l oi dal di spersi on. For sel f-assembl ed struc-
tures such as equi l i bri um vesi cl es, the si ze di stri buti on
i s i nti matel y rel ated to characteri sti c bi l ayer parameters
i ncl udi ng the spontaneous curvature,
1,2
the Hel fri ch el asti c
modul i ,
1-4
or even mol ecul ar parameters such as the
extended l ength of a surfactant chai n, the area per
headgroup, and soforth.
4-8
Whi l e a number of theoreti cal
vesi cl e si ze di stri buti ons are avai l abl e i n the l i terature,
1-11
there i s a l ack of correspondi ng experi mental si ze di s-
tri buti ons, especi al l y for submi cron equi l i bri um vesi cl es.
1,3
Accurate si ze di stri buti ons of vesi cl es and other sel f-
assembl ed submi cron structures, whi l e theoreti cal l y pos-
si bl e, cannot be obtai ned wi th any rel i abi l i ty from l i ght
or other scatteri ng methods. I magi ng techni ques, such as
cryo-transmi ssi on el ectron mi croscopy, provi de the most
real i sti c method of obtai ni ng the compl ete si ze di stri bu-
ti on.
1,2,12,13
However, al l el ectron mi croscopy techni ques
can suffer from sampl e preparati on and el ectron i magi ng
arti facts and need to be rei nforced by other methods.
Here we show the si mi l ari ti es and di fferences between
the mean radi i , standard devi ati ons, and pol ydi spersi ti es
of catani oni c equi l i bri um vesi cl es measured by cryo-
transmi ssi on el ectron mi croscopy (cryo-TEM), freeze-
fracture el ectron mi croscopy (FF-TEM), and quasi -el asti c
l i ght scatteri ng (QLS). We used the si ze di stri buti on
determi ned from cryo-TEM and a theoreti cal expressi on
deri ved from Hel fri ch el asti ci ty theory and a si mpl e mass
acti on model to determi ne K ) + j/2, the sum of the
Hel fri ch mean ()and Gaussi an (j)curvature modul i , and
R
0
, the spontaneous radi us of curvature for vesi cl es made
from cetyl tri methyl ammoni um tosyl ate (CTAT) and so-
di um dodecyl benzene sul fonate (SDBS). For 7:3 wt CTAT/
SDBS at 1%total surfactant i n water, K ) + j/2 ) 0.15
( 0.03 k
B
T and R
0
) 55 nm ( 10 nm. For CTAT/SDBS,
w/w, at 2% total surfactant, K ) 0.54 ( 0.05 k
B
T and R
0
) 36 nm ( 1 nm. The l ower val ue of K i n the CTAT-ri ch
sampl e i s l i kel y due tothe hydrophobi ctosyl ate counteri on
i ncreasi ng the area per surfactant headgroup.
* To whom correspondence shoul d be addressed. Joseph A.
Zasadzi nski , Department of Chemi cal Engi neeri ng, Uni versi ty of
Cal i forni a, Santa Barbara, Santa Barbara, CA 93106-5080.
Phone: 805-893-4769. Fax: 805-893-4731. E-mai l : gori l l a@
engi neeri ng.ucsb.edu.
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5632 Langmuir 2003, 19, 5632-5639
10.1021/l a034311+ CCC: $25.00 2003 Ameri can Chemi cal Soci ety
Publ i shed on Web 06/06/2003
We al so present a compari son of the cryo-TEM resul ts
wi th QLS measures of the mean hydrodynami c radi us
and pol ydi spersi ty and the mean di ameter and standard
devi ati on determi ned from freeze-fracture el ectron mi -
croscopy. The mi croscopy methods show a surpri si ng
agreement i n the mean di ameter and the standard
devi ati on of the vesi cl e si ze di stri buti ons, once the fracture
process i s factored i nto the si ze di stri buti on, as both are
number-average methods. QLS shows a systemati cal l y
l arger mean vesi cl e si ze that i s i nvari ant wi th concentra-
ti on rati o wi thi n a gi ven vesi cl e l obe. The l arger mean
si ze i s to be expected as the hydrodynami c radi us gi ven
by QLS more heavi l y wei ghts the l arger vesi cl es i n the
di stri buti on. However, when thi s wei ghti ng i s taken i nto
consi derati on, the mean radi us and pol ydi spersi ty mea-
sured by QLS are consi stent wi th that measured by
el ectron mi croscopy. Asecond di sadvantage of QLS, whi ch
i s not often appreci ated i n compl ex surfactant systems,
i s that a vari ety of shapes, such as di sks, cyl i nders, and
spheres, may coexi st i n a surfactant sol uti on. QLS
averages over al l such shapes, and both the mean radi us
and pol ydi spersi ty of each structure mi ght be qui te
di fferent from the average determi ned by scatteri ng. The
mai n advantage of QLS i s the mi ni mum amount of sampl e
preparati on and the qui ck data accumul ati on, whi ch
makes QLS i deal for i ni ti al screens of a l arge number of
sampl es. However, the combi nati on of QLS, freeze-fracture
TEM, and cryo-TEMcan provi de rel i abl e si ze di stri buti ons
of equi l i bri um vesi cl es that can then be fi t to theoreti cal
expressi ons to extract bi l ayer parameters.
1,14
Materials and Methods
CTATpurchased from Si gma (St. Loui s, MO)and SDBS (hard-
type) from Tokyo Kasei (Japan) were used as recei ved. Sampl es
were prepared by fi rst mi xi ng stock sol uti ons of the surfactants
wi th Mi l l i pore-fi l tered water tothe desi red wei ght fracti ons. The
stock sol uti ons were then combi ned i n the appropri ate amounts
and al l owed several weeks for equi l i brati on. Phase di agrams of
the vari ous catani oni c vesi cl e mi xtures have been publ i shed
el sewhere.
15-20
Whi l e i t i s possi bl e toprepare metastabl e vesi cl es
by sheari ng l amel l ar phases,
21,22
there i s no i ndi cati on of a bul k
l amel l ar phase at the concentrati ons of i nterest here, nor that
shear has i nfl uenced the formati on or si ze di stri buti on of the
structures shown. Vari ous zero-shear sampl e preparati on meth-
ods i ncl udi ng di al ysi s, i sothermal counterdi ffusi on of twomi cel l ar
di spersi ons, and i n si tu surfactant synthesi s have shown that
catani oni c vesi cl es, i ncl udi ng the mi xtures of i nterest here, form
i ndependentl y of the method of sampl e preparati on.
23
Si mi l ar
catani oni cvesi cl es recover thei r si ze di stri buti on after soni cati on
or heat treatment, confi rmi ng that the si ze di stri buti on i s an
equi l i bri um feature of the di spersi ons.
23
Cryo-TEM. To prepare sampl es for cryogeni c el ectron mi -
croscopy, a thi n (<1 m) l ayer of the surfactant-water mi xture
was spread on a l acey carbon gri d (Ted Pel l a, Reddi ng, CA) i n
a temperature-control l ed chamber
24
saturated wi th the sol uti on
of i nterest. The gri d was pl unged i nto a mi xture of l i qui d ethane
and l i qui d propane cool ed by l i qui d ni trogen.
25
The frozen sampl es
were transferred to a GATAN (Pl easanton, CA) col d stage and
i maged di rectl y at 100 kV usi ng a JEOL 100CXI I transmi ssi on
el ectron mi croscope. Bri ght fi el d phase contrast transmi ssi on
el ectron mi crographs were recorded usi ng standard l ow-dose
procedures ei ther on fi l m or wi th a GATAN CCDcamera. Vesi cl e
radi i were measured from the outer edge of the dark ri m i n the
i mage to obtai n the best representati on of the true radi us of the
vesi cl e (see Fi gures 2 and 3). Hundreds of i ndi vi dual vesi cl es
were measured from the di gi ti zed i mages usi ng a commerci al
i mage anal ysi s package (I mage-Pro Pl us versi on 4.1, Medi a
Cyberneti cs, Si l ver Spri ng, MD) to determi ne the si ze di stri bu-
ti ons. Sampl es for cryo-TEM were prepared and exami ned over
the course of several weeks to ensure that nei ther the structures
nor the si ze di stri buti ons were changi ng wi th ti me.
Freeze-Fracture Electron Microscopy. Freeze-fracture
sampl es were prepared by fi rst deposi ti ng a fi l m of sampl e l i qui d
approxi matel y 100 mi crons thi ck between twocopper pl anchettes.
The sampl es were frozen by pl ungi ng the sampl e i nto a l i qui d
propane/l i qui d ethane bath cool ed by l i qui d ni trogen. The frozen
sampl e was transferred under l i qui d ni trogen tothe sampl e bl ock
of a JEM Cryofract freeze-fracture devi ce. The bl ock was then
qui ckl y transferred tothe vacuum chamber of the Cryofract devi ce
vi a an ai rl ock. After temperature (-170 C)and pressure (<10
-7
Torr) equi l i brati on, the sampl e was fractured and the two
resul ti ng surfaces were repl i cated wi th approxi matel y 1.5 nm of
pl ati num deposi ted at a 45 angl e, fol l owed by about 15 nm of
carbon deposi ted normal to the surface. The repl i cas were then
removed from the vacuum and warmed to room temperature.
The copper pl anchettes were di ssol ved i n chromerge (a mi xture
of chromi c aci d, sul furi c aci d, and water), and then the repl i cas
were washed i n water and al l owed tostand i n ethanol for several
days to di ssol ve any remai ni ng surfactant. The repl i cas were
col l ected on Formvar-coated TEM gri ds (Ted Pel l a).
26
A Gatan
CCD camera was used to record di gi tal bri ght fi el d i mages usi ng
a JEOL 100CXI I transmi ssi on el ectron mi croscope. Hundreds of
i ndi vi dual vesi cl es were measured from the di gi ti zed i mages usi ng
a commerci al i mage anal ysi s package (I mage-Pro Pl us versi on
4.1, Medi a Cyberneti cs) to determi ne the si ze di stri buti ons.
Quasi-Elastic Light Scattering. QLS was performed at 25
Cwi th a Brookhaven model BI -200SMgoni ometer, a Brookhaven
model BI -9000ATcorrel ator, and a Lexel 300 mWAr l aser. Most
experi ments were performed at 90, and the autocorrel ati on
functi on was anal yzed by the method of cumul ants.
27
The
apparent hydrodynami c radi us was obtai ned from the measured
di ffusi on coeffi ci ent usi ng the Stokes-Ei nstei n rel ati onshi p as
descri bed bel ow. The pol ydi spersi ty i ndex, whi ch i s rel ated to
the wi dth of the si ze di spersi on, normal i zed to the mean vesi cl e
si ze, was determi ned from the second cumul ant of the fi t to the
autocorrel ati on functi on usi ng the software provi ded wi th the
correl ator.
Results and Discussion
The fi rst reported systems of spontaneous catani oni c
vesi cl es were mi xtures of CTATand SDBS.
15
The tosyl ate
counteri on of CTATi s hydrophobi c and remai ns strongl y
associ ated wi th the surfactant aggregate i n sol uti on.
I nserti ng the tosyl ate i nto the membrane effecti vel y
i ncreases the area per surfactant headgroup, whi ch i s
predi cted to reduce membrane ri gi di ty.
18-20,28-30
SDBS
(14) Jung, H. T.; Lee, Y. S.; Kal er, E. W.; Col dren, B.; Zasadzi nski ,
J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15318.
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A. N. Science1989, 245, 1371.
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A. J . Phys. Chem. 1992, 96, 6698.
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VesicleSizeDistributions via Electron Microscopy Langmuir, Vol. 19, No. 14, 2003 5633
(hard-type)i s a branched chai n aromati c surfactant; such
branchi ng can i nfl uence hydrocarbon packi ng i n the
bi l ayer i nteri or, whi ch may i n turn affect the bendi ng
ri gi di ty. I t i s theoreti cal l y predi cted that branched hy-
drocarbons pack more l oosel y i n the bi l ayer and thus are
more accommodati ng to the gauche hydrocarbon confor-
mati ons associ ated wi th membrane deformati ons.
28,29
Mol ecul ar rel axati on (chai n rearrangement and fl i p-fl op)
i n the bi l ayer coul d be enhanced by the presence of
branched hydrocarbons. Any of these effects coul d resul t
i n a strongl y reduced bendi ng ri gi di ty rel ati ve to that of
equi val ent l i near chai ns. However, chai n mi xi ng of
di fferent surfactants by i tsel f i s al so predi cted to l ower
the bendi ng ri gi di ty.
28,29
The ternary phase di agram of CTAT/SDBS/water at 25
C i s domi nated by two l arge and hi ghl y stabl e vesi cl e
l obes for total surfactant concentrati ons bel ow about 3 wt
%. One l obe consi sts of CTAT-ri ch vesi cl es, and the second
consi sts of SDBS-ri ch vesi cl es; the vesi cl e l obes are
separated by a preci pi tate formed at equi mol ar concen-
trati on.
15
The upper l i mi t of surfactant concentrati on
corresponds roughl y to the cl ose packi ng of uni l amel l ar
vesi cl es about 100 nm i n si ze.
Cryo-TEM Results. A quanti tati ve si ze di stri buti on
i s necessary to appl y theoreti cal model s rel ati ng equi -
l i bri um vesi cl e si ze di stri buti ons to fundamental bi l ayer
el asti c parameters.
1
Fi gure 1A shows a representati ve
cryo-TEM i mage of CTAT/SDBS, 3:7 w/w rati o, at 2 wt %
total surfactant i n water. I n cryo-TEM i mages, vesi cl es
appear as dark, ci rcul ar ri ngs on a uni form bri ght
background. The i nsi de of the ri ng i s somewhat darker
than the background, and the wi dth of the ri ng i s al ways
greater than the expected bi l ayer thi ckness. There are
vesi cl es of many di fferent si zes, as wel l as some cyl i ndri cal
i n shape, whi ch may be due to sl i ght sheari ng effects
duri ng sampl e preparati on. However, the curvature
energy of suffi ci entl y l ong cyl i nders i s l ess than that of
the equi val ent area sphere i f there i s a fi ni te spontaneous
curvature, so coexi sti ng cyl i nders and spheres may
be possi bl e for certai n val ues of the Hel fri ch el asti c
modul i .
10,14
These vesi cl e i mages can be easi l y understood as the
projecti on of the di fferent scatteri ng densi ty of the bi l ayer
rel ati ve to the water background. As el ectrons pass
through the sampl e, they must travel through vari ous
l engths of the spheri cal shel l of the vesi cl e; the fracti on
of el ectrons scattered depends on these projected l engths.
Near the edge of the vesi cl e, these projected l engths are
l arge; near the center, the projected l ength i s si mpl y twi ce
the bi l ayer thi ckness. The rate of vari ati on of the projecti on
of a spheri cal shel l depends on the shel l thi ckness rel ati ve
to the shel l radi us. I f the shel l thi ckness i s l arge i n
compari son to the shel l radi us, the projecti on wi l l vary
sl owl y, l eadi ng to a l ow contrast, broad ri ng. On the other
hand, i f the shel l thi ckness i s smal l i n compari son to the
shel l radi us, the projecti on wi l l be much hi gher i n contrast
and appear much sharper. Si mpl e cal cul ati ons of the
projected thi ckness of a spheri cal shel l as a functi on of
the radi us are shown i n Fi gure 2 for a 4 nm thi ck bi l ayer
shel l , wi th an i nner radi us of 80 nm (Fi gure 2A) or 20 nm
(Fi gure 2B), whi ch are typi cal of the range of vesi cl es seen
i n the i mages. The absol ute contrast shoul d be greater for
l arger vesi cl es than smal l er vesi cl es, whi ch i s consi stent
wi th the i mages. However, i n ei ther case, the apparent
wi dth of the ri ng i s si gni fi cantl y greater than the shel l
thi ckness. The greatest vari ati on i n contrast occurs at
the exteri or ri m of the shel l ; the change i n contrast marks
the outsi de radi us of the vesi cl es and i s the best-defi ned
l ength scal e associ ated wi th the vesi cl e. The absolute
contrast i s compl i cated by phase contrast, defocus, and
other i magi ng effects, but the outer di ameter of the dark
ri ng i s very cl ose tothe true outer di ameter of the spheri cal
vesi cl e.
Fi gure 1B shows the di stri buti on of the outer vesi cl e
di ameter for approxi matel y 2000 vesi cl es taken from cryo-
TEM i mages of CTAT/SDBS, 3:7 w/w, at 2% total
surfactant i n water. Consi stent wi th thermodynami c
predi cti ons, the di stri buti on i s uni modal and roughl y
Gaussi an. The si zes of equi l i bri um vesi cl es are determi ned
by a subtl e competi ti on between the entropy of vesi cl e
and surfactant mi xi ng and the curvature el asti ci ty of the
(30) Herri ngton, K. Phase Behavi or and Mi crostructure i n Aqueous
Mi xtures of Opposi tel y Charged Surfactants. Ph.D. Di ssertati on,
Uni versi ty of Del aware, Newark, DE, 1994.
Figure1. (A) Cryo-TEM i mage of 3:7 w/w CTAT/SDBS, 2 wt
% total surfactant i n water at 25 C. I n cryo-TEM i mages,
vesi cl es appear as dark, ci rcul ar ri ngs on a uni forml y bri ght
background. The area i nsi de the ri ng i s usual l y somewhat
darker than the background. The wi dth of the ri ng i s al ways
greater than the expected bi l ayer thi ckness due tothe curvature
of the vesi cl e as shown i n Fi gure 2. Extended cyl i nders are
often found i n these mi xtures and may be due to shear duri ng
sampl e preparati on, al though they do not appear to be
preferenti al l y al i gned and thei r numbers are al ways l ow. A
sphere to cyl i nder transi ti on i s al so possi bl e at equi l i bri um for
certai n val ues of the el asti c modul i (ref 14). The nonspheri cal
objects were excl uded from the si ze di stri buti ons shown i n (B).
(B) Hi stogram of vesi cl e si zes from cryo-TEM i mages of CTAT/
SDBS, 3:7 w/w, at 2 wt % total surfactant i n water. The fi t of
the bendi ng ri gi di ty model was opti mi zed vi a the reduced Chi
test, gi vi ng R
0 ) 36 nm and K ) 0.54 kBT. On the basi s of raw
numeri cal data, the mean vesi cl e radi us was R ) 30 nm and
/R ) 0.32, whi ch are i n excel l ent agreement wi th the FF-
TEM data. For these broad di stri buti ons, the mean vesi cl e i s
smal l er than the l owest curvature energy vesi cl e (R <R0)due
to entropy, whi ch favors smal l er aggregates.
5634 Langmuir, Vol. 19, No. 14, 2003 Coldren et al.
bi l ayers.
3,5,31,32
The curvature energy per uni t area of
bi l ayer, f
c
, i s
9,11,33
R
1
and R
2
are the pri nci pl e radi i of curvature; i s the
curvature modul us, and j i s the saddl e-spl ay modul us.
The spontaneous curvature, 1/r
0
, i s nonzero onl y i f there
i s asymmetry between the di fferent si des of the bi -
l ayer.
9,11,33
Equati on 1 i s an appropri ate descri pti on when
the membrane thi ckness
9,34,35
(here about 3-4 nm
1
) and
the Debye l ength for i oni c surfactants
34,36,37
(al so about
1-3 nm
1
) are smal l compared to R
1
and R
2
(about 20-60
nm, see Fi gure 2). For the spheri cal vesi cl es studi ed here,
R
1
) R
2
) R, and eq 1 can be si mpl i fi ed:
11,33,38
R
0
i s the radi us of the mi ni mum curvature energy vesi cl e
(whi ch i s di fferent than r
0
, the spontaneous curvature
radi us), and K i s an effecti ve bendi ng constant.
11,33
The
di stri buti on of surfactant between vesi cl es of aggregati on
number M, correspondi ng to the mi ni mum curvature
energy radi us, R
0
(M )8R
0
2
/A
0
, i n whi ch A
0
i s the mean
mol ecul ar area), rel ati ve tovesi cl es of aggregati on number
N and radi us R, i s di ctated by a bal ance between the
entropy of vesi cl e mi xi ng and the curvature energy, whi ch
can be expressed as a mass-acti on model :
5
X
M
,
M
0
and X
N
,
N
0
are the mol e fracti on of surfactant and
the standard chemi cal potenti al per mol ecul e i n vesi cl es
of si ze M and N, respecti vel y. Equati on 3 assumes i deal
mi xi ng of the vesi cl es (not the mol ecul es wi thi n the
bi l ayers)and i s val i d for di l ute vesi cl e di spersi ons i n whi ch
the Debye l ength i s smal l i n compari son tothe i ntervesi cl e
di stance.
39,40
The chemi cal potenti al di fference i s due to
the change i n curvature energy per mol ecul e for surfactant
di stri buted between vesi cl es of di fferent radi i :
I nserti ng eq 4 i nto eq 3 and substi tuti ng M ) 8R
0
2
/A
0
and N ) 8R
2
/A
0
gi ves the vesi cl e si ze di stri buti on as a
functi on of R
0
and K:
1,2,5
C
M
()X
M
/M)and C
N
are the mol ar and number fracti ons
of vesi cl es of si ze M and N, respecti vel y. A consequence
of eq 5 i s that vesi cl es wi th K k
B
T have a much broader
si ze di stri buti on than vesi cl es wi th K . k
B
T. Thi s i s the
opposi te of vesi cl e si ze di stri buti on model s that do not
i ncl ude a spontaneous curvature.
3,4
A best fi t of eq 5 tothe
si ze di stri buti on of CTAT/SDBS, w/w, at 2% total sur-
factant measured by cryo-TEM resul ts i n K )0.54 (0.05
k
B
T and R
0
) 36 ( 1 nm, wi th a reduced Chi test ) 1.8
(Fi gure 4). On the basi s of the raw numeri cal data, the
mean vesi cl e radi us R ) 30 nm and /R ) 0.32.
A cryo-TEM i mage of 7:3 w/w CTAT/SDBS at 1 wt %
total surfactant i n water i s shown i n Fi gure 3A. Fi gure
3Bshows the vesi cl e si ze di stri buti on generated from thi s
and other cryo-TEM i mages of about 2000 vesi cl es. The
di stri buti on of si zes i s uni modal and was fi t to eq 5 to
(31) Morse, D. C.; Mi l ner, S. T. Phys. Rev. E 1995, 52, 5918.
(32) Si mons, B. D.; Cates, M. E. J . Phys. I I (France) 1992, 2, 1439.
(33) Safran, S. A.; Pi ncus, P. A.; Andel man, D.; MacKi ntosh, F. C.
Phys. Rev. A 1991, 43, 1071.
(34) Duboi s, M.; Zemb, T. Curr. Opin. Colloid I nterfaceSci. 2000, 5,
27.
(35) Safran, S. A. Statistical Thermodynamicsof Surfaces, I nterfaces
and Membranes; Addi son-Wesl ey: Readi ng, MA, 1994.
(36) Fogden, A.; Ni nham, B. W. Adv. Colloid I nterfaceSci. 1999, 83,
85.
(37) Fogden, A.; Carl sson, I .; Dai ci c, J. Phys. Rev. E 1998, 57, 5694.
(38) f
c and f are rel ated by a constant term i ndependent of the vesi cl e
curvature.
(39) The mi ni mum i oni c strength of these catani oni c sol uti ons i s
equal to the concentrati on of the mi nor surfactant component, and the
maxi mum i s the total surfactant concentrati on. For these experi ments,
the i oni c strength ranges from about 10-100 mM. Thi s l eads toa Debye
l ength of 1-10 nm (see Chapter 12 of ref 4), a smal l fracti on of the
average i ntervesi cl e spaci ng (as seen i n the mi crographs) of 75-100
nm. Hence, el ectrostati ci nteracti ons are negl i gi bl e between the vesi cl es
and the i deal mi xi ng approxi mati on i s justi fi ed.
(40) Chi ruvol u, S.; I srael achvi l i , J.; Naranjo, E.; Xu, Z.; Kal er, E. W.;
Zasadzi nski , J. A. Langmuir 1995, 11, 4256.
Figure2. Projected thi ckness of a spheri cal vesi cl e shel l 4 nm
thi ck for (A) a spheri cal shel l of 80 nm i nsi de radi us and (B)
a spheri cal shel l of 20 nm i nsi de radi us. Note that the projected
l ength i s a maxi mum at the i nsi de radi us of the shel l and that
the projecti on tapers off sl owl y toward the i nsi de of the vesi cl e.
The contrast i n cryo-TEM i mages, whi l e compl i cated by phase
contrast and other el ectron opti cal effects, i s rel ated to the
projected thi ckness: the greater the thi ckness, the darker the
i mage. The apparent thi ckness of the bi l ayer shel l i s al ways
greater than the actual thi ckness of the shel l due to the
curvature of the bi l ayer. The most appropri ate measurement
from cryo-TEM i mages i s to l ocate where the contrast vari es
the most, whi ch i s the outsi de radi us of the vesi cl e. Hence, the
outer ri m of the dark ci rcl es i n Fi gure 1 gi ves an accurate
measure of the outsi de radi us of the vesi cl e.
f
c
)
1
2

(
1
R
1
+
1
R
2
-
2
r
0
)
2
+ j
(
1
R
1
R
2
)
(1)
f ) 2K
(
1
R
-
1
R
0
)
2
2K ) 2 + j (2)
R
0
)
2 + j
2
r
0
X
N
N
)
{
X
M
M
exp
[
M(
M
0
-
N
0
)
k
B
T
]}
N/M
(3)
(
N
0
-
M
0
) )
4R
2
f
N
)
8K
(
1 -
R
R
0
)
2
N
(4)
C
N
)
{
C
M
exp
[
-8K
k
B
T
(
1 -
R
0
R
)
2
]}
R
2
/R
0
2
(5)
VesicleSizeDistributions via Electron Microscopy Langmuir, Vol. 19, No. 14, 2003 5635
determi ne that the opti mi zed K ) 0.15 ( 0.03 k
B
T, and
R
0
) 55 ( 10 nm, wi th a reduced Chi test ) 2.3. The
uncertai nty i n R
0
i s qui te hi gh when worki ng wi th such
broad di stri buti ons because the model i s onl y weakl y
sensi ti ve to R
0
. The rel ati vel y poor fi t qual i ty al so can be
attri buted to the mi smatch between theory and experi -
ment for R < 10 nm. The si mpl e Hel fri ch theory does not
account for hi gher order curvature terms that are i m-
portant when the bi l ayer radi us i s onl y a few mul ti pl es
of the bi l ayer thi ckness. On the basi s of raw numeri cal
data, R ) 42.6 nm and /R ) 0.59. For these broad
di stri buti ons, R
0
> R, as entropy favors smal l er ag-
gregates.
4,5
Comparison of Mean Vesicle Sizes Measured by
QLSand Cryo-TEM. Al though the data are di ffi cul t to
i nterpret quanti tati vel y, QLS i s a conveni ent tool for
qui ckl y compari ng vesi cl e si zes when deal i ng wi th a l arge
number of sampl es. I n addi ti on, QLS does not requi re any
speci al sampl e preparati on except for fi l teri ng dust and
other parti cl es. The vesi cl e si zes we observed by QLS are
general l y i n good agreement wi th vesi cl e sampl es of
si mi l ar composi ti ons publ i shed previ ousl y.
16
For both
CTAT-ri ch and SDBS-ri ch vesi cl es wi thi n each vesi cl e
l obe, there i s novari ati on i n vesi cl e si ze wi th SDBS/CTAT
rati o. For CTAT-ri ch vesi cl es, we observe a hydrodynami c
radi us R
QLS
) 57 ( 2 nm, and a pol ydi spersi ty i ndex (PI )
of 0.25 ( 0.03. For SDBS-ri ch vesi cl es, R
QLS
) 49 ( 3 nm
and PI ) 0.2 ( 0.03. The l ack of si ze dependence wi th
composi ti on i s consi stent wi th the i dea that the actual
vesi cl e bi l ayer composi ti on i s rel ati vel y fi xed and that
excess ani oni c or cati oni c surfactant i s expel l ed to coexi st-
i ng mi cel l es or sol uti on.
30
I t al so suggests that the bi l ayer
el asti c parameters and spontaneous curvature do not
depend on composi ti on wi thi n a vesi cl e l obe. The pol y-
di spersi ty i ndex i s a rough measure of the wi dth of the
si ze di stri buti on, normal i zed to the mean hydrodynami c
radi us, but shoul d not be confused wi th the standard
devi ati on of the di stri buti on.
27
Qual i tati vel y, the QLS data
agree wi th both the cryo-TEMresul ts that show the SDBS-
ri ch vesi cl es to be smal l er and more monodi sperse than
the CTAT-ri ch vesi cl es. However, the hydrodynami c
radi us measured by QLS i s si gni fi cantl y l arger than the
number-average radi us measured by both cryo-TEM and
FF-TEM.
Thi s i s because QLS does not gi ve a number-average
mean radi us; rather, the hydrodynami c radi us measured
by QLS i s skewed toward l arger si zes due to the depen-
dence of the scatteri ng i ntensi ty on the si ze of the object.
The i ntensi ty of the scattered l i ght from a vesi cl e wi th a
mass M
i
i s proporti onal to M
i
2
. QLS measures the
z-averaged di ffusi on coeffi ci ent, D
z
, as
41
where x
i
i s the number fracti on of vesi cl es of radi us i, M
i
i s the mass of si ze i, and D
i
i s the di ffusi vi ty of si ze i. For
a vesi cl e, M
i
i s proporti onal toR
i
2
as the vesi cl e i s a hol l ow
shel l . Hence,
and i nserti ng the Stokes-Ei nstei n rel ati on between si ze
and di ffusi vi ty, D
i
) k
B
T/(6R
i
), we have
The mean hydrodynami c radi us, R
DLS
, can be extracted
(41) Berne, B. J.; Pecora, R. DynamicLight Scattering; J. Wi l ey and
Sons: New York, 1976.
Figure3. (A) Cryo-TEM i mage of 7:3 w/w CTAT/SDBS, 1 wt
%total surfactant i n water at 25 C. Onl y spheri cal vesi cl es are
observed, and they are l arger and more broadl y si ze di sperse
than the SDBS-ri ch vesi cl es i n Fi gure 1. Smal l bl ack spots are
frost contami nati on. (B) Hi stogram of vesi cl e si zes from cryo-
TEM i mages of CTAT/SDBS, w/w, at 2 wt % total surfactant
i n water. The fi t of the bendi ng ri gi di ty model was opti mi zed
vi a the reduced Chi test, gi vi ng R
0 ) 55 nm and K ) 0.15 kBT.
On the basi s of raw numeri cal data, R ) 43 nm and /R )
0.59, whi ch are agai n i n excel l ent agreement wi th the corre-
spondi ng FF-TEM data.
D
z
)

i
x
i
M
i
2
D
i

i
x
i
M
i
2
(6)
D
z
)

i
x
i
R
i
4
D
i

i
x
i
R
i
4
(7)
D
z
)
k
B
T
6

i
x
i
R
i
3

i
x
i
R
i
4
(8)
5636 Langmuir, Vol. 19, No. 14, 2003 Coldren et al.
from thi s expressi on for D
z
by agai n usi ng the Stokes-
Ei nstei n rel ati on, R
hyd
) k
B
T/(6D
z
), yi el di ng
From eq 9, the hydrodynami c radi us can be ap-
proxi mated as the fourth moment of the vesi cl e si ze
di stri buti on di vi ded by the thi rd moment. Whi l e i t i s
i mpossi bl e to determi ne the ful l si ze di stri buti on from
QLS to compare to the cryo-TEM si ze di stri buti ons, the
hydrodynami c radi us can be cal cul ated from the si ze
di stri buti on measured by cryo-TEM.
For the CTAT-ri ch vesi cl es, the number-average radi us
determi ned from the cryo-TEM i mages i s R ) 43 nm.
From the si ze di stri buti on i n Fi gure 3B, we can use eq 9
to cal cul ate an equi val ent hydrodynami c radi us, R
hyd
)

i
x
i
R
i
4
/
i
x
i
R
i
3
) 63 nm, whi ch i s cl ose to the R
QLS
) 57 (
2 nm measured by QLS. The cal cul ated hydrodynami c
radi us i s very sensi ti ve to the fracti on of the l argest
vesi cl es; i f the l argest 1% of vesi cl es i n the cryo-data i s
l eft out of the cal cul ati on, R
hyd
drops to 59 nm. Hence,
whi l e QLS can cl earl y gi ve a consi stent measure of the
average radi us, thi s average shoul d al ways be greater
than that determi ned by TEM and i s domi nated by any
l arge parti cl es that may be i n the sol uti on. For the SDBS-
ri ch sol uti on, the number-average radi us of the spheri cal
vesi cl es i s R ) 30 nm. From Fi gure 1B and eq 9, R
hyd
)

i
x
i
R
i
4
/x
i
R
i
3
) 37 nm, whi ch i s qui te a bi t smal l er than
the 49 (3 nm measured by QLS. Thi s di screpancy poi nts
out a l i mi tati on of QLS rel ati ve to di rect i magi ng. I n the
cryo-TEM i mages, Fi gure 1A, of the SDBS-ri ch sol uti ons,
there are l ong, cyl i ndri cal bi l ayer aggregates i n addi ti on
to the spheri cal vesi cl es. QLS gi ves an average hydro-
dynami c radi us for al l of the structures i n sol uti on; the
cyl i ndri cal vesi cl es l i kel y skew the mean si ze and the
pol ydi spersi ty toward l arger val ues.
Freeze-Fracture Results. Si ze di stri buti ons from
cryo-TEM sampl es can be compl i cated by vesi cl e segrega-
ti on duri ng sampl e preparati on. The water fi l m i n a cryo-
TEM sampl e vari es i n thi ckness from a few mi crons to a
few hundred nanometers over the TEM gri d, thi cker near
the supporti ng hol ey pol ymer fi l ms of the gri d and thi nner
i n the gaps between the pol ymer fi l ms. Thi s shoul d not
si gni fi cantl y affect the si ze di stri buti ons shown i n Fi gures
1A and 3A as the vesi cl es are smal l compared to al l the
sampl e thi ckness. However, toconfi rm the cryo-TEM si ze
di stri buti ons, i t i s useful to compare the mean radi us and
standard devi ati on between sampl es that have the same
mean radi us and pol ydi spersi ty when measured by QLS
by a techni que that does not requi re such thi n water fi l ms.
Hence, we used freeze-fracture to measure the si ze
di stri buti on i n sampl es wi th sl i ghtl y di fferent composi ti on,
but that had the same mean radi us and pol ydi spersi ty as
measured by QLS.
However, eval uati ng a mean vesi cl e si ze from a freeze-
fracture el ectron mi croscopy i mage i s compl i cated by the
fracture process. I n freeze-fracture, vesi cl es appear as
hemi spheri cal domes or craters wi th di sti nct shadows
(absence of pl ati num). The fracture pl ane can pass through
any part of the vesi cl e, skewi ng the apparent si ze
di stri buti on toward smal l er si zes. By accounti ng for the
probabi l i ty densi ty of cross fracture pl anes, Hal l ett et al .
42
determi ned si mpl e correcti ons to gi ve better esti mates of
the mean and standard devi ati on of a spheri cal vesi cl e
di stri buti on. I n a freeze-fracture repl i ca, fractured vesi cl es
can be i nterpreted as the i ntersecti on of a random fracture
pl ane wi th a sphere of radi us R. The i mage then appears
i n the mi crograph as a ci rcl e of radi us r eR. I f the cl eavage
pl ane i s equal l y probabl e at al l posi ti ons wi thi n the vesi cl e,
the probabi l i ty that the apparent radi us i n the i mage i s
between r and r + dr i s G(r) dr, whi ch, for a vesi cl e of
radi us R, i s
42
I f H(R) i s the actual vesi cl e si ze di stri buti on, then the
apparent di stri buti on of radi i determi ned from a freeze-
fracture i mage i s I (r):
The i ntegrati on goes from r to because the true vesi cl e
radi us, R, i s never smal l er than the apparent radi us on
the freeze-fracture i mage, r. As can be seen from eq 11,
eval uati ng the si ze di stri buti on requi res a pri ori knowl -
edge of the functi onal form of the di stri buti on. Even i f we
assume the functi onal form gi ven by our spontaneous
curvature theory, eq 5, a compl ete deconvol uti on of the
freeze-fracture si ze di stri buti on i s a practi cal i mpossi bi l i ty.
Fortunatel y, the fi rst twomoments of the si ze di stri buti on
can be determi ned from FF-TEM data independent of any
functi onal form of the actual si ze di stri buti on. Si nce
eq 12 rel ates the mean radi us measured from the freeze-
fracture i mages, r, wi th the true mean radi us of the
di stri buti on, R: R ) 4r/. By determi nati on of the
average apparent radi us of the fractured vesi cl es, the
average vesi cl e radi us can be cal cul ated by a si mpl e
mul ti pl i cati ve factor.
The standard devi ati on of the vesi cl e radi i can al so be
determi ned from the freeze-fracture i mages i n a si mi l ar
way. The second moment of I (r) yi el ds the mean square
val ue, r
2
:
From eqs 12 and 13, the standard devi ati on of the vesi cl e
si ze di stri buti on can be easi l y cal cul ated:
Al though freeze-fracture i mages cannot easi l y gi ve a
compl ete representati on of the vesi cl e si ze di stri buti on,
the mean and standard devi ati ons of the si ze di stri buti on
can be compared between the cryo-TEM and freeze-
fracture sampl e preparati ons. As freeze-fracture sampl es
(42) Hal l et, F. R.; Ni ckel , B.; Samuel s, C.; Krygsman, P. H. J . Electron
Microsc. Tech. 1991, 17, 459.
R
hyd
)

i
x
i
R
i
4

i
x
i
R
i
3
(9)
G(r) )
r
R(R
2
- r
2
)
1/2
(10)
I (r) )

r
r
R(R
2
- r
2
)
1/2
H(R) dR (11)
r )

rI (r) dr )

H(R)
[

0
R r
2
R(R
2
- r
2
)
1/2
dr
]
dR
(12)
r )

RH(R) dR )

4
R
r
2
)

r
2
I (r) dr )

H(R)
[

0
R r
3
R(R
2
- r
2
)
1/2
dr
]
dR
(13)
r
2
)
2
3

R
2
H(R) dR )
2
3
R
2

2
) R
2
- R
2
)
3
2
r
2
-
16

2
r
2
(14)
VesicleSizeDistributions via Electron Microscopy Langmuir, Vol. 19, No. 14, 2003 5637
are typi cal l y 10-100 mi crons thi ck, probl ems of si ze
segregati on, compressi on, and ori entati on that troubl e
cryo-TEM are not present i n freeze-fracture. Whi l e both
techni ques may suffer from freezi ng arti facts (whi ch are
mi ni mi zed i n cryo-TEM sampl es due to thei r smal l er
thi ckness and can be eval uated by di ffracti on todetermi ne
i f there has been i ce crystal formati on, see refs 25 and 26),
freeze-fracture sampl es are not damaged by el ectron beam
i rradi ati on, and the contrast i n the i mage i s generated by
di fferent processes. Hence, agreement between the two
methods i s good evi dence that the si ze di stri buti ons
determi ned by both methods are accurate.
Fi gure 4 shows vesi cl e si ze di stri buti on hi stograms
determi ned from freeze-fracture TEM i mages (not shown)
for CTAT/SDBS, 2:8 w/w at 2 wt % (Fi gure 4A) and 8:2
w/w at 1 wt % total surfactant i n water at 25 C (Fi gure
4B). The si ze di stri buti on shows the apparent vesi cl e
radi us, uncorrected for the effect of random fracture
pl anes, and thus i s skewed to smal l er si zes. Fi gure 4A
shows CTAT/SDBS 2:8 w/w at 2 wt % total surfactant i n
water at 25 C. The corrected R )30 nm and /R )0.34
are i n excel l ent agreement wi th cryo-TEM of CTAT/SDBS
3:7 w/w at 2% total surfactant of R ) 30 nm and /R
) 0.32. For the 8:2 w/w CTAT/SDBS at 1 wt % total
surfactant i n water at 25 C, the apparent si ze di stri buti on
i s shown i n Fi gure 4B. As expected from the random
fracture pl anes, the di stri buti on i s bi ased toward smal l er
radi i than the cryo-TEM di stri buti ons. After deconvol u-
ti on, R ) 37 nm and /R ) 0.59, i n good agreement
wi th the cryo-TEM whi ch gave R ) 43 nm and /R )
0.59. As di scussed previ ousl y, QLS shows that the
hydrodynami c radi us and pol ydi spersi ty do not change
si gni fi cantl y wi th composi ti on over thi s range, sothi s good
agreement speaks to the consi stency between al l three
methods of measuri ng the si ze di stri buti on. Apparentl y,
nei ther the smal l di fference i n vesi cl e composi ti on nor
the FF-TEM deconvol uti on i ntroduced si gni fi cant errors.
Furthermore, the twosets of i mages were generated years
apart from di fferent surfactant stocks, i ndi cati ng the
repeatabl e properti es of these thermodynami cal l y con-
trol l ed vesi cl e systems.
For both vesi cl e composi ti ons i n the CTAT/SDBS/water
system, cryo-TEM and FF-TEM si ze di stri buti ons are i n
excel l ent agreement once R and /R are properl y scal ed.
However, cryo-TEM remai ns the onl y techni que for
unambi guousl y determi ni ng the ful l si ze di stri buti on of
pol ydi sperse fl uctuati ng vesi cl es and, hence, esti mati ng
el asti c properti es. Neverthel ess, FF-TEM i s cl earl y a
rel i abl e tool for determi ni ng R and /R as wel l as
veri fyi ng a uni modal vesi cl e si ze di stri buti on. Further-
more, FF-TEM shares cryo-TEMs advantages over scat-
teri ng i n the abi l i ty to i gnore dust and generate number-
average data rather than hi gher moment averages. There
are even advantages of FF-TEM over cryo-TEM: a much
l ower probabi l i ty of shear-i nduced arti facts and arti facts
due toevaporati on/condensati on. Thus, FF-TEMand cryo-
TEM shoul d be vi ewed as compl ementary techni ques that
shoul d be used i n paral l el to generate unambi guous
col l oi dal si ze di stri buti ons.
Conclusions
Cryo-TEM, freeze-fracture TEM, and quasi -el asti cl i ght
scatteri ng are compl ementary methods todetermi ne mean
radi i , pol ydi spersi ty, and even the compl ete si ze di stri bu-
ti on of submi cron col l oi dal parti cl es i ncl udi ng spontaneous
vesi cl es. Cryo-TEM and FF-TEM show surpri si ng agree-
ment i n both the mean radi i and the standard devi ati on
of both CTAT-ri ch and SDBS-ri ch spontaneous vesi cl e
sampl es, once the FF-TEM data are corrected for the
fracture process. Moreover, both are number-average
methods so they general l y are more sui tabl e for com-
pari son to theoreti cal si ze di stri buti ons. QLS shows a
systemati cal l y l arger mean vesi cl e si ze, but compari sons
of the rel ati ve si zes and pol ydi spersi ti es between the
CTAT-ri ch and the SDBS-ri ch sampl es are consi stent wi th
both mi croscopy methods. For the CTAT-ri ch sampl es that
onl y form spheri cal vesi cl es, the cryo-mi croscopy data,
when wei ghted appropri atel y for the scatteri ng i ntensi ty,
gi ve a mean radi us i n good agreement wi th the hydro-
dynami c radi us measured by QLS. However, the agree-
ment i s l ess good for the SDBS-ri ch sampl e. Thi s poi nts
out a di sadvantage of QLS that i s not often appreci ated
i n compl ex surfactant systems. Surfactant aggregates can
take on a vari ety of shapes i ncl udi ng di sks, cyl i nders, and
spheres. QLS averages over al l such aggregates i n sol uti on,
Figure4. (A) Hi stogram of vesi cl e si zes from freeze-fracture
TEM i mages (not shown) of 2:8 w/w CTAT/SDBS, 2 wt %total
surfactant i n water at 25 C, uncorrected for the effect of random
fracture pl anes i n the FF-TEM i mage. After correcti on, R )
30 nm and /R ) 0.34, i n excel l ent agreement wi th the
correspondi ng cryo-TEM si ze di stri buti ons of the SDBS-ri ch
vesi cl es (Fi gure 1A). (B) Vesi cl e si ze di stri buti on hi stogram
from FF-TEM i mages (not shown) of 8:2 w/w CTAT/SDBS at
1 wt % total surfactant at 25 C. Si zes are raw FF-TEM data,
uncorrected for the effect of random fracture pl anes, and thus
are skewed to smal l er si zes. After correcti on, R ) 37 nm and
/R )0.59, whi ch are i n good agreement wi th cryo-TEM data
above. Apparentl y, nei ther the smal l di fference i n vesi cl e
composi ti on nor the FF-TEM deconvol uti on i ntroduces si g-
ni fi cant errors. The two sets of i mages were generated years
apart from di fferent surfactant stocks.
5638 Langmuir, Vol. 19, No. 14, 2003 Coldren et al.
and both the mean radi us and pol ydi spersi ty of each
structure mi ght be qui te di fferent from the average
hydrodynami c radi us. The mai n advantage of QLS i s the
mi ni mum amount of sampl e preparati on and the qui ck
data accumul ati on, whi ch makes QLS i deal for i ni ti al
qual i tati ve compar i sons between l ar ge number s of
sampl es. However, the combi nati on of QLS, freeze-fracture
TEM, and cryo-TEMcan provi de rel i abl e si ze di stri buti ons
of equi l i bri um vesi cl es that can then be fi t to theoreti cal
expressi ons to extract materi al parameters.
Wi th the excepti on of systems contai ni ng l onger fl u-
ori nated chai ns (>7 carbons),
1
equi l i bri um vesi cl es formed
from catani oni c surfactant mi xtures of unequal chai n
l ength are typi cal l y pol ydi sperse. Wi thi n the confi nes of
our thermodynami c model i ncorporati ng spontaneous
curvature, thi s woul d i ndi cate that these catani oni c
surfactant mi xtures form bi l ayers of very l ow ri gi di ty (of
order k
B
T) regardl ess of di fferences i n headgroup chem-
i stry (sul fate, sul fonate, carboxyl ate, etc.), counteri on
(bromi de, chl ori de, tosyl ate, etc.), chai n l ength (6-16
carbons), and degree of branchi ng (SDBS). The tosyl ate
counteri on i n the CTAT/SDBS system al so l eads to a
decrease i n the effecti ve bi l ayer ri gi di ty. For SDBS-ri ch
bi l ayers, the effecti ve el asti cconstant K )0.54 k
B
T, whi ch
i s si mi l ar to that of l i near-chai n bi l ayers i n cetyl tri m-
ethyl ammoni um bromi de (CTAB)/sodi um octyl sul fate
(SOS) and CTAB/FC5, whi ch al so have K 0.5 k
B
T.
1
Hence, i t appears that hydrocarbon branchi ng does not
reduce membrane ri gi di ty si gni fi cantl y over si mpl e chai n
mi xi ng. However, CTAT-ri ch vesi cl es have an effecti ve
el asti c constant K ) 0.15 k
B
T, si gni fi cantl y l ower than
those of CTAB/SOS, CTAB/FC5, or SDBS-ri ch bi l ayers.
I n fact, i t i s si mi l ar to that of equi mol ar dodecyl tri -
methyl ammoni um chl ori de (DTAC)/SOSo bi l ayers (K )
0.12 k
B
T, tobe publ i shed). Si nce the DTAC/SOSobi l ayers
compri se ful l y di ssoci ati ng hydrophi l i c counteri ons and
much shorter unbranched chai ns, we may concl ude that
the tosyl ate counteri ons i n CTAT-ri ch bi l ayers probabl y
do remai n associ ated wi th the bi l ayer, i ncrease the area
per surfactant head, and dramati cal l y reduce the mea-
sured ri gi di ty. I n that case, the rol e of tosyl ate i n the
bi l ayer i s anal ogous to that of short-chai n fatty al cohol s
that al so promote the reducti on i n measured membrane
ri gi di ty.
43
Hence, i t appears that much of the reducti on
i n bi l ayer el asti ci ty common to spontaneous vesi cl e
bi l ayers i s due to si mpl e chai n mi xi ng, regardl ess of the
type of chai n bei ng mi xed. However, i ncreasi ng the area
of the headgroup can reduce the el asti ci ty even more,
l eadi ng to more pol ydi sperse vesi cl e popul ati ons.
Acknowledgment. The authors thank J. I srael achvi l i
and E. Kal er for ongoi ng col l aborati ons and di scussi ons
and Dr. D. Dani no for trai ni ng R.V.Z. i n cryo-mi croscopy
techni ques. The authors grateful l y acknowl edge support
by Nati onal I nsti tutes of Heal th Grant EB-00380 and
Nati onal Sci ence Foundati on Grant CTS-9814399 and the
use of equi pment supported by the NSF MSERC Program.
LA034311+
(43) Chi ruvol u, S.; Warri ner, H. E.; Naranjo, E.; I dzi ak, S.; Radl er,
J. O.; Pl ano, R. J.; Zasadzi nski , J. A.; Safi nya, C. R. Science1994, 266,
1222.
VesicleSizeDistributions via Electron Microscopy Langmuir, Vol. 19, No. 14, 2003 5639