A high pressure cell for simultaneous osmotic pressure and x-ray diffractionmeasurementsBéatrice L. L. E. Gauthé, Andrew J. Heron, John M. Seddon, Oscar Ces, and Richard H. Templer Citation: Review of Scientific Instruments 80, 035107 (2009); doi: 10.1063/1.3089826 View online: http://dx.doi.org/10.1063/1.3089826 View Table of Contents: http://scitation.aip.org/content/aip/journal/rsi/80/3?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells Appl. Phys. Lett. 104, 251107 (2014); 10.1063/1.4884420 Spectroscopic infrared near-field microscopy and x-ray reflectivity studies of order and clustering in lipidmembranes Appl. Phys. Lett. 89, 233906 (2006); 10.1063/1.2403184 A microfluidic setup for studies of solid-liquid interfaces using x-ray reflectivity and fluorescence microscopy Rev. Sci. Instrum. 76, 095103 (2005); 10.1063/1.2040187 Effect of hydration on the structure of oriented lipid membranes investigated by in situ time-resolved energydispersive x-ray diffraction Appl. Phys. Lett. 86, 253902 (2005); 10.1063/1.1952583 High pressure cell for small- and wide-angle x-ray scattering Rev. Sci. Instrum. 68, 4588 (1997); 10.1063/1.1148436

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

A high pressure cell for simultaneous osmotic pressure and x-raydiffraction measurements

Béatrice L. L. E. Gauthé,a� Andrew J. Heron,a�,b� John M. Seddon,Oscar Ces, and Richard H. TemplerDepartment of Chemistry and Chemical Biology Centre, Imperial College London,London SW7 2AZ, United Kingdom

�Received 24 November 2008; accepted 8 February 2009; published online 23 March 2009�

In this paper, we report on a novel osmotic cell, developed to simultaneously subject a sample toosmotic stress and measure structural changes by small angle x-ray diffraction. The osmotic celloffers many advantages over more conventional methods of osmotically stressing soft materials tomeasure their structural response. In particular, a full osmotic analysis can be performed with asingle small sample �25 �l�. This reduces sample handling and the associated systematic errors, aswell as enabling tight control and monitoring of the thermodynamic environment during osmosis,thereby increasing measurement precision. The cell design enables control of osmotic pressure to�0.04 bar over a pressure range of 1–100 bar, and temperature control to �0.05 °C. Under theseconditions, the lattice spacing in lyotropic structures was resolved to better than �0.005 Å. Usingthe osmotic cell, we demonstrate good agreement with previous conventional measurements on theenergy of dehydrating the fluid lamellar phase of dioleoylphosphatidylcholine in water. © 2009American Institute of Physics. �DOI: 10.1063/1.3089826�

I. INTRODUCTION

The physical properties of lipid membranes are nowknown to play an important role in modulating the functionof a significant number of membrane associated proteins andin enabling trafficking across cell membranes.1–5 There ishowever a gap in our knowledge of the physical parametersof many biologically relevant lipids. This is in large part dueto the fact that experiments that probe these lipid propertiesare difficult and time consuming. In many cases, the rarity ofthe lipids are also a hindrance to such studies, where largesample volumes are needed. This paper is concerned with theuse of osmotic stressing coupled to x-ray diffraction �XRD�structural measurements for measuring both the energy ofinteraction between membranes and the energy associatedwith bending or stretching the membrane interface.6–9

Polymer stressing is commonly used to osmoticallystress lyotropic samples.6 In this method, a sample is held inequilibrium with a polymer solution �e.g., polyethylene gly-col� of known osmotic pressure, where the two are typicallyseparated by a semipermeable membrane, and the samplegains or loses water to achieve osmotic equilibrium with theexternal solution. Polymer stressing is widely employed forstudying lyotropic samples for a number of reasons includingthe following: �1� the necessary materials are cheap andreadily available, �2� it is easy to implement, and �3� is wellsuited to applying low osmotic pressures, which is importantfor studying the energetics of lyotropic systems. However,there are some significant disadvantages to the method: �1�

The approach is time consuming and labor intensive, requir-ing long equilibration times of many days, many XRD mea-surements, and weighing-drying-reweighing water contentmeasurements of many samples. �2� The method requiresrelatively large quantities of sample ��10 mg� for eachstressing point, which is not easily recovered. �3� The tech-nique is also prone to many random errors, which arise fromthe handling and transferal of sensitive samples and from themultiple measurements on many samples.

Here we describe a high pressure “osmotic cell,” devel-oped as an alternative means of osmotically stressingsamples. The osmotic cell uses physically applied pressure toosmotically stress samples and is integrated into a smallangle x-ray beamline for simultaneously acquiring onlineXRD structural measurements of a sample subjected to os-motic stress.10 While some attempts have been made in thepast to integrate polymer stressing based devices into x-raybeamline equipment for online structural analysis,11 to thebest of our knowledge, this has not been attempted beforewith a physical pressure based system. Primarily intended foruse on lyotropic samples, the osmotic cell offers many ad-vantages over conventional osmotic stressing techniques.The instrument we describe is automated, reduces systematicerrors, and increases the precision of this technique signifi-cantly, while using small sample volumes. Additionally,while we have only studied lipid systems, the instrument canin principle be used to probe the energetics of any watersolvated system as long as a structural signal can be re-corded.

The use of a physical force to drive water out of asample for osmotic stressing purposes is not a new conceptand has been applied in a number of quite different ap-proaches. One method, the “osmomanometer,” uses gravity

a�These authors contributed equally to this work.b�Author to whom correspondence should be addressed. Electronic mail:

andrew.heron@chem.ox.ac.uk.

REVIEW OF SCIENTIFIC INSTRUMENTS 80, 035107 �2009�

0034-6748/2009/80�3�/035107/10/$25.00 © 2009 American Institute of Physics80, 035107-1

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

to osmotically stress a sample by varying the height betweentwo open reservoirs interconnected by a semipermeablemembrane.12 The osmotic pressure accessible by this methodis however very small ��10 bar�, limited by the practicalworking height difference. The pressures produced are insuf-ficient to probe the energetics of lipid systems. Another com-mon method of applying a pressure difference to a sample isto directly squeeze the sample with a piston against a sup-ported semipermeable membrane.6,13 Under the applied pres-sure, water is squeezed from the sample through the semi-permeable membrane. An approach such as this is able toachieve much higher osmotic pressures than the usual 10–100 bar range available using stressing polymers but becauseof friction at the piston interface the approach is unreliable inthe low pressure regime. By replacing the piston with a flex-ible nonpermeable membrane, we have reduced friction tolevels where it can be practically eliminated, allowingsamples to be osmotically stressed between 0.1 and 100 bar.

The schematic outline of this osmotic cell is shown inFig. 1. A pressurizing liquid medium of known pressurecompresses the sample against a semipermeable membranesupported on a rigid porous frit. Under compression, thesample exchanges water with the equilibrium bulk solution�at atmospheric pressure� across the semipermeable mem-brane until osmotic equilibrium is reached. The sample isseparated from the pressurizing liquid by an elastic nonper-meable membrane. It is important that this membrane has alow elastic modulus to minimize the extent of back pressureapplied, which is particularly crucial for the low pressureregime when measuring the curvature elastic energy of lyo-tropic samples. A certain level of back force from the elasticmembrane is inevitable but can be quantified and kept to aminimum by careful material selection.

When integrated into an x-ray beamline, three simulta-neous measurements can be acquired �Fig. 1�, which are asfollows: �1� the pressure applied to the sample, �2� structuralinformation from XRD data, and �3� the temperature of thesample. The osmotic pressure need not be calibrated, and itcorrelates directly with the applied physical pressure differ-ence across the sample �i.e., the measured applied pressure P

of the pressuring liquid minus atmospheric pressure for thebulk solution�. This osmotic cell design allows a full osmoticrun to be carried out with a single sample that can be recov-ered for reuse if required.

The design requirements for the osmotic cell were �1� asmall sample volume for rare and expensive samples �lessthan 25 �l�, �2� a sample holder with low x-ray absorbanceproperties to enable online diffraction measurements to betaken, �3� a temperature controlled environment, �4� a pres-sure network capable of generating osmotic pressures in therange of 0–100 bar, �5� automated pressure generation toeliminate pressure fluctuations, and �6� minimum back pres-sure from the elastic membrane separating the sample fromthe pressurizing medium.

II. DETAILS OF THE APPARATUS

The osmotic cell assembly is illustrated in Fig. 2. It isheld in a thermal jacket that both contains and clamps theseparate components of the internal sample cell �Fig. 2�a��.The thermal jacket itself is a copper block with a central borein which the cell’s components are fitted and which containsadditional openings for the x-ray path. The exit port, conicalin shape, allows for 2� diffraction angles of up to 30° to berecorded. In addition, the thermal jacket has an internal net-work of water channels, which can be connected to an exter-nal water bath to provide temperature control to the cell. APt100 temperature probe fitted to the thermal jacket providestemperature feedback for a more precise temperature controlof the assembly.

The sample cell assembly within the thermal jacket iscomposed of two working halves �Fig. 2�b��. The bottomhalf includes a polycarbonate sample cell in which thesample is loaded and through which the x-rays pass. Thesemipermeable membrane �SPM�, supported on a sinteredsteel frit, is compressed flush against the inner surface of thepolycarbonate cell and locally compresses a custom madesealing O-ring �SO� to create a seal at the sample’s interface.This half separates the sample from the displaced water heldat atmospheric pressure during osmotic stressing of the

FIG. 1. �Color online� Schematic outline of the osmotic cell. Physical pressure �of known pressure P� is applied to compress water from a sample across asupported semipermeable membrane. The device is held in a cell of known temperature T, and simultaneous x-ray measurements provide structural infor-mation.

035107-2 Gauthé et al. Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

sample. The top half of the cell includes a brass membranesupport ring which supports and compresses a custom madenonpermeable elastic membrane �NPM� against the top ofthe polycarbonate cell. The membrane support ring also ori-entates the osmotic cell in the thermal jacket and positionsthe sample in the path of the x-rays by means of an orienta-tion rod �not shown�. The components that make up the os-motic cell are clamped together and secured within the ther-mal pressure jacket. Standard O-rings are used around thethermal jacket to ensure appropriate sealing of both the pres-surizing water network and the displaced water network. Forfurther details of the assembly, mechanical drawings can befound in the supplementary material.14

A. Polycarbonate cell

Polycarbonate was chosen for its strength and rigidity,required to hold the pressure in the cell for extended periodsof time, as well as for its low x-ray absorbance coefficient.These aspects allow thin window sections to be machineddirectly into a single polycarbonate cell for the x-ray path,precluding the need for separate x-ray transparent windows,and thereby eliminating sealing issues that might arise underthe high pressures involved. Wall thicknesses of 0.5 mm inthe path of the x-rays are thick enough to withstand the 100bar of pressure exerted and thin enough to minimize the levelof x-ray absorbance.

The sample channel in the polycarbonate cell was de-signed with careful consideration toward minimizing thedepth of the sample channel. A factor important in increasingthe efficiency of equilibration and minimizing sample vol-ume. The sample channel is a slot 1.5 mm wide ��z�, 5.5mm long ��x�, and 3 mm deep ��y�. These dimensions,respectively, provide �1� optimal width for good XRD from

lipid water mixtures, �2� sufficient length to aid the elonga-tion of the nonpermeable membrane, and �3� a minimaldepth to reduce sample volume, improve the sample volumeto semipermeable membrane surface area ratio, and ease theintroduction of sample. The volume of the sample channel is23.3 �l. The incoming x-ray path has been drilled with a1.5 mm diameter end mill. This allows for a certain amountof tracking across the sample by the �300 �m x-ray beam,which is necessary to ascertain the level of homogeneity in asample. The exit path of the x-rays was constrained to a slot2 mm high by 5 mm wide. As a result, wide angle diffractionis partly occluded in the �y direction �2�max=13°� but thefull range of wide angle diffraction can be captured in the �xdirection �2�max=30°�.

B. Nonpermeable custom made membranes andsealing rings

The NPM and the SO seals were both custom developedas no commercial products were suitable for the unique di-mensions of the cell. Casting the seals in custom molds, asillustrated in Fig. 3, allowed control of the size and shape ofthe seals, while careful material selection allowed control ofthe elasticity of the seals. Among the many synthetic elas-tomers available,15 silicones were found to exhibit excellentelastic properties and offered good chemical and aging resis-tance. The SO between the sample and the SPM was castfrom Ecoflex 0040 silicone �Smooth-On, Pennsylvania,USA�. The NPM, by necessity, was a hybrid of two silicones.The outer O-ring edge was cast from the less compressibleSylgard 184 poly�dimethyl siloxane� silicone �Dow-Corning,Michigan, U.S.A�, while the softer and more elastic Ecoflex0040 silicone was used to form the stretching inner mem-brane part of the NPM seal. Ecoflex 0040 is an ideal material

Membrane support ring

Pressurising water

a

c

5 mm

Pressure deformedNon-PermeableMembrane (NPM)

Semi-PermeableMembrane (SPM)&Moulded SealingO-ring (SO)

Porous Sintered SteelMembrane Support

Equilibrium DisplacedWater

Orientation rod

X-Ray Path

Sample (S)

(S)

Y

Z X

(NPM)

(SPM)(SO)

b

FIG. 2. �Color online� �a� Schematic view of the assembled osmotic cell clamped within a thermal pressure jacket. �b� Schematic of the “working” samplecell. �c� Enlargement of the working section of the sample cell.

035107-3 Osmotic cell for x-ray studies Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

for the elastic component of the NPM, with elastic propertiesthat approach those of latex; 900% elongation at break for alower tear and tensile strength �30%–50% that of latex�.Testing of the hybrid NPM seal revealed that less than 0.1bar was required to fully stretch the membrane, allowingosmotic stressing to be carried out in the low pressure re-gime. The NPM seal was also able to perform with no leak-age at pressures up to 100 bar.

C. Semi permeable membrane

The purpose of the semipermeable membrane is to sepa-rate the lipid sample from the equilibrium displaced water/buffer. It allows water and solutes to pass in either directionacross the membrane to satisfy osmotic equilibrium condi-tions under the applied pressure, while retaining the lipidbehind the membrane through size exclusion.

A number of alternative membranes were considered,from standard regenerated cellulose dialysis membranes tomodern synthetic polymer membranes �e.g., polyvinylidenedifluoride, polyethylene sulphones, and cellulose acetates�but the only varieties that are suitable in terms of their sizeexclusion properties are those used in dialysis or ultracen-trifugation applications. For the purposes of this develop-ment, we used the cheap and readily available regeneratedcellulose dialysis membranes. Regenerated cellulose mem-branes are available with a range of pore sizes �from �15 to�50 � and have a symmetrical pore structure that allowsmigration of small molecules in either direction across themembrane. They are also extremely strong and exhibit goodlong term stability in a range of solvents, over a suitable pHrange, and a wide temperature range, which makes them suit-able for long timescale osmotic stressing experiments. Thereare however a number of disadvantages with regenerated cel-lulose membranes. The membranes need to be prewashed toremove trace sulfurs and heavy metals, and once wetted themembrane cannot be allowed to dry out as this alters the porestructure. Cellulose membranes are also susceptible to cellu-lase activity.

We used Spectra/POR 6 �Spectrum Medical Industries,USA� 3500Da regenerated cellulose membrane in this work.The average pore size of this membrane is �15 Å. In theosmotic cell, the regenerated cellulose membrane needs to besupported in order to withstand the pressure applied by thepressurizing medium. This is achieved by resting the cellu-lose membrane upon a porous sintered steel frit that isclamped in place in the polycarbonate cell �Fig. 2�b��. Thefrit SIKA-R40 AX �GKN Sinter Metals Filters GmbH, Ger-many� with 40 �m pore size is highly porous and simplyserves the purpose of preventing any deformation of thesemipermeable membrane under high pressure.

III. PRESSURE NETWORK

Figure 4 shows a schematic layout of the pressure net-work constructed to control the osmotic pressure applied tothe sample. Pressure networks of this type can be found inthe literature16–18 for pressure control of sample environ-ments.

The system is based around a hydraulic pressure genera-tor �PG, HiP High Pressure Equipment, Pen, U.S.A� with an11 ml stroke capacity. Water was used as the pressurizingmedium due to its low compressibility19 and to prevent con-tamination of the sample should failure of the NPM occur.The generator acts as a screw-driven piston. The generator isconnected to a water reservoir and to the osmotic cell bymeans of valves and fittings. Included in the pressure net-work are manual �PM, 0–120 bar, Fluid-Air Components Ltd,U.K.� and electronic �PE, 0–100 bar, Ascroft, Dresser EuropeGmbH� pressure gauges, one safety valve �EV, with an es-cape setting of 100 bar� and one extra reservoir �PC�. PE isconnected to a microcontroller �ScM, Scorpion VM-1 Em-bedded Controller, Microrobotics, Cambridge, U.K.� to pro-vide feedback for automated control of the applied pressurevia a stepper motor attached to the pressure generator. Anadditional 150 ml reservoir �PC� was fitted to the pressurenetwork increasing its total volume to 210 ml. Revolutionsof the pressure generator therefore produce smaller changesin pressure, allowing finer control of the applied pressure �14bar per screw revolution�. Valves V1–3 are used to isolate thevarious components of the pressure system for differentphases of operation. The system is at atmospheric pressurewith all valves opened. With valve V1 closed and valves V2

and V3 open, the system can be pressurized. Should the tar-get pressure not be reached within one stroke of the genera-tor, closing valve V2 and opening valve V1 enables the gen-erator to be refilled.

Automation of the pressure generator was implementedto prevent pressure fluctuations. Small changes occur whenthe sample changes volume under stressing but more prob-lematically we found that changes in room temperature pro-duced substantial pressure fluctuations due to volumechanges in the pressurizing network �between 20 and 30 °C,a 1 °C increase in temperature resulted in a �1 bar increasein pressure�. To implement automation, the pressure genera-tor was fitted with a 25:1 ratio gearbox �718–874, Ra-diospare, U.K.� and 1.8° hybrid stepper motor �191–8340,Radiospare,U.K.� which was connected to the Scorpion mi-

FIG. 3. �Color online� Custom molded nonpermeable membrane �NPM� andsealing o-ring �SO�. �a� The photograph shows the hardened steel mold forthe two components. ��b� and �c�� Enlargements of the finished moldedNPM and SO respectively, with their extraction “wings” that are cut offbefore use in the osmotic cell.

035107-4 Gauthé et al. Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

crocontroller. The microcontroller manages the set pressurevia feedback measurement with the electronic pressure trans-ducer.

IV. PERFORMANCE AND RESULTS

A. Pressure control

The electronic pressure transducer has a rated sensitivityof 0.1% over the full pressure range �0–100 bar� and outputsa voltage between 0 and 10 V. The signal is acquired by themicrocontroller using an input channel with 10 bit resolution.Although the microcontroller’s resolution is marginally bet-ter than that of the pressure transducer, signal noise causespressure readings to vary by about �0.5 bar, with occasionalrandom noise events of as much as �1 bar. Precision isimproved through a custom pressure determination routine inthe microcontroller’s software. The routine averages manyindividual readings while excluding any readings beyond acertain cutoff range due to excessive random noise events.Averaging 100 raw readings and applying a cutoff of �0.35bar to artificially high or low values improved the resolutionbeyond the �0.1 bar of the controller’s input channel to lessthan �0.025 bar �Fig. 5�.

Automated control of the pressure is managed by theScorpion microcontroller, which measures the pressure viathe electronic pressure transducer and regulates pressure bydriving the stepper motor connected to the pressure genera-tor. A pressure routine runs a continual loop checking theset pressure against the measured pressure, adjusting it as

required to maintain the pressure within �0.04 bar of theset pressure �Fig. 6�. The system changes the pressure at�0.7 bar /s and produces little overshooting �less than �0.1bars for �10 s� when ramping. The automated pressure sys-tem can regulate stable pressure control for continuous pres-sure runs of many weeks without failure.

As the elastic nonpermeable membrane produces littleback force ��0.1 bar is required to elongate fully�, the pres-sure measured in the pressure network has very little system-

100 bar

RS

RS

Osmotic Cell

RS Water Refill Reservoir

GB Stepper Motor Gearbox

V1-3 Isolating Valves

PE Pressure Gauge (Electronic)

PM Pressure Gauge (Manual)

ScM Scorpion Microcontroller Box

SM Stepper Motor connected to Scorpion

EV Pressure Escape Valve

PC High Volume Pressure Chamber

PG Pressure Generator

PG

EVV1 PE PMV3 V2

PCScM

GB

SM

FIG. 4. �Color online� Schematic layout of the pressure generation equipment.

FIG. 5. Precision of pressure measurements. Comparison of pressure valuesas a function of software averaging, showing mean pressure, standard de-viation as the error bar, and minimum and maximum values. Comparisonsshow raw individual Scorpion channel readings �raw�, averaging of 30 rawreadings �Av30�, averaging of 30 raw readings discarding raw readings of�P�0.35 bar �Av30_x�, and averaging of 100 raw readings discarding rawreadings of �P�0.35 bar �Av30_x�.

035107-5 Osmotic cell for x-ray studies Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

atic error, and we apply no further corrections in the mea-surement of the osmotic pressure.

B. Temperature control

An external water bath provides temperature control tothe cell by recirculating water through the thermal jacket. APt100 temperature probe fitted to the thermal jacket and con-nected to the microcontroller is used to account for any tem-perature offset with the water bath. An averaging routine inthe microcontroller software, averaging ten raw values anddiscarding any value differing by more than 1 °C from theprevious reading, provides temperature acquisition with aprecision of �0.05 °C.

The water bath is able to maintain a set temperature towithin �0.1 °C over the course of an entire experiment.Figure 7 shows the temperature stability over �100 h of an

osmotic stressing run. This level of control is important formaintaining the stability of the sample, which is essential forthe high resolution XRD measurements made using the os-motic cell. For a lamellar 1,2-dimyristoyl-sn-glycero-3-phosphocholine �DMPC� sample in excess water, a tempera-ture change of 1 °C is enough to change the lattice-spacingD by 0.2 Å.20 In contrast, with conventional polymer stress-ing methods, it is difficult to control the temperature whentransferring samples between the stressing solutions and thex-ray beamline, and changes of many degrees can occur es-pecially when working at temperatures more removed fromroom temperature.

C. X-ray absorbance

X-ray transmittance tests were carried out on polycar-bonate pieces to ensure that satisfactory levels of XRD couldbe acquired in useful timescales through the osmotic cell.Measurements were made using a silver behenate samplewith and without 1.0 mm of intervening polycarbonate ma-terial equivalent to the total thickness of polycarbonate in theosmotic cell’s x-ray path. We found that polycarbonate has anegligible effect on the scattering profile and transmits77%�3% of the incident x-rays.

D. XRD measurements

The osmotic cell is integrated into a custom small-anglex-ray beamline based about a Bede Microsource x-ray gen-erator �Durham, U.K.� with integrated polycapillary optics�XOS, Albany, New York, USA�. XRD was acquired with animage-intensified charge-coupled device x-ray camera �Gem-star HS, Photonic Science Ltd, U.K.� using custom software.X-ray images were analyzed with custom software that ac-curately determines the center of the diffraction images tosubpixel accuracy and fits diffraction peaks with Gaussian

FIG. 6. Automated pressure ramps. �Left� Two separate pressure ramps overlaid �10→80 bar, and 80→10 bar�. Dashed lines indicate the set pressure.�Right� Enlarged regions of the two ramps, showing a small overshoot and subsequent fine adjustment to achieve set pressure to within �0.04 bar.

Temperature(°C)

Time (hours)

FIG. 7. Temperature stability in the osmotic cell. The graph shows �100 hof online temperature values recorded every 30 min for a set temperature of37 °C.

035107-6 Gauthé et al. Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

functions, producing D-spacing measurements with a preci-sion of �0.001 Å for high intensity well-defined powderimages. Given a stable sample, the precision of D-spacingmeasurements can also be improved by the acquisition oflonger images with better defined features. Sample changesresulting from osmotic stressing are not fast, thereby allow-ing longer exposures to be acquired. XRD acquisitions every30 min were easily sufficient to resolve changes in thesample under applied osmotic stress. Figure 8 shows the im-provement in D-spacing resolution as a function of acquisi-tion time for a changing sample under an applied osmoticgradient. Acquisitions were additions of multiple shorter 30or 60 s exposures. This allowed the acquisition of extremelywell-defined images without overexposing the higher inten-sity features. Image addition of multiple short exposures ishandled automatically by custom analysis software. Ulti-mately, acquisitions of about 5 min in total were employed,which allowed D-spacing changes to be resolved to better

than �0.005 Å for well diffracting samples. This representsan improvement of two orders of magnitude on the �0.5 Åprecision usually reported in the literature. Measuring andtracking XRD changes to this level of precision is made pos-sible by the consistent control of the thermodynamic param-eters �pressure and temperature� as well as the absence ofsample handling.

The precision with which small changes can be trackedis illustrated in Fig. 9. This example shows tracking of aD-spacing change of only 0.2 Å in a lamellar dioleoylphos-phatidylcholine sample, which is losing water in response toa small change in applied pressure.

Continuous monitoring of the sample also allows theuser to know when the sample has reached equilibrium. Atworst the equilibration curves can be extrapolated to obtainreasonable equilibrium value estimates.

E. Sample homogeneity

For the precision of the XRD measurements to have anyrelevance, stability and homogeneity of the sample is essen-tial. Inhomogeneous samples that contain a substantial levelof defects, such as excess water trapped in grain boundaries�see the review of Nagle and Tristram-Nagle9 and referencestherein�, would lead to erroneous XRD measurements. Os-motic stressing does generally remove most excess water de-fects and improves homogeneity considerably but the issuecan still be problematic in the low pressure regime. Subject-ing samples to a physical pressure is an excellent method ofhomogenizing samples, and pressure cycling can easily becarried out with the osmotic cell. Repetitively squeezing wa-ter from the sample then allowing it to reabsorb again canreduce the number of defects and improve the measurementsin the low osmotic pressure regime. We examined a range oflamellar systems, including dioleoylphosphatidylcholine�DOPC� and dioleoylphosphatidylserine �DOPS�, and foundthat sample homogeneity was good after an initial equilibra-tion period. All the lamellar samples showed practically no

Time (minutes)

D-Spacing(Angstroms)

single 20 s acquisitionssingle 60 s acquisitions4 x 30 s added acquisitions10 x 30 s added acquisitions

FIG. 8. Precision of D-spacing measurements. D-spacing measurements ofa DOPC sample in the osmotic cell, calculated from images of 20 s, 60 s,four additions of 30 s, and ten additions of 30 s. For the 10�30 s imagesthe D-spacing precision is approximately �0.005 Å.

FIG. 9. Example of continuous tracking of D-spacing in the osmotic cell. The graph shows a lamellar DOPC sample losing water in response to successiveincreases in applied pressure. The enlargement on the right shows a small �0.2 Å change in D-spacing, resolved to �0.01 Å, resulting from a �3 barpressure ramp at low sample hydration.

035107-7 Osmotic cell for x-ray studies Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

deviance in homogeneity horizontally �parallel to the semi-permeable membrane�, with all D-spacing values within�0.01 Å for a given vertical height. Vertically the homoge-neity varied to a greater extent and depended on the state ofequilibration at the time of measurement. In a stable state atosmotic equilibrium, the samples were vertically homog-enous to within �0.1 Å. Away from osmotic equilibrium,when the system was gaining or losing water, the homoge-neity showed a linear variation in water content reflecting thewater concentration gradient. Figure 10 shows vertical ho-mogeneity scans up a lamellar DOPC sample. The samplestarted from dry inhomogeneous conditions before reabsorb-ing water under low applied pressure. Enlargements showrepresentative homogeneity scans for the sample when at ornear osmotic equilibrium �bottom right� and when awayfrom osmotic equilibrium when absorbing water �top right�.The small vertical water gradients observed when not at os-motic equilibrium show that the rate of internal water ex-change within the sample is limited to some extent. Althoughno detailed measurements were carried out to gauge the rateof internal equilibration between regions of different watercontent, water gradients were observed to fully equilibrate�when under equilibrium osmotic stress� in a few hours.

F. Sample equilibration

While the semipermeable membrane functioned cor-rectly and allowed water to pass in both directions, the rateof equilibration was slow. Under large applied pressures�theoretically enough to drive most water from the sample�,no measurable sample changes were observed over a periodof a few hours, and equilibration times were of the order ofseveral days �see Fig. 9 and supplementary Fig. 2�.14 Withtraditional polymer stressing techniques, a typical lamellarsample can be fully dehydrated from excess water conditions

in about 24 h, removing a couple of microliters of water andreducing the D-spacing by more than 20 Å. In comparison,for the osmotic cell, although the sample volume to semiper-meable membrane surface area ratio may be anything up tofive times less than that for polymer stressing techniques, theobserved equilibration rate was at least ten times slower thanthis. For various lamellar lipid systems tested, regardless ofthe pressure difference applied or the ultimate change insample D-spacing, the maximum rate of D-spacing changewas never more than 2 Å/day in our system, and the waterexchange never more than �0.5 �l /day. This indicated thatthe semipermeable membrane itself was limiting the ex-change of water, and we suspect that the semipermeablemembrane suffered from substantial pore blockage. Giventhat the dimensions of the pores ��15 Å� are similar to thelength of a single lipid, it is possible that the lipid phase canbe forced into the pores of the membrane to some extent.However, it is unlikely that lipids can be squeezed throughthe pores as the dimensions are too small to allow clusters oflipids to pass, and the hydrophobic cost of passing a singlelipid would be prohibitive.

G. Comparative performance

The final examination of the osmotic cell’s performancewas made by making measurements of the compressibility ofDOPC bilayers that could be compared to previously pub-lished data �Fig. 11�. The raw data for the full DOPC stress-ing run can be found in the supplemental material �supple-mentary Fig. 2�14 along with the equilibrated values for Fig.11, which have been tabulated in supplementary Table I,14

and were primarily determined by single-exponential fits toequilibration curves, as detailed in supplementary Fig. 3.14

Figure 11 �right� shows how DOPC data obtained fromthe osmotic cell compares with published data obtained us-

FIG. 10. Vertical homogeneity scans up a lamellar DOPC sample. The sample starts from dry inhomogeneous conditions �filled squares� but homogeneityimproves as the sample reabsorbs water under low applied pressure �empty circles�. �Top right� Sample homogeneity when the sample is not at osmoticequilibrium �no applied pressure� and is taking up water �empty triangles�. �Bottom right� Homogeneity when the sample is held at or near osmotic equilibrium�applied pressure sufficient to maintain water content� �filled triangles�.

035107-8 Gauthé et al. Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

ing the polymer stressing technique.21 In the calculation ofDw� �water thickness between bilayers�, we used values forthe bilayer thickness DB� determined by Tristram-Nagleet al.21 �Dw� =D−DB��. Figure 11 �right� shows that the os-motic cell data is approximately parallel to the data ofTristram-Nagle et al.21 and a little less than 1 Å lower inD-spacing. The data of Tristram-Nagle et al.21 was acquiredat 30 °C while our data was acquired at 37 °C. It is esti-mated from temperature dependence studies of DOPC in ex-cess water conditions20 that this 7 °C increase might in-crease the deviance from the data of Tristram-Nagle et al.21

by no more than 0.5 Å. Nevertheless, overall the �1 Å dif-ference between the data is comparatively small when con-sidering that the published D-spacing values for DOPC varyby as much as 3 Å, both within individual studies and be-tween different workers, e.g., DOPC at 25 °C has been re-ported to have an excess water D-spacing of between 61 and64 Å.22,23 The osmotic cell DOPC results also compare wellwith other published DOPC and Egg yolk phosphatidylcho-line osmotic stressing data.9,24

In summary, we have demonstrated an osmotic cell thatoffers many advantages over conventional osmotic stressingmethods, including the use of small samples ��25 �l� andthe ability to dramatically increase the precision of stressingmeasurements. Using the well characterized DOPC system,we have shown that structural changes in a sample understress can be resolved to better than �0.1 Å, and the finalstressing measurements are in good agreement with pub-lished work using conventional stressing methods. The majorlimitation of our system is the rate of equilibration, with fullstressing runs taking many weeks to complete. Future workwould be needed to optimize the cell design to improve therate of equilibration and implement a system with multiplesamples in parallel to improve throughput.

ACKNOWLEDGMENTS

The authors would like to thank Mr. Mike Sheehy aswell as the team in the High Energy Physics Workshop at

Imperial College for machining the numerous parts thatmake up the osmotic cell and the osmotic beamline. Thiswork was funded by the BBSRC through a project Grant No.28/B15299 and the EPSRC through a Platform Grant No.GR/S77721/01 which supported B.L.L.E.G. and a Ph.D. stu-dentship to A.J.H.

1 S. L. Keller, S. M. Bezrukov, S. M. Gruner, M. W. Tate, I. Vodyanoy, andV. A. Parsegian, Biophys. J. 65, 23 �1993�.

2 L. R. Fisher and A. R. Malloy, Annu. Rep. Prog. Chem., Sect. C: Phys.Chem. 95, 373 �1999�.

3 G. S. Attard, R. H. Templer, W. S. Smith, A. N. Hunt, and S. Jackowski,Proc. Natl. Acad. Sci. U.S.A. 97, 9032 �2000�.

4 A. R. Curran and R. H. Templer, Biochemistry 38, 9328 �1999�.5 C. E. Morris and U. Homann, J. Membr. Biol. 179, 79 �2001�.6 V. A. Parsegian, R. P. Rand, N. L. Fuller, and D. C. Rau, MethodsEnzymol. 127, 400 �1986�.

7 R. P. Rand and V. A. Parsegian, Biochim. Biophys. Acta 988, 351 �1989�.8 S. M. Gruner, V. A. Parsegian, and R. P. Rand, Faraday Discuss. 81, 29�1986�.

9 J. F. Nagle and S. Tristram-Nagle, Biochim. Biophys. Acta 1469, 159�2000�.

10 A. J. Heron, Stored Curvature Elastic Stress in Lipid Membranes and ItsEffect upon the Activity of CTP: Phosphocholine Cytidylyltransferase �Im-perial College London, London, 2005�.

11 A. Squires, Bending Energetics and Phase Transition Kinetics in Type IIMesophases Formed by Hydrated 2LA/DLPC �Imperial College London,London, 2001�.

12 E. Raspaud, Eur. Biophys. J. 32, 402 �2003�.13 V. A. Parsegian, N. Fuller, and R. P. Rand, Proc. Natl. Acad. Sci. U.S.A.

76, 2750 �1979�.14 See EPAPS Document No. E-RSINAK-80-015903 for supporting infor-

mation containing further details of the DOPC experiments and the os-motic cell assembly. For more information on EPAPS, see http://www.aip.org/pubservs/epaps.html.

15 B. Dzikowicz, Latexes �R. T. Vanderbilt Inc., Norwalk, CT, 1998�.16 J. Woenckhaus, R. Kohling, R. Winter, P. Thiyagarajan, and S. Finet, Rev.

Sci. Instrum. 71, 3895 �2000�.17 P. M. Duesing, R. H. Templer, and J. M. Seddon, Rev. Sci. Instrum. 67,

4228 �1996�.18 P. T. C. So, S. M. Gruner, and E. Shyamsunder, Rev. Sci. Instrum. 63,

1763 �1992�.19 R. A. Fine and F. J. Millero, J. Chem. Phys. 59, 5529 �1973�.20 S. C. Costigan, P. J. Booth, and R. H. Templer, Biochim. Biophys. Acta

1468, 41 �2000�.

FIG. 11. �Color online� Osmotic cell stressing of lamellar DOPC. �Left� A plot of the equilibrium DOPC D-spacing as a function of the applied pressuredifference across the sample �i.e., the osmotic pressure�. The data set includes points from all three sections of a rehydration:dehydration:rehydration stressingrun. �Right� A plot of log �applied pressure� against water spacing Dw� from the osmotic cell, overlaying published DOPC stressing data from Tristram-Nagleet al. �Ref. 21� carried out at 30 °C.

035107-9 Osmotic cell for x-ray studies Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51

21 S. Tristram-Nagle, H. I. Petrache, and J. F. Nagle, Biophys. J. 75, 917�1998�.

22 L. J. Lis, M. McAlister, N. Fuller, R. P. Rand, and V. A. Parsegian, Bio-phys. J. 37, 657 �1982�.

23 S. M. Gruner, M. W. Tate, G. L. Kirk, P. T. C. So, D. C. Turner, D. T.Keane, C. P. S. Tilcock, and P. R. Cullis, Biochemistry 27, 2853�1988�.

24 R. P. Rand, Annu. Rev. Biophys. Bioeng. 10, 277 �1981�.

035107-10 Gauthé et al. Rev. Sci. Instrum. 80, 035107 �2009�

This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://scitationnew.aip.org/termsconditions. Downloaded to IP:

131.230.73.202 On: Sat, 20 Dec 2014 22:28:51