AN EPIFLUORESCENCE MICROSCOPIC SURFACE BALANCE TO OBSERVE MONOLAYERS AT T HE A IR - WATER INTERFACE .
by
I<aush ikNag
AThesi ssubmi tted in par tia l fulfilment ofth e requiremelit for the degree of
Masterof Science
Departm ent of Biochemis tryI
Memorial Univer s ityof Newfoundl a nd, St.Johnls,Newf ou n dland AlB 3X9.
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ABSTRACT
The details of thedesignand construct i on of an epifluores c e nce mi c r os c op ic surfacebalance and so me preliminar y observations onli p i d monomolecul arlayersperformed usin g t.hi s balance are di scus s ed. The balance cons istsof aTeflon tr oug h ....it h a computer- controlledmovable barriermounted ona vibrationreduc i ng platform. The balan ceis equippedwith an epifluorescencemicroscopicattac hmentby whi chvisual observationof fluorescentprobes in the monolayer is pos s i bl e. Barrier movement can be ut il i ze d to givemonolayer comp r ession and expansionvelocityfrom 20mrnZ/s e c up to600 mmZ/ sec . A Wilhelmy dipping plate connectedto a force transducer is used to measure surface tension in the mcnoLayez-during compres s i on and expans ion. Theepifluorescencemicroscopeis coupledto a cha rg e couple device in tandem witha nrceccnannea plate Whichpe rmits observation of the low li ght levelfluorescence from the monolayer. Images of the monolayerundercompression we r e visualized, sto r e d , digitized and proce ssed usi ng a videouni t and operator interactive software .
Using the balance preliminarystudies of phase behaviourhave beenperformed formonolayers of dipalmitoyl phosphatidylcholine (OPPC) and other lipidS. The phasecha ng e s were observed by incorporating1 moltof a fluorescentprobe,
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NBD-PC . int othe lipid formingthe monolayer. Thispro be had no app reciableeffectonthe isothernsand surface tens i on pro pe rt i e sof DPPCin.onola yers. DPPC lia sobservedto undergoa number of phase transitionswhen compr e s sed inmon o l ayer3. The liquid- expanded to liquid-co nde nsed pha s e transition wa s visualized as the formationof dark. p3tc he 5 (domains) frolll a fluoresce ntback.qround. The domainsincreasedin ave r a gesize tr om 100 IJ2 to 1500 p.2ina nonlinear manner asa fu nct ionof ar e a oc c up i ed perecrecmeof theDPPC. whe n mono layerswere compr essedat a rateof 20 M 2/sec. Th e size, shapeand gro....th of these domai ns"'e re quantitat i velycharacteriz ed and foundto be depe nd e nt; on compressio nra t es. At a fas t spe e dof compression(4A2/molecul e/sec) the domains were smaller 1n si ze . more irreqularlysha pe d, anduniJllodallYdi s t r i bute d. At asIa....
compressionspeed (0.13 Az,tllol e c u l e/s e c l the domains....ere large r insize and Illor e regularin sha pe. The shapes of thedomai ns werefo und to cha nge dramaticallywhen asmall amount (2 1110 1\) of cholesterol was incorporated in thel,ono l a ye r . No domain tonation was observedinJ:l.onola ye r s tor lipi dswithoneor two uns atu r ate d chains.
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ACKNOWLEDGEHP.NT
I would like to extend my sincerest gratitudeto my supervisor, Dr.K .M.W.Keough. for his patience, supervisionand encouragement during the course of this work.
I am extremely grateful to Dr. N.H.Rich for the in1 t1aldesigning and help in construction of the surface balance, and to Mr.C.Boland for writing the progranunes needed to control the instrument. I wouldalso like to extend my sincerest thanks to Dr. D. Pink, st.FrancisXavier University, Nova Scotia for suggesting a method for image analysis and toOr . P. Davis for constant enccur aqement; , periodical moral boosts and in helpful comments in revisionof the thesis.
I appreciate the help received from the Technical services, MUNespecially from Mr. a.Parson, D.FHlierand G.Brown, in construction of the various parts of the instrument.
I am specially indebted to Mr.R.Ficken of the Biology department ofMUNfor initial help in photography and experimentation with different methods of image acquisition. I would also like to thank Dr.M.Morrow of the Physics department, MUNfor acquiring the video camera.
I am extremely gratefUl to my friendDoreen and to Dr.
S. Mookerjea for constant motivation and moral support during the course of this work. Final ly I wouldliketo express my appreciation to SchoolofGraduate studies for financial support
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i-.the tormot afellowshi p, andW.Heckle for some valuable adv ice.concer n ingvisualizationof the monolayers.
(vi
TABLEOF CONTENTS
ABSTDCT ACINOWLEDGEMENT LIST 01' ABBREVIA T IO NS LIST OF FIGURES INTRODOCTION
I . Surface active monolayer 11. Pulmonarysurfactant
III. physical properties of insoluble monolayers IV.Instrumental design and development v.Visualization of the monolayer VI. Monolayer architecture VII.Objectives of the work
DESIGN ANDCONST ROCTIONOFTHEAPPARATOS I. princ i p l e s of construction II. Surface balance XII. Data collection IV. Epifluorescence and optics v,Ima g e acquisition VI.Image processing MATERIALS AND ME'l'HODS I.Materials
II. System calibration and procedures III. Ima ge analysis
(vii
i i iv viii ix
13 1S 22 23 25 25 28 Jl
"
35 37 41 41 42 43
RESULTS
A.Pr obecha r a cte ris t ics 45
a.oeserv eta cn of the mono l ayer 51
C. Ph as etra ns ition of DPPC in llIo nolaye r s 54 D.Qua nt itati v e analys isof the sur face ar chit e c t ur e 63
E. Other monvlayers 67
DISCUSSION 79
I. The EHSB: Performance 79
II. TheEMSB :App licatio n perspec t i ve 83 II I- Later al compression of amph i phi les:Domai nstru c t u re87 IV. Qua nti ta tiveanalysisof the surfacetextu re 95 V.Applic a bil i t ytolung surfactant study 99
CONCLUSIONS 1.0 2
REPERENCEB 104
APPENDIX 123
A. Name s and st ructuresof lip ids 123
B. Progra mme TEMP 6 125
C. Progra mme lMAGEPRN 157
D. Fo rmul a fo r conversionof thermisto r out putto 'K 161 E. Publ icatio ns arh.;"ng outofthis work. 162
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LIST OF ABBREVIATIONS A - Angstrom
AOt - Area Of Interest
AROS- Adult respiratorydistre s s synd ro me OPPC- Olpa1 mitoylphosphat i dy l ch oline OMPA- oimyristoy l phosphatidi c acid DOPe - Oioleoyl ph osph atid ylcholine
EMSB - Epifl uores =enceMicroscopic Sur f a ce Balance FT-IR- FourierTr an sformInfra r e dspectros c opy 1 - surfacetension
LE/LC - liquid expanded toliquid conde n sed MHz - megaHertz
mN/m- mil liNewto n per nUOIt e r po - micrometer
NBD - 7-Nitr o- 2- 1 , 3- benzod i a zol- 4 - y1
11"- sur fa c e pressur e
PC - phosphatidy1choline PE - phosphat idy1ethan01 a mine PG - phosphatid ylg1y c e r ol PI - phos ph a tid y linositol RDS- Re spiratoryDi stres s Synd r ome SAM - Surfa c eactivemateri al
SOPC - 1-s tear oyl, 2-01e o ylphos pha t idylcholine T. -gel toliquid- crystall inetransitiontemper atureof
bilayors
TLC - Thinlayer chrom a t og ra phy (viii)
LIS'l'OJ' FIGURES
1. Diagrammatic representationof possible route of secretionofpUlmonarysurfactant to the air alveolar interface(after wright 1966).
2. Examplesof surface pressu r e~molecular area isotherms sho....inglatera l phase transitions for four differentlipid species compressed in monolayers (after Cadenhead198 5 ).
3. Diagram showing the possiblemolecular arrangement 11 of phospholipid molecules in thedi f f e r e nt phases of the monolayer duringcompression (after Cadenhead 1965).
4.
5.
Sho ....s the possiblearrangementof molecules in monolayers in (a) surface balanceand (b) a bubble.
DiagralMlaticoutlineof surface balance and horizontal viewof thetro ug h .
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15
17
6.
7.
Diagram of the balanceou tli n i n g the attachment ofthe epi f luores cencemicroscopeto thesurfa c e balance.
Schematicblock diagram of the balanceshowing interface connectionsof the different parts with the computer.
26
27
8. Photograph of a DPPC+2mon NBC-PCmonolayer 36 takenwith ast il l camera.
9.
10.
11.
13.
Photographof theEMSB showing theva r i ous accessorypa rtsneededto control the funct i on i ng of the balance .
Fluorescenceexcitation andemi s s i on spectra of NBD-PC inethanolsolution.
SurfacetensionYo§.%pool are a plots of DPpe, and DPPC+va r iousmol t NBD-PC.
Isothermsof DPPCand DPPC+2 mo1% NBD-PC.
Photograph of TLC plateshowing the migratio n ofDPPCand thepr o b e s.
ex,
38
46
47
49
50
u .
15.
Photographsof a) DPPC and b) DMPE monolayers
with 2mol ' of the probeNBD-PCas se e n under the epifluorescencemicroscop e.
a) Surfacepressuren area/moleculeisotherm 53
55
16.
17.
re
ofaDPPCmono l a y e r at 26 ·C. b) su r f a c e ten sion:i§t of pool areaplotof the sam" monolayer .
Isotherm of a Dl'~Cmonolayer at 24'Ccomp re s s ed witho u t barrier st op p a g e at a rateof 4 A2/molecule/sec.
a) Isotherm of DPPC+1 mol';:~ I'\-PCco mpressed at a rate of 4Al/molecule/ sec obtainedat a temperatureof26 'C.b)imag es of themo no laye r obtained at different sur f a c e pressures.
Frequency distribut i onof domainsizeobta ined 57
5'
60 fromana l y z i ng theimaS' ~ srecorded from twomonol a yers at amole c ula r ar e aof 52 A2/ molecule.
1 .
a) Isotherm of DPPC+lmol Ii:NBD-PC compre s sed at a rate of 4A2/ molecule/sec .b) The averagearea of the domainsplottedas a functionoftime afterbarrier stoppage.
(x i)
62
20
21
2'
a) Isothennof DPPC+1mol tNBD-PCcompressed in steps at a rate of o.nA2/molecule/secanda temp era t u r e of20·C. b)Typical frame
grabbedimages fromvarious regions of the is ot h e rm.
c) Frequencydistributionof size analyzed from20 images obtained at surface pressures A to E.
Frequency distributions of domain si z e obtained fromtwo independent experiments.
Average domainsize plottedas a functionof molecularareaof two monolayerscompressed at slowspeed of 0. 13Al/molecule/sec.
64
.5
sa
as 23 Ca ) Frequencydistribu tionof domainsiz e
obtai nedfrom analyzing 20 randomlyselected imagesof a mono layercompressed at 4 A2/molecule/sec.
b) frequency di s t r i b ut i o n of domainsize obtained from the same molecularareas of a mono layercompressed at a slow speed (0. 13 A2/molecule/sec).
24 Frequencydistributionof domain sraee calculated fr omtwo experiments compressed at different speeds.
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71
25 Linear regression of Jomain size as a function 72 of molecular area obtained from four independent expQriments.
27
2B
29
Histogram of average value of percent coverage by dark domains plotted against molecular area.
Isothet'll\ ofsape+1moltNBO-PCcompressed at slow speed and a temperature of24"C.
Typical image ofOPPC+2mol t cholesterol obtained at a surface pressure of5mN/m, from theLE/LCphase transition region.
Diagrammatic representation of a model showing 74
76
77
"
the possible arrangement ofOPPC in theLC (darkareas ) and theLEregions (light areas) of a monolayer.
30 sequential images showing the growth of individual 92 domains from a surface pressure of11mN/m (A) to 18 mN/m (C).
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INTRODUCTI ON
I.Surfa ce ac tive monol aver
Surface tension of a fluid can be consideredas a force acting per unit length at its surface. The moleculesat the gas- fluidinterface compared to the onesinthe bulkphasehave a non compensatedattractive force acting on them directed towardsthe bulk phase. This imbalance of forces at the interfacecauses the surfaceof th e fluid to behaveli ke a stretched filmunder tension. Th e force exertedcan be measuredby dipping an inert thin metal plate into the fluid interface . A cleansurface of water would register a force acting on the plate. This force, measured per unit length in milliNewt ons /meteror dynes/centimetre , is known as surface tension. At 37'C the surfacetensionofwat~ris observed to be 70 mN/rn.
Ala r ge number of biological and synthetic amphiphilic molecules when spread on a clean water surface spontaneouslyform a monomolecularlayer of thic kne s s of several Angstrom units These monola:r ..rs can counteractthete ns i l e forces acting on the fluid interface and thus reduce the surfacete ns i o n . The amphiphilic molecules range frombiological membrane const ituents such as phospholipids to fatty acids, certain proteins and a numbe r of other organic species.
During the early part of this centuryan instrumental apparatus,today knownas a surf a c e balance,was devisedby Langtlluirby which the physical pr ope r tie sof monolayers could be studied (r ev i ewe d by Gaines 1966). The propertiesofmo no layer s werest ud i e d by compre ssingthe monolayerlaterally at the interfaceand by moni t ori ng thesurfa ce pressure exe r t e donit (Ga i nes 1966, Adam1968). Thesurfac e pressureis definedtobe the value of the differencebetween the surface te nsion of the cleansurfaceofthe subphaseand ';hatof the inte rfacewiththe monolayer. Sev e ra l fattyacidswere used to studythe area occupiedby the moleculesat the interface and howth i s would affectthe surface pressure during a decrease of monolayerarea (Ga i ne s 1966 ) . Surface balanceshave been modified inthe last five decades,thoughthe bas icprinciple of construction remains thesa me. The balanceconsistsof a trough, made out of chemicallyinert ma t e r i a l , inwhichthe fluidor wateris contained. Us i ng a dipping plate in combinationwitha force measuringdevicethe surfacetensionis measured. Other teChni ques usingdi f f e r e nt typesoffloa t s and threadson the su r face havebe e nused tome a s ur e thesu r fa c e pressure. A monolayerof the materialto be studiedis spre a d at the sur f a ce troma solution of known conc e ntra t i on of the SUbstance. The solution ismadein a hiJhly evaporative solvent. The evaporativesolvent is chosen for quick deposition of the material byspreadi ng the solut i o n at the surface. After evaporation of the solventth~monolayer canbe compressed and
the surface pressure monitored as a function of molecular area (Gaines 1966, Adams 196B).
II. Pulmonary Surfactant
., number of workers have shown that the type II cells of the alveoli secrete a surface activematerial to the alveo l ar air-fluidinterface (BUckingham 1962, Ring 197 2 , Clements 1976 ).
The surface activema t e r i a l is foundto consistof a numberot lipid species and some proteins (King 1972,Hawgood 1985). The surfactant extractedfrom mammalianlung by lavaging the alveolar fluid was foundto consist of about 74%phosphatidylcholine (PC). 10% phosphatidylglycerol (PG), 5% lyso lecithin and sphingomyelin, 4%phosphatidylethanolamineand 2%
phosphatidylserine int of total phosphorous assayed (Ki ng1972, Van Golde1988 ) . Somehyd r ophob i c proteins,re f e r r e d to as SP-B and SP-C (Suzuki 1982), and a hydrophillic protein SP-A were al s o found later by other workers (King1973). I tha s been sugge sted that these lipids in conjunctionwithsome ofthe proteins actas the pUlmonary surface active materia l (King 1972,Goerke 1974, Suzuki 1982). The surface active material collectivelyknown as pulmonarysurfactant has be e n suggested to form a monomolec ular layer at the air- alveolar fluid interface (PattIe1960 ) . Figure 1 shows a diagram outlining the processof sur-fa ctant;secretion by type II cell to form a monolayer atthe air alveolar interface. The monolayer of surfactant undergoing compression duringexpirationreduces thesurfacete ns i o n ofthe alveolar
Fiqure1. Diagra mmatic represent ationof pos sibleroute of secr e t i o n of pul mo narysurfa cta nt tothe ai r alve o l a r int er fac e indic a ted by thearrows. The surf a cta nt is synt he sizedand packagedintola mella rbodies (LB) , secreted tothe alv eol a r flu i d astubularmye lin (TM)and fina llyad s orbe dtothe air alveol a r inte rfa ce as amo nol a ye r. (redrawn from wr i ght 19BB)
Re.J..ya.W'"
·h oMvJ... i \j-ht
wa~4.
fluid (Sc hu r ch 1978 , Goerke1974). It wasfound laterthatat physiologicaltemperatureof n"cthesurface tensionatthe air alveolarsurfaceat maximal expirat ionis about 1mN/ n (Schurch 1984). Thi sdecre a seof surfacetensi on in combinatio n withthe tensile prope rty of the alveolireduc e s lung comp lianceandthus the"'orkofbre ath i ng (Sca rpell i 1988 ).
Themajo r compo ne nt ofthe surfa c tant (about 45\ of to t al weight) was found to be a zwitterionicphOSPholi pid 1,2 dipalmitoyl phosphatidylcholine [OPPe l (Jl:ing1972). This amphi phile wa s alsofound to spread ina monolaye r ~and re duc e surface tensionof waterto near 0 tnN/mWhe n compre s s e d to the minimal mol e cular area (Galdstone1967, Notte r 198 0). Dire c t determination of surf acetensionof thealveolarfl uid interface at low lungvct ueealso showedsuch low va l ues (Schurch1978 ) . OPPCha s be en studiedextensive l y inthe lastthir t yyearsusi ng su r f a c e bala nc etechn iques,and itssurf ac t ant and other biophys ical prope rtif!s char acterised (Chapman 1967 , Ph il li ps 1968, Not t er 1984 ). Tho ug hDPPCseelllsthe ideal material for actingas asurfac ta nt during there s pirat o ryprocess, it cannot ma i ntai n a con stant monomo l e c u l ar laye r durinqre s pir a t ory cycles. Due to the ma teri a l s slo..,respread ingand reads o rpti on propertiesat the interlace ,lossof DPPC at the surfac e during the increaseand decreaseof alveolar int erf ac i al areawould depl e t e the monolaye rof mate r i al. other surf a c t a nt co mpon e nts suc h as mixed acyl cha inphospha tidylglycerol ,
phosp ha t idy llnositol(Notter 1980 ) and hydrophobicprote insSP-B
andSP-C (Su zuki1982, Ha wgo od 1989 ) have bee n suggest edto play animportantrolein surf a c t a nt mono layer home osta s i s (King 1912, Notter 1980 , Keoug h 1984). Thoug hthe phy s icalproc e s s bywh ich the pUl monary su r f ac ta nt formsmonola yersat the air- alv e ol a r int erfa c e is not cleartodate thest udy ofits individual compo nen ts 1n...Y.1..t..can yield inf orm ati on rel ev an t tono rmal pu lmon a ryfunc t ioning(Goerke 1914 , Ba ngh a m1979, Ke oug h198 4).
Absenceof sur facta nt in thealv eol iwas shownto causea particular syndr ome duri ngwhich the lun g s of prema t u r e neonate s collapse dur ing cyc les of breathing (Avery195 9). In neona testhis condition is termed as Resp i r ato r y Distress Syndrome[ROS] (Hildebran 1979, Enho r ninq 1990). Another syndrome knownas adu l t re sp i ratory distresssynd r omeor AROSis suggested to oc c urin ad u l tsduetoleakageof somepla s ma prote i ns into th e alve ol arfluid causi ng hindera nce innormal surfact a nt activi t y(Ashbaugh 1967 ). Medical re s earch has be e n intensive in the last de cadetofind anar ti ficia l su rfaceacti ve ma t e r i a l (SAM) which co u l d beappliedexoge nouslyinpat i e n ts SUf fe ring fr om ROS(Hal lman 198 4,Oblade m1990). It issugg ested th atthecompos i tion ofsuch ar tificialSAM shou l dhavethe propertiesoffa st resp readi ngand adsorptio n tothe int erface , loweri ng of the sur f a c ete ns i o ntoabout 0 mN/mof the alveolar flu idinte r fa ce during max i ma l expirati on and ab ilityto forma stable film for a si g ni ficant pe r i od of ti me (Cl e me nts1976, Bac hofen1987). The materi alshc uf d alsobe easil ydeliver edto the air-a lveolarinte rfacefromanexogenoussou rce. Itis yet
not totally clear which componentsof SAM are responsible for these biophysical processesi.n...Y.iY2., but a number ofcl i n i c a l trials....ith differentartificia l lipid andli p i d- pr o t ein mixtures have shown a certain degree of successin alleviatingpatients sufferingfromthe syndrome (Fujiwara80, Muller1990). These findings have renewed inte rest in studyi ngsurfactant monolayers
.in...Y.i.tJ;using versions of the class icalLangmuir technique,and
by current modifiedmethods of visualobservation of the monolayer. Other techniques of direct inf r aredspectroscopy of the monolayerat the interface (Dluhy 1989 ) havegi ve n a new approachto study thesurface phenomenaof surfactants. Thes e techniquesprov ide in f o rma t i onon themole c ula r organization of thesu r f acta nt inthe mono layer .
III. Physicalproperties of i05 0 ]ublemonolayers
Phospholipidand other amphiphilic molecu leswhen spread at the air- waterinte r f a ce form a monomolecularlayer, in whichthe hydro phobicmoietyof the molecule orients itsel f towardsthe air andthe hydroph ilic or polar headgroup dissolv e s in the water (Gaines1966). Whensucha molecularlayeris compressed laterallythe surfaceshows a non ~line ar reduction ofsurf a c e tensionor an increase of surfacepressurein the monolayerup to the point of monolayer collapse. The surface pressureplottedagainstthe molecu lararea atth e relevantstate
ofcompress ionand at afixe dte mp eratu r e, givesindirect inf o rma tionaboutthephy s i c a lsta t e and theorientationofthe mol eculesat the inte r fac eand is knownas a pressu re-a rea isotherm. suc hplotsare shownschematicallyin figure. 2.
Is ot he rms of thephos ph olip id , OPPC,below 41·C displaya "l ate ral phasetransi t i on " ora disorderedto an orderedstateof molecularpack i ng in the monolayer over a range ofmolecu lar areasandsu r fac e pres sure s (Phillips 1968 , Gershfeld 1976, Albrecht 1978 ). As shown bytheis o t he rm II in Fi gure 2the sur facepressure inc reas es ina nonlinearmanner duri ng compl:ession anddecr eas eofthemo l e c u l a r inte rfac i alar ea ofthe amphiphiles inth e mono layer. The isothermsbecome horizontal at a part i cu la rmole c u larareas, indicat inga change ofphy s i c a l sta teof the OPPCmono layer. Thischan ge of state occ u r s dueto the change ofpa c k i ng and dynami cs of the mole c u l e s at the interface and is termed as a"phas etr a ns i ti on". At large molecularareas and euerece pr e s s ur e s near0 mN/m. themon ola ye r be ha ve s like atwo -dimen s i o nal gas (Gin isot herm II, Fig.2) in which it is assumed the molec u lesare separa tedby large inte rmo l e c ula r distances. Wit hdecreasingareapermole cu l eand inc r easi ngsu rface pr e s s ureth e gas phase changestoa li qui d- l ikepha s e . For amphiph ilessuc h as OPPCtwo liquid- li ke pha s e , liquid - exp an ded (LE in Fi g . 2 ) and liqu i d -condense d (LC inFig.2) may exist (Albrecht 197 8 ). Atr an s i t i on from LEtoLC occurs as the areaper moleculeis decreased . It hasbe en sugge ste dtha t in theLCphasethe chains of the molecu les
Figure 2. Examplesof surface pressureYJi.ee r e e u r a r area isotherms showingis othe rma l phas e transitions for four dif f e r e nt li p i d species compressed in monolayersat room temperature . I. Stearic acid, II.OPPC,III. DOPC andIV. diolein. The individu al phases are indicated as gas (G), liquid -exp a nde d (LE), liquid - condensed(Le) and solid (S) on th e isotherms. (after Cadenhead 1985)
SURFACe PRESSURE
=
. .
are orientedmore re gu l arl y andperpe nd i cular totheinterfa c e th a nin the LE phase (se c xn en 198 7 ). Furthe r Inc r-easeof surface pr essure is suggestedto form a solid (S in Fig.2) phaseuntil themono l a ye r collapses (Gershfeld1982).The isotherm I in Figure2 showsa gas to sol id tra nsi tionasseenduri ng compressionof various fa t ty acidmonolayers suchas stearicand palmiticacidsat room temperature. The isotherm III in Figure 2 disp laysa gas to li qu i d - expandedtransitionas observed during compression of phos pho lip idshavinguns a t ur a t e d chainssuch as DOPC,and isotherm IV!i.IOWS a continuousgaspha s e as observed for monolayers of diolein and triolein (Cadenhead1984). The phaseregionsfor DPPC are alsoencv.:in th e isotherm in Figure 3<., andthe p'::lssible molecular confo rmation 1nthese regions as suggested by a number of workersare shownin figure 3b (Albrecht 197 8 , Sackman1987). The ectecu rar de t a il s of these transitions are not quite clear at present and several th~ o ret i c a lmodels for mo l e c ul a r orienta tioninthe different phases of th e monolayer havebe e nsuggestE.~ (Albrecht 1978,Georgallas198 2,Zuckerma n 19 82) . These theoretical modelssugnest thatbe l ow their cha in meltingtemperaturethe conformations (gauche, trans) ofthe hydrocarbonchains of the eacur-at.ed phos pho l i p i d moleculeschange duringth e expanded to condensedpha s etr a n s i t i o n of the mo nol a y ar. The changeinconformationofthehydroc a r bon chains ca us e s an orderi ngof the molecu les in the condensed phase.
Infrared spectroscopicdatasuppo r tsthis suggestion of acyl chain orderingduringthe phasetrans i t i on of DPPC in
10
Figure3. Diagram showing the possib lemoleculararrangement of phosPho lipidnokecu j.es(b) in th e diffe rentphasesof the monola yerduringcompression.The phases arepointedon theDPPC iso therm (a) as G (gas), LC~LE (liquidexpanded to liquid condensed) and S(s Ol i d ). (after Cadenhead 1985)
11
so
w~20
'"
~JO lC;::=LE
G
60 110
AREA/MOLECULE:.
a
• 4eY r i AIR
...
WATER.~'fW 'e".
IVWW·
LE" ~[
...
_WIll alllR
u.;dc.a...LC.II1U "S
Solill~tta
b
h.--.:.(l..,...1.~i.$.V\ t~:-\"v\
C.,,<b,,l,,,,,,~ 1'185
monolayers (Oluhy 1989).
Mostat the biophysical studies otphos phol i p i dS have beenconducte d in relationto the factthat a monolayer is a simple model of a biological membrane (Oanei11i 1934). The externa lsurfacearea of a set of erythrocytes, and the area obtained fromextracted lipid materialswhen spread and compressed in a monolayer were comparedby some workers (Gorter 1935 ).They found that the surface area oftheli p i d monolayer was close to twice that of the erythrocyte and hence proposed a bilayer st ructurefor cell membrane. The earlymodels of the bilayermembrane were flawed by not assuming that proteins arean integral part of biomembrane, and were latermodified by others.
The currentlyacceptedbi l a ye r model ofthe cell membrane (Singer 1972) is infa c t designed part lyfrom initia ldata ofthe earlie r stUdies. Research on monolayers as a model forbiological membranes has provided useful biophysical information, especially onthe potential lipid orientation (Cadenhead 1980, 1995), lipid - lipid (Cadenhead 1984), and lipid- protein interactions (Nakagaki 1982, Mohwald 1990 ) in biornembranes. Information from monolayerstUdies also shed light on the nature of bilayer organ izationof moleculessuch as chainordering (Birdi 1988), headgroup electrostaticint e r a c tio n , electrical properties
(Korenbrot 1980, El Mashak 1982) and some immunological processes suchas cell surfaceantigen binding (Subramanium1986) and receptor-substra tecoup ling(KrUll 1990 ) .
12
IV. Instrumentaldesig nand development
In thela st fe w decades monolaye r physical properties have been studied usingseveralmodifiedversionsof Wilhelmy balances (Gaines1966 ) , by bubbleandbalance type s of surfactometers (Enhorning 1977) and thr oug h transfer of the mo1ecuhrhyer onto a solidsubs trateby the Lanqrnu ir-Blodgett technique (Fischer 198 5 ). The stud iesconductedusing either the surfacebalance orthe bubblemethodhave furnis heda great amount of information on pUlmona rysurfactant dynamics. These met hodssimulate the respiratory process~as the mono laye rscan be compressedand expanded rapidlyand their dynamic propert iesstudied wit h ease. Ind iv i d ua l components of the pUlmonary surfactantandtheir binary andternary mixtures have beencharacterizedusing these methods (Notter 1980), al thoughthe reliabi lityofthe surface balance techniqueto study pUlmonarysurfac ta nt has been questionedby others (Bangham
198 7 ).
Though th ebubblesurfactometer technique may apply the most relevant con di t ions to surfactantsas found in alveoli it has certaindisadvantages. The technique can create conditionsthat best simulat ea brea thinglung such as 100%
humidityfo rthe monolayer environment, th e monolayers have inte r f a c i al curvaturein the bubble similarto that expected in the alveoli, and the cyclingspeeds performedare reflectiveof the re s p i r a t ory process. The or ientationofthe monolayer inthe
13
sur f a c e balanc e and the bubbleareshown in Figure4aand 4b re s p e c t ively.The process by which the surfac e tens i onof the int erf a c e is lIeasu redinthebubble is shown in Figur e 4b. The pressure PllIeasu re~to hold thebubb leopenis related to surf acetension by the Laplac e equ ationdi s c us sed inthe legend ofFiqure4b,and should not beco nf u s ed with su rfacepressure
('Jl"). Dis a dvan tage.of the bubblete c hn i qu e are tha t it can only
yie ldminitna l inf orm ationaboutthe conc entrationof the material on the surf a c e (Enho r n i ng 1977) and it doe s no t allow for sepa r ation of proc e s s e s of adsorptio n andsur f ac eref in i ngvery easily. Thus it isdifficult toana lyzewi thprecision the respreadingpropertie s of binaryandothe rmixt u r e sof the surf a c t antcomponentsusing this method (No tte r 1984). The Wilh e lmy te c hniqu e doesgi ve in f ormationabout sp eci fic conc entrationof themat e rial inthe 1lI0no iayer , and alsosurface pr e ssur e - area, surfacepressur e - tillede pe ndency ofthe mono layer. The major disadvantageof thete ch n i qu e is found to be monolayerleakage aroundthe barriersat high surface pressur e. A combinatio nof informationfromboth te c hn i qu e s is be stsui t e d for analyzingpulmonarysurfactant and its biop hysica l properties.
The basicdesign ofa Langmuir type sur face ba l an c e consistsofatroughmad efrom soli d Te U o nblockin whicha aub ph a a e wi th asurfacemonolayeris co nta i ne d. Amov ab le ba r r i erwi t htap e rededges is us ed tocompr essthe monolayer . A con t inuous Teflonstri p ora rhomboida l fra me can also beus e d to
14
Flqur e 4. Shows the possiblearrangementof molec ulesin monolayers in (al surface balance and (b) a bUbble. Surface tension('O orsur f a c e pressur e(lf) during compressionof the monolayer in the balance can be represented as a functio nof mo no l a yer area as shown by the qraph X. The surface tens ion ('r) in the bubb leint e r f a c e is measur e d from th echanqeof the pressu reacrossthe bubbleint e r f a c e (P:-P1or AP as shown in theinset qraph'l) and radiusof the bubble (r)durinq inflation ofthe bubble by Laplaces equation;.ll.P..21/r. The arrows indicatedirection of compression of the monolayer in (a). and thedire c t ion of decreasing pressure to keepthe bubble open in
(b)•
15
a
Pressure transducer
15,
b
comp r e s s the monola yer . By using the latter methodmo nolaye r leakage around barrierscan be avoidedor reduced. Monomolecular films arespreadov e r a water surface from aso l ut i on of the material in which the solvent eva po r ate s ina short period of time ,leavingbehind a metastableorst a bl e monolayer at the ai r - water interface. The surfa cetens ioncan be continuously monito r e d byadipping plate incombi na t i on with strain gage. The outlineof the instrumentalset up usedin thiswor kis shown in Figure 5.
The molecularareaca n be deduced fromthe geometr i c dimensions of the troughandca l c ul a ted for the particular molecular spe cies usingthe fo rmula-
MolecularAr ea (A~/molecul e)... [( l e ngt h of trough inA) * (br e a dt h ofthe troughinA)]/ (Amount of ma t e rial on th e sur f a c e inmole s* 6.023*lO~J) .
The surface pressurecan be calculatedfrom the force act i ng on a dippingplate of neglig iblethickness by the forrnul a-
Surfa ce Pres suren(roN/ ml '" (Sur f a ce tensionof water (mN/m) - Surfacetensio n of waterwith monol ayer (mN/m) ]
nteA"f'""fo- "fwhere"fo- sur fa c e tensionof distilled
water
l' - surfacetensi o n of water with monolayer Anal t e r na tive method of empl oyinga force transducerin combination withcomputerized programmewhichcan measure
16
Figure s. Diag r amma t i coutline of a) surf a c e ba l a nce and b) horizontalvie w ofthe trough. The monolayer is spr e adon the trough andcomp resse d by a barrier. The sur f a c e tensi on can be moni t ored bya platinumdippingplateincombinati o nwitha fo rce tra nsducer ,anddisplayedwith a digitaldisplayunit. b) Shows the trough wit hcyl indr ica l collarat tac hme nts toreduc e mono l a ye rconvec t ivemoveme nt .One of thecylindricalhol e can be usedfor visual obs e rv a tio n and the ot he r tor the dipping plate . Theholes are conn ectedto the bu lkof the subpha s e bycha nne ls. Thecylindrical co l l ar at t achment is usedin sur fac e ba l a nc e s in which vi sua l observa tion ofthemonolayer is pos sible.
17
TODICITAL DISPLAYUN IT
DIR ECTION OF' COIIIIPFIESSION
~
ii
I
[- -I---- ---e....-
ReJ...,.""" ;" 0""'
Poll"'t Iq~T'70
differential force (Cadenhead 1984 )or surface tens ionchange shown in the equation-
AF '" AIoI •g .. p'*0.1.cos B
where6101-measured weight change of the plate
g- acceleration due to gravity p-perimeter of the plate B- contact angle of thesub s t r a t e
meniscuson the plate. can also he used.
When the'plate is totally wetted the contact angle B is equalto o and thus cos8..1and the equationbcccne a -
Il =(Aw'*g)/p.
For precise measurement of these parameters a microprocessor based system can he used in which the barrier movements, pressure and temperature measurement and time dependent changes ca nbe monitored by a computer (Albrecht1983).
V.Vis ualizati on of Monolayers
Attempts to visualize monolayers transferred onto a solid substrate by electron microscopy lead to conflictingviews on monolayer surfacearchitecture (Fischer 1985, Neumann 1984 ).
The major breakthrough in this field came in the early part of this decade when a group of workers reported observing
18
visu a llythe phasetransition region oflloDPPCllIonolayer using fluo resce ncemicroscopy (von Tscharner19 8 1 ). Thetechnique useda fl u ore s c en t lipidanalogueinth eIllo nolayerand observed the fluorescencefrom the monolaye r duringcompre ssi on. Using an epifluorescencemi croscope the yobserv ed blackpatches against a fluo rescent ba c kg r ound attheLEtoLCphasetrans i t i on regionof the isotherJI. Epit'l u ore s c en cecan be describedas a microscopic arrangemen t ofle ns e s by'~i1 i chus ing a singl e obj ect ive le nsthe excitationand obs e rvat i onof fluoresce nce fromtheObjec t can be performed. It wassu gge s t e d that theprob e molec u le s pa r ti t i o ne d betweenthe di f fe r e nt phases. The dark patcheswere late r co ns i de redasli qu id-conde nsed pha s e and thefluoresce nt area asliqu i d- expanded phase (McConne ll1984. Losche19 8 4 ).
Anumber of othe rworkers SUbs ta ntiated the pre liminaryobservationsintheyears tofollow. One groupof workerspublishedthe firstvi sual bla ges of the phase ccexf snence reqion while attemptingtome a s ur e translational diffusion speedsof OMPCandDPPC in monolayers(Pe te rs1983). A compl e tedescriptionof the technique waspubLf ahed
si mUlta ne ous lyby others (Losche 198 4 , Burghardt1984). The te c hn i que was extensiVelymodified in thelast few years using comput e r - assis tedvideo mi c roscopy (Melle r 19 8 8 )anda differ en t typ eof le ak proo ftroug hdesign (Moj t a ba i 19 8 9).
Each ind ividua l instrumenta ldesig nwasbas ed on the typeof inf o rma t i o n neede d to beobta ine d about the monolayer.
SomestUdieswereaimedatvisuallycharacterizing th e
phase 19
transition of fat tyacid lTIonolaye rs(Moore 1983, Knobler1990 ) , whereasothe rshavetr i e dto studythe effect of ions on the phase transition processof a charged phospholipid monolayer(Eklund1988). Others, by modifyingthe technique, could measureth eelectrostaticinte ractionbetween phospholipid moleculesat theair - water inte rface (Thompson 1984,1988). To datea single report exists on the quantitative analysisof the monolayer architectureof DPPC (FlorSheimer1989). These workers (Florsheimer 198 9 ) attempteda quantitativeevaluation of the surface arc hitecture in order to determine if the features observed had equilibrium properties andtheir efforts had met with limited success (Florshelrner 1989). Th.... visual architecture observed by fluorescencetechnique was also observed by other techniques such as charge decoration electron microscopyand electrondiffractionof DMPA and arachidic acid monola ye r s (Fi s c he r 1986). The architectures observed in electronmicroc opy of DMPA monolayers were similarto the ones observed by the fluorescence technique of DPPC monolayers. The process of enzyma ticdegradation of DPPC and DMPC monolayers by hydrolytic action of phospholipase Ai was also visualized for the first time using the fluorescence technique (Grainger 1989). Lipid - protein interaction (Heckle1985) and ligand- receptor interactions (Kr u ll1990) have also been visualizedby this technique.
The basic design and construction of surface balances for visual observation of monolayers are simila r. The
2.
instrumentsconsistof a rectangu lar tr oug h wit ha movable bar r ier or compressionsystem and anep ifl uo r escencemicroscopic at tachment . Th e fluoresce nt pr obe is excitedeithe r fromabove (von Ts ch arner 1981, Mel ler 1988 )orfrom below (Losche198 4) the monol aye r using alight sourceand app ropriate filt e r combination allowi ng a fixedwavelength(s) of lightto be incident on the mono layer. The emission of fluorescencefrom the monolayer is vi s ualiz edei t her us ing st il l photog r a p hy (McConnell 198 4)or by using a lowli g ht level video camera (Losche 198 4 , Moore198 6 , Mel ler 1988). The surface pressure- molecularareais simUlta ne ou s l y monitored. Thiste c hn i qu eallows forvisual inspectionof each specificreg i onof the is othe rm. Th e visual dataisquanti f ied using digital image process. '9 and image ana lysissystems (r.cecne19 88, Seu l 199 0 ) . The surfacepressure -molecular area data can accordingly be correlatedwith quantitat i v e visual inf orma tion.
The method ha s beenus e dto observe the phase tr a nsiti o n process of vari ous ampiphil icmoleculesin the monolayers suchas stearic acid (Moore 198 6 ), DPPA (Losche1988) . DMPE(Helm 1988) andDPPC (McConne ll 1984, Florsheirne r 198 9 ).The informati onobtained by the above mentionedworkersis of releva nc e tomode lling molecula r pa c kingin bioll1embranes. None ofthe studieswereaimedat observ ingthe mono layerduring dynamiccompression -expansioncycl eswhichare rele v a nt to lung surfacta nt recyclingj" the alveol i. The variousdesigns discussedabove were modified byusto attempt to simu latesuch
21
conditions.
VI.Monolaye r Arc~
Usingthe epifluorescence balan c e diff e r en t regions of th e DPPC isot herm showed distinctlydifferentvisualarchitecture
(vonTscharner 1981 , Peters198 3, Losche 1984). At large molecu larareas wherethe surface press ure wa s near 0 rnN/m large dark "g a s e ou s" circular areaswere observed to coexist wi t h fluor esce nt"l i quid " outlines(Losche 1984) . These images resembled foam like structures andweretheore t icallyspeculated to be either the coexistence of gas an dli qu i d phases (Losche 19 84 , Berge 1990) or to be areas inthe monolaye r devoidof any lipids coexisting with lipid containingareas (McConnell 1984).
Thesepatt ern s werealso observedin monol a y e r s of pe nt a de c a no i c acid (Berge 1990 ) and stearicacid (Moore 19 83 ). When the DPPC monol aye rwas compressedbe yo ndthe point ofgasph a s e the surfacewas observedtobe homogenously fluo r e s c e nt from surface pr ess.z -ee of 2-5InN/muptoth e break point of the isot herm (McConnell 19 84). The breakpoint of the is othe rm (sh ownby pointBh Fi g ur e 2) wa s observed visually as consistingof II non homoge nous bla c k patches or nUcleation sites "appeari ng from th e fluorescent background andwas re ga r d e d as the onset pointot the main"lat e r a l phasetransitio n " (vo n Ts cha r ne r 1961 , Mohwald 1990 ) . These patchy areaswere foundto growto a limitedsize wi t hla t eral compressionofth e monolayerandto exhibi t diffe rent shapes (mainl y cirCUlarorell ipt i c al ). They were.
te rmed as " condensed phase lipid domains" (McConnell 196 4). 2Z
Thesedomainswere found to have lo ng rangeelectr ostaticorder andwere arranged in the monolaye r with periodicsymmetry (McConnel l 1984 ). The domainswere also fo undto have certain equil i bri um pr op e rt i e s and wer econsidere das li qu id crystalsby a certai nqroup (Hohwald 1990 ). Thesedomain structureswere alsoobservedin DMPC ,DMPA(Losche 1988 ) and in DLPEmonolayers (Helm 1988 ) compressedat very slowspeedsbelowthe th e rma l transitiontemperatu re (Tel of these molecules.
VII.Objectives of this work
Themain objectives of thi s work were to designand constr uctan epifluorescencemicroscopic surfacebalanceand studyingmo no laye rs visually at the air - wate r interfaceusing such a balance. The Lne't r-ument;was designedto perform compr ess ionand expansionof monolayerswithdifferent speeds so th at sloW'and fast processes occurringin the monolayerduring phasetr a ns i tio ncoul d be studied .
Thecomponents of pUlmonary surfactants, especially DPPC has beenstudiedin surfacebalances, andsurfacepressu re - area, pressure - time dependency of such mono layersquite well charac te r isedby a number of workers (reviewedbyNo tte r 198 4) . Thoughthese studiesgive us a fair idea ofDPPC mono layers phys i c al behaviour atthe air - water interface, the actual pr o .::e s s bywh i c hthese behavioursoccur could not be directly visua lized by earliertechniques. Our second objective was to studyDPPC in monolayersusing ou r balance. The s e studies ca n
23
als o yi e ld informat i on on theefficiencyofour balance to study compo ne nt s of thepulmonarysurfactant. As mentioned earl ie r monol ayers need tobe compr essed and expanded rapidlytorelate to respiratory ratesand pulmonary surfactant functio ni ngin
~. We alsowanted to obs e rv e ourins t rumen tsca pab ilityof performing fastcompre ssionsof llIonola ye r s, and whether the proce s s esoccurring at theinte r f a ceduring suchcomp ress ions cou l d be visuallyobs e rv ed. Checking th esecapabilitieswould alsoyield inf ormat i o non the me r i t s and demer i t sof the instrumentand theli mits of itsperformance.
Our fina l aim wastovisu a llyob s erve the LEto LCpha s e tran s ition of DPPCin i'lrmolaye rs, and possibl ysome higher surfacepre ssureregimeof the DPPC iso t h e rm and to ch aracter iz e quantitat i v elythe featuresobserveddur ingsucha tra ns iti o n.
Th e quantitativeinformatio n ofthevisua l architecture s observed during this transitionisext re me ly limited at present. Thuswe wa ntedto study theLEILeph ase vi sua lly andana l yz e thedo ma i n st ruc tures obs erved in relationto someot he r phys i c a l prope rt ies of themo no l aye rssuc h assurfacetension , sur f ace pre s s ureand molecularareas. Weal s o wanted to visu a lly observe some other lipidsin monolayersunder compr ession to gai nan understanding mo l e c u l a rme ch a n ism of domain formation inmono l aye r s.
21
DESIGNJ.NDCOHSTRUC'rIOHorTHE APPARATUS
I.Prin cipl esofco~
The principlesof design and const ruc tionwereba s e d upon earlier reported designs of epifl uo rescencemi croscopic surfa ce balances (Peters 1983, Losche 1984,Meller 1988 ) and from initialexperimentsperformedby us witha small Teflon trough and a mi croscope. Each ofthese experiment alsys temsdiffe r in the irfuncti onalaspects. The de s i g n of aco llar , as usedby some workers (Pe t e r s 1983) to decrease translationalmotionon the monolayersurface ....as also employed inour design. A microprocessorand inte rfacingde s ignwas tested and built into the system. A sof twa re programmeto operate the system was also constructed ,mod ifiedand used.
The basicmechanicalcomponentsof theba lanc ear e re pr e s en t e d in Figure6. The main componentsare the Teflon tr ough , a Wilhelmyplate ,a fo r c etr a ns duc e r mounted on a XYZ translator, the microscope and optical system, and the anti- vibration bloc k. Aschematicblock diagram ofthebalancewith its computer-controlledbarrierdrive and data acquisition components is representedin Figure7. The balanceport ionof the trough is attached tothe anti-vibrationblockseparate l y fr omth e microscope. The motormovements arecontrolledby a steppe r board, and da t a collectedby a data acquisitionsystem.
2'
Fiqure 6.Diag r a lll outlining the6tt a c hme ntsof the epifluorescencemicrosco peto the surfacebalance.A.Teflon tr ough , B.aluminum ba s e , C.Tefloncollar,D.p lasticcover with sealing,E.movableTeflo n barri e r, F.ste pper moto r,G. limit switch fo r motor cont ro ls, H. X-'i-Z tr a ns l ator, I.vi br a t i on isolationsupport,J.grani tebase, K. forcetra nsduc e r , L.
fluorescence conden ser, M. li g ht source, N. image int e ns i f i e r, O.
CCDcamera, P.trinocular tube.
2.
2••
Figu re7. Schematicblock diagram of thebalance showing interface connect ionsof the differentparts withthe computer . Image sarerecordedby the camera into the VCR ordirectly into the computer by the framegrabber board.The computer rece ive s digital informationfrom thepressure transduce r vi a thepre s s ur e gauge and dataac qu is i t i o n interface (DAS-S) board. The mo t or control isperformed by signalstrollthecomputerconverted throughtheHSTEP-5 int e r f a c e board.
Z7
~
These systems are interfaced to the same computer, and the experimentaloperationcontrolledby programme written in C language.(Appendix B)
Fluore_scent probes are excitedby a SOW mercuryvapou r li ght source and emissionis monitored by usingan
epifluorescence filtercOmbination. Images arerecordedbyus ing a charge coupled device (CCO) chip and microchannel plate intensifier either directly onto video tapeor by digitizingand recording directly in the computer by"frame-grabbing". Image s from the video tape can be subsequently digitizedby "frame- grabbing". The frame - grabbed images can be analyzed by an image analysis system and plotteddirectly from the memory.
II.SurfaceBalance
Initial experimenta tionwith varioustroughs of small area was conducted in ourla bo r a t o r y. To observe mono layers at high surface pressures in thesetroughsan increased loadof material on the surface had to be used. This allowed for a partial construction of thesur f a c e pressure- area isotherms.
It was also observed that domains in a monolayers couldonlybe visualizedwhen compressionwas initiated from large area per molecules. Thusto visually observethe monolayer of a complete isothermfrom large molecular areas to thepoi nt of film collapse a la r getr ough was chosen. The large troughallowed greater
28
ac curacy inmeasuri ngthe area permolecul eand increasedth e exten t to wh ichmonola yerscouldbecOlllpressedintheba lanc e.
A solid Teflon block 35 emx15CII.wa sus e dto construc t the trough {Fiq. 6 CAJ] havingcavity dillensions of22 C1Il x 7.8c. X 1.5ca , AnalWlli numbas e (Fig. 6 (B))with milled chan ne l s toallow wa te r flowwasat tachedinorder to control temperature of the aqueousphas e. The wholeunit we i gh e d10Kg and could not be mount edon a ordi n arymi c roscop ebase.
Tempe r a t ure contr olwith time iswi t hi napproxi ma t e l y l"ene a r room temperature ina givenregion ofthe trough, although variation s over the whol e troughran ged over ±l"c. Thermal co nd uc tionthrough Teflonis low, and some unev enness in milling ofthe tro ugh base couldcaus e the se localized te mperat ur e diffe r ences. Tempera turemon i t ori ngwas ac hievedby a thermistor in con tactwith ene watersurface. The m1l1io hms output of the thermisto r is convertedto temperature val uesin deg reeke lvin by a form u lashown in append i x D. Surfacetension and optical obse rva tio ns were made in co nfi ne d reg ions as in a "collar"
design [Fl g.6 ee)]Wh i c h was suggested ear lier tored u c ela ter al pres suregradie ntsand di f fusi ongra di entsin the ecncteyee (Pe t e r s 19 8 3). Surface te nsion measureme ntsof the mo noli.,yerwere notaffected by the col lar, as thesurface tensionvalue s were the same forisothermsconstructed in thetroughwith and wi thout the collar. The trough is enclosed bya pl a stic
cover [Fig. 6( 0 ) ) which isabo ut4Clllabove it s surface and 29
cont a i nssma llopenings fo r themicroscopQ ob j ective ,trans d u cer arm andthe mot or sha f t forthe mova ble barrier . Theopeningfor the object ive alsoallowsfor cle an i ngthe obj ective dur i ngthe experimen ts without changing thefocallength. Thecover is sealed at the ends to avoid air tur bu le nce,toimpr ove te mperature equilibration, andtominiaizllIevap o r a tion. vari ous desig ns of ecvebj.ebar rie rs wereconstruc ted and tes ted. xe rre e a material Which, likeTeflo n, doe s not we t well, wasfound to be uns u i t a b lefor the movab l e bar rier const r uct i on due its high elast icity. A Tetlondamwith ta per e d edges [Fig . 6 (E)]and adjus tabletightening attachmentwas found su i t ab le. At high surface pres s ure s (> 55 mN/m) we observedsomeev i dence sugg e sti ve of a small amount leak ag e of material out of the monol a ye r. The mov able barrier is connected ,via a 12 inch threaded shaft, to abi d irectio na l linear actua t or steppermotor (Air pax , L9 2411- P1, Cheshire , CT)whichopera tes in 0.001inch increme nts [Fig.6 (F)]. It s tra ns i t isconstrainedby two roll er-typ e limit swi tches {Fi g. 6 (G)) at tachedtothe trough fram e . The linear actuatormotor iscontrolled by a stepper motor controll er board (MetraByte , MSTEP-5Taunton, MA) in combinati on wit ha accessory bo a r d (Hetra Byt e ,STA-STEP). The combi na tion provide s twoinde pe ndent channelcon t ro l , bidirectional stepmovement atcons tantspeed orac c e leration or de c e l e r a t i on. ThGcombinatio nals oha s 5limi t s'Witch inputsand programmable inte r na l and external clocksou rc e s. Thi s combina tio n re s ultsinawide ra nq e of moto r sp e e ds.
3 .
For control of theva r i ou s accessories ,an IBM-AT compatiblepersonal computer (TATUNG7000 ) with640 Kbyte RAMand 20 Mbyteharddisc memoryisus e d. Theco ntrol of the motor movementis performedby modifying theMSTEP-S software assho wn inthe progralMlBTemp6(Appe ndi x B). The operatio nof the instrument isco nt r oll e d bya clocksetting of 10 MHz.
The trough assembly is mountedon an XYZ tra n slator [Fi g.6 (H)) which allows itspo s i t i o n i ng, inclUd i ng focu sing , underthe epifluorescence microscope . The translator could withstanda load of maximum 10 kg. To avoidstraining thesyst em a ball bearingisat t a ch e d to the Z arm of the translator. Thi s helps to smoothenthe verticalmovement dur i ng focusing and during other vertical movement of the translator. Duringan experiment , movementof the troughis restrictedto about3mmin the X and Ydi r ect i o n by the coverofthe trough. The restriction occursbecause the coverisdesignedto fit fairly closelyaroundthe microscope objective . The troughan d the translatorare fastenedtoala r ge granite base which isin tur n mounted on vibration Iacj.atIonsupp o r ts [Fig.IS(J)j. The trouqh can be easily removed for cleaning.
III. Data Collection
A forcetransducer (GRASS,5FSSPO, Quincy, MA.) of
31
displacement sensi t i v i ty20mm/Kg . in combinati onwi tha stra i n gauge DCampli f i er (de s i gne dand built by Technical servi ces, Memorial. University ofNewfoundland )isus e d tomon i to rsurface tensionof the eubphaaeof the mo nolay e r . The sur fa c e tension is me a s ur e d by a rectan g ularpl a t i num dippi ngof2.5cm in length, 1 emin breadth and 0.25mmthickness. This sy s t emis int erfa c ed through adata acquisitionsys t e m (Metra Byte ,DAS-8) tothe comput e r . The CAS-8 sys tem has a limitationof re a di ng analog si g na l s in thevoltage scale. A 9vol t D.C . amp li fier usedto boost the mill. ivo l t signal s fromthe pre s sure gauge is connec ted to thean a l og todig ita l (A! D) converter. Theachannel A!D con v ert er all o wsda tacol lectione!tici ency at 4000data points / sec ,whichis an adv antag e dur inghighspe e dcompressions.
compre s sion-expansioncyc les arecont rolled bya high leve l program,(TEMP-6 ) co mbining the si gna l fromthetxa nsduce x andthe motor st e pinforma tion into sur f acearea~surf acepre ssur e (or surfaceten sion) for rea l-ti me gra phica lou tput (rea l -t i me displa y of, su rface pres s ureY.§t imeisalso possib le). The programmealso contains inf ormatio n about total area ofthe trough, the are a compr essedbythe ba r r ie r andti me re qu i red for one complete compres sion expansio n cycle. The control of the programme is pe r f orm ed via a mo u seand curse r. Th.eAIDconve r ter sc an rate is set at 100 0 conv e rsions/ s ecandthesoft wa r e ave ragesfrom50- 250 conver s i on s/da t a pointdur ing compress io n.
In additio nto data fi l e contr ol , theprog r a m pr ov i des
32
forinput of volume and concentration of the material used for spr eadingthe monolayer which permitscalc ul a t i o n of, and direct di s pl a yof,thearea permoleC"uleof theli p i d on theXaxis of an isotherm. Percentageof total pool area can also be displayed on the abscissa.
Speeds of compression from 20-600mmZ/sec (7 t - 219 t of the maximummono laye r area Imin) are attainable. The barrie r can be stepped in small or largeincre me nt al distancesand stoppe dafter any desired incremen t. Thisallows foruseof pressurejumpsfol l owed byreal-time monitoring of changes in the sur f a c e.
IV. Epifluorescence andOptics
Anep i f l uo r esc e nc e condenser (Zeiss, typeIVFI) (Fig.
l(L)] was attachedto ast a nda r d ~e i s soptical microscope base fi xe dto the anti-vibrationblock (a 70kg gra ni teslabof dimensions90cm X 38 cm X 7 cm). The stage of the micros cope was removed,and the troughpositioned under the objectiveby the XYZ translator.
various typesofobjectivelenses were investigated.
ZeissPlanar ,Neofluar and Epiplanar lenses of magnification16X, 40X and60X weretried. The customary objectivelensesusedfor observing fluorescentsamplescould not beusedas theserequir e acov e r s l i p over thesample. The Lew magnification objective
33
(16X) had largewor k i ng distance butwa sfo u nd to have 10\0'light gathering efficiencydue to the large distance from the monola ye r surface. Lenses withlIa g n i fica t i o n of 60X had too short a working distance and werealso fol.1nd unsu itablefor observing a mo v i ng liquidsurface . An 40Xobjective (Zeiss £piplan 40/0.8 5 W) was foundappropri atefor currentapplicati ons . Thi s le ns does not require acove r s lip over the salllple and alsohas II large aperture whichenhancedthe light collection efficiency of the mi c r os c o p e . The ob jec t i ve andthe trinoculartube [Fig. 6(P) l in combination with the camera anda 12 inchmonitorgavevi sua l magnification up to about 5000ti meli. The hi g hre s ol ut i o n of the instrumentha s an advantageof allowingobservationof anysha p e or structural changes that occur in individualdomains.
A 50Wattme r cu ry vapour lampwith housing (zeissHBO SOW) [Fig.6(M)] was found sui table fo r excitation. The low lightleve ldecre a s edphotobleachingof the probe and thus al l owe d an inc r e a s e in the observati on tilDeof themono l ayer. An red attenuation til tQr was us ed inthe cond e ns e r of the mi c r os c o pe to block the infraredand redwavelengthsof the excitation light. An advantage of loW' inte nsi t y il lUllli n a tionwas that surface photo rea ct ionsdue tohigh excit at i o nint e nsi ty, which might cause fluctuationsin domain sh ap e(Se u l 1990)could be avoided. For Observing ep LfLuc eeacience from the surface, a fluores c e nc e filtercombination (Zeiss,type £516) suitabletor observingfluorescencefrom NBD-phosp hol! pidswas used. The
34
combination of filtersconsistedof a barlJ pa s s 485/20, excitation FT510,wide emissionLP520 fil ters,whichwere suitable for observationof qreenfl uo r e s c e nc e and compatible with the Zeissepifluorescence condenser.
v. Image Acquisit ion
still photographywith a Zeiss camera (WinderM 35 mm )to collectimages during growthof domainsre s u l t e d in inadequateima g e resolution due tolowli g ht levels andmono layer movement during longexposure times. We could successfully applystill photographic techniquesusing a superfa s t film (TMZ 5054 Kodak)with maximum clarit yonlyat higher sureece pressures,where the monolayerwas relativel yimmob ile. A still photograph obtainedat 1 second exposuretime and film speed of 30,000ASA at surfacepressuresof 20mN/mis re pre s e nt ed in Figure 8. Inorde r to allow forobservation under dynamic compressio nandunde r quasi -equilibriumconditionsat low and intermediate sur-race.pres sures a low lightleve l video camera [Fig.6 eN)] (FairchildCorp.CCO3000, PaloAlto,CAl in tandem wi t h an image intensif ier [Fi g . (6) OJ(Varo, 25 mm Hep, Garland, TX) was used. The intensifierwas controll",dbya custom built- power sourcewith manualcontrol features. This al lowedfor amplifyi ng the signalsfromthe camera during observations of low fluorescenceintensity from the monolayersur -ra c e and impro ved
35
Figure S. Ph o t o g r a ph of a DPPC+2 molt NBD-PC monolayertaken witha st i llcamera ....ithsuper fast speed film (30,000 ASA) at a surfacepressure wher e th e monolayer is relatively immobile. The fluorescent areas are represen tedas white inthe photographand the condensed areasas dark spots.Scale baris 151-1.
"
15~
3••
the quality of the images considerably. The CCD chipof the camera was attached to the eyepiece of the trinocular tube. The siliconchip of the intensifier reduced the noise and was not easily damaged by excessive light. During monolayer compression the fluorescenceintensity varied at different regions of the isotherm, but by using automatic qain control the observed fluorescence and quality of images could be controlled. The signal to noise ratio of the images weresa t i s f a c t o r y for subsequent image analysis. A photograph of the epifluore.scen ce microscopicsurface balance with it s various accessories such as the computer, video recorder andpower source controls isshown in Figure 9.
VI.Image Processing
Image processing and analysis was controlled by menu - drivensoftware (J a ndel Scientific,JAVA,CorteMa dera, CA) in conjunction with an image - grabber board (Truevision, TARGA-M8), a digital VHS video cassette recorder (JVC,HR-D700V, Ja pa n) and a monochrome monitor [panaaon Lc , WV54-10, 'ioklohama,Japan). The image capture board featureda spatialre s o l u t i on of up to 512by 482 pixels with a-bitresolution and monochrome inputfa c ilit i e s.
It alsohad hardware-implementedzoom and pan features, real-time digitization, and the ability to display256 colourfrom a pallet of over 16 million, for falsecolour if desired.
37
Piqure 9. Photographotthe EHSBsh ov i ng the various accessory parts needed: to control thefunct i o ningot the balanc e .
..
38.
Images could be either captured directly from the camera through the TARGA board and stored through a specially written macro programme, or recorded with the VCR and digitized from the tape. Initial size calibration was done using a micrometer scale giving 0.2 #.1m/pixel of spatial resolution.
In routine operation, recording was initially done directlyto video tape from the CCD camera. During video recording the surface pressure - area information displayed on the computer screen could be overlaid on the video images, and the combined image recorded. These frame overlays were used during the recording period to distinguish the length of recording time for each individual step of compression. Recorded monochrome images were played back: from tape using the digital still-frameand single-frameadvance features of the VHS recorder. This allowed the operator to choose from a vtce selection of images from the tape. Storage of a large number of digiti..~dimages Iotas unnecessary When they were recorded on VHS tape since they could be digitized subsequently from the tape for analysis.
An area of interest (AOI) was defined within the spatial resolution of the display and was maintained constant for all images used in any analysis of a series of images from any given isotherm. Suitable images, randomly chosen at a given surface pressure, were isolated and captured to the display memory of the frame - grabber board. Some preliminary image
3'
processing was usua l lynecessary to enhance the cont rast in the AOI. Suc hoperationsinclud edcontras t enhancement, filte ring, an dsmoothing . AnInten s i t y RangeOf Int e r e s t (IROI) was chosen soas tode f i n e the objectsof inte r estinthe AOI endfor object arei!l and perimeter de t e rm i n a t i on . Randomnoise and partia.l boundary objectswere eliminatedfromthe AOI and da t a for remaining objects tra n s f e rred to a dataspreadsheet. This process was repeated for a number of chosenframes in eachofthe recordingperiods and theresultingdataand images were stored and ana Lyzed ,
4.
MATERIALS AND METHODS
1,2M dipalmitoylphosphatidy lcholine (DPpe), cholesterol, 1-stearoyl,2-o1eoyl phosphatidylcholine (SOPC) and stearic acid werepurchasedrxc n siqma Chemicals [St.Louis ,MOl.
The fluorescentlipid analoques N-4-nitrobenzo,2 -oxa-l -Jdiazole phosphatidylethanolamine (NBC-PE) , I-pa l mi t oyl- 2- ( 12- ((7-nitro-2- 1,3 -benzoxadizole-4 yll amino]dodecanoyl) phosphatidylcholine (NBD-PC), NBC-Stearic acid and I-pa lmitoyl,2 -parinaroyl phosphatidylcholine(Pa-PC) were purchasedfr o m Avanti Polar Lipids (Pelham,AL) and used as probeswithout further purification.
DPPC and the probes were dissolvedin chloroform- methanol (3:1,v/v) andstoredinthe freezer at -20·C. The concentrationof the solutions were checked periodicallyby a modified Bartlett phosphate assay method (Keouqh 1987). The lipidswere jUdged to be pureas they showed a single spot on charrinq with 75 %:SUlphuricacid after thinlayer chromatography usinga solvent front of chloroform -methanol- water
(Ei5:25:4,v/v) . The probes were characterized bythe i r emission andexcitationspectra in ethanolandchloroform - methanol (3:l,v / v ) using a Shimadz uSpectrofl urime ter (RF-S40 ,
Shimadz ucc,, Japan).
Thesub ph as e on whi ch the monolayer was spreadwas 0.9\ (0.15M) NaCl. Thesaline wa spreparedby dissolving NaCl in deionize d,double- distilledwater , these c o n d distillation be i nc;r frolldilutepermangana tesolution. The pH ofth e subph a s e was adjustedto6.9 usinc;rO. 5N HaOH sol uti o n . Thesali ne was turt he r tilteredusingaMi llipore tiltersyst em(gvwm0.22pm fil te r,Mill iporeCo.,Missisuag:a,Ontario) .
OnemMstock sol u t i o not theprobeand DPPCwe re mixed atva r i ous molarra t i os.The etfectof theprob e onis o therms at' DPPCwas te sted ona Ki mr ay su rt ac t omete r (Kimr a y Inc.,Ok lahoma City,OL).Thetroug hot' thesurfactometer wasclean e dthor o ughly with ch loroformmetha nol (2:1,v/ vl and ri nsed withdo ubl e distilled wa ter.
XI Systemcalibra tionand Proc e d ure'
Inthe epitluore sce nc emi c ro s co p i csurf a c e ba l an c e preventi on ofleaka g e ar o un d the ba rrier s was attemp ted by using a Lantha num-OSPC coat i ng ofth e wallsand barrierof the trouq h, asus edbyanothergrou p(Go e rk e 1981 ). Th e Lanthan um DSPC coating is as sumedtoform aso f t coating betwe e n the tr ough and theba rrierwhichsomeh ow re du ces lea kage (Go e rke 1981 ).We encoun t e redsome problems using this tec h n i qu e (discus s ed in secti o n I of dis cu s sion). Anumber at' stear i c aci dmo no1ay e rs weretes t e d onthetrough to observeanyle akaq e at lower
..
su r f acepressures.
The plat inum di pp ingplat ewa sroughe n e d and heated over a oxidizi ng flame. Thefla g was attac hedto the tip of the tra n s d uc er system andits weight in milligrams arijusted using the strai ngaugeampli f i er. The surfaceof the saline subphase was cleanedby compressionand suctionprocedures, unti l a clean surfacewithsurface tension~70 mN/mwas obt a i ne d. About 20 nanomolsof DPPC+1mol%: of theprobe was spreadonthe interfaceusing a 23pl Hamiltonsyringe. The monolayer spread in th i s waywas kept in darkness under th e plasticcoverfor30 mi n before any compression was performed.
The llIono l a ye r s werecompressedat spee dsranging betwe e n 0.13A2/ molecule/secand 4 A2/molecule/secin ast e pwise manner. The barrie r movement was stoppedat each stepand visua l observationswere recorded on a VMS videocassette. Any surfa ce tension changes duringthe visual Observation time were also monitored. Isotherms of DPPC were alsoconstructedby compressingrnonolaye rswithout barrier stoppage. These isotherms of DPPC were compared withthe ones constructed forthe visual observation expe riments.
III Image analysis
After initial processing of the images the areas and perimeters of the individualdomains were measuredinsquare
"
micronsand microns respectively using theJAVAsoftware . The JAVA menu for object Number was used to countthe numbe r of objects inthe frame and the informationwas save d in a data worksheet. Other.pa r ame t e r s of theimage s suchas intensity T'inge of the imagesand number of pixelsin the AOIwereal so sa v e d . A number ofimage s were analy z ed (- 20 ) foreac hof a nUmber of selected su r f ac e pressures. Th'!1 frequ enc yof doma i ns within certai n sizelimits wa s cal culatedperframe by div i d ing the totalnumbe r of Objects in all framesme asure dby the to t al number of fra mes . The fre qu e nc y di str ibutions of doma in si ze wereconstructed byus in g 50squa r emicro n group ing s.
To det ermi netheaccuracyof the imageana l ysis , computerge ne r a t e d imagesofdoma i ns of known distr ibutio n producedbyDr.Da v i d Pin k , oepartment of Phys ics, St. Francis Xavie r Univ ersity ,Nova Scot i a weredigit i zed and analyzed. The frequency di s t r i bu tioncons t r uc ted fromthese images werethe ones expected fromthefun c t i ons us edto generatethe images.
