Ultrathin supporting films in electron microscopy

of unstained biological macromolecules

DARIA STE\'E .BOCCIAR F.LLI anù FRA :"i CO TA:'iG UCCl

/)~partm~nt 1Jf Physirs

Summary. - A simplc t rchnique is described , in which the increase in thicknrss of a carbon fi lm is controlled. Films np to 10-15 A in thickness, which arf' hoth continuous und s trong enough for use as supporting fìlms in electron microscopy, can be cus ily obtain('Ù by this routine method.

lmages a re s h 0 \\11 of uns tained rihosomial subunits and tropocolla~en

molecnlcs on 15 A curhon ti lms.

Riassunto (Film supporto ultrafini per fu microscopia Plettronica di macromolecole biologiche 11011 colorate). - ' i descrivr un metodo pt•r control­lare lo !~pcssore di un film di carbone durante la sua deposizione. Il m e;: todo t onsentc di ottenere film di 10- 15 A di spt>ssore che sono sufficientemente resistenti per essere usati come film supporto a l microscopio elettronico.

Si riportano immagini di subunità ribosomiali ' c di tropocollagene non colorati.

INTRODUCT ION

Con tras t in electron microscopy images of biologica! molccules or small molecular aggregates is extremely low, due to the unavoidable prcsf'ncc uf the supporting film , which contrihutf'S to thc three effects which appea r to be superimposed in the formation of these images: elastic scattering, phase cont rast, inelastic scattering.

Ca rbon films 100-200 A t hick are gcnerally used as supporting fìlms; but thesc are too thick if biologica} molecules with dimensions of only 10-20 A are observed. Moreover, tbey are usually obtained by are discharge between two carhon clectrodes: hy this t<'chnique thc strata are not homo­geneously produced , due not only to their zona} structure (KAKINOKI et al., 1960), but a lso to tbc frequent deposition on them of graphite microcrystals cmitted from the source. These a re easily visihle undcr thc electron micro­scope, if cross scctions of cariJon films (emhcdded in araldite and cut in thc ultramicrotome) are observcd.

.~nn. 111 • .'iuptr. Sanitd ( I!IH) lt, 183-1

18~ F.SPERIE~Zt: E RICERCHE

lo this paper a simplc routine method is descrihc·d for thc prep aration of ultrathin films ( up t o 10- 20 A in thicknl.'ss). which are continuous an d strong enough for usc as supporting films in high r<'solution electron micru­scopy of low a tomic wcight materia!.

Exampl<'s are gi,·en of imagc~s of unstain eJ biologica! macromolecult•s laid down on films about 15 A thick.

~IATElllALS <\.~JJ ,\lt:TllvlJ::.

Shadotcing plant.

A Spccdivac shadowing apparatu!:> (Edward;,. High Yacuum Lttl.. 12 E6/ l 707) was useù, cquipped with thl' electron gun dcscribetl by CHOPIU

and RA NDLEIT (1966) sourcc and a liquid air trap bctwcen thc chamiJer aod the oil diffusion pump. As showed in Fig. l , the work chamhcr of tlw apparatus was lengthened !Jy means of a metal cylinder with a water cooliug system, composed of a copper tube sealed arouncl the cylindrical waU. A glass \\ indow \\ as sealed into this waU an d h\ o rot.b \\ ere inserted t o enablc thc screens to be moYctl into pos ition. After which, thc total lc•ngth of the bell jar measured 60 crn, and tbc work ch arnber coultl be pumped down to 10-~ torr in 5 min. Tbe glass or mica surfact• useù to support thc carbon

~ ,.

~~ !l ~

~l q l

Il . . "

Jl .. ,,

l. ' ..

{] <- ~

l

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Il ., ,, '•

i ~

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Fig. l. - Schemi' of tb<> work chambrr: the lengtbcning metal cylin­der with t he wnter rooliug system and the ";ndo"·· the electron gun, tbc screeu~ aud the film supporting surface are outliued.

il'"'- f il. :>upu .• <:anit<i (19; J J l O, l ~3-1 90

STEVE BOCCIARELLI E TANCUCCI 185

&ba wa.s 15.6 cm far from tbe emitting point of the carbon electrode. The teJDperature of the supporting surface, measured with a copper vs constantan t)aerrnocouple, never rose ahove 125 °C, even after severa! bours of evaporation.

Tbe supporting surface was rotated at approximately 7 RPM, normally to the axis of the systcm, in order to ensure more uniform deposed layers.

The geometry of tbe system and tbe working conditions of tbc electron gun were rigorously stundardized; an external electrometer (3440 Hcwlett­Pack.ard digitai electrometer} was inserted to increase tbc accuracy of rC'adout,

80 that thc curren t in the gun could be maintained b ctwecn 18.5 and 19.5 mA with 4 kV as a high voltage. Under thesc conditions no sparking from tbe source occurs.

The apparatus was uccuratcly outgassed beforc evaporation, tbe electron gun being excited. The carbon dcposition started when the Yacuum reached 2.10 -o torr: this pressure lasted tbrougbout the entire period of deposition.

Carbon electrode.

Spectroscopically pure carbon rods were obtained from Ringsdorff­Werke GMBH (Godesbcrg), as graphitic carbon rods (RW1). Evcn if amor­pbous carbon would be preferable to tbc graphitic ty pe on account of its higher resistivity (60.Q mm•jm, ins tead of 8 .Q mm•jm), its property to absorb large quantities of gas and vapours makes it unsuitable for use in the vacuum plant. The rods were 6.15 mm in diameter; one of tbeir tips was sharpened by latbe to a 60° point, tbe end being rcduccd to 1.2 mm diametcr. Tbc total length of tbc carbon clectrodc mcasured 29 mm.

Supporting surface.

The carbon film for use in thc intcrferential microscope was formed on glass slides, wbilst tbat for use under the electron microscope was formed on freshly cleavcd mica. The metbod (introduced in our Laboratory Ly A. K . Kleinscbmidt) of covcring a glass slide witb a thin collodion film and using it as a supporting surfacc for the carbon film is unsuitable, as the collodion hacking the film is never completely destroyed in thc ovcn, cven after severa! hours at 200 °C.

To check this point a specimcn grid was covercd by a carhon film and a collodion layer was laid down on a portion of tbe carbon film. The ohser­vations before and after tbe oven treatment showed that the horderlinc of tbc collodion film was clearly visihlc in both cascs. On tbe contrary, tbe collodion film alone, unbacked by the carbon film, was easily destroyed. The chemical treatment with a mixture of alcohol and etber 50 : 50 was able to destroy tbe collodion fùm backed by tbe carbon in 10 sec, but only strong carbon films could endure tbc strain due to tbis procedure .

. ltul. 111. Sr;per. 8a11itrì (IOH) lt, 1:!3- 100

<.arl•ou film .. 1111 l t··~ d 10n 11111 A 111 lltwl..u•··· 1'1111 e·a·i" 111' <kt:wlwel fru lli Llw mit·a :-urfan·. u11 .trt·ounl ,,f th,·ir llll'l' IIJIIÌl·al re•:,isL:IIIrt•, "hidt i t- c·onl pnra l iYl'l~ hi~h: 1 ... 1 .. ,, l hi~-o l i m i 1. rore· li H l t- t 1... Luk<'u in handlin~

lllt'lll, aud ln•lo\\ 50 .\ il ia- '•·r~ d iflkult lo dc•Lc·t·l llw film '' lwn il fl oa t-. ull' tllt• ruica. f l i ~-o ••aJo. i,•r lu tiNudt :u ul lo "''" il u hc•a , ;,.,. <·urh<ell c•ntporatinu Ìl' macll' ou an ,.dg•· ( \\ II. I. IAJ\1 ' ~ C t.At:~ EII, 1972} .

.'·Hcpporting ftrid~ •

• \.Hthc 6lms n}, tairu•J "l'lo ]11- l:i \ tltil.. t uultl l w l ''l'~e·d up by uwr•·l~ 111urhi11!! thr m. from tlw uir .,uJ, .. \\ itlt a 1(111 me• .. h t·opp•·r 1-rrid.

Tl~trlml'.~~ m l'a~~~ Tf'flll' tll'.

A l .eitz tranEmi,.. .. ioll Ìlll•·rfc•rf'n t i.t l mic-rut.l'llfW '' ..... 111-•'d in liiOnll· •·bwmotic· liglJl (). = 5~ hO A). nn d t h•· ho l t t~ h :uJ,. mc· tiHt~l \Ht~:> appli•·d (SI\U TII. 195-k P r.llT4. 1971: Sr.orr. 1971 ) .. nnw ronlrnl,., ,,<·n· mnclt- "ith n \\ ilJ rf'6('cliou Ìllterfe•re•ntiol mirtc)i-C'UJII'.

T n tt\'nid . callen•d liçln from ùw gla:-1- l'>lidt. tlw 11111 mt·::-h g ritl cm r n ·cl ~~ ~ l h t • film w1Òt: r Jlli'8Hi r• ·m•·n l \\ ~ huppurtc·tl 1,) 11 JWrfur;.Ht>d m e tu l pia Il' . 'l'hl' measnring '' a" m od•· in a unifnrul li•·ld: u purt iun uf a squar•· uf tiH· J! ritl "h i cL "a" p ortiall) t'O\ t>rt•c l hy 1 hr fìl111. n11cl 111 \d11d1 a har app••nrt·cl. "at- r l'nterNI in Lh •· mÌ C'rii"I'OfH·. Thr pltu~o c · c·ompt•fl!-lllur ' ' :J!' srt 6r~ l <•f ull

""' tlwt the• film ::mJ tht• h.tr uf tlu• grirl 'lhll\\l' tl l''lll'll) tlll' ... anw hrightnf',.:,, tlllll tfH'II , thr :H'<'OIHI I ÌIIII'. \\Ìih thr htu ·k~riiUIIIl ( 1. e•. tlu• Ztlllt' lliiCCI\f'l'l'tJ

l') tlu· fìlm ) sho'' ' Ìil;! th t· 8anlt' hri~hllH't'S ~~~ tlw t.ar. 'l'lw ""' rc>nding::; gi' ,. tltt• optit·nJ puth in thr filut , if <·uli!Jralinu of tlu• l'lllltpt•n;mtor sys tl.'rn hns

ltt•t•n tmtdl'. T o c·\·aluatf· tlw thit·kn•·",; nf tlw lil111 . tlw n·frur ti \t' mdc·' uf tLt• l'arlmll

hlnt anu•l lw knc•wn . Thi~ 'alm· ran hl' t 'u!!il) ul•t aiuo•d tf. in acldi tion In lhr 1 r<llt~ lllÌ!!:<Ìon intl'rfr n ·ntinl rnicruH·or"· a rc•fl (·C' tl llll mlt·rft·n•nltal mit:ru~CIIJlt' , ... ·" ailable: in our r :t:.t· thi c·kn r<.~ nwn,.u rt'Dit'Uh \\(' rt m ad.· l1oth "itll tlu­L f't lz nod thP \Yild appara tu<. on a film aht~ul )1111 \ thtt l. . h) th•· frin~.-­lit•ld ml'thc)d.

Tht optical pat h iu tlw c•arhnu film ~ "., .. li r .. l IIII 'U•ttrc·d uudt·r ll tt· lrUIIl'>rrtÌ !ol' Ìon interff' rt•ntial IIIÌcro!lcol"'· aud tlwn lht· uplirul patb io a tbkk­"''"" nf air <'qual tu lbut ,,f thr t•arhnu film '' U" utt•a,u n•d Wltler th•· rl' fl l'ctwn tlillt·n ·ul ial micro:.COJ ~~'· T•l t·nhaurc· th•· rt ·flc·rt i' sl~ uf th<• s tralu:. a ~< il ' t' r

lil111 ''"" e\ apornl<'tl lwth 011 th1· carhon Lìlrn aod 1111 tlw gJa .... ~urfoct• ~u p­porlillg it. Thc difTt·r<'rtt'!' iu tht• optical path uf tht• ltf:!ltl bcam (ì = !HbO \) r<' flt·ct<'d ou the t'arlwll film and that n·flt't'ted on lite s upporting surfaf •· giHs L\\ ict' th•• opl iral putll 8. io thc nir. <' liTri' purulin~ lo th t• tbicknl'l.:l­

of thc film. lt is t•asily ~ho" 11 tbat tlu· rl'frac tiH ind•·' ;... gh "" hy

'Il 8, , ~. + l.

STEVE BOCCURELLI E TAl'iGUCCJ 187

By tbis metbod, roeasurement for tbe rcfrnctivo index of carbon film give 0 = 2.5. Corre~:~pondingly tbc minimum tbickness measured by tbe ba){-ùade motbod on a carbon film is 22 ± 15 A. It is not easy, bowever, to reacb thts limit, oo account of the difficulty in judging '";tb sufficient certainty tbc cqual brigbtness. RcUable and rapid measurcments can be ronde on film not less tbao 100 A thick.

Electron microscopy.

AJ1 AEl microscope '~as uscd with a higb volugo of 80 kV and douhlc condenser, ilOO IL diapbragm in the cooden ser und 150 !L in tho objective lens, magnifi~tion 7.500 x. Alteroatively a Siemcns Elmisk:op lA was used, with n high \'Oltagc 80 kV, a single condeoser (K1 unexcitcd), 200 !L diapbragm in tbe condeoser, 50 1.1. in tbc objective lcns, mngnificatiou. 8.000 x, following tbe techoique introduccd in ottr Laboratory hy A. I<. Klei.nschmidt. The a(h·nntage to use the microscopc at very low magnilìcation ~;eems to be due to the vcry Jow intensity of illumination requestcd in this conditioo. [n fnct, to rencla at 80.000 x the same intensity of iUumioation as at 8.000 x, a lwum current one hundred times bigher is ncccssary. That meana a diffuse buckgrou.nd intensity and a contanùnation rate proportionally higber.

The i m age w ere rt-corded o n Koclak E lectron I ruage vtates, '~w cb w ere dc' doped by undilutcd D 19 in 2 min.

RI::SLì.T

l t was possihle to obtaju n ' 'elocity of setlimentation of thc curbon lìJm whicll remaioecl constant for many hours (.f.' jg. 2), by ensuring the stnhiHty

1 4~--r-~r--,--~--~---r---r--~--~--,

h

Fig. 2. - Thiek:nesses of eurbon film io .\ vnro& cvnporlllion timu iu houn: lhe velocity of ll.-dim~ntutioo

r t< nlaiu"' cou-tanl for muny hours.

·' " " · Tll. S"l"''· 8•onitll ( IOil) Il, 1~3-1011

188 E S PERlE="ZE E RICERCH E

of the carbon cvaporation procedun•. This wa achirntl l1 y s tandardizing the geomf'try of th e system. thoroughly cleaning the work chamhcr , accu­rately outgassing bcforc the evaporation process. achieving a good vacuum (2.10 - a torr) during ••vaporatiou , nnil con6nuously controUing tht' currr nt in the electron gun.

As can be seen from Fig. 2, thc time vcrsus thiclmrss relationship is represt•utcd by n straight linc, which passes through thP origin. so that il can br assumed that tht• rclationship remains lincar until cxtremely thin .6lms are obtain<'cl , even if the direct m e11suremrnt of thr thicknrss of t hP film becomes un eertain brlow 100 A.

If th•~ work charnbrJ· is d ismantled . ali t h l' piccrs cleaned and thc carhon changcd in thf• electron gu n, thll d<'rivative of tim<'-thickness function m ay cha11gc as much as 10- 20 %: so that un error of this magnitude must hl' taken intn eous icleration in tllf• rstimation l1y this method of the thickncsl> from the evap oration time.

lt may hl' ohscrved from Fig. 2 tha t tbr time required. for is lance. to rcach a thickn ess of 100 A l1y this dcYicc is mu ch longc·r t.han the timc: required in tlw usual shadowing apparatus (2h 30 min againr; t few seconds): hut it must be considl'rcd that. lo obtain ultratbin films, this ti_m~ Ìì' pro­port ioually redu ced and in auy cast' the unpredict ablr time TCCJUired to measure and selcct vcry tl1in lilrns ohtnincd with coil\'entional methotls is completd y climiuatcd (sec•, for instancc, thc laborious measurcmcnt teeh­nique employed hy WJJITINC & 0TTENSil'1EYER, 1972).

On the basis of thcsc rcsult~, attcmpts h aYr lwcn mudc• to cvnluntf' tbc minimum thickness of a carbon film consistcnt wilh tl1c mechanical stahility necdcd for u sc: as a supporting film in electron microscop)' . It has been scen tbat down lo 10-15 A tb t• film can be manipula ted, JJut bdu"­this tbickness the film. st:rms to disintegrali.' whcu de tacbed from tbc· sup­porting surface, floating on water. This finding is in agreemenl ,,-iùt somr data obtained hy HAYEK & ScHWABE (1970): according t o tlJesc AA., tbc carbon film evaporat('d at temperature brtwcen 25 anù 350 °C grows from an island stage to a porous structurc, and reachcs thc coalcl\ccncr. stag!' at a thickness hetwecn 10 and 15 A when f'\·aporated at 260 °C. In our case the t emperature of thc supporting surfact" du.ring cvaporation rOSf' from ahout 50 °C (nft('r the outgassing) to ahout 100 °C for ultrathin fìlms , reaching 123 °C for 75 A thin fìlms and rt•maining a t this ,·alut> for longer evaporatiou t i m es.

lmagcs of un~ ta ined biolog1cal specimcns 0 11 15 A carLon fìlms urc shown in Plates la, h, c. In P late la, 50s rihosomcs cau be secn. 'fhc spccimco was obtained by ARACO et al. (1971) from E. coli, buffered with lO-~ M tris­HCl with 10- s M Mg acetatc. The aspects of the unstained rihosomc units

visible in thc picture are in keeping witb thosc ohservcd by SPIRIN et al.

A nn. 111. S upu. SanUà (1974) lt, 18:! l!Jtl

STEVE BOCCIARELLl E TANGUCCl 189

(1963) in a staincd specimen. Preparations from a solution of tropocollagen molecules are seen in Pia te lb ande. Finely divided collageo from n rabbit teo· don was treated witb 0.5 p. c. acetic acid for 24 hours a nel purifìed by precipita· tion with 5 p. c. NaCl solution accordiog to Cox e• al. (1967). Plate lb showes the unstaioed specimen: it appcars as an irregular network made up of strands 30-40 A thick and 300-3000 A long, which could correspond to the « Fast­scdimenting component » obtained by BErER & ENGEL (1966) at low tem­perature. In Plate le the same preparation, negatively s tained -w-itb phospho­tungstic acìd (pH 7.4), is seen. Long filament, up to 1000 A in thickness, ure ubserved, which are n cver visihle in the unstained materiaJ. This ob­scrvation supporta tbc view of 0LSEN (1965) on tbc possihle packing action uf pbosphotungstic acid on tropocoUagcn molccules, when used as a negative stain.

CONCLUSIO:'\S

The method outlincd in this paper enablcs carbon films of known thick­ness to be made with an accuracy of 10-20 %, bavìng a high mechanical s tability, up to approximately 15 A in thickness . As the aim of this work is to ohtain extremely thin supporting fìlms for electron microscopy, the precision in the determination of the thickness is unesseotial: it is important, on the contrary, to establish a method with which it is possible to 6x in advancc the thickncss of the supporting film depending on the size and type of tbe specimen under study. Such a condition is irrelevant when nega· tively or positively stained specimens are ohservcd; hut it is of fundamental importance when unstained materia} is under observation.

The results obtained in pictuns as those shown in Plate la, b, c confìrm this assertion: to our knowlt>dge, it is the fìrst time that ribosome subunits or tropocollagen molecules bave hcen secn with su ch a good contrast and resolution without staining.

As can be seen from a comparison of Platc l e whh Plate l h, an unstained spccimen may give an important indication on the packing arrangemcnt of macromolecular units in the ahsence of environmental factors , other than the adhesion to the supporting 6Im.

W e are indebtcd to Riccardo Crateri for his skillfull tecbnicul ussistance io lbe arron­gemcnt of the shadowing apparatus.

Received Oecember 16, 1974.

A cceptcd December 28, 1974.

Alltl. l1t. Super. Sanità ( IUH ) 10, 183-100

J90

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umma&ry. - \ntiH c·ollagrn hbriJs, mcchanjcnlly clt·todat'cl •·ithu frmu

l r~o: trmlon uf ruhllits ur frnrn tuil t('oÙun or aortic rull'natiti11 uf adult r a ts

Wl'rt' uluwr' .. ,J ut tht• ··lt•t· tron mit•roscopt> by 'urÌIIll i! t N·hnicul mrthcu.l~.

Tlu· ll t•ut~ ity Jl4~1t·rn ~t rP~·ordl'cl alo ng ood ucrosfl inlt'l.(t'r lilnih;, lui<l

clu,,n un uhrnthin C li lm i:i , t> lww t h ut tlw drnsity •·utio uf thr liglat lo tiH• l!t·n!W lmndi! i1< mttc•h highl'r tbun that c:xprct cd from t lw ~t·m• ru lly tu·c·rrn•·cl IJIIArll·r "'"f(~t· r urran~t·nwn t uf cullagrn unitic>:..

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:t munuiU)I't. h•" in~ lht• furm nf a long ribbno ... pirnll~ \•11111111 aruuntl tbt•

lun!liltltlannJ u,i,.. ur tllf' i rH t·~t·r nhriJ.

m~ .. UJIIO ( lltra.-trulltlrO tltl rolltr&tnl' n•llitu ). Ftltrilli· rollag··nc

n;tll\ ,. ,Ji .. t.tn·~Ht• mt'C'I'Illlit•aml'nlt· dal tt>odint· clt·lla t..tlllJHI tl c·l t•uni!-llio

upp urt• tlul t t•ndint· tl,•lln coda u tlall'odt•tntit io .IOrtu•à tlt•l rutw ,ulultu,

.. , .. w .. t:llt• ., .. rn att• al mirro,.t·upu.l dl'ttronico impit•~auùu 'ari tnt•tutlr tl i

pr•·p aru?iun.-.

Lt• tl t•nsiltlllll'trit· fu ll•• lungo lìhrillt· intt·grt• u lrn;,' t•r.,nl llll' lll l' .111 t'!l~ t',

(t'"'ct· ntl u I JIII'~II· dt•poRII• 'ili film di r. ultrnsottili) mu~otrn ntt d11• il rapport(l

1 ru In tl••n!'itù ti ..Ili' huntlt' t•hinrP t• IJ111' lln drll t• huml11 ..,, ... , ... è ll!>l'llli p iì1 PIPvalu

d i cpu•llu rich il's to cJul t'IIIHIIIII' Illl'lllt• ucc•t•ttato uwtl <• lln (lfll ll rt•·r !it:l~~··r

t~rra~) tll'lln • li ~pn-.it.illnt• d t•;!li .. tPnw nti unitari d t•l c•t~ll u~rnt• ut•lla lihrilla.

'··llt· hltrillt• rigoulìc• >~ i """iCn unn lunghi lìlouwnti '" c•u t i c·irnt ')()A

di "l"'""' ' r ''· dlt' ~nno clu ritcUt·r~~i ~li dementi <'osti tut l\ i t lt•lln hlmlla . La Jll' flllllll·st:l C'nrll ltt>rl'>lÌC'll cl.-1 C'OJia#!('OI'. dj l.'in•a (} ~0 \. Cltll\ ,.j fl,._t'r\ U mai

in ' lut•..,ti hlamrnll. ma io ~?ÌM """t•rvahile in gruppi tli due· o l re· t li , ..... i. L'o • ., .. ,... o/Ìtlllt' 1li fìhriJI,• parzi.tlmPratt' elisfatte fa :.upporrc> tlll' •tur,ti hlaml'nt i

t•le•nu•nt urt "' ·•~!!n·ghmo in mooo,..trato, e c br CJUP:~lo "'" •" 'ulto ...... piral~ a tturnn ull'ao< t' l ungi tmlinali· cl.-lla fthri lla.

192 E.SPElUF::vu ; f: JIICEIICII It

C•>llagen 6bril;, a r (• mainly furmed by n pr•lll'iu, Lrupucollagen , wllich

hns a moJccular weig ht 11f nhuut 300.000. From X - ray diffractinn datu

(H.AMA COANDRAN, 1967) it is known thatthis molecul•· Ì!! madt· up Lo 3 helical

}JOiy peptid chains "rapprrl in a r·i~ht hantlf·d up(•r- hl'lix , thl' "holr moleculo·

bring formrd by al,out 1-J A iu cro~s Sl'ctinn. 1'111' fìJ, ri ll' a re pa ra- cry.s tal­

liut~ arrays of these long chnius. Thc arrangt>mt:'nt g i,·cs a l'•'riodical t! lru('lun·

wbich slaows up clrarlr a t thr t' lrrtrou microsc('P' '· lw tlt in pns iti' ,. nn d o(•gatin stain .

A larg<' amount nf work wos in voh rJ ìu urul<'rs tuutliug h ow tltc tropo­

coUagcn (TC) moleculc., art' pnckcd iu ordcr t u produc{' tlù pcriodic.al

strocture, givcn t.hr imvortant roh• tha t colJagt•n bas iu ronnecth·c tissu•·

properties aod disord('r s. AU thl' samc tbc problem is tilJ opcn. Th••

Jimitations in our knowlcdgc of f1brillar tmr tur•' rcAl•ct t t><'Lnica1 rPstrict iou .

In fac t, low anglc X- ray diffrac tion tl·chniqut· givl's a poor rcsolution b t'caust·

of tbt> iuexact packiug lattice of collagcn: physiCt)- chcmical data an' uo l

suliicicnt to sctt.lc a mode! of d~o packiug urray. so that tlw most s triking

rf's.ults wcr <' obtaincd from electron microscupy. But by this tecbniquc tlw

dircct iuvestigation of native unstaiawd collagl·n 6hril t> under thc clt~ctron

microscopc is prevented Ly lhl• low c•mlrat' l givcu by Liologicul materia!.

Conscqucntly electron microscopc data art· basf'd on 13 tudics and discussious

of imagcs of collagenous materia! in nt·gativc lllu.l positive stain . But tht·

int craction of tropocollagcn moltoculcs witl1 thc s1 ainiug salts caunot b o.:

c)j n •gardeù, a :> it may Lt• !lf'Cu (STI::n~ 8 oCCtAH ELl.l & T ANC\:CCt. 1974)

Lhat dùs materia! can undl'rgo clHI'c r{'nt o~gre"'atinu statl's if ohsrn (•d ' ' itl1

nr 'dthout negati' c s l!liuing. With electron microscopy a conapari<son '' us mndt• lwtw<·t·n t h l' irnagf's

of11atin collagen fìbril8 and tho.st' of artifì c- ial aggregate.-, tht' :L~' or segmcnt­long-spacing wbich haYc hecn througlat to bt· a pa ra lll'l nrray of ÙH· T C: molccu1es polarized in ùae samr dircction and wi lh rcgion of corrcsponding chemica1 properties in register ( BoDCl: & MTTH, 1960: 0 LSEN. 1963). SLS frequently W<'re formed adjacent lo r<'cons tiluNl collng<"n fibrilli'. ~tpanning

.~ p eriods of the fibrils in u reproducihlt' position, ancl tla.is ohservation led to tb(• cooclusion that TC molrcult's nr<' packl'd iu oativr roUagen 61JriJ;; foUowing a « quartcr-stagg rr » array. Later on, tbil' modr l w as modified alter a comparison of thc length of SLS and tlac leogth of anothcr arti6cia l aggregate, tbc FLS or fìhrous-long-spacing. This was considcrcd to bf· a pa:rallc•l array of TC dimers made of molcculcs attacbr d end- to-eml. Tbis comparÌSOD showed that in nath.'f~ fibriJs thc molccult'S ar(' nol attachcd jns t cnd- to-end, but with t hn superimposition of about 1/10 of their

STEV·~ BOCCURELLI 193

lcogtb. Jn conclus ioo, tbt• final pattero of the s lructure of native 6brils scemccl to Le that of Fig. l. Unfortunately some difficu ltics a rose: Lhis t )'pc of packing produces a monolayer shee t , but it is geom etrically impns ible in thrce dimensions (Mc G RAVI:"i, 1964). This difficulty may be ovcrcome if a random choice in comhining t be bonding zones of tbc macromulrculrs to form the na t ive collagen fihril s is arct!pted (Cox et

nl.. 19b7). \ ~econù difficulty is tha t the tJueaùlike s tructurcs or fil umr nts com posing

t h c Cl)llagen fibrils seem gcnerally too thick to IJe cons idercd lincar polimers uf individua i TC molectÙl'S. In fact , according to OL E:-1 (1963), they a re 15- 30 A in d iume ttì r a nd it is nssumed t hat d tcy rrpresent from one to four rrc mnlcculc~. The t~ame is true of tbc fìlaments forming thc SLS or FLS. 'l'hrse d imens iona nrt• in agreemcn t wit h the data obtuinrd frum soluhilisation expf'r im«>nts, which sugge t tbat tbc fi la ments could be huilt from 4 or 5 m olf•cull' . Thcse mny be s uppost>d to be ass«>mlJled hy tlw quartM- s t aggcr mr thml nnd havc a hclicttl arrangcment (VEIS t>t al., 1967) . MILLE n & WnAY ( l 971) pNfo rmed a detailed ana lysis nf t he information which they could nbtaine with tha (•quatoria l X- ray diffract ion pa ttr rn of collag~>n la ttice und tlt('y concludr d tha t the fìlam en t is a fìvc· - strandeù rope (« coilcd coilcd roil »), lmt they bave not defined the la ttice of fila rnents in na tive librils. MIT II (1968) reachcd the same cooclusion on tlilfercnt experimeotal ~o-rrounds und SECREST & CuNN l NGHA \l (1973) aftr r indirect noa lytical npproaches.

ff i t is ncccp trd tha t collagen fìbrils are not made !Jy ingle TC molecules !Jut by thin « ropes » of '~ or 5 .,r thcm , tbc prohlcm is agaio open of how Lhe fibrils a re built f.rom these .ropes so as t o bave tbc wcll known period icity of about 640 A.

Fig. l. - Dingram of tbe q u.orter-stagger arrangement of eollagrn monorrtl'r unite.

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t;Sl'ER i t:~ZE E III CERClfl·.

In 1971 PEASE & BOliTEll.LE, applying tbc medwd nf « Ìlll'rt J,,_ hydration » . ohserwtl secti(tns of unfixt-d collagc-n front rat aortic odventitio

aod showeri that tlw individuai filamenti:'. that is thf' structural unities of dt<' fiucr, arr· arrangril in a .hdicnl pattf'm. Tlte samt• result wa~ rt'cc·ntly

ohtaincd by HA YNS (1974) aftcr ha\oing applied l hr fret>ze facluriug teclmii]UI' lo cardine collugen: 111~ fouud , moreover , that this filamcn t arra y is consiRLenll) right handcd.

Nor musl tbc Jlrcst'DCC' of carbohydrutes in or arountl th r fìLers 111'

disgregarded. as thf•y seem to hc bound to the coUagcn, ev<'n if their umount is estimuted Hl ht· vcry low in vrrtebralt·, nf the order nf' l % (EASTOE, 1967).

By means of ruthcnium red ~:~elcctive l' taining MYERS et al. (1969} founcl th c fullowing different typrs of r cd- positi' ,. strut· tures associated with syno­vial collagen: l ) amorpbous coats surrouuding inJividual fihrils; 2) tran~''·ersr

helts overlyiug tbc major pcriodic bands; 3) fine latcral fìlamcnts ext•'nding outwards from the fibril s; ·1) intcrmedialt: fibrils iotr rmindcd with colla~r·n fihrils. PEASE & Do UTEILLE (1971) , using thc acid PTA statu, gavc evitlenc1· of carhohydratc, in an amorphous space-filling matrix which is placed ht' twt'CII discretr filamcnts, ali along the fibrils.

Botb tbc Jindiug of MYERS et al . (1969) anù thosc of PEASE &. BouTEILLE (1971) show an1owtts of carbohydratcs \,·hich srem to bt· tno

high to h1· i11 agrecmcnt with hiochl'micaJ estimat(·s. lmt thi discrepancy may b e e'<plained (PEASE &: BoUTEILLE, 1971) by a \'ery favouraltl r ratio hetween the amount of staining and that of stained materia!.

lu our laboratory a method bas hccn d<'Yelopcd for tLr prepara tion

of ultra thin carhon films (up to 15- 20 A) which are continuuus and stron~ •·nough to L1• cmployrtl as supportiog fìlms (STEVE BoCCJARELLI & TA~ ·

CUCCI, 1974). Their usr gives thr possibilit)r of reaching a comparativcly strong coutras t in thc imagcs of uustaincd hioJogical muterial, in both clcar and dark fìeld micruscopy. With tbis 1.echnique density patterns p('rformrd un thc electron microscop<' imagcs acquirc significauce, as the thickncss of the supporting film is ncgligihle in respf'ct to that of the spccimen , which, in tbc pre8ent case, is of thtl ordcr of 1000 A. Moreover, partiaiJy disrupted

ftbrils may be observcd without any intc: raction with staining salts. E,·en if the images are n ot equally brill.iant, somt: results may h•! ohtainc•d whicla

cannot be reached by tbc use of staining method. This report is conceroed ,,;tb tbc contribution made hy the results oh·

tained in our lahoratory towards solviog tbe problem of the asscmbling of native collageo fihcrs. These results bave bcco briefly reported clscwher<' (STEVE BocCIARELLI, 1974).

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~T'F. n ; BO CC I.\ IU::LI,I 195

\1.\TERI.\L- _\\Il \ IET II Ur>.

<~) SttS[II' IISÌ<III of llfiiÌ ve r·nllnge11 j ibrils. Bun!llr!< uf nati·q • r ollagt•n wen• t".:<·i~rfl from l··~ tentlon of ra!Jbits

11 r frnm tnil tl'n tl•>n ur uortic wh-entitin nf ndult rats . The drafts '"'rt' cut in lt1 !'mali piccN! untl collagt'll til.rils 1\'f'n• .., ,, il'tly ~ <' [l<lrntt·d b v rrwrhaniral

l t·a!' in~. io hidis ti lled wnter .

b) P11rtiully tliu ttptrd fibrils . .:\ !>nlutiun uf 0 .05 p .<·. nct'li<: a cid in bidis ti iJrd \Hlll'r \\' tHI prl' fHlrt'd , a

:-mali quantity uf the suspensinu of tiLrils fro m rabbi t tr11don, in w all'r. "as

placetl cm a s litlc aml CO\' t' red wiLh a drop of tbc act•tic ncid !iolut inn for u fcw

miuutrs (l- 5). l.c.Jlagcn t'ro m rat tail t entlon "ns found partially swo1Jeu after th r nwchanical t1•asing in bidistillell "atrr, antl n solution 0.005 p.t·. was

nl·t•tled tn obtaiu n prcp a ration s ho"-iug lihrils almos t ali part ially fi Wtt llPn.

<') Snlutin11 nf tropocollagn1.

Tlw ~usp(·n~inn nf rnllagell fiuril s in watf·r was dinlyz(' d fot· :!· ~ h in l p.e. ammonium accta lP n t f.uc. Tu the clialyzed suspen~ion an Pf[Ual

'olumr nf U.+ p.c. ari'lir acitl "as addl'd and tbe mixturt• was lc ft 24 l1 al ·~ of.. T bE' mixturc wa~ fiJterrll through a micrnporr f! lass fillt•r 100- 200 if tno viscous, tbc snlutinn was clilutr·d with 0.2 p.c . ac<' tic al'id, in nrt ll'f

111 acedcratf' the proet·c.lurE'. Tlw ti1tra1t' was purifiecl hy prrcipita tion wi th 'luCI snlntion (!l p.c. fina l ~aCl concentratiuu). TlH' pn·cipita te was rc·n­

trifugecJ 15 min at 1000 rpm a nd rctl issvlvcd in hidifltilled watcr. Tbr

~uspension was again dinly:a:d , an c' qual volurrw of O.tl· p .c. acdic acid was

atldr ù . an d tbc mixturt· was Id t 24 h al ·~ °C.

d) Thi n .~ect iort tJj r.ollngm.

Frrsb tissur!' wt·re imrncrsrtl in 1 p.c. OsO. in ace tatc "\t'ronal bufl'er p ll 7A (Mich arli$ huA'rr) for l h , deh ydratrd in eth y l alcohnl serirs ancl

t·mhedrlt•d in t\.raldite. Sl'ctious wen· stainec.l with l p.c . PTA or 2 p. c.

urany l Ol' l' l atr or c·lsc \\ ith dou l, lc· s tain ing (ur anyl acctatc and lt·ad citratt·) .

••) Freezc l'tchi n g.

Frrsh ti~su t.•s wrn· immerscd iu 20 p.c. colei glyccro l for 30 mio , fmzcn in liquiJ Frcon 22 anJ transfcrrt•cl in th r Balzer freeze-et ch ing apparatus. Scctions wcre ohtained ut - lOO 11C and s ublimation lime was 1 rnin 30 sec.

The replicas '' ere clcan('Ù iu sodium hypocloriùt> for 2 h an d washNI in num eroub changcb uf \\ alcr.

f) 1\regative sta.ining. A tiOlution 11f 2 p.c. JJbototungstic acid in hidistill<'tl watt r, \dtich hud

heen adjus ted top H 7.3 or 7.4 by the ndflit ion of KOH ( BREN NEH & lioRNE,

1959) was used as a nt'gatin· s tain.

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196 E SPERIENZE E RICl:RCDl:

g) Ultrathin supporting fìlms.

Amorphous carbon was evaporated on freshly cleaved mica sheet, in a modified Edward shadowing apparatus, as previously described (STEVE BoccrARELLl & TANGUCCI, 1974). Carbon stra ta 15- 25 A thick were used as supporting film.s for clear and dark 6eld nucroscopy of unstained specimens.

h) Electron microscopy tcchniques.

As the supporting film is very fragile it is impossible to put a drop of the suspension to be examined on the coated grid, using a micropipette. To preserve the carbon film it is preferable to pass the coated grid gently, face down, over the surface of a drop of the suspension, and leave it to dry . If a negative staining is wanted~ the suspension must be mixed ,.,.;th th(' stain and the same procedure may b e folJo•Ned, but usually an ultrathin supporting film is not used in tbis case. Sometimes bowcver a sort of autonegative staitùng of tbc specimen is found, as smalJ quantities of residua! fats or other materia} may be sufficient to act as such a stain, given the extr(:· me thinness and trasparency of the supporting film and of tbc specimen itself.

A Siemens 102 electron microscope was used, ·with a high voltage 80 kv and douhle condenser , 200 ruapbragm in the condenser and so in tbe ohjective lcns. Pbotograpbs were taken usually at magnification 8000 x~ to reduce the background intensity and tbe contarnination (STEVE Bocci.A.RELLJ & TANGuccr, 1974), somctimes at 40.000 x, when stained specimens w~r('

observed. Dark 6eld microscopy was performed by the sourcc tilting meth od .

i) Density patterns.

A J oice-Loebl densitometer was used and density patterns along and across integer fibrils werc recorded.

RESULTS

a) Integer fibrils in cle4r fteld microscopy.

Fibrils isolated from leg tendon of rabbits or from tail t endon of rata wcre observed by transparency and when tbey appeared clean and with a neat banrung, images like tlbat of Fig. 2 were obtained. Thc pictures were processed at a Joice Loebl densitometer and density patterns along and a cross the fihril a.x:is were recorded. Sin ce the supporting carbon film, l 0-25 A in thickness, makes no appreciable contribution to tbe density of tbe image, in objects wrucb, like tbese fibrils, are DO less than 2000 A in thickness, tbere is no doubt that the density patterns so obtained are a recording of the dcnsity distribution in the fibrils.

.ilnt1. 111. Swp~r • .Snnild (IOH) 11, lQl-202

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ST&VB BOCCLUIEU..l 197

Both in 6brils isolated from rabbit tendon and in tbose from rat tendoo tM deu•ity ratio of tbe ligbt to tbe dense banda as measured in the centrai .... 0 ( bao<Ù, was, aa a mean, a little higher than 90 %· Tbis result has to be compared with tbat giveo by tbe quarter-stagger modd of the tropo-

.. coDagen assembling of the collagen fibrils: from Fig. 3 a ratio of 80 % is easely evaluated.

Tbe clifference cannot be abscribed to experimental crrors in tbc measu­rements of the density ratios, because if a density pattern across tho fìbrils was recorded along both a clear and a dark band, tbe right distribution of density to be expected from a cilindrical homogeneous structure was found; morcover, the density value of 80 % io respect to that recorded nt the centcr of the dnrk bands was properly found, in the same bande, at a distaoce from the axis wbich is 60 % of the radius of tbc fibrils cross section: tbis value was òirectly compared, as a contro), with tbc donsity ut tbc center of ligbt bunds.

Tbc t•xcessive value of tbc density ratio of tbe ligbt to tbc dense bands could be due lo a retraction of lhe fibrils duriog tbe dehydration on the carbon fìlm, or to a contrihution in density coming from a materia! sur­rounding lhe fibrils or embedding tbem. The first bypothesis may be dis­cardcd lJccause tbe banding in tbe fibrils appeaTs to corrcspond to the wcll knowo periodicnl array of about 640 A, wilh no alteration io the lengtb of the light bands in respect to tbe dark ones. Jf the second hypothesis is right, an cnvelope, made for rnstance of carbohydrates, surrounds tbe fìhrils or at lcast the light banda, or else emheds tbem. If an envelope surrounds the whole fibril it is easy to sbow tbat if it may change the dent1ity ratio of the ligbt to the dense hands from 80 % to more than 90 %. it has to be about of the sa me tbickness as the fìber itself, if its density is about tbe sa me.

As a matter of fact, fìhrils surrounded by an amorphous coat werc frei'JUf•otly observed, and lhey were clearly cvident in dark 6eld micro­scopy (Fig. 3). Moreover lhe presence of a carbobydrate envelope around tbc wholo fìbril hns heen recognized by many AA. Acid mucopolisuccarides l1ave been demonstrated in sinovial collagen by MYERS et al. (1969), and in various connective tissues by KAJIKAWA et al. (1970). B OUTEJLLE & PEASE (1971) identilìed with carbohydrates the tbin pcripheral zone of collagen which in tbc sections of aortic adventitia embedded after « inbert debydration » appears enphasized by the acidic PTA staining. Similar rcsults may be obtaincd in osm.ium fìxed fibrils from rabbit tendon, after ncutral PTA staining, as showo in Fig. -l; even with the freeze-etchiog Lechni­ttue such an envelope is observed around the fibrils (Fig. 5 a and Fig. 5 b).

Tbe tbickness of these cnvelopes scems to be no more lhan l /10 of the cross section of tbe 6brils, and equaUy distributed along the fibril, so that a significative contributjon of the envelope in determining the c.lensity

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198 ESPERIENZE E RléERCUE

ratio from light to dense bands seems to be excluded. All the same, cxperi­ments were performcd to remove carbohydrates: eollagen fihrils werc treated with jaluronidase in phosphate buffer pH 5.4 for 4 h , washed io the same buffer, and density patterns were obtained from the fìbril picturcs. Thfl density ratio wa.s evaluated as described ahove, and the same value, that is more than 90 %, was obtaineù.

b) Th-P building units of tlw fibrils .

Aher a mild treatment in a week solution of acetic acid, tbc fìhrils are swollen and their structuraJ units appear as long thin fìlaments. Thesc fìlaments may be separated without complete disruption of the basic spacc ali along the fìhrils, as shown in Fig. 6 with negative staining. Similar results bave been obtained by BAIRATr et al. (1970) in eollagco fibrils from buman dermis.

Measurements performed on these fìlaments, in images obtained in negative staining, show that they are from 35 to 50 A thiek. Their length is indefinite, but it seems to epan more than 4 or 5 collagen bands. Rarely the end of a filament is seen in partialJy disrupted fìbrils: if this happens, a small knob ahou t 70 A in diameter may be seen attached to it.

Filaments seem to be flexiblc and are never emooth: fine granulr·s {about 50 A in diamet er) are usually seen along them. Pictures obtainod from unstained specimens (Fig. 7) or in autonegative contrast (as explainod in 2, b) (Fig. 8) confirm these results.

It could be supposed that Lhe minimum thickness of about 35 A measurcd in these pictures could be given by tbc technique itself. To check this point TC molecules werc prepared and observed without any stnining, both in clear field as the previous specimens, and in dark field microscopy. Fig. 8 shows tropocoUagen molecules in clear fìeld microscopy. The contrast given by single molecules ie extremely poor becausc the thickness of tbe specimen (15 A) is just a little less than that of the supporting film {20 A). In this condition the physical characteristics of tbc photograpbic emulsion makc the tbickness mea.suremcnts worthless. The results in dark fìeld microscopy are no more satisfactory. Ali the eame in images like Fig. 9 tropocollagcn molecules appear so faint in comparison to the 6Jaments seen by the same technique, for instance in Fig. 7 that we are bound to admit a difference in tnickness between the two structurea.

We may conclude that the filaments are made by the latera) aggre­gation of no less than three tropocollagen moleculee. If we considcr that 50 A ie a very frequent diameter of the fìlaments, tbe numbers 4 or 5 secm equaUy probahle.

These fìlaments appear to be the building unities of the fìhrils. They could correspond to the tetramers postulated by VEJS et al. (1967) whioh

.Jn11. 111. S rtpu. So11itd (19a ) lO, lVl - 002

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STEVE BOCCIAR ELt.l l99

are supposcd to be u right handed helica1 arrangcment of four tropocol­lagen molccules displaced succossively so as to preservo the quarter-stagger ordering of Fig. 3. or else the pentamers suggested by SJ\UT.R (1968). The beaded appearance could be in agreement with a helical arrangement, but a periodicity of about 70 A seems too high a value to correspond l o the helical arrangement of four of five wound elongated molecules about 14 A thick. On the otber band, collagen banding of 640 A periodicity, wbich may be recognizable cYen in small groups of filaments, never appear (Fig. 6). in single fùaments so that wc have no data to confìrm tbc hypothesis of a quartcr­s tagger arrangement of tropocollagen molecules i11 thcse s truclures.

c) Parking arrangement nf the filamen ts in 11ative fibrils.

In nl'gatively s tained swollen fìbrils, as already observcd, single filaments never show the banding whicb is supposed to be the basis of the pcriodicity of about 640 A founJ in tbe native collagcn fìbrils. When the picture shows the heginning of tbc disruption of a band, an attempt may be madc lo u11tlerstand how the filaments are linkeù to eacb other to make up a band. It appears that the handing is preserved even if groups of very few fìlaments are left , may be two or tluee. The latera! connection between tbc fìlaments scems to be mad e without iuterrupting or clsewise perturhing the course of tbc filaments thcmselves: small knobs (ahout 70 A in diameter) seem to be. the bincliug struclures, and they appear in a r ow on tbe bordelinc of the clear band. Gramùes of the same dimcnsion may be found apparently free in swollen fibers, or at tbc end of detached fi.Jaments.

Pictures obtained without any staining, from partially disrupted fibrils laid down un ultrathin carhon 6lms, obviously give a poorer contrast in the image. But in images of this type the density recording of the ohject is reported , so tbat a relief cffcct may result. From some pictures (Figg. 10 and 11) portions of the fìbers appear which suggest that the filnments may be assembled in a monolayer having the form of a long ribbon wound in a spiral around the longitudinal a.-ris of the fibrils. In these pictures zoncs in which the originai lattice of the fìhril is preserved are alternated with zones in which the monolayer is spread out. This effect may be due to thc adhaesion of tbese las t zones to the supporting film, aided by tbe action of the water surface tcnsion during the drying of the specimen. The periodicity of 640 A of the native fibrils is clearly evident not only in tbe rollcd up portiona but even in those wich are stretched out. This banding seems to be inclined a few degrees (no more t han 5° or 6o) in respect to transversal axis of the monolayer: this condition is geometrically important because only a band having an inclined periodica} banding is able, after rolling up into a t ight helix, to produce an elongated structurc having a transversal periodicity, that is a structure similar to the lattice of native collagen 6hrils.

AnN. hl. llrtptr. Snnild (1974) IO, 101-20

200 E SPE RIENZE E RI CEIIC::RE

DISCllSSION

From tbc experimental results reported in tbis p aper e , ·idence seems to Le given that collagen fibrils can be formed by filaments 35- 50 A thick attaching themselves to one other Jaterally in the form of a long n arrow sh ect, whicb then rolls up to form a helical structure.

A structure of this type was predictecl by Ramacltandran antl Sasi­sekharan as an explanation of a series of peaks obtaincd in small angle diffraction patterns by Cowa n and ColJ. from wet specimcns of tendon (RA­MACRA NDRAN, 1967) . These AA. rca ched the conclusion of tbc occurrence of a cylindricaJ la ttice with a finite number of shcets, or a roll st ructurC'. The elementary nnit of this structure was t bc triple-h elix of tropocollagen molecuJe. Tbe whole aggregate in natura! t endon had a d iameter of nppro­:J..'Ìmate 200 A: it was named « cryptofibril ». MnLER & \ \'nAY (1971) on a similar experimental basis seemed to confirm thesc resul ts. On th(:

coutrary, a t the electron microscope it is unnsual to see in p artiall y disruptcJ fihrils elongated s tructures about 200 A in diam(!ter and showing the cha­ractcr istic 640 A axial period as requested by R amachandran and Sasi­sek aran assumpt ion. When the fibril begins t o b e dismantleò (Fig. 12), t he more frequent aggrega te which appears and is mainta ioed in murc ad ­Yanced stages (Fig. 6) is a packing of no more thao 1- 5 tropocollag<'o mu­lecules . On the other ban d from low an gle diffraction expcriments MtLLER & WRAY (1971) dcduced that a latera! packing of tropocollagen in a five-stron. dcci rop e, as suggested b y SMITH (1968), was con sistent with their resul ts .

A helical winding of 6.lamentous substructures in colJagen fihrils secms to b e confirmed by other experimental evidences. BouTEILLE & P EASE (1971) in unfìxed collagen from aorta adventitia of tbc ra t , « inhertly dehydratPd », emhedded in hydroxypropylmetacrilate, sectioned and s tained with uranyl acet atc and or lead citra tc, obscrved filamen tous structures 30-35 A thick , arranged in a helicaJ pa ttern . Thesc AA. affirm that the fìbrils prcpared by this technique were « expa.ndcd », so tha t a clcar visualjzation of fib r illar fila ment could be achjc,w cd.

W e b ave tricd to observe collagen from aort ic adventitia of tbc rat with

thP freeze- etching t ecboique and h aYe obtaioecl (Fig. 5 b) some imagt>s

confì rming the cxistencc of sp iralized fila men ts. On thc.' cont rary, fibrils

from rabbit t cndon whicb , in our experiencc, a re: more 1·esistant to cherucail

agents thnn aortic adventitia or t ail t endon of rat, never show n spira lized

course in tbc filaments in gHcerinated frecze- etched spccimcus (Fig. 5 a).

We may concluùe t hat fi lamen ts ar ranged in a h elical p attern ,,·ithill

fibrils aro sccn only when the whole s tructurc is a ltereù in a suitahJc way. If wc consider t b c cxper imentnl t ecbniques uscd to prep are tbe specimens,

we may suppose that the results obt ained by RAY!\'S (1974-) and quite

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STEVE BOCCJA.RELLI 201

.._tly by BELTON el al. ore in agreement with this assumption. But if * apiralized filaments appear in unrolling structure.s, ~ ,these structures tlae 640 A banding has to be found inclined in respect to the longitudinal -. 0 { the fibrils, as tbc fìlaments seem to be gènerally normal to the cross banding of the fihrils. The effect, actually, may be observed in the pictures of inhert dehydrated fibr.i.ls puhlished by BOUTEILLE & PEASE (1971}: the 640 A banding seem to be inclined to tbe transverse piane at a few degrees. 0n the contrary, when the native fibrils are integer, so that the monolayer is tightly rolled up, even in aortic adventitia collagen the banding appears, normal to the longitudinal axis of tbe fihril, as it may be seen in Fig. 13 in negative staining.

The problem of the packing of tropocollagen to form the filaments or « ropes » seems moreover to be worthy of discussion: from email angle diffract ion patterns the resolution required to solve this problem cannot be attained, and after morphological observations tbe quarter-stagger assem­bling of the « ropes » from tropocollagen molecules seems no t t o be confirmed. The 640 A banding, indeed , never is observed in siogle filaments, but it may be observed when two or three fila.ments are bound together. If this is so, we bave to assume that peculiar latera] bindings are presen t and we could identify them with the small spots wicb are clearly evident in partial dis­rupted fihrils (Fig. 6) .

If the quarter-stagger model eannot be supported either by morfological evidence on single fìlaments, or by density measurements along integer fihrils, or by X- ray diffraction pattel118, we may ask if i t is really necessary: lateral bonds acting between filaments may justify the higher density measured in the so-ealled dense bande in comparison with the others, and the proposal of the quarter- stagger assemhling of TC molecules, which was made to explain just why this axially repeating band pattem may be discarded .

CONCLUSIONS

The experimental clata reported in this paper, comparecl with those obtained by others, may be explained if the following model for the n ative collagen fihril is accepted: the triple helix tropocollagen molecules are as­sembled in filaments made up of the latera] packing of 4-5 molecules. These filaments are assemhled in a monolayer, a long narrow ribbon showing the 640 A banding charact eristics of coUagen fìbrils . The monolayer ie rolled up to form a helical st:ructure which appears to be loose when the fihril is swoUen, but is tightly wound when the fihril is integer.

The hypothesis of the quarter- stagger array of TC molecules is unnecessary and seems not to bave experimental confìrmation on the basis of morphological and X - ray diffraction pattem data .

• bn. 111. Su]Hr. SaftiliÌ (11174) lt. 191-202

202 ESPEBlENZE .E RICERCHE

Thc Author wish lo thank C. R umoui, F. llosati, F . 1'1Ulgucci, P. Trovalu•ci for the excellenl teehnical 8$Si&tonce, and Prof. C. Quiutorelli for his criticai rtuding of t be rnonu· llCript.

Reuived Decernbcr 16, 1974 .

.Acupltd l>ecember 28, 1974

DAtRATJ, A., M. G. P ETBUCCJOLI & L. 1 'o l1Rt 'fANELLI, 1970. 7~ ... Congr. lnt . l'rlierouop. Elutron., Grenoble, voi I , 635.

BELTON, I. C .. D. Mrc UA.ELI & H. Huc n FcDENBERC, peraonal commuuicution . BouTEILLE, l\1. & D. C. PEASE, 1971. J. Ultra&l . R es., 35, 314. BRENNER, S. & R. W. IIORNE, 1959. B ioth. Bioph . .Acta, 34, 103. Cox, R . W., R. A. GBANT & R. W. HOBNE, 19fi7. J. Roy. Microsc. Soc., 87, 123. EASTOE, J. E ., 1967. In: RAMACUANDBAN G. N., 1'rtatise on Collogm, vol. I , p . l, Acad. Press,

New York. HoDCE, H . T. & F. O. S&uTT, 1960. Proc. U. S . .Acod. Sci., ~. 186. IU.JTXAVA, K. I. N.U::AN ISDI. I. HoBI, Y. MA.TS UDA & K . KoNDo, 1970. }. Elrrlronmicro~e.

19, 3477. Mc G RAVTN, S. 1964. J . Mol. Hiol., 9, 601. MILLER, A. & J . S. WRAY, 197 1. Nature, %30, ·l3i. MYERS, D. B., T. C. HICIITON & D. G. RAYNS, 1969 . ./. Ulmrstrucl. RP~., 28, 203. OI.SEN, B. R., 1963. Z. ZeUforsc., 59, 199. PE.ASE, D. C. & M. BouT.BILI.E, 1971. J. Ulluulruct. Rts., 35, 339. RuJACltANDJUN, G. N. 1967. Treatise on Collaf{en. RAYNS, D. G., 1974 . .). Ultrostrucl. Re1., •s. 59. S ECBEST, J . P. & L. W. CUNNINCllAM, 1973. B iopolyrrurs, 12. 825. SMTTU, J, W. 1968. N01ure (London), 219, 157. STEVE BOCCIARELLI, D., 1974. Eigh' lnl . Congr. on Eleclron Micro&c. , Canberra, vol. II, 692. STEVE BoCCU.IIELLl, D . & F. TANCUCCI, 1974. Ann. l sl. Suptr. Sanilà, 10, 183. VEJS, A., J. ANESEY & S. ~hJSSEL, 1967. N alt.,c, 215, 931.

Fig. 2. - Uosta.ioed fibril, from rabbil t endon, obstrved b)· traspnrtncy in clear 6elù micro&copy.

Arrn. 111. S u per • .Suit.t (1074) l t , 101- ::1!

STEVE BOCCURELLJ

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Fig. 3. - A par l i:• li) disrul'1 l'd fìlnil iu clnrk fil'lclmi,·ru«t"OI'} : nu uuuorJihuu' o·11u1 ;, nh''' " ~·l uround I li<" inll'p:c•r porliuu o( lht' fibril .

STEVE BOCClARELL l

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Fig o 4o - Sccl i un of osmiuna fixcd fìbril~ frorn rahiJit Lt'ndon, ~~ uilll·d "ith PT A, p H i .4 o

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Fijt. so. - Fre•lt t i..-ur from ruubit lendou, prepnred "itb l hl' frerze-rtchiug trt•hniiJUt'. Au uruorphou• root ~1'1'1113 to •urrnuud the fiunl,.

Fig. 5 b. - Fn·~IJ ti ~~u!' frou1 uortir aJventitill of odult rut ~, prrpurl'd with the frecu ­ctrleiug tcchniiJut•. A spirulizcd cour•~ iu thc filnmc-uts is sccu.

STEVE BOCClARELLl

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Fig. 6. - SwoUen fibrilli from rubbil tendou, negotively slnined witJt PTA, pH 7.3. Rough fìlaments 35- 50 A tltick und of indefinite lengbt secrn to be t be st:ructurol uni lb of the fihril ~.

STEVE BOCCIARfJI. LI

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STE\"fi BOCCIARELLI

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STEV.E BOCCIARELLT

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STEVE BOCClARELLI

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