J Cell Biol 1981 Pease 287s 92s

8/10/2019 J Cell Biol 1981 Pease 287s 92s

1/6

El ect r on Mcroscopy

and

U trarni crotomy

DANEL

C

PEASEand KEITHR

PORTER

The ol d adage ` t o travel

hopef ull y

i s better

than t o ar r i ve

scarcel y

appl i es

t o mcroscopy,

because i n a sense

sci ence

never

a r r i v es , t he roadgoi ngon

and

onf romany

t emporar y

st oppi ng

pl ace Moreover, hope al one i s not enough except somet i mes

t o

count eract

despai r

W need

tenaci ty and the w l l t o cl i ng

on

agai nst odds to reach

somethi ng

we bel i eve

t o

be i mpor t ant

.

I rene

Manton

1975

( 1)

I t i s commonpl ace t o recogni ze that the depth t o

which

we

expl ore

oursel ves

and

our envi ronment

i s f requentl y deter-

mned

by t he devel opment of newi nstruments andthecreati on

of techn ques

f or

thei r use Usual l y i n suchdevel opments

one

can recogni ze

a ti mewhen

f ragments

of

i nf ormati on,

acquired

previ ousl y,

are

ready

tobe used to sat i s fy a concept or anurge

t o do or see what hadnot seemed

poss i b le

before So

i t

was i n

the earl y 1930s that a

group

of physi ci sts and engi neers,

mostl y

i n Berl i n,

found condi t i ons ri ght

t o create an

el ectr on

mcro-

scope

MaxKnol l andhi s s tudents ,

Ernst

RuskaandBodovon

Borr i es, had avai l abl e

t he knowedge

that el ectrons

woul d

move through avacuum

andbe def l ected

i n

thei r moti on

so as

to

be

focused by

sol enoi d l enses I t was mostl y engi neeri ng

s k i l l s

that were needed

to

generate a

mcroscope

I nt er es t i n

the appl i cati ons

of the f i r s t mcroscopes

natural l y fol l owed

and by the

late

1930s,

el ect ron mcrographs

of r ecogni zabl e

val ue

t o

b io logi s t s were beingpub i shed

Any

consi derati on

of t he

pace

at which

bi ol ogi cal

el ectr on

mcroscopy thendevel oped

must

take

i nto

account worl dwde

events and const rai nts

rel ated

t o t he outbreakandprosecuti on

of Wrl dWar I I H t l er i nvadedPol andon

September

1, 1939

Thevery f i r s t

Siemens

Halske

AGl ectronmcroscopemade

f or commerci al sal e was del i vered i n that year, onl y

a

few

months before t heWar actual l y

started

2)

However si nce

t he

pol i t i cal a i gnments of Axis and Al l i ed count r i es

had been

substant i al l y establ i shed t he year bef ore, af ter t he annexati on

of Czechosl ovaki a s

Sudeten and i t i s

not

supr i s i ng

that not a

si ngl e

Siemens

Halske mcroscope

was ever del i vered

t o

count r i es out s i de of Axis control Nonewas i n Al l i ed

hands

unt i l one

mcroscope

was captured i ntact

and

brought

t o

Engl and

aft er

the 1944

Normandy

i nvasi on

I n t he

Uni ted States,

t he Radi o

Corporati on

of Ameri can

wasnot

ready

t o

del i ver

i t s f i r s t

commercial l yavai l abl e

el ectr on

DANELC

PEASE

Department of

Anatomy School of Medi ci ne,

Uni -

versi ty

of

Cal i forni a, Los

Angel es,

Cal i f orni a

KEITH

R

PORTER

Department of

Mol ecul ar, Cel l ul ar, and Devel op-

ment al Bi ol ogy,

Uni versi ty of Col orado, Boulder,

Col orado

THE JOURNAL OF CELL

BOLOGY

VOLUME

91

NO

3

PT 2

DECEMBER

1981

287s-292s

he Rockefel l er Un versi ty

Press

0021-9525/81/12/287s/06

1 . 00

mcr oscopes t he RCA-EMBmodel s

unt i l

1941 The

Lend-

Lease

program

had

started

earl i er

i n the year

anddeveloped

qui ckl y as amassi ve ai d programto

Engl and

About

one- thi rd

of

al l RCA-EMB

i nstruments ever

madewere shipped t o

Engl and

About

40Siemens

Halskemcroscopes seem

to havebeen

manufactured

duri ng thewar years, andabout 60

RCA/EMBs

2)

On

bothwarri ng

si des,

most

of the

appl i cati ons

of the

new

i nstr uments were di rected towards t he research

needs

of the

m l i t ar y We are aware of onl y

one

RCA-EMB

i nstrument i n

the

Uni ted

States that was avai l able

pri mari l y fo r b io logi cal

research amcroscope at t he Massachusetts

I nst i tute of

Tech-

nology

i n t he

l aborat ory

of Ceci l E Hal l , who

was

already

recognzedas an important pi oneer i n the

ori gi nal

devel opment

of

prototype i nstruments at t he Uni versi ty

of Toronto

A

second

EMB

nst r ument ,

i ns t al l ed i n the

research

l aboratori es

of I nterchemcal Corp

nNewYork was

made avai l able i n

1943 t o Al bert Cl aude

and Kei th

Porter

at t he Rockefel l er

I nst i tute

3)

StewardMudd

a bacteri ol ogi st

at

t he

Uni vers i ty

of

Pennsyl vani a

al so had substanti al

access t o RCA

i nstr u-

ments at

t he

RCA manufacturi ng pl ant

i n Camden New

J ersey,

where

Thomas

F

Anderson

worked

on

bi ol ogi cal

prob-

l ems

as anRCA

Fel l ow

The

s i tuat i on

i nGermany fo r bi ol ogi s t s duri ng

t he earl ywar

years

seems

not

t o have beenmore advantageous

I n Apri l of

1940, Siemens

Halske

ponsored an

i nterd i sci pl i nary

meeti ng

where

the most promnent users of the new

el ect ron

mcr oscopes

as

wel l

as sci ent i sts who had had

access t o

pro-

totype i nst ruments ,

reviewedt he achievements of

nonml i tary

appl i cati ons RS ebeck

di scussedmedical appl i cati ons i nGer-

many ( 4 ) , and there

were other revi ews of

botani cal

and

bacteri ol ogi cal

appl i cati ons Bi ol ogi cal work

necessari l y

had

been l i m t ed

mai n y

t o

examni ng si l houett es

of bacteri a,

vi -

ruses, f i b rous

protei ns,

and

other organi c obj ects that

coul dbe

studi ed

i n

toto By

thi s t i me, both

bacteri al f l agel l ae

and

t he

repeati ng

per i odi ci ty

of

col l agen

had

been

seen

Metal l i c

mesh

s ui t abl e f or

gri ds,

was

avai l abl e f romphotoengravers and

col l od i on

support

f i l m hadbeen i ntroducedbyHel mut

Ruska

i n 1939

5)

I n t he

Uni ted States,

Canada andEngl and

bi ol ogi cal

di s -

coveri es closel y parall el ed the

German

efforts at

f i r s t

E F

Burton and

H Kohl

6) reviewed

the

appl i cati ons of

el ect ron

mcroscopy

that

took pl ace duri ng t he war

years on

thi s s i de of the Atl anti c Wrkwth bacteri a

and

vi ruses

was

soon underway

part i cul ar l y at the RCA

Laboratori es, under

t he

i nspi rat i on

of L

Marton Mudd

Anderson

and

M

27s

Published December 1, 1981


2/6

Stanl ey The

I

. T

Laboratory group, consi sti ng at

f i r s t

of

Hal l , Franci s

Schmt t andMari eJ akus, part i cul arl y

pursued

studi es

of

proteins that coul d

be

i sol ated

by

t i ssue

f ragmenta-

ti on

(col l agen

and muscl e)

But the war

years al so saw

two

i mpor tant

techni cal devel opments

f i r st , i n 1942,

the

repl i cati on

of

surf ace

t opography w th Formvar

f i l m by Schaefer

and

Harker

7 ,

and

second,

i n 1944, shadowng by

Wl l i ams

and

Wyckhof f

8 Then, i n 1945, Porter et al 9

demonstrated

that

whol e-cul tured ti ssue

cel ls

coul d

be

brought

t o the stage

of

the

EMand

examned

profi tabl y

Whi l e

bathed

i n

bal anced

sal t

sol uti on

(Tyrode s,

pH

7

. 4 , the cel ls i n these earl y

studi es

were

fi xed

w th vapors of OsO4 The advantagesof th i s reagent

f or the fai thful preservati on of cul tured cel ls had been de-

scri bed

i n

1927

by Strangeways and

Canti

(10) For several

years after

1945,

and

unti l thi n- secti oni ng became a real i ty,

these th in ly spread cul tured cel ls

provi ded

the onl y access t o

knowedge

of cel l f i ne structure, and cont ri buted t o observa-

ti ons

on t he

endopl asmc reti cul um

and

the

i ntr acel l ul ar pres-

ence of

vi rusli ke parti cles

i n cel ls from

chi cken tumors and

mouse

mammary

t umors

Qui t e apart from theoreti cal

consi derati ons,

the l imted

avai l abi l i ty i n v i t r o of many ki nds

of t i ssue cel ls

convi nced

bi ol ogi cal l y ori ented el ectr on

mcroscopi sts

of the need f or

ul trathi n

secti oni ng

I n

1934,

Marton 11

had

examned os-

mumf i xed, 15- ti n secti ons

of

pl ant mater i al

Natural l y

he had

l i t t l e success,

even

at

a

magni f i cat i on

of

onl y x450

Later, von

Ardenne 12 att empted t o

cut taperi ngwedges of

t i s sue so

that

at l east some parts of thei r

areas woul d

be adequatel y

thi n

Ri chards

et al

13 and

Sj bst rand(14)

cont i nued

th i s approach,

but onl y theRi chards

group

had suf f i cient success t o warrant

publ i cati on of mcrographs

consi derabl e

hi stori cal hi atus i n

secti oni ng t echni ques f ol l owed,

l asti ng

unti l 1948,

as

t heWar

and the recovery years t ook thei r tol l There

was,

however , one

di versi onduri ng that

i nterval i nto

ul tr a- hi gh- speed

mcrotomy

I n 1943,

O Bri en

and

McKi nl ey

15 devel oped thehypoth-

esi s

that,

at hi gh

secti oni ng

speeds, speci men i ner t i a

shoul d

restri ct stra in di stri buti on so as

t o

l ocal i ze

i t

very

closel y

t o

the

kni f e

edge

They

reasoned

that

there

woul d

be no

t i me

f or

pl ast i c f l ow and that t hermal expansi onwoul dbe negl i gi bl e

They therefore desi gned amcrotome w th a

steel

kni fe

sup-

port ed

j ust beyond

the ci rcumerence of an

8- i nch

wheel

The

wheel

ori gi nal l y was

dri ven

at

12, 500

rpm

and produced a

cutt i ng

speed equi val ent t o

140 f eet per second

The

bl ock

was

moved

i nto

this

whi rl i ng bl ade

at a rate cal cul ated t o

del i ver

0. 1 - t i n

secti ons

Thei r

ori gi nal ar t i c le

was not i l l us t rated w th

mcrographs

of

successf ul sect i ons

However ,

accordi ng

t o a

report by

Gessl er

and Ful l am 16 , a year

l ater,

at the 1944

Annual Meeti ngof the El ectr on M croscopy Soci ety of

Amer

i ca, O Bri en and McKi nl ey di d

show some mcrographs

of

reasonabl y

good

sect i ons, which

i nspi red

Ful l amand Gessl er

t o begi n thei r

own work

w th hi gh-speed mcrotomy They

al so

report ed

that

by

1944,

O Bri en

and

McKi nl ey

had

al most

doubl edthe

ori gi nal

speed of thei r mcrotome t o 22, 500 rpm

Subsequent l y,

Ful l amandGessl er

17

producedamcrotome

whi ch

operated

at

57, 000 rpm del i veri ng a

cutt i ng

speed of

1100

f eet per

second

They used f ragments of razor bl ades as

kni ves,

and sect i oned a

vari ety

of pl as t i c s, as wel l as ti ssues

They

demonst rated

considerabl e success i n cutt i ngthe

pl asti cs,

and even

some

i n secti oni ng ti ssues 3, 17 I n vi ewof

this,

i t

i s curi ous that they di d not expl ore the potenti al of embeddi ng

w th pl asti cs

I nstead,

they f ocused thei r att enti on on embed

di ng medi a

that

woul d vol a t i l i ze af t er secti oni ng was

com

pl eted,

and experi mented w th such subst ances as camphor,

288s

THE

J OURN L

O

CELL

I OLOGY

VOLUME

91

1981

resorci nol , naphthal ene, etc

and

eutecti c

mxtures of

these

and

rel ated

compounds al most al l w th

boi l i ng

poi nts below

85C,

and

some

w th

boi l i ng

poi nts as l ow as

32C They

di d t r y

paraff i n embedments

but recogni zed

probl ems i n

i t s subse-

quent

extracti on from the

sect i ons

Thei r

f i rst

ar t i c le was

i l l us t rated

w th

onl y one

mcrograph

of at i ssue secti on,

that

of

l i ver

fi xed w th

osmum

tetr oxi de

Fai rl y severe arti fact was

evi dent

Ernest

F

Ful l ams

venture i nto bi ol ogi cal

mcroscopy fol-

l owed

an

earl i er

acquai ntanceshi p w th

Cl aude

and

Porter,

who

then were devel opi ng an i nt er est i n the potenti al of

el ectron

mcroscopy

f or

cytol ogi cal

research Cl aude wanted

especi al l y t o

i denti fy

the cytopl asmc ori gi n of mcr osomes I n

1945, Cl aude and Ful l ampubl i shed a j oi nt

paper

18 , i l lus-

trati ng osmumf i xed

l i ver that was

sect i oned

at hi gh speed

(49, 000 rpm

Thei r embedment was spec i f i cal l y characteri zed

as a

eutecti c

mxture of camphor and naphthal ene w th a

mel t i ngpoi nt of 32. 5C

Dry

sect i ons, which

l i t e r a l l y

f lewfrom

the

kni fe,

were

col l ected

on a

s t r i p

of

copper mesh that

was

coated w th a

Formvar f i l m

Areas of

promsi ng secti ons were

sel ected w th

a

l i ght mcroscope, and then sui tabl y posi t i oned

gri ds were

punched

out of the

mesh

They strove f or

secti ons

0

. 3

t o 0

6

pi n thick The publ i shed

mcrographs showed

sub-

stanti al

arti facts,

i

e

many

art i f i c ia l l y-created

hol es

The

au-

thors at l east par t i al l y recogni zed

this,

and

t ook

t he

posi ti on

that the

maj or

probl ems

l imt i ng ef f ecti ve

bi ol ogi cal

ul trathi n

mcrotomy

rel ated

not so

much

t o t he secti oni ng i tsel f as t o

a

need f or

refi ned fi xati on

and

embeddi ng

Al though the

War

ended i n 1945,

and

RC

was

ready

t o

begi n marketi ng

thei r

newy

desi gnedEMU

seri es

mcroscopes

at the end of that year,

few i nstruments were

avai l abl e

t o

bi ol ogi s ts unt i l wel l i nto the 1950s

By today sstandards, those

machi nes

were

rudi mentary

the

f i r s t

of the newRC mcro-

scopes

di d not even have a bi ased gun Obj ect i ve apert ures

werenot i nt roduced unti l 1950 Al though H l l i er andRamberg

(19) had

recogni zed

theneed

and

means f or l ens corr ecti on as

earl y as

1947, st i gmators were not added t o product i on i nstru-

ments

unti l

1953

external l y contr ol l abl e

compensat i on

was

not

avai l abl e f or RC mcroscopes unti l Canal coCo of Bethesda,

Maryl and, marketeda

ki t

i n 1956

and Si emens di d not i ntr o-

duce

i ts

wel l - equipped,

postwar Elmskop unti l 1954 Thus,

i t

t ook about

a

decade after

t he

end

of the

War

f or

el ectron

mcroscopes

t o

evol ve

t o i ncl ude f eatures

we now regard

as

absolutel y

essenti al

f or

bi ologi cal work, such

as stabl eperf orm

ance,

asti gmat i c l enses, and

excel l ent contr ast

Only

at the

mdpoi nt of

that decade

di d ul t ramcrotomy and i ts associ ated

techni ques

al so

mature

i mages

of secti oned materi al

were

produced

that woul d s t i l l

be regarded as

acceptabl e

Earl i er,

theresi mpl y were toomany di sparate probl ems t o permt rapi d

progress t owards a total

sol uti on

Dr Cl aude s

1945

experi ence w th Ful l am

convi nced hi m

that

very

hi gh-speed

mcrotomy

was

not goi ng

t o

be

essent i al

He

therefore

started

work

w th

J oseph Bl um

(then D rector

of

the

i nstrument

shop

at The Rockef el l er I ns t i tute f or

Medi cal

Research) t o devel op a mcrotome that operated at amore

modest

speed The prototype, descr i bedby Cl aude 20 , i ncor-

porated

some

of the desi gn

f eatures

of the earl i er

Ful l am

Gessl er i nstr ument, but was operat ed

si mpl y

by

hand- turni ng

a

f l ywheel

The ar rangement

of t he pul l ey systemundoubtedl y

produced speeds

that

nowwe

woul d

regard as excessi ve How

ever , i n t hi s i nstr ument , t he kni f e di d not move

past

the

f i xed

speci men, as i n the earl i er hi gh-speed mcrotomes ;

i nstead,

the

speci menwas

mount ed

at t he edgeof aturni ng and

advanci ng



3/6

di sk,

whi ch

advanced bysmal l

i ncrements towarda

f i xed

kni fe

Thi s permtted t he use of a trough i n

associ ati on w th the kni fe

so

t hat

sect i ons coul d be col l ected as a

ri bbon

on

a f l uid

surf ace

Cl adde s publ i shed report of thi s

mcrotome

was

not

i l l ustrated

w th

mcr ographs

and he re-emphasi zed that t he

task

ahead

i s

to

f ind

bet ter ways f or the preparati on

and

preservat i on

of the

speci men.

I n J anuary, 1948, i n New

York,

Cl aude

del i vered a Harvey

Lecture on Studi es on Cel l s, i n

whi ch he

summari zed

hi s

ongoi ng

efforts,

i ncl udi ng

hi s

work

on thi n- secti oni ng tech-

ni ques to

i mprove electr on

mcroscopy

of cel l s The l ecture

reached

mainl y

an audience from

that

c i tyand t he manuscri pt,

unfort unatel y,

di dnot appear

i n pri nt unt i l 1950

Thus, Daniel

Pease

and Ri chard

Baker

(21),

worki ng

at

the Uni versi ty of

Southern Cal i f orni a, had

no

i nkl i ng

of t he work i n progress

at

The Rockefel l er I nsti tute when they

publ i shed thei r

own

ac-

count of some success

w th l ow speed

mcrotomy

L

H Bret-

schnei der (22) i n Hol l andwas al so

attempti ng

to

produce hal f-

mcron

sect i ons,

apparentl y

unaware of the

devel opments

at

The

Rockefel l er

Pease and

Baker

( 21)

were i nf l uenced

i n

thei r efforts

to

obtai n ul trathi n sections

f or electr on mcroscopy by a

sugges-

ti on of Prof

F Ki ss

f rom

Hungary, who had been associ ated

w th

Prof

St

Apathy

The

l att er,

worki ng

at

Cl uj ,

Rumani a,

duri ng

the

l ast

years

of

t he ni neteenth century and the earl y

years of t he twent i eth,

contri buted much to the devel opment

of conventi onal

mcroscope

t echni ques,

i ncl udi ng

doubl e

embedments

of paraff i n and

col l odi on

I n a personal commu

ni cati on,

Dr

Ki ss

i ndi cated that

i t was

al most

commonpl ace

f or

members of

that

school to secti on smal l , doubl e-embedded

bl ocks

i n

t he submcron

range

of thi ckness by usi ng conven-

t i onal

mcrotomes

at normal operati ng

speeds Thi s encouraged

Pease and

Baker

(21)

to

change

rather

si mpl y

the

advance

mechani smof a standard Spencer

820

mcrotome

by

a f act or

of ten so

t hat

thenomnal

i ncrement

of

speci men advance was

reduced

to 0. 1 [ , m

More i mportant

f or

success, however, were

t he

real i zati ons

that an adequate embedment had

to

be much

harder,

and

offer

more

support ,

than conventi onal paraff i n,

and that

secti on

size had to be reducedby at l east an order of

magni tude from t hat commonl y empl oyed f or conventi onal

secti oni ng Thi s l ed them

f i r s t

to inf i l t rate t i ny ti ssue bl ocks

w th as much col l odi on as

possibl e,

and

t hen, second, to add

hard paraff i n

Subsequentl y,

Pease (23) hardened

bl ocks

s t i l l

f urt her by a tr i ple-embedding procedure,

whi ch

i nvol ved

i n-

corporati ng Damar resin between the ni tr ocel l ul ose and wax

i nf i l t rat i on

st eps

Al so,

the

paraff i n

was

hardened addi ti onal l y

w th bayberry

or carnaubawax

At f i r s t Pease and Baker (21) col l ected dry secti ons i ndi vid-

ual l y

w th

a camel s hai r brush,

and

so transferred t hemto

gri ds

The

sect i ons

then were f l ushed w th xyl ol i n order to

remove onl y t he paraff i n component , thus l eavi ng the ni tr o-

cel l ul ose network

i n

pl ace

to provide

speci men

support

(The

part i al extracti onwas

deemed

necessary f or want of an ef fecti ve

stai n

to provide adequate

cont rast

I n retrospect, the

resi dual

col l odi on was

i nadequate

to prevent

fa i r ly

seri ous

col l apse

of

f i ne- str uctural

detai l

However , i t seems

fai r

to say

that

t hese

resul ts,

when

f i r s t

publ i shed, f i nal l y

demonstrated that

ade-

quatel y

thin secti oni ng coul dbe

achi eved

w th relati vel y si mple

i nstrumentati on

and w th

l ow

cutt i ng speeds

Thus,

the

work

served as

a

sti mul us f or other l aboratori es, and wt hi n t he next

f our

years, a rashof modi f i cati ons of ol d

mcrotomes,

as

wel l

as rather

si mpl y

desi gned

new

mcrotomes,

were announced

At t he same

t i me,

Bretschnei der (22) i ndependentl y

began

efforts

to

achi eve ul trathi n secti oni ng wthout resort i ng to new

i nstrumentati on

or hi gh speeds

He real i zed

that

the

uni t

of

advance

of the Cambri dge Rocki ng

M crotome

mght pro-

duce secti ons

as

thi n

as 0. 6 j an

or, w th simpl e

modi f i cat i ons,

even thi nner The basi c desi gn of thi s f undamental l y

si mpl e

and mechani cal i nstrument dates from1885, and i s

attr i buted

to H Darw n ( 23)

Lat er , t he desi gners of t he emnentl y

successf ul Port er- Bl umul tramcrotome unw tti ngl y i ncorpo-

rated some of i t s desi gn f eat ures i n t he

mechani sm

whereby

the

speci men

arm

was

suspended and advanced

Bretschnei der real i zed, as had Pease and Baker, t hat a

pri nci pal probl emw th ul trathi n

secti oni ng

l ay i n the soft ness

of t he conventi onal embeddi ng

medi a

He t heref ore used

par-

af f inw th a mel ti ng poi nt of 65C and operated

hi s

i nstrument

at 10C

The mcr ographs

he publ i shed

i ndi cated

successf ul

secti oni ng i n the submcron

range

Unfortunatel y, he had not

preservedhi s ti ssue

w th osmum

tet roxide,

but

rather wth the

mor e

conventi onal fi xati ves of t he

day,

i ncl udi ng

Boui n,

Champy,

Carnoy,

bi chromate- formol , al cohol i c subl i mate,

etc

Al so,

as a

f i nal step, he extracted

the paraff i n

Thus,

al though

the speci mens demonst rated

some

el ectr on t r ansparency, they

were ful l of art i facts At l east two other

European

l aboratori es

(Danon and Kel l enberger 1251 i n Geneva, and Oberl i ng, Gau-

t i er

and

Bernhard

[261

i n Par i s )

al so

made

seri ous

efforts to

use rocki ng mcrotomes f or

ul trathi n

sect i oni ng, and had

enough success t o warrant

publ i cati on

I n 1952, Bretschnei der

(27) publ i shed a

comprehensi ve

revi ew of ul tramcrotomy,

whi ch

i ncl uded refer ences i n

t abul ar

form

of

what he thought

to be the enti re

l i terature through 1951 on the resul ts

of

ul trathi n secti oni ng

t he

l i s t

i ncl udedonl y 36 papers

I n the crit i cal

years i mmedi atel y af ter 1948, other key de-

vel opments

permtted

fa i r ly

rapi d t echnol ogi cal advances The

i ntr oducti on

i n

1949by

Newman

Borysko, and

Swerdl ow

(28,

29) of polybutylmethacryl ate

(and

l ater,

mxtures of butyl and

methyl

methacryl ate as an

embeddi ng medi umserved as a

great st i mul us,

al though the

bot ani cal

materi al i n thei r

mcro-

graphs general l y

was

not

wel l

preserved Ori gi nal l y, t hese

i nvesti gat ors

advocated

the

extracti on

of the

pol ymeri zed

meth-

acryl ate

by

an

organi c sol vent

such

as acetone,

tol uene,

or amyl

acetate

At t he

t i me,

the

l atter step

seemed necessary to provi de

adequate

cont rast

i n l i eu of

any

ef f ecti ve

stai ni ng procedure

other

than that provi ded

by an

initial

fi xati on w th osmum

tetr oxide

Another i mportant advance was t he 1950 i nt roducti on of

gl ass

kni ves

by

Latta and Har tmann ( 30) These i mmedi atel y

repl aced

theuse of steel

kni ves,

whi ch

had

al waysposed

seri ous

and l argel y unresol ved probl em Apparentl y, most i nvest i ga-

tors hadbeenusi ng

di sposabl e

razor bl ades

whi ch

wereground

w th

such an acute angl e as t o be undesi rabl y fl exi bl e Heavy

kni ves, made f or conventi onal mcrotomy, had to be

reshar-

pened

bef ore every use, at l east

i f

exposed to a

trough

f l uid

that

vi sibl y

di scol ored (oxi di zed)

edges w thi n

a

f ew

mnutes

Very l i t t l e had been publ i shed

about how

heavy

kni ves

mght

be

sharpened

easi l y and rel i abl y

(but

see

H l l i er

[311

and

Ekholm

et

al

[321)

Perhaps

thi s

was

because

f ew

i nvesti gators

bel i eved

they had

achi eved

anythi ng

approachi ng

perf ecti on

At

best,

the i nherent

grai n

structure of

steel

presumabl y

woul d

al ways

have l i mted true uni formty and st andardi zati on Fer-

nndez- Morn s

(33)

i ntr oducti on

of di amond kni ves i n 1952

ul ti matel y became an i nteresti ng success story, but t hese have

proved to be more of a

convenience than a

necessi t y

I n

1950,

Gettner

and

H l l i er ( 34) f ormal l y i ntroduced the

useful and i mportant technique

of spreadi ng

and col l ecti ng

PEASE

N

PORTER

El ectr on

Mcroscopy

and

U tr amcrotomy 2895



4/6

secti ons

on and from

aqueous

surf aces i n t roughs att ached

t o

kni ves,

al though

Cl aude (20) had suggested

this

t echni que

earl i er

Duri ng this peri od al so, vari ous

l aboratori es experi -

mented

w th

heavy

metal stai ns

but

had

onl y

l i mted success

However ,

i t

became obvi ous that

phosphotungst i c aci d

was

useful as a

stai n, especi al l y

after OS

4

f i xati on,

and w thout

the necessi ty t o

extract methacryl ate embedments

The

aci d

gai nedw despread

use i n anal cohol i c

sol uti on

The usefulness

of phosphotungsti c aci d had

been

par t i al l y

real i zed

and ex-

pl oi ted

earl i er,

notabl y

by

the

group

at

the

Massachusett s

I ns t i tute of

Technol ogy,

i n

work

w th

whol emount s of

f i brous

protei ns,

etc. However , i t was not unti l 1955

that

Hal l [ 35]

recogni zed, and

del i beratel y

used, phosphotungst i c aci d as a

negat i ve stai n.

Fromthese earl y attempts at mcrotomy i t became

apparent

that singl e-pass mcrotomes werea necessi t y , i n order t o take

advantage of methacryl ate embedment s secti oni ng w th gl ass

kni ves, and

the

col l ecti on of secti ons on f l ui d surf aces

.

Other-

wse, sect i ons of ten were

l ost on the

return stroke

of the

mcrotome,

or the

f ace

of t he

bl ock was damaged

Thi s

i n f lu -

enced al l subsequent

desi gns of i nstr uments

made spec i f i cal l y

f or

ul t ramcrotomy

Many

i ndi vi dual

efforts

t o devel op mcrotomes spec i f i cal l y

f or

ul t ramcrotomy

were

made

i n

the

earl y

1950s

These i n-

cl uded modi f i cati ons of

convent i onal

mcrotomes, and

al so

some

i ngeni ous

ori gi nal desi gns

t o mni mze or el i mnate

prob-

l ems w th the beari ngs and

l ubri cati ng

f i l m of movi ng parts.

Thus,

f l exi bl e rods and

l eaf

spri ngs

were someti mes i ncorpo-

rated in to

the design t o permt

movements

w thout

bear i ng

surf aces

Substanti al efforts

were

made t o i ncrease the mass

and decrease the

elast ic i ty

of the machi nes

Desi gn

f eatures

that were

f i n a l l y

to

appear

i n commerci al

mcrotomes

i ncl uded,

i n addi ti on t o

mechani cal

advance mechani sms, t hermal ex-

pansi on

syst ems

that

ori gi nal l y were i nt roduced by Newman

Borysko, and Swerdl ow ( 28, 29) The l i s t of ul t ramcrotome

desi gns that havebeenpubl i shed, but

never

reached commer

ci al devel opment, i s l ong I n hi s 1955 paper, S i t te 36) appended

an

extensi ve

bi bl i ography of

the pert i nent

i nf ormati on

avai l -

abl e at that ti me, and i n 1956 Gettner and

Ornstei n

(37) wrote

a spl endi d revi ew Porter

(38),

i n 1964, andSj bst rand

(39),

i n

1967,

publ i shed consi derabl e

detai l ed

i nf ormati on on the de-

si gn features

of

earl y mcrotomes, part i cul arl y

of

those

that

reached commerci al

product i on

For

the trul y

rapi d expansi on

of t he devel opi ng f i e l d of

ul t ramcrotomy t o occur, a sui tabl e,

commerci al l y

avai l abl e

mcrotomewas an

obvi ous

necessi ty Thi s was

real i zed

i n 1953

w th

the i ntr oducti on of the Port er- Bl um i nstrument

w th

a

mechani cal advance,

manuf actured

and eventual l y market ed

by I van Sorval l , I nc of Norwal k, Connect i cut (40) Thi s was

f ol l owed, al so i n 1953, by t he Sj dst rand ( 41) thermal l y ad-

vanced

mcrotome, manuf actured by

L

. K. B. - Producter AB

Stockholm

For

a

t i me

the

l atter

mcrotome

domnated

the

European

scene

but

eventual l y

was t aken out of

producti on) ,

whi l e

the

Por ter- Bl um

i nstrument

became w del y used i n the

Uni t ed

States andel sewhere The

simpl i ci ty and the r e l i a b i l i t y

of the MT 1

Por t er - Bl um

mcrotome soon made this

t he

i n-

st rument of

choi ce,

and i t i s

s t i l l

manuf act ured t o t hi s day,

despi te the compet i t i on of second- and thi rd-generati onmcro-

tomes that are f u l l y automated As mght be

expected,

this

mcrotome

went

t hrough

several model

changes

before the

commerci al design was

establ i shed

The

most i nteresti ng of

these i ncorporatedahor i zontal steel bar , whichwas suspended

i n a

gi mbel

at oneendandhel d the speci men i n achuck at t he

2905 THE

J OURNAL

O CELL

BI OLOGY

VOLUME 91 98

other

I t

had no

mechani cal

advance,

but rel i ed on t hermal

expansi on

w th heat froma

readi ng l amp

t o move

t hespeci men

t oward

the

kni f e

I n i ts si mpl i cit y,

i t i s s t i l l

a charmng and

rel i abl e i nstrument

Duri ng the w nter of 1954, an ext raordi nary

workshop on

mcrotomy was hel d at the New

York

Academy of

Sciences

Desi gners of mcrotomes fromup and down t he East

Coast

came

t o the

meeti ng

w th thei r creat i ons Al together,

10

or

12

di f f erent

i nstr uments

wereshown I reneManton 25 years l ater,

recal l ed

the

occasi on as f ol l ows

:

I t

was

my

pri vil ege,

soon

after

arr i val

i n

New

York, t o att end a

meeti ng

at theNewYork

Academy of

Sciences at whi ch an array of

devi ces f or

th in

secti oni ng were

displ ayed,

somecrude, others

al most

comcal l y

compl ex,

but

onl y

t he

Port er - Bl um

behaved

perfectl y, cutt i ng

a cl ean

r i bbon of

seri al secti ons

of the

ri ght thi ckness t o order,

from

a

methacryl ate bl ock 1)

I n 1955, H

S i t te desi gned a

t hermal - advance mcrotome,

whi ch then was

manufacturedandmarketed by Rei chert

AG

Vi enna

;

i ts deri vati ve

commerci al

model s

have

enj oyed

a con-

ti nui ng

success

Four

years

l ater,F Huxley (42) i nt roduced

a

mechani cal - advance

mcrotome,

whichwas f i rst produced by

the

Cambri dge I nst rument

Company anda cosmeti cal l y i m

proved

andmotor - dr i ven versi on

cont i nues t o be bui l t and

sol d

by

L

. K

. B

I n addi ti on

t o

these

mcrotomes

of

earl y

desi gn

that

reached commerci al producti on

most

of whi ch are

s t i l l

bei ng

manufactured) , i nevi tabl y others

were

i ntroduced,

onl y t o di s-

appear

w thout l eaving an i mpor tant

heri tage

We

knowthat t he

foll ow ngmcrotomes

f o r ul t rathi n

secti oni ng were

advert i sed

as bei ng i n commerci al producti on H stor i cal l y

f i r s t i n

t he

late 1940s, was t he Ful l amand Gessl er

very-hi gh-speed

mcrotome,

advert i sed

w th

the suggesti on that t he i nvesti gator coul d

protect

hi s

i nvestment

by an easy

conversi on

to an ul tr acentr i f uge TheAmeri can

Opti cal Co of

Buf fal o then

marketedaversi onof t he

adaptor

f or thei r

Spenser 820 rotary mcrotome

that had

been

devel oped by

Pease

and

Baker

Af ter L H

Bret schnei der s useof t he Cambri dge

Rocki ng

Mcrotome, the device was advert i sed specif i cal l y as an

i nstrument

sui tabl e

f or the el ect ron mcroscopi s t s

M nor

mcrotomes,

r ede-

si gnedaccordi ng to pl ans

by

B B Geren

and

DMcCul l och,

were sol d

f or

some t i me by

the

I nternat i onal

Equi pment Co of Boston

Other mcr otomes

of substanti al l y newdesi gn

thenbegan

to appear

on

t he

market

A

J Hodge,

H E Huxley,

and

D Spi ro produced

prototype i nstruments that were

i ntended f or manufacture by t he

Scientif ic

Equi pment

Corp

Wal tham Mass For a

number

of years,

Ernst Lei tz, of Wetzl ar, Germany,

marketed

a successi on of model s

based upon a desi gn of H Ferni ndez-MOri n,

and

i ts

subsequent

i mprovements

Phi l i ps,

I nc

Ei ndhoven,

produced mcr otomes de-

si gned by H B Haanst ra

J

L Farran

t

and

S

E

Powel l devel oped a

mcrotome sol d through Schuco

Scienti f i c Co NewYork

B

von

Borri es, J Huppertz, and

H

Gansl er

i ntroduced amcrotomemanu

f act ured

by

Sartori us-Werke of G6t t i ngenD

Damon

at

t he Wei zman

I nsti tute of Sci ence, Rehovoth, I srael , marketed a commerci al mcro-

t ome

through

the

Y

.E.D.A.-ResearchandDevel opment Co

associ ated

w th t he

I nsti tute

M

E

Gettner

made

an

effort

to

sel l

a

mcrotomeof

hi s desi gn

through

the Process and I nstruments Co

Brookl yn, N. Y

Georg

J acob KG Lei pz ig, of f ered a

mcrotome patterned

after an

i nstrument f i r s t bui l t

by

N kl owtz

I n

addi ti on

to thesedesi gns of i nvesti gati ve

sci enti sts,

theengineeri ng

staff of LKB- Produckter, Stockholm anonymousl ydevel opeda succes-

si on of substanti al l y di ff erent desi gns TheSorval l D vi s i on

ofDuPont

I nstruments Co

Newtown

Conn

i s nowbegi nni ng

to do

l i kew se,

and,

at least tw ce,

t he

J apan

El ectr onOpti cs Laboratory Co of Tokyo

hasmarketed ul tramcrotomesw thout desi gn credi ts

I n

addi ti on,

J ose

Del vi l l e, Sai nt Germai n-en-Lave,

France,

has recentl y

i ntroduced

a

newi nstrument

I t i s

possi bl e that s t i l l

other

mcrotomes

of

whi ch

we

are unaware may have appeared i n t he market- place I t i s evi dent,



5/6

As one revi ews thepubl i shed

mcrographs of

the

earl y

years

of ul tr athin secti oni ng, i t

i s

apparent ,

i n retrospect,

that poor

f i xati on-of ten borderi ng on

t he utt erl y

i nadequate-was

a

maj or source of di ff i cul ty

There

were

almost

no gui del i nes

except perhaps

f or

t hat of Hei denhai n

i n

awel l - known essay

i n 1911 on Pl asma

and

Zel l e,

he had emphasi zed that

osmum

tetr oxide

was

the

onl y known fi xati ve that preserved

del i cate ti ssues

such

as

nerve axons w thout

enormous

shri nk-

age 43)

Al so,

the f ai thful ness of osmumtetr oxide

had

been

dramati cal l y

demonstrated

i n 1927

by Strangeways and

Canti

10)

i n

thei r studi es of cul tured cel l s by dark-f i el d l i ght mcros-

copy

I t

was

thi s di spl ay

that

l ed Porter to use Os i n

t he

f i xati on

of cul t ured cel l s i n 1945

Cert ai nl y

these consi derati ons

al so

i nf l uenced Pease and Baker s

21)

ori gi nal choi ce of thi s

fi xati ve

Subsequent uses

of

osmum

tetr oxidewere i nf l uenced

by the qual i ty of those earl y preparati ons Nonethel ess, howto

use i t to

best

advantage i n the

fi xati on

of

ti ssues was

not

i mmedi atel y

evident

I t s

poor penetr at i on

through ti ssue

was

al ready

not or i ous

and

i t

di d not perfuse

wel l At

f i r s t

overl y

l arge

ti ssue

sampl es were used,

whi ch then

were

i mmersed i n

unbuf fered sol uti ons The di ff i cul t i es w th

thi s f i xat i ve

were

recogni zed and,

i n

1952, when

Pal ade

44)

f i r s t

reported

and

demonstrated the val ue of

pH

contr ol ,

thework was heralded

as

a

l andmark

by

al l

i nvesti gators

i n

t he

f i eld

I n

retrospect,

no

doubt t he Pal ade

Pickl e worked as

wel l

as

i t

di d

part l y

because he refi ned

ti ssue- mnci ng

to

produce t rul y

smal l bl ocks

whi l e the

ti ssue

was

i mmersed

i n

t he

fi xati ve

I n di ssol vi ng

osmum

tetr oxide

i n Veranol buf f er

sol uti on, Pal ade

made no

attempt to

empl oy

a physi ol ogi cal l y compati ble vehi cl e, f or he

di d

not thi nk i t

was

i mportant Most subsequent i nvesti gators

have

al so i gnored physi ol ogi cal compati bi l i t y, even when

se-

l ecti ng

other buf f ers By contrast, i n t he md-1950s, Rhodi n

45) , Zett erqvi st 46) , and Sj dstrand 47) advocated the use of

a bal anced sal t sol uti on w th onl y

mnor buf f eri ng

propert i es

as the fi xati ve vehicl e,

and

al so succeeded

i n f i xi ng ti ssue

remarkabl y

wel l

f or the

t i me

Unfortunatel y, si nce then thi s

approach

has been

used onl y

sporadi cal l y

In the

years

1952-54,

t he

var i ous

U

S

Nati onal

I nsti tutes

of Heal th

came

to

recogni ze

t hat theessenti al tool s f i nal l y were

avai l abl e

to

uti l i ze ul trathi n secti oni ng techniques ef f ecti vel y

to expl ore

cel l ul ar structure

and

functi on

The

NI H became

generous i n

establ i shi ng new

l aborat ori es and i n support i ng

exi sti ng

ones

El ectr on-mcroscope i nstall ati ons

prol i f erated

Many

tal ented

young i nvesti gators

changed thei r

research

di -

recti on I n

J anuary 1954,

a

new j ournal ,

t he f i r s t desi gned

specif i cal l y to accommodate the

expandi ng i nformati on rel at-

i ng to

cel l ul ar

fi ne structure, was

l aunched

under t he

aegi s

of

The

Rockefel l er

I nsti tute

Thi s

was

t he J ournal of

Bi ophysi cal

and

Bi ochemcal Cytol ogy J BBC) , l ater

to become

the J ournal

of Cel l Bi ol ogy J CB) I n J anuary of 1956, Kei th Porter orga-

ni zed a Conferenceon Ti ssue Fi ne Str ucture,

whi ch

had the

f i nanci al

support

of

the

Morphol ogy

and

Geneti cs

Study

Sec-

t ion of t he Nati onal I nsti tutes of Heal th Thi s meeti ng pro-

duced

anextr aordinary vol ume, publ i shed i n 1956 as asuppl e-

ment to

Vol ume

2 of the J BBC

The

conference presented a

good overvi ewof what

had

been accompl i shed i n the short

peri odof t i me si nce

sati sfactory

mcrotomeshad

become com

however , that very

f ew

ul t ramcrotomes

have

had l ong

compet i t i ve

exi stences,

and even t hese

w th

t i me, have undergoneextensi ve

mod-

i f i cat i ons The i mprovements have added

t o

aut omati onand to

costs

but not

necessari l y

t o

the ul ti mate

qual i ty of

the secti ons

they

have

produced

merci al l y avail abl e,

osmumtetr oxi de fi xati on

had

become

reasonabl y wel l understood,

and

methacryl ate embeddi ng

had

become routi ne

One- hundred and

ni ne

i nvesti gat ors

parti ci-

pated,

i ncl udi ng many

from

abroad, and 75 papers

were

pre-

sented Despi te the hi gh

qual i ty

of many of the mcrographs

presented, two

papers

spoke of i mpendi ng probl em Borysko

48)

had

come to recogni ze pol ymeri zati on damage t hat

somewhat capri ci ousl y, but seri ousl y, coul d change cel l ul ar

fi ne structure I n addi t i on, Morgan, Moore,

and

Rose

49)

showed

convi nci ng

evi dence

that

t he

subl i mati on

of

methac-

ryl ate in t he el ectr onbeam previ ousl y recogni zed),

coul d resul t

i n severe

cytol ogi cal

art i facts,

i ncl udi ng

damage to

both cyto-

membranes

and

protei n part i cul ates

However ,

t he f u l l

extent

of the

l i mtations

of methacryl ate embedments was not- and

coul d

not

have

been- f ul l y appreci atedunti l

comparati ve

eval -

uati on was

possi bl e, af ter the devel opment of cross- l i nked

pl asti cs as embeddi ngmedi a Before

t hat devel opment,

vari ous

pal l i ati ve

measures

were devi sed to

mni mze

ti ssue

damage

i n

methacryl ate

These

i ncl uded the

part i al

polymeri zati on

of

methacryl ate

mxtures before

i ni tati ng

the

embedment Bor-

ysko

and

Sapranauskas

[50]

t he use of more exoti c

catal ysts

than the

ori gi nal

benzoyl peroxi de azodi i sobutyroni tr i l e, Shi p-

key and

Dal ton [51])

; and t he

i ncl usi on

of traces

of

substances

that

coul d

possi bl y serve

as

nucl eat i on

cent ers

(uranyl

ni tr ate,

War d

[52])

I n

addi t i on,

to

prevent ,

or at

l east

to

mnimze,

subl i mati onart i facts,

Watson

53) proposedsandw chi ngmeth-

acryl ate secti ons between two supporti ng f i l m

I t

was, however, the work w th epoxy resi ns,

begun

by

Maal oeand

Bi rch-Anderson 54) , that f i nal l y di scl osed t he ful l

l i mtati ons of methacryl ate

embeddi ng

I t became

apparent

that these cross-l i nki ng resins do not

l i qui fy

or

decompose

i n

the el ectr on beamas does polymethacryl ate,

and

that poten-

t ial l y

destr ucti ve surf ace- tensi on forces coul d be avoi ded en-

t i rel y

through thei r use Ori gi nal l y,

Maal oe

and Bi rch-Ander-

son used an unspeci f i ed hi ghl y vi scous epoxy compound,

w th

di ethyl ene tri amne as t he

hardeni ng

agent Soon

after,

G auert et al 55) i ntroduced Aral di te

M

At

about

t he same

t i me,

Kel l enberger

et

al

56)

started

to expl ore

cross-l i nking

pol yest er

resi ns as embeddi ng medi a, whi ch l ed

Ryter

and

Kel l enberger 57) to

settl e onVestopal

s

thei r fi nal

choi ce

Al l of thi s

ori gi nal

work was

concerned

wth i mprovi ng t he

preservat i on

of bacteri a, and

i t mght have

had a

more

i mme-

di ate

i mpact i f

more

compl ex cel l ul ar morphol ogy had

been

presented

part i cul ar

diff iculty arosew th

Aral di te

M f or the

Ameri can-made product

turned

out to be di f f erent fromthe

Engl i sh one,

and

there were

probl em

wth

i t s

i nf i l t rat i on i nto

ti ssue Thi s al so del ayed

i t s

general acceptance as an

embed-

ment ,

and

i t

real l y

was

not unti l Luf t 58) i ntroduced

Epon

812 i n 1961 as the res in of choi ce that el ectron mcroscopi sts

worl dw de

had

an easi l y obtai nabl e and reasonabl y

rel i abl e

cross-l i nking

embedment

I nvesti gators

faced

another

probl em

when they

started to

use

epoxy

resi ns, that

of

i nadequate

speci men

cont rast

pri nci pal di ff i cul ty

was

t hat cured epoxies are themel ves very

dense

subst ances,

and

there i s no subl i mati on of materi al

duri ng

el ectr on

bombar dment to

enhance

a

cont rast

di f f eren-

t i al

Furthermore,

cured

epoxy

res ins are qui te hydrophobi c,

so that the aqueous, heavy-metal stai ns that

had

been used

successful l y wth pol ymethacryl ate di d not al ways penetr ate

wel l

and

di dnot produce adequate contrast Fort unatel y, Wat -

son 59, 60) i ntroduced an al kal i ne l ead stai n i n 1958 that

proved to be hi ghl y ef f ecti ve w th

epoxi es,

and, wt h i ts

var-

i ants, i s

s t i l l

by

f ar

the most val uabl e general -purpose stai n

PEASE

N PORTER

El ectr onMcroscopyand U tramcrotomy 2915



6/6

Ac t u a l l y , Wat son di d a l l of hi s

o r i g i na l

work w th

methacr yl ate

embedment s

I t

seems

t o have been si mpl y f o r t u i t i o u s

t h a t

t h e

a l k a l i ne - l ead s t a i n

worked s o we l l w th

t h e

epoxy

embedment s

These

embedment s

and

an e f f e c t i v e s t a i n i ng t echni que wer e

t he n ex t - t o - l a s t s t e p t oward reachi ng the goal t h a t we

now

recogni ze

as

st andard operati ng

procedure

.

The f i n a l s t e p was

t o

be

f i x a t i o n

. I n

s p i t e

of

t h e c y t o l o g i c a l

d et a i l

t h a t

obvi ousl y coul d

be preserved

w th

osmum

e t r o x i d e,

i t

was

s u s p e c t f o r

a

number

of

r e a s o n s

I t s

chem cal

r e a c t i v i t y ,

p a r t i c u l a r l y i n r e l a t i o n t o pr o t e i n s , was

poorl y

understood,

even

though

P o r t e r

and

Kal l man

61

and

Bahr

62

had

r e p o r t e d on numer ous model experi ment s These had

made

i t

c l e a r , however , t h a t by no means a l l cytopl asm c macr omol e-

c ul e s

wer e rendered s u f f i c i e n t l y i ns o l u bl e t o w thstand l e ac h i n g

i n

subsequent

processi ng

s t e p s

Furt hermore, i t was g en er a l l y

recogni zed t h a t

OS0

destroyed

e s s e n t i a l l y

a l l

enzymat i c

a ct i v -

i t y

so t h a t cytochem cal r e a ct i o ns

coul d

not be

demonst rated

a f t e r i t s use Sa ba t i n i e t al

[ 6 3 ]

Thus,

pr o t e i n conf i gurati ons

wer e recogni zed as bei ng s e ve r e l y

damaged A l s o ,

and qui t e

un f o r t una t e l y , t h er e was no other f i x a t i v e known i n the 1950s

t h a t coul d

be

used f o r compari son w th OS0

4

t o

hel p eval uate

t h e q u a l i t y

of

i t s u l t r a s t r u c t u r a l

p r e s er v a t i o n

Thus, f ormal de-

hyde had

proved t o

be

compl etel y i nadequat e i n methacr yl ate

embedment s, and al though

a cr o l e i n ,

as i ntr oduced

by Luft

64 ,

was

recogni zed

as

an i mprovement, i t s

noxi ous

t o x i c

p r o pe r t i e s di scouraged i t s

w despread

use and

del ayed e xp er i -

mentati on

The di scovery of

gl utaral dehyde as t h e

pri mary f i x a t i v e

of

choi ce by

S aba t i n i ,

and

Bensch,

and Barrnett 63 i mmedi -

a t e l y

demonst rated

c o n s i s t e n t l y good

and

uni f orm

t i s s u e prep-

a r a t i o n, p a r t i c u l a r l y of

p r o t ei n s

Cytol ogi cal s t r u c t u r e s no t

g en er a l l y

seen

b e f o r e , such as

cytopl asmc

m crotubul es, now

wer e r o ut i ne l y

observed

Many

t i s s ue s

coul d

be r e a d i l y

p e r -

f u s e d

because gl utaral dehyde

does not

c o n t r a c t

vascul ar

smooth

muscl e

as di d OSO A f i r s t

approxi mat i on

of t he

p r o t e i n chemstr y i nvol ved

i n

gl utaral dehyde

f i x a t i o n

appeared

t o

be

r e l a t i v e l y

si mpl e and

understandabl e

.

To

a consi derabl e

e x t e n t , p r o t ei n s

and other

macromol ecul es

o f t e n wer e sogentl y

denatured

t h a t

hi stochem cal

and i mmunol ogi cal

s p e c i f i c i t i e s

wer e preserved

Fort unatel y,

gl utaral dehyde coul d

be used

w th osmumt et r ox i d e, as we l l as w th uranyl s a l t s s o t hat

doubl e

f i x a t i o n

w th

t he ad di t i o n

of

heavy metal s proved

t o

be

p os s i b l e ,

and

demonst rated p a r t i c u l a r l y

wel l - preserved

c y t o -

membrane

systems

Wth

t h i s

somewhat b el a t e d recogni ti on

of

t h e

g r e a t

val ue

of

gl utaral dehyde, ul tr am crotomy

f i n a l l y coul d

be

s ai d t o

have

compl et ed at

l e a s t

t h e f i r s t phase

of

i t s h i s t o r i c a l

devel opment

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Documents

J Cell Biol 1981 Pease 287s 92s