acquired by FIB/SEM. - Vision Day · 2) Take image of fl at surface (SEM) 3) Remove thin slice...
1
Why FIB/SEM? Why not cryo? Sample preparation References Acknowledgements University of Copenhagen B. Lubelli, D.A.M. de Winter, J.A. Post, R.P.J. van Hees, M.R. Drury, Cryo-Fib-SEM and MIP study of porosity and pore size distribution of bentonite and kaolin at different moisture contents, Applied Clay Science, 2013, 80-81, pp. 358-365 J.C. Riemersma, Osmium Tetroxide Fixation of Lipids: Nature of the Reaction Products, J Histochem Cytochem 1963 11: 436 OsO 4 fixation Using the previously described preparation method images were taken showing three different phases: Liquid phase (lipid) Bulk phase (chalk) Air bubbles Verifying the observations: Air bubbles indicate the observation of a fixed liquid Elemental contrast due to different density of lipid and bulk Information can be gathered from 2D images as well as from 3D image stacks acquired by FIB/SEM. Future possibilities: Direct observation of fluid distributions for different saturations as well as the possibility of elemental mapping. Thanks to the NanoGeoScience group at Copenhagen University for enabling this work in the framework of the P-cubed project. Special thanks to Zhila Nikrozi and Klaus Qvortrup from the Core Facility for Integrated Microscopy (CFIM) Copenhagen. Imaging liquids in pores - beyond X-ray tomography Lipid Fixation in porous samples Results Ralph Harti, Henning Sørensen, Kim Dalby, Susan Stipp Nano-Science Center, University of Copenhagen, Universitetsparken 5, 2100 København, Denmark Focused Ion Beam - Scanning Electron Microscopy (FIB/SEM) FIB/SEM sample preparation before and after removing slices using the Focused Ion Beam - the direction of the imaging electron beam is indicated by the arrow FIB/SEM is combining the imaging capabilities of the Scanning Electron Microscope with the sample manipulation opportunities of the Focused Ion Beam. Imaging steps: 1) Cutting a flat surface of the sample (FIB) 2) Take image of flat surface (SEM) 3) Remove thin slice (FIB) 5) Continue with step 2 e-beam Often tomography is used to acquire 3D images of porous materials (see central image). Drawbacks of this technology however are the limited number of imaging modes and the expensive and complex image acquisition. This work presents a new sample preparation method for imaging lipids in porous systems using FIB/SEM, overcoming the limitations of tomography and tackling some challenges of the classical SEM approach. FIB/SEM offers: Fast image acquisition (can be done at all FIB/SEM setups) High spatial resolution Multiple imaging modes (BSE, SE, EDX) However FIB/SEM is a destructive method limiting the data collection to a one-time possibility. The direct observation of the pore space, made possible by FIB/SEM, and its content eliminates the need for image reconstruction and offers a direct view on the area of interest. Immobilizing liquids by freezing them is a common approach to image liquids in the Scanning Electron Microscope (SEM). However combining it with the ion milling process of the FIB/SEM is, especially considering porous rock samples, a challenging task. The most popular approach for cryo-SEM imaging of rocks is a fracture process including fracturing the sample inside the SEM by the use of a blade (B. Lubelli et al., 2013). Fracture technique: No 3D information Topographic contrast due to the not sufficiently flat surface after fracturing Fixating fluids without the need of freezing them (using the technique illustrated in this work) enables the use of the Focused Ion Beam providing the capabilities of 3D information as well as flat surfaces without topographic contrast. Original image Bulk coloured Pores coloured Air bubbles coloured Osmium tetroxide (OsO 4 ) fixation is widely used in Life Sciences to immobilize lipids by reacting with unsaturated bonds in fatty acids to form diols. Oxidation of OsO4 leading to the fixation of unsaturated lipids (after J.C. Riemersma, 1962) Osmium tetroxide (OsO4) Reaction steps: Opening up double bonds Cycloaddition to form osmate ester Rapid hydrolysis leading to vicinol diol The sample To take advantage of the principle of Osmium Tetroxide Fixation the sample has to be flushed with a lipid containing a sufficient number of unsaturated fatty acids. Sunflower oil is a suitable choice. Emerge sample in Sunflower Oil Heat up to around 80°C for an hour Take the sample out Put sample in Osmium Tetroxide gaseous environment (~ 2 days) After following this procedure the lipid inside the pores is fixed and can be treated as a solid. Basic knowledge about the investigated chalk sample (Aalborg outcrop) is gathered by data analysis on tomographic images (see central image). Porosity: ~ 50.9% Specific Surface Area: ~ 0.621 m 2 /g Permeability: ~ 32.24 mD The permeability is calculated using the Kozeny-Carman equation, i.e. based on the porosity and suface area. 3D image data set with fixed liquid inside of pores
Transcript of acquired by FIB/SEM. - Vision Day · 2) Take image of fl at surface (SEM) 3) Remove thin slice...
Wh
y F
IB/S
EM
?W
hy n
ot
cry
o?
Sam
ple
pre
para
tion
Refe
ren
ces
Ackn
ow
led
gem
en
ts
Univ
ers
ity o
f C
op
enhag
en
B.
Lub
elli
, D
.A.M
. d
e W
inte
r, J.A
. Po
st,
R.P
.J.
van
Hees,
M.R
. D
rury
, C
ryo-F
ib-S
EM
an
d M
IP s
tud
y o
f p
oro
sity
and
pore
siz
e d
istr
ibu
tion
of
bento
nit
e a
nd
kaolin
at
diff
ere
nt
mois
ture
conte
nts
, A
pp
lied
Cla
y S
cience
, 2
01
3,
80
-81
, p
p.
35
8-3
65
J.C
. R
iem
ers
ma,
Osm
ium
Tetr
oxid
e F
ixati
on o
f Li
pid
s: N
atu
re o
f th
e R
eact
ion P
rod
uct
s, J H
isto
chem
Cyto
chem
19
63
11
: 4
36
OsO
4 fi
xati
on
Usi
ng
th
e p
revio
usl
y d
esc
rib
ed
pre
para
tion m
eth
od
im
ag
es
were
ta
ken s
how
ing
thre
e d
iffere
nt
ph
ase
s:
Liq
uid
ph
ase
(lip
id)
Bulk
phase
(ch
alk
)
Air
bu
bb
les
Veri
fyin
g t
he o
bserv
ati
on
s:
Air
bu
bb
les
ind
icate
the o
bse
rvati
on o
f a fi
xed
liq
uid
Ele
menta
l co
ntr
ast
due t
o d
iffere
nt
den
sity
of
lipid
and
bu
lk
Info
rmati
on c
an b
e g
ath
ere
d f
rom
2D
im
ag
es a
s w
ell a
s f
rom
3D
im
ag
e
sta
cks a
cquir
ed
by F
IB/S
EM
.
Fu
ture
possib
ilit
ies:
Dir
ect
ob
serv
ati
on o
f fluid
dis
trib
uti
ons
for
diff
ere
nt
satu
rati
ons
as
well
as
the p
oss
ibili
ty o
f ele
menta
l m
ap
pin
g.
Than
ks t
o t
he N
an
oG
eoS
cience
gro
up
at
Cop
enhag
en
Univ
ers
ity f
or
enab
ling
this
work
in t
he f
ram
ew
ork
of
the P
-cub
ed
pro
ject
.
Sp
eci
al th
anks
to Z
hila
Nik
rozi
and
Kla
us
Qvort
rup
fro
m t
he C
ore
Faci
lity f
or
Inte
gra
ted
Mic
rosc
op
y (
CFI
M)
Cop
en
hag
en.
Imag
ing
liq
uid
s in
pore
s -
b
eyon
d X
-ray t
om
og
rap
hy
Lipid
Fix
ati
on in p
oro
us
sam
ple
s
Resu
lts
Ralp
h H
art
i, H
enn
ing
Søre
nse
n,
Kim
Dalb
y,
Susa
n S
tip
pN
ano-S
cience
Cente
r, U
niv
ers
ity o
f C
op
enh
agen,
Univ
ers
itets
park
en 5
, 2
10
0 K
øb
enhavn,
Denm
ark
Focu
sed
Ion
Beam
- S
can
nin
g
Ele
ctron
Mic
roscop
y (
FIB
/SEM
)
FIB
/SEM
sa
mple
pre
para
tion
befo
re
and
aft
er
rem
ovin
g s
lices
usi
ng t
he F
ocuse
d I
on
Beam
-
the
dir
ecti
on
of
the
imagin
g
ele
ctr
on b
eam
is
indic
ate
d b
y t
he a
rrow
FIB
/SEM
is c
om
bin
ing t
he im
agin
g c
apabilit
ies o
f th
e S
cannin
g E
lectr
on M
icro
scope w
ith
the s
am
ple
m
anip
ula
tion o
pport
un
itie
s o
f th
e F
ocused Ion
B
eam
.
Imag
ing
ste
ps:
1)
Cu
ttin
g a
flat
surf
ace
of
the
sam
ple
(FI
B)
2)
Take
imag
e of
flat
surf
ace
(SEM
)
3)
Rem
ove
thin
slic
e (F
IB)
5)
Con
tinue
wit
h s
tep
2
e-b
eam
Oft
en t
om
og
rap
hy is
use
d t
o a
cquir
e 3
D im
ag
es o
f p
oro
us
mate
rials
(se
e c
en
tral im
ag
e).
Dra
wb
ack
s of
this
tech
nolo
gy
how
ever
are
the lim
ited
num
ber
of
imag
ing
mod
es
and
th
e
exp
ensi
ve a
nd
com
ple
x im
ag
e a
cquis
itio
n.
This
work
pre
sents
a n
ew
sam
ple
pre
para
tion
meth
od
for
imag
ing
lip
ids
in p
oro
us
syst
em
s usi
ng
FIB
/SEM
, overc
om
ing
the lim
itati
ons
of
tom
og
rap
hy a
nd
tack
ling
som
e c
halle
ng
es
of
the c
lass
ical S
EM
ap
pro
ach
.
FIB
/SEM
off
ers
:
Fast
im
ag
e a
cqu
isit
ion (
can
be d
one a
t all
FIB
/SE
M s
etu
ps)
Hig
h s
pati
al re
solu
tion
Mu
ltip
le im
ag
ing
mod
es
(BS
E,
SE
, ED
X)
How
ever
FIB
/SEM
is
a d
est
ruct
ive m
eth
od
lim
itin
g t
he d
ata
colle
ctio
n t
o a
one-t
ime p
oss
ibili
ty.
The d
irect
ob
serv
ati
on
of
the p
ore
sp
ace
, m
ad
e p
oss
ible
by
FIB
/SEM
, and
its
conte
nt
elim
inate
s th
e n
eed
for
imag
e
reco
nst
ruct
ion a
nd
off
ers
a d
irect
vie
w o
n t
he a
rea o
f in
tere
st.
Imm
ob
ilizi
ng
liq
uid
s b
y f
reezi
ng
th
em
is
a c
om
mon a
pp
roach
to im
ag
e
liquid
s in
th
e S
can
nin
g E
lectr
on
Mic
roscop
e (
SEM
). H
ow
ever
com
bin
ing
it
wit
h t
he ion m
illin
g p
roce
ss o
f th
e F
IB/S
EM
is,
esp
eci
ally
consi
deri
ng
poro
us
rock
sam
ple
s, a
challe
ng
ing
task
.
The m
ost
pop
ula
r ap
pro
ach
for
cryo-S
EM
im
ag
ing
of
rock
s is
a
fractu
re p
rocess incl
ud
ing
fra
cturi
ng
the s
am
ple
insi
de t
he S
EM
by
the u
se o
f a b
lad
e (
B.
Lub
elli
et
al.,
20
13
).
Fra
ctu
re t
ech
niq
ue:
No 3
D in
form
ati
on
Top
og
rap
hic
contr
ast
due t
o t
he n
ot
suffi
cientl
y fl
at
surf
ace
aft
er
fract
uri
ng
Fixati
ng
flu
ids
wit
hou
t th
e n
eed
of
freezin
g t
hem
(usi
ng
the
tech
niq
ue illu
stra
ted
in t
his
work
) enab
les
the u
se o
f th
e F
ocu
sed
Ion
Beam
pro
vid
ing
the c
ap
ab
iliti
es
of
3D
info
rmati
on a
s w
ell
as flat
surf
ace
s w
ith
out
top
og
rap
hic
con
trast
.
Ori
gin
al im
ag
eB
ulk
colo
ure
dPo
res
colo
ure
dA
ir b
ub
ble
s co
loure
d
Osm
ium
tetr
oxid
e (
OsO
4) fixati
on is
wid
ely
use
d
in L
ife S
cience
s to
im
mob
ilize
lip
ids
by r
eact
ing
wit
h
unsa
tura
ted
bond
s in
fatt
y
aci
ds
to f
orm
dio
ls.
Oxid
ati
on o
f O
sO4
leadin
g t
o t
he
fixati
on o
f u
nsa
tura
ted lip
ids
(aft
er
J.C
. R
iem
ers
ma, 1
96
2)
Osm
ium
tetr
oxid
e
(OsO
4)
Reacti
on
ste
ps:
Op
en
ing
up
dou
ble
bond
s
C
ycl
oad
dit
ion t
o f
orm
osm
ate
est
er
Rap
id h
yd
roly
sis
lead
ing
to v
icin
ol d
iol
Th
e s
am
ple
To t
ake
ad
van
tag
e o
f th
e p
rinci
ple
of
Osm
ium
Tetr
oxid
e F
ixati
on t
he
sam
ple
has
to b
e fl
ush
ed
wit
h a
lip
id c
onta
inin
g a
suffi
cient
num
ber
of
unsa
tura
ted
fatt
y a
cid
s. S
unfl
ow
er
oil
is
a s
uit
ab
le c
hoic
e.
Em
erg
e s
am
ple
in S
unflow
er
Oil
Heat
up
to a
round
80
°C f
or
an h
ou
r
Take
the s
am
ple
out
Put
sam
ple
in O
smiu
m T
etr
oxid
e g
ase
ous
en
vir
onm
ent
(~ 2
d
ays)
Aft
er
follo
win
g t
his
pro
ced
ure
the lip
id insi
de t
he p
ore
s is
fixe
d a
nd
ca
n b
e t
reate
d a
s a s
olid
.
Basi
c kn
ow
led
ge a
bout
the invest
igate
d c
halk
sam
ple
(Aalb
org
outc
rop
) is
gath
ere
d b
y d
ata
analy
sis
on
tom
og
rap
hic
im
ag
es
(see c
entr
al im
ag
e).
Poro
sity
: ~
50.9
%
Sp
ecific
Su
rface
Are
a: ~
0.6
21 m
2/g
Perm
eab
ility
: ~
32.2
4 m
D
The p
erm
eab
ility
is
calc
ula
ted
usi
ng
th
e K
oze
ny-
Carm
an
eq
uati
on,
i.e.
base
d o
n t
he p
oro
sity
and
sufa
ce a
rea.
3D
im
age d
ata
set
wit
h fi
xed liq
uid
insi
de o
f pore
s
