Remo ing he e ec s o he “da k ma e ”in omog aphy
Lionel C. Gon a dQ1
Ins i u o de Ciencia de Ma e iales de Se illa (CSIC), 41092 Se illa, Spain
a icle in o
A icle his o y:
Recei ed 16 Oc obe 2014
Recei ed in e ised o m
10 Ma ch 2015
Accep ed 18 Ma ch 2015
Keywo ds:
Tomog aphy
Elec on omog aphy
Til se ies
Image p ocessing
abs ac
Elec on omog aphy (ET) using di e en imaging modes has been p og essi ely consolida ing i s posi-
ion as a key ool in ma e ials science. The fideli y o a omog aphic econs uc ion, o omog am, is
a ec ed by se e al expe imen al ac o s. Mos o en, an un ealis ic cloud o in ensi y ha does no
co espond o a eal ma e ial phase o he specimen (“da k ma e ”) blu s he omog ams and enhances
a e ac s a ising om he missing wedge (MW). He e we show ha by simple p ep ocessing o he
backg ound le el o any omog aphic il se ies, i is possible o minimise he nega i e e ec s o ha
“da k ma e ”. I e a i e econs uc ion algo i hms con e ge be e , leading o omog ams wi h ewe
s eaking a e ac s om he MW, mo e con as , and inc eased accu acy. The conclusions a e alid i -
espec i e o he imaging mode used, and he me hodology imp o es he segmen a ion and isualisa ion
o omog ams o bo h c ys alline and amo phous ma e ials. We show examples o HAADF STEM and BF
TEM omog aphy.
&2015 Published by Else ie B.V.
1. In oduc ion
Elec on omog aphy (ET) in ma e ials science is a echnique
based on he acquisi ion o a se ies o p ojec ed images om a
sample by il ing he specimen using a mic oscope goniome e
and can be combined wi h spec oscopic da a [1–4]. I p o ides
impo an 3D s uc u al and chemical in o ma ion ha makes i
possible o co ela e he syn hesis, s uc u e, and unc ionali y o a
di e si y o nanos uc u es e en a a omic esolu ion [5–16].ET
applica ion ollows a se ies o s eps (Fig. 1a). In ansmission
elec on mic oscopy (TEM), usually ‘single-axis il ing’is used, in
which he specimen is il ed abou he eucen ic axis o he spe-
cimen holde od. A e accu a e alignmen o he omog aphic il
se ies, econs uc ion, segmen a ion, and isualisa ion o he 3D
olume o omog am a e he s eps necessa y o ex ac quan i a-
i e mo phological in o ma ion om he 3D omog ams [17–20].
Fo op imal esul s, he specimen mus be il ed o e a il ha
is as close o 790°as possible, bu in p ac ise he maximum il ing
ange a ely exceeds 775°. This is he well-known p oblem o he
missing wedge (MW) o in o ma ion ha esul s in a dec ease o
he spa ial esolu ion and he p esence o a e ac s in he omo-
g am [1–4]. Fo ce ain ypes o sample geome ies, he use o
double- il ing, ull- o a ion holde s and adap ed p epa a ion
me hods can be used o dec easing he MW [21,22]. Impo an
p og ess in ET has esul ed om digi al p ocessing o omog aphic
il se ies be o e 3D econs uc ion by accu a e in e pola ion o
sinog ams, edge de ec ion, con as enhancemen , and accu a e
alignmen o il se ies [23–26]. The nex s ep is he econs uc ion
o he omog am, ypically using weigh ed back-p ojec ion (WP)
o he simul aneous i e a i e econs uc ion echnique (SIRT)
[3,24]. None heless, much e o is being pu in o no el e-
cons uc ion algo i hms which can no ably imp o e he quali y o
omog ams, e en wi h low sampling and limi ed angula ange,
using ei he he disc e isa ion o in ensi ies, he calcula ion o si-
nusoidal ajec o ies in sinog ams, he inco po a ion o geome ic
p io knowledge, o comp essi e sensing [27–31]. Tomog ams
ob ained a e he econs uc ion a e 3D da ase s con aining a
con inuous ange o in ensi y le els, and he e o e in o de o
ex ac use ul in o ma ion, isualisa ion and segmen a ion a e
equi ed in combina ion wi h he applica ion o sophis ica ed al-
go i hms, such as aniso opic non-linea di usion, o educe noise
and enhance local s uc u e wi hou wo sening he esolu ion o
s uc u al in o ma ion, adap i e h esholding, o equalisa ion in
eal space [32–35]. In he p esen wo k, we ocus on he e ec s o
an expe imen al pa ame e : he minimum alue o he in ensi y
used o acqui e a omog aphic il se ies. Ou esul s show ha by
op imisa ion o he backg ound le el o a omog aphic il se ies i
is possible o d ama ically imp o e he quali y o he omog am.
2. Me hods
In ou expe imen s we use a comme cial so wa e package o
acquisi ion and econs uc ion o omog aphic il se ies called
Explo e3D, which is he s anda d o TEMs manu ac u ed by FEI.
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Con en s lis s a ailable a ScienceDi ec
jou nal homepage: www.else ie .com/loca e/ul amic
Ul amic oscopy
h p://dx.doi.o g/10.1016/j.ul amic.2015.03.017
0304-3991/&2015 Published by Else ie B.V.
E-mail add ess: [email p o ec ed]
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎
The file o ma is a e sion o he o iginal one.m c c ea ed by he
Medical Resea ch Council..Wi h his so wa e, he pixel in ensi ies
o he il se ies o images a e eco ded as numbe s wi h he o -
ma o in ege signed and 16 bi s, meaning ha pixels can ake
disc e e alues in he ange be ween 32,678 and þ32,677. In
o de o op imise he backg ound o he il se ies, he minimum
alue o he ma ix I
min
is ound and he alues o he ma ix a e
downshi ed by an amoun gi en by he di e ence be ween
32,678 and I
min
. The esul ing ma ix is sa ed back in o he FEI
o ma o subsequen omog aphic econs uc ion.
I is wo h men ioning he e ha he basis o omog aphy, he
Radon ans o m defines a p ojec ion unc ion (an image in TEM)
as an in eg al om ze o. Simila ly, in o de o con e ge o a co ec
solu ion i e a i e econs uc ion me hods equi e he alues o
in ensi ies o be posi i e, i.e., ha he in ensi ies mus be equal o
abo e ze o [36]. Because he so wa e o FEI econs uc s
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Fig. 1. Tomog aphic il se ies and minimum in ensi y. (a) Typical
Q2
s eps pe o med in elec on omog aphy. A s ep consis ing in he op imisa ion o he backg ound le el o a
il se ies is equi ed be o e econs uc ion. (b) Di e ence be ween he bi esolu ion o he de ec o and he ange o in ensi ies used while adjus ing I/C. (c and d) Model o
one sphe ical P pa icle suppo ed on a C film su ounded by acuum and he co esponding p ojec ed in ensi y images wi h and wi hou backg ound shi . (e) In ensi y
p ofiles measu ed along he a owed lines shown in (c) and (d). In he ideal si ua ion, he acuum le el coincides wi h he absolu e minimum alue o he eco ding sys em
(blue cu e wi h squa es). In p ac ise he images o a il se ies a e o en eco ded wi h a di e en minimum ( ed cu e wi h ci cles). The shi is equi alen o he p esence o
a laye o ma e ial ha does no exis . (Fo in e p e a ion o he e e ences o colou in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎2
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
omog aphic se ies co ec ly we assume he e ha he ange o
signed alues used in he FEI so wa e is only o s o age and i-
sualisa ion pu poses, and ha he econs uc ion algo i hms mus
escale in e nally he in ensi ies so ha hey a e unsigned o e-
cons uc ion pu poses.
The omog aphic HAADF STEM se ies shown in Fig. 3 was ob-
ained using a Tecnai F30 wi h a field-emission gun (FEGTEM)
ope a ed a 200 kV. Images in Fig. 4 we e acqui ed using an
abe a ion-co ec ed Ti an TEM (FEGTEM) ope a ed a 300 kV and
a came a leng h co esponding o inne de ec o semi-angles o
E90 m ad o enhance he con as be ween he co es and he
shells o he pa icles while minimising di ac ion. Images we e
acqui ed using a single- il omog aphy holde model 2020 ( om
Fischione Ins umen s). Alignmen and omog aphic econs uc-
ion o he il se ies we e pe o med using he SIRT algo i hm
wi h 15 i e a ions using Inspec 3D so wa e om FEI. The same
so wa e was used o in e he con as o he BF TEM se ies o
Fig. 5. Visualisa ion using ol ex and o hoslices, segmen a ion,
isosu ace ende ing, and Sobel fil e ing o he 3D da ase s was
pe o med using A izo so wa e. The omog aphic BF TEM se ies
shown in Figs. 5 and 6was acqui ed using he same Ti an mic o-
scope and a CCD came a. Image con as is defined as
CI I I I/
max min max min
=− +
Fo he op imisa ion o he backg ound o he il se ies and
algo i hm was implemen ed using Ma lab. An aligned il se ies o
images is loaded in o a ma ix. The ma ix con ains he in ensi ies
o e e y pixel o he omog aphic se ies. The in ensi ies ha o m
an image as measu ed by a de ec o sys em mus be ans o med
in o he digi al domain by analogue- o-digi al con e sion so ha
pixel in ensi ies ha a e eco ded in an image ake disc e e alues
wi hin a ange ha depends on he bi esolu ion o he de ec o
sys em.
3. In ensi y o a il se ies and backg ound op imisa ion
When an expe imen al omog aphic il se ies o images is
acqui ed du ing an expe imen , i is necessa y o fi s adjus he
minimum alue and he ange o alues o pixel in ensi ies ac-
qui ed by a de ec o by a ying he O se and Gain (O/G) pa a-
me e s (which co espond wi h he imaging pa ame e s in ensi y
and con as ). No mally, he adjus men o O/G is done by check-
ing ha he image is co ec ly eco ded a a fini e numbe o il s
(e.g. 0°,730°,770°). Ne e heless, because an use ul il se ies
equi es he acquisi ion o many mo e images ( ypically wi h a il
s ep o 2°), he O/G adjus men made o some images may no be
alid o he whole se ies o images because he e will be hickness
changes and a ia ions in di ac ion con as ha canno be
con olled a p io i. I he expe imen is pe o med such ha he
in ensi y le el is oo low o i he con as ange is oo wide, he
pixel in ensi ies o some images can all ou side he dynamic ange
o he de ec o , and pa s o he images will become o e sa u a ed
o will be unde sa u a ed a se e al il s.
The e o e i is a common p ac ise o adjus he O/G le el wi hin a
ange o alues ha is na owe han he ull a ailable bi esolu ion
o he de ec o (o o he image o ma used o s o ing he images)
(see Fig. 1b). The d awback o his s a egy is ha he backg ound
le el o e e y image o he expe imen al il se ies will be shi ed
by an indefini e cons an owa ds highe alues o in ensi y. This is
ce ainly he case o mos specimens when he e is no a ailable a
acuum egion o define a ze o e e ence le el o in ensi y. On he
o he hand, mos o en when pe o ming ET o a nanos uc u e, we
a e no in e es ed in i s su ounding ( he suppo ing film o a
con aining ma ix). Hence, we p opose he e o minimise he
backg ound le el o in ensi y o he images o a il se ies a he
expense o no econs uc ing he space su ounding de nanos-
uc u e. Such s ep elimina es he unce ain y o lacking o a e-
e ence le el o in ensi y while i imp o es he fideli y o he e-
cons uc ion o he nanos uc u e o in e es .
Fig. 1c and d shows a simula ed HAADF STEM image ( e-
p esen a i e o one image o a omog aphic il se ies) o a model
o a high-densi y sphe ical pa icle suspended on a lowe densi y
film and su ounded by acuum. In ensi ies can ake alues be-
ween 0 (black) and 1 (whi e). In Fig. 1c he minimum in ensi y
( ha o acuum) coincides wi h 0, he minimum alue a ailable
wi h a hypo he ical de ec o sys em. In Fig. 1d he minimum in-
ensi y ( ha o acuum) is shi ed o a alue 40. Fig. 1e shows he
in ensi y p ofiles along he cen e o he images in Fig. 1c and d.
The backg ound shi (see dashed o ange line) is equi alen o
adding new ma e ial ha is no eal (o ange colou in Fig. 1d) o
he model o he specimen. Bu i a il se ies o images such he
one in Fig. 1d is used o omog aphic econs uc ion, he o ange
laye will show up in he omog am as a eal ma e ial ha will
in oduce a e ac s. I is known ha i a ca bon film used as sup-
po o omog aphy is hick, hen i can in oduce a e ac s in a
econs uc ion such as he op–bo om e ec [37].
Howe e , he p oblem desc ibed in his pape is a wo se si-
ua ion because he o ange laye in Fig. 1d is p esen in e e y image
o he il se ies, as a hick laye ha is no limi ed in space and i will
show up in e e y oxel (3D pixel) o he omog am.Fig. 2a–c com-
pa es he omog aphic econs uc ion o an objec model (a
“phan om”) shown in Fig. 2a wi h a mo e ealis ic econs uc ion
wi h a MW o 40°and using p ojec ions o he “phan om”wi h
and wi hou backg ound shi (Fig. 2b and c espec i ely). Fig. 2d
shows h ee in ensi y p ofiles along he cen al lines o he
“phan oms”. I is clea ha in he case o using a il se ies wi hou
a shi o he backg ound le el (blue line, I
min
E0) he con as o
he econs uc ion inc eases, he in ensi ies quan i a i ely ma ch
he in ensi y ange o he o iginal “phan om”mo e accu a ely, and
he e a e ewe a e ac s ou side he phan om. In summa y, i an
inapp op ia e backg ound le el abo e ze o is chosen an a ificial
ex a hickness is added o he suppo o he sample in he ex-
pe imen al omog aphic il se ies.
4. Expe imen al esul s
4.1. HAADF STEM omog aphy: Imp o ing con as and educing
a e ac s
Fig. 3a is an expe imen al image acqui ed a 0° ep esen a i e
o a HAADF STEM il se ies o a sample o BiPO
4
s a -like pa icles
[38]. The il se ies was acqui ed wi h a minimum in ensi y alue
o 619. This alue is shi ed by a la ge amoun wi h espec o he
absolu e minimum alue allowed by he de ec o sys em, ha is,
32,768 o 16-bi esolu ion. The same il se ies was p ocessed
o fline and i s backg ound le el was downshi ed by sub ac ing a
cons an alue o þ32,016 om e e y image ( he di e ence be-
ween 32,678 and 619). Fig. 3b shows he cen al line p ofiles
o in ensi ies measu ed on he images a 0° il o he wo il
se ies wi h and wi hou backg ound op imisa ion. Tomog ams
we e econs uc ed om bo h il se ies and hei his og ams
calcula ed (Fig. 3c). Bo h his og ams display a maximum peak o
coun s a low in ensi ies. These peaks co espond wi h oxels (3D
pixels) ha su ound he pa icle. The his og am o he o iginal il
se ies (wi h an upwa ds backg ound shi a 619) con ains
coun s a in ensi ies o he le o he maximum peak. So oxels o
acuum a ound he pa icle in a e non-ze o in he omog am and
blu he pa icle in Fig. 3d. In con as , in Fig. 3e he con as o he
omog am is much highe , and blu ing a ound he pa icle
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L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎ 3
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
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Fig. 2. SIRT econs uc ion and backg ound in ensi y. (a) Model o “phan om” ha would be compu ed in an ideal omog aphic expe imen wi hou MW. The ull in ensi y
ange is [0,1]. The backg ound le el is ze o (I
min
¼0), and he con as o he omog am is C¼1. (b) Simula ion o a econs uc ed “phan om”wi h a MW o 40°and il s ep o
2°using SIRT wi h 50 i e a ions. I
min
o he il se ies shi ed o 0.3. The con as o he omog am is now C¼0.63. (c) Recons uc ed “phan om”unde he same condi ions
whe eby (c) and he I
min
o he il se ies a e se exac ly o ze o. The con as o he omog am is 1 and a e ac s ou side he phan om a e minimised. Below he omog ams,
he co esponding di ac og ams a e shown. (d) Line p ofiles o he in ensi y o he omog ams (b) and (c) compa ed o he ideal case (a).
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎4
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
disappea s. Mo eo e , s eaking a e ac s cha ac e is ic o he MW
ha e almos disappea ed, as shown in he o hoslices in Fig. 3d
and e. No e ha he o hoslices we e no manipula ed and hey a e
displayed wi h he ull dynamic ange [32,678, 32,677].
4.2. HAADF STEM omog aphy: imp o emen o segmen a ion
Blu ing and a e ac s in omog ams make hei p ocessing o
isualisa ion and accu a e segmen a ion di ficul , leading o a e-
qui emen o dedica ed human in e en ion ha is subjec i e and
ime-consuming [17,19,25,35,39].Fig. 4 shows an example o he
imp o emen ha can be ob ained in he p ocess o segmen ing a
omog am when he backg ound o he il se ies is op imised. The
sample consis o bime allic Au@Ag co e–shell nanopa icles, used
as ags in bio ecogni ion, ha we e deposi ed on a hin ca bon
suppo o hei in es iga ion using HAADF STEM [40]. When he
backg ound is op imised (wi h a alue close o 32,678), he
edges and su aces o he pa icles a e much be e defined (see
o hoslices in Fig. 4c). Fig. 4d shows he same o hoslices a e
applica ion o Sobel fil e ing, which is a common image-
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Fig. 3. Imp o ing con as and educ ion o a e ac s. a HAADF STEM image a 0° il ep esen a i e o a il se ies o images o a BiPO
4
s a -like pa icle acqui ed a 300 keV
wi h a ange o il s be ween 70°and þ70°(high il s ep o 1°abo e 60°and 2° il s ep below 60°). (b) The minimum in ensi y le el eco ded was a 619. A e
op imising he in ensi ies, i was se o 32,635. (c) His og ams o he econs uc ed omog ams be o e (in ed and ci cles) and a e backg ound op imisa ion (in blue and
squa es). The his og am o he unp ocessed se ies has in ensi y alues o he le o he maximum peak (a ea in o ange unde he ed cu e) ha blu he omog am. (d) and
(e) show he ol ex isualisa ion and h ee o hogonal cen al sec ions o he omog ams. I he backg ound o he il se ies is shi ed, he omog am will be su ounded by a
cloud o in ensi y ha does no co espond o eal ma e ial (“da k ma e ”) (Video S3 suppo ing in o). (Fo in e p e a ion o he e e ences o colou in his figu e legend, he
eade is e e ed o he web e sion o his a icle.)
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎ 5
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i

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Fig. 4. Imp o emen o segmen a ion o omog ams. (a) HAADF STEM image a 0° il ep esen a i e o a il se ies o 39 images o bime allic Au/Ag co e–shell nanopa icles
wi h a il ange om 60° o þ70°, a high il s ep o 2°, and a low il s ep o 4°in s eps wi h a il swi ch a 50°. (b) His og ams o he omog ams using he o iginal il
se ies ( ed) and he one wi h backg ound op imisa ion (blue). (c) XY o hoslice o he omog am ob ained using he unp ocessed il se ies (le ) o he one wi h backg ound
op imisa ion ( igh ). The con as is enhanced and blu ing disappea s. (d) The same as in (c) a e applica ion o a Sobel fil e . (e) Segmen a ion o he omog ams using he
anges o in ensi ies in b acke s shown in (a). (Video S2 suppo ing in o). (Fo in e p e a ion o he e e ences o colou in his figu e legend, he eade is e e ed o he web
e sion o his a icle.)
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎6
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
p ocessing ope a o used in edge de ec ion algo i hms ha em-
phasises ansi ions o in ensi y maps by calcula ing local g a-
dien s. I is e iden ha he e is an imp o emen in he quali y o
he omog am when he backg ound o he il se ies is op imised
(Fig. 4d, igh ). Using su ace ende ing, Fig. 4g and h compa es he
di e ences in quali y o he segmen a ion o he omog ams o
sepa a ing he Au co es om he Ag shells using he in ensi y
anges shown in b acke s in Fig. 4b. In he case o he omog am
using a il se ies wi h he op imised backg ound, he e is no gap
be ween he wo anges o in ensi ies used o he segmen a ion.
Compa ing Fig. 4e wi h he image a 0° il in Fig. 4a, backg ound
op imisa ion pe mi s a mo e ai h ul segmen a ion/ isualisa ion in
e ms o he sizes and shapes o he Ag pa icles and he Au co es.
This is a clea indica ion ha he econs uc ion imp o es, because
less blu ing also implies a be e sepa a ion o in ensi y anges
co esponding o dis inc ma e ials o phases p esen in he
specimen.
Supplemen a y ma e ial ela ed o his a icle can be ound
online a doi:10.1016/j.ul amic.2015.03.017.
4.3. BF TEM omog aphy: imp o emen o esolu ion.
BF TEM is no mally used o ET o amo phous and so
ma e ials, which is ypically he case o biological samples [21,41].
Fig. 5a shows a BF TEM image acqui ed a 0° il o a powde o
Vulcan XC-72R, a comme cial ca bon black ha find applica ions
in he e ogeneous ca alysis and ha lacks long- ange o de
[19,20,42]. Fo omog aphic econs uc ion, he in ensi ies o he
il se ies we e in e ed (I
x,y
-I
x,y
), as shown in Fig. 5b, because
i acili a es u he segmen a ion o he omog am al hough his
s ep is no necessa y in gene al. The minimum in ensi y alue o
he in e ed se ies is ound o be he alue 5060, which is
g ea e han he absolu e minimum o 32,678 o he image
o ma used in ou expe imen s. Hence, backg ound op imisa ion
o he BF TEM il se ies is a c i ical s ep in o de o econs uc
be e omog ams o compensa e o he esul ing o se o he
acuum le el. The co ec ion is applied by downshi ing he in-
ensi ies by an amoun o 27,618 ha is he di e ence be ween
32,678 and 5060. Fig. 5c shows a model o ca bon black made
o a o uous 3D agg ega ion o sphe ical nanoc ys alli es. Fig. 5d
and e a e he ol ex isualisa ion o he omog ams wi h and
wi hou backg ound op imisa ion o he il se ies. As in he case
o HAADF STEM omog aphy. Fig. 5 is he same as Fig. 5d bu wi h
he isualisa ion op imised in he ange [1678, 1000] o emo e
he e ec o he blu ing. In he case o he ol ex isualisa ion o
he omog am ob ained using he op imised il se ies, only was
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Fig. 5. Backg ound op imisa ion in BF TEM omog aphy. (a) Rep esen a i e image om a omog aphic il se ies o 71 images o a powde o ca bon black deposi ed on an
amo phous holey ca bon film and acqui ed in he angula ange be ween 70°and þ70°wi h a s ep o 2°. The in ensi y o he acuum in (a) is no sa u a ed (I
accum
¼
5060osa u a ion alue¼þ32,677). (b) In e ed e sion o (a) o omog aphic econs uc ion. (c) Model o ca bon black as a 3D agg ega ion o hollow sphe es wi h
concen ic laye s o ca bon. (d) Th ee-dimensional isualisa ion o he omog am o he sample. ( ) In o de o emo e he cloud o in ensi y in he acuum ha obscu es he
sample, i was necessa y o adjus he in ensi y ange o [1678, 1000]. (e) Th ee-dimensional isualisa ion o he omog am o he sample ob ained using he il se ies wi h
he backg ound op imised. (Video S3 suppo ing in o).
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎ 7
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i
necessa y o se he uppe ange o 0 o display pu poses. Com-
pa ing Fig. 5e and i is clea ha when he il se ies is op imised
he ca bon film and he ca bon black a e be e sepa a ed and
he e is less blu ing in he z-di ec ion.
The sphe ical c ys alli es in mos comme cial ca bon blacks
ha e an a e age o app oxima ely ou g aphi e concen ic laye s
wi h a hollow cen e [42].Fig. 6a and b compa es YZ o hoslices o
he omog ams o he ca bon black specimen using he un-
p ocessed il se ies wi h he backg ound op imised. In his case,
a e ac s a e absen o a g ea ex en , and de ails a e clea ly en-
hanced (spa ial esolu ion imp o es). This is mos e iden in
Fig. 6e, in which he c ys alli es show a b igh im wi h a da k
cen e, as one would expec o he hollow sphe e c ys alli es o
his ca bon black.
5. Conclusion
In gene al, an absolu e e e ence le el o in ensi ies is no
a ailable in ET unless an a ea o he sample in acuum is exposed.
I he minimum le el o in ensi y o a il se ies ha co espond
wi h a eal phase o he ma e ial is no adjus ed so ha i coincides
wi h he absolu e minimum alue o he dynamic ange o he
elec on de ec o sys em (o o he image o ma used), he
omog am will display ypically in ensi ies in places which should
co espond o acuum. These in ensi ies will blu he ull olume
and will enhance he a e ac s in oduced by he MW. In such
cases, he fideli y o a omog am ob ained using i e a i e e-
cons uc ion me hods can be g ea ly imp o ed (wi h mo e con-
as , ewe a e ac s, and be e accu acy) easily i he backg ound
le el o he il se ies is downshi ed be o e i s econs uc ion.
Mo eo e , his s ep acili a es u he segmen a ion and isuali-
sa ion o he h ee-dimensional omog am. We belie e ha his
ype o p ep ocessing should always be pe o med in o de o
ob ain op imal esul s i espec i e o he imaging echnique used.
I is also complemen a y wi h he use o o he me hodologies o
minimising he MW e ec s. And we en isage ha his simple
me hodology can be also an e ec i e way o h esholding an
embedding ma e ial o a low-densi y suppo ing hin film ha a e
no pa o he sample unde examina ion bu ha a e ypically
used o sample p epa a ion o biological samples o as suppo s o
nanos uc u es espec i ely.
Suppo ing in o ma ion

Th ee ideos S1–S3 showing he isualisa ion o he omo-
g ams be o e and a e backg ound p ocessing.
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Fig. 6. Imp o emen o esolu ion in backg ound-op imised BF-TEM omog aphy. (a) ZY o hoslice o he omog am ob ained using he unp ocessed il se ies shown in Fig. 4.
(b) The same o hoslice om he omog am calcula ed om he backg ound-op imised il se ies. (c) His og ams o he wo omog ams. (d) and (e) a e de ails o he fideli y
o he econs uc ed nanoc ys alli es. When he backg ound is op imised, blu ing la gely disappea s in ZY o hoslices and he hollow cen es o he ca bon sphe es a e
clea ly ecognised. ( ) In ensi y p ofiles measu ed along he line p ofiles indica ed in (d) and (e) wi h a owed lines. When he backg ound is op imised, he o hoslices o he
omog am co ec ly show he b igh ing s uc u e expec ed om a hollow sphe e.
L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎8
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i

A p og amme o so wa e w i en in Ma lab o pe o ming
backg ound op imisa ion o .m c files o omog aphic e-
cons uc ion using FEI so wa e is a ailable a www.lcgon a d.
es. The file called BKG_Op imisa ion_Tomog aphy_LCG_2015.exe
is an execu able and he e o e i equi es he p eins alla ion o
Ma lab MCR abo e e sion 2012b.
Acknowledgemen s
The au ho wishes o hank di e en collabo a o s ha p o-
ided he samples o suppo , P o . Ra al E Dunin-Bo kowski, D .
Ana Bece o, D . Dogan Ozkaya and D . Cuong Cao. The au ho is
also g a e ul o P o . Asunción Fe nández as leade o he Eu opean
P ojec REGPOT-CTAl-NANOFUNC and o he Eu opean Union
Q3
un-
de a Con ac o an In eg a ed In as uc u e Ini ia i e 312483-
ESTEEM2 o unding.
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L.C. Gon a d / Ul amic oscopy ∎(∎∎∎∎)∎∎∎–∎∎∎ 9
Please ci e his a icle as: L.C. Gon a d, Remo ing he e ec s o he “da k ma e ”in omog aphy, Ul amic oscopy (2015), h p://dx.doi.
o g/10.1016/j.ul amic.2015.03.017i