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An in e ac i e and in ui i e
isualisa ion me hod o X- ay
compu ed omog aphy da a o
biological samples in 3D Po able
Documen Fo ma
Ma ké a Tesařo á
1, eglan ine Heude
2,3,4, Glenda comai
3,4, Tomáš Zikmund
1,
Ma ké a Kaucká
5,6, igo Adameyko5,6, Shah agim ajbakhsh
3,4 & Joze Kaise
1*
3D imaging app oaches based on X- ay mic ocompu ed omog aphy (mic oCT) ha e become
inc easingly accessible wi h ad ancemen s in me hods, ins umen s and expe ise. he syne gy o
ma e ial and li e sciences has impac ed biomedical esea ch by p oposing new ools o in es iga ion.
Howe e , da a sha ing emains challenging as mic oCT iles a e usually in he ange o gigaby es and
equi e speci ic and expensi e so wa e o ende ing and in e p e a ion. He e, we p o ide an ad anced
me hod o isualisa ion and in e p e a ion o mic oCT da a wi h small ile o ma s, eadable on all
ope a ing sys ems, using eely a ailable Po able Documen Fo ma (PDF) so wa e. Ou me hod is
based on he con e sion o olume ic da a in o in e ac i e 3D PDF, allowing o a ion, mo emen ,
magni ica ion and se ing modi ica ions o objec s, hus p o iding an in ui i e app oach o analyse
s uc u es in a 3D con ex . We desc ibe he comple e pipeline om da a acquisi ion, da a p ocessing
and comp ession, o 3D PDF o ma ing on an example o c anio acial ana omical mo phology in
he mouse emb yo. ou p ocedu e is widely applicable in biological esea ch and can be used as
a amewo k o analyse olume ic da a om any esea ch ield elying on 3D ende ing and CT-
biomedical imaging.
One o he o midable phenomena in de elopmen al biology is how he shape di e si y obse ed among li ing
o ganisms is de ined and con olled du ing de elopmen and g ow h. Emb yonic pa e ning is a highly dynamic
p ocess implica ing mul iple molecula mechanisms and cell in e ac ions a he basis o o gan o ma ion. In he
human emb yo, de ec s in such cellula p ocesses can a ec he de elopmen al p og am leading o congeni al
diso de s. Congeni al de ec s ha e an incidence o 3% in he human popula ion1 and hey a e causal o up o
one-qua e o all epo ed neona al dea hs2. Thus, con ex ual isualisa ion o emb yonic de elopmen is c i ical
o elucida e he o igins o mal o ma ions.
Mul i-disciplina y collec i es composed o clinical doc o s, biologis s, enginee s and imaging expe s a e
cu en ly pushing o wa d he unde s anding o biological ques ions using h ee dimensional (3D) app oaches.
While he analysis o his ological sec ions emains a mains ay in de elopmen al biology, he econs uc ion o
3D olumes om 2D slices has p o ided impo an in o ma ion o unde s and mo phogenesis in mouse and
human emb yos3–6. Non-des uc i e echnologies such as 3D imaging by con ocal mic oscopy and ligh shee
me hods7–9, op ical p ojec ion omog aphy (OPT)10–12 and mic o-compu ed omog aphy (mic oCT)13–16 cons i-
u e eme ging powe ul me hods o analyse he opog aphy o de eloping s uc u es in 3D con ex , o gain insigh
in o he pa hogenesis o congeni al diso de s. Howe e , one majo challenge o 3D app oaches is sha ing o com-
plex da ase s e ec i ely and in ui i ely be ween colleagues om di e en ields o discussion, and ul ima ely
1Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech Republic. 2Depa men
Adap a ion du Vi an , Museum na ional d’His oi e na u elle, CNRS UMR 7221, Pa is, F ance. 3Depa men o
De elopmen al and S em Cell Biology, S em Cells and De elopmen Uni , Ins i u Pas eu , Pa is, F ance. 4CNRS UMR,
3738, Pa is, F ance. 5Depa men o Physiology and Pha macology, Ka olinska Ins i u e , Solna, Sweden. 6Depa men
o Molecula Neu osciences, Medical Uni e si y o Vienna, Vienna, Aus ia. *email: kaise @ me. u b .cz
open
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p esen ing hem in a publica ion o ma . Gene ally, 3D da ase s need special so wa e o isualisa ion o a e
educed o 2D pic u es in which impo an in o ma ion migh be los .
The s udy o he de eloping head cons i u es a good example o he need o 3D app oaches gi en i s com-
plex ana omy. The c anio acial egion is buil o di e se emb yonic cell ypes gi ing ise o ha d and so issues
including skele al, muscula and ne ous componen s17. The inal shape o he ace s ongly depends on he
geome y o heskele al elemen s and hei in e ac ion wi h adjacen so issues such as muscles and he ne ous
sys em15,16. Nume ous congeni al c anio acial abno mali ies a ec he o m and unc ion o he ace and explana-
ions o  hese mal o ma ions s ill awai he undamen al unde s anding o he unde lying ailu e o mo phogene-
sis18. The mic oCT app oach ollowed by 3D econs uc ion has enabled high-quali y in o ma ion o he complex
mo phological aspec s o head and ace de elopmen 15,16.
To acili a e assimila ion and isualisa ion o mic oCT da ase s, in e ac i e 3D Po able Documen Fo ma
(PDF) has been used in di e en ield including in de elopmen al biology6,19,20, in human physiology and ana -
omy4,21–23, in en omology24 and ma ine biology25,26. Howe e , a majo limi a ion o p e iously published me hods
is he equi emen o ad anced p og amming skills and/o ins alla ion o u he p epaid so wa e packages21.
Mos o hese app oaches depend on he use o Adobe Ac oba P o Ex ended so wa e4,6,19,23–28 o Ja aSc ip
p og amming language20,27.
He e, we p o ide an al e na i e, use - iendly way o c ea e in e ac i e 3D PDF iles om mic oCT da ase s
aking he complexi y o mouse c anio acial ana omy as a model example. Ou inno a i e and e icien pipeline
comp ises mic oCT da a acquisi ion, segmen a ion and inal es ablishmen o a 3D PDF using a combina ion o
ee and p e-paid so wa e. The esul ing ile can be iewed wi h s anda d PDF iewe s and o e s an in e ac i e
in e ace o mic oCT da a sha ing, analysis and p esen a ion.
Pipeline o he C ea ion o In e ac i e 3D PDF
To c ea e an in e ac i e 3D PDF om mic oCT da a, he c i ical s eps a e: (i) chemical con as ing o biological
samples i so issues a e o be isualized, (ii) da a acquisi ion and (iii) CT i ual econs uc ion (Fig.1). This is
ollowed by da a p ocessing, he mos c i ical s ep consis ing o segmen a ion, su ace ex ac ion, adjus men o
segmen ed 3D models, and con e sion in o a inal in e ac i e PDF ile. He e, we p o ide a de ailed desc ip ion o
each s ep o he pipeline (see also Supplemen a y Ma e ial1).
Figu e 1. O e iew o he me hod pipeline desc ibed in his s udy.
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X-Ray Mic o omog aphy Measu emen
Mic oCT is an es ablished echnology o imaging mine alized issues in animal specimens. Howe e , i s use in
compa a i e mo phology has been limi ed by he low in insic X- ay con as o non-mine alised so issues. To
o e come his p oblem, me hods ha e been de eloped o inc ease issue con as , including chemical ea men
wi h con as ing agen s29,30, phase-con as imaging31–36 o dual-ene gy compu ed omog aphy (DECT)37.
Chemical con as ing ea men s ha e mos ly been applied o he cha ac e iza ion o musculoskele al issues
on small ixed samples. Fo in i o CT imaging including clinical diagnoses, non- oxic iodine-based con as
agen s (e.g. iohexol o hexab ix) a e used o he analysis o he ca dio ascula sys em and ca i ies, bu no o
di ec analysis o muscle issues38,39. Clinical CT imaging o he musculoskele al sys em is commonly pe o med
wi hou a con as ing agen , he eby limi ing he analysis o disc e e so o gans40. The e o e, on small ixed sam-
ples, mul iple con as ing p o ocols ha e been used, each wi h i s own ad an ages and limi a ions41,42. The s ain-
ings based on iodine, osmium o he oxic phospho ungs ic acid (PTA) a e he mos commonly used29,30. Fo he
pipeline p oposed he e on he s udy o c anio acial mouse de elopmen , we p opose he use o PTA con as ing
ea men ha pe mi s high con as imaging o a wide a ie y o so and mine alised issues composing he
mouse head (Fig.2). Howe e , mic oCT da a ob ained wi h o he con as ing agen s o in hei absence, such
as clinical da a o da a om non-biological specimens, will be equally ameneable o he subsequen s eps o he
3DPDF pipeline.
Da a P ocessing
Following he acquisi ion o high-con as omog aphic slices, he segmen a ion o complex c anio acial s uc-
u es cons i u es a majo challenge. Fully-au oma ic app oaches ha e been ied and we e gene ally unsuccess ul43.
Thus, he manual app oach is usually he only me hod a ailable o achie e p ecise and accu a e segmen a ions44.
Howe e , some semi-au oma ic me hods such as local segmen a ion o in e pola ion be ween manually seg-
men ed slices can be used45,46. Fo e i ica ion o he accu acy o manually segmen ed s uc u es, Fig.2 shows
bo h o iginal and segmen ed omog aphic slices. This app oach pe mi ed he segmen a ion o so issues such
as eyeballs, ex aocula muscles and he cen al ne ous sys em, as well as o ha d issues including he chond o-
c anium, bones and u u e ee h (den al placodes) (Fig.3).
Fo 3D olume ende ing, i is hen mo e con enien o use 3D mesh o ma s a he han a s ack o 2D images.
3D mesh o ma s ep esen a se ies o 2D polygons ( ypically iangles o quad angles) linked oge he o ec e-
a e he su ace o a 3D objec 28. I encodes he 3D model’s geome y, colou s, ex u es, e c. Fo u he wo k, he
segmen ed masks need o be ans e ed o he meshes. Mos so wa e can ans e he mask in o a wide ange o
Figu e 2. Tomog aphic measu emen s and segmen a ion o c anio acial s uc u es in a mouse emb yo 15 days
pos - e ilisa ion. Colou planes on 3D models (le panels) indica e he posi ion o he aw omog aphic slices
(middle panels) and some segmen ed s uc u es ( igh panels) including he cen al ne ous sys em (pu ple),
he lens (da k blue), he den al placodes (ligh blue) and ex aocula muscles ( ed).
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equi alen o ma s. Howe e , only some o he o ma s con ain in o ma ion abou he colou (e.g. OBJ, VRML
and STEP) which is impo an o he isualisa ion o se e al s uc u es in one model. O he o ma s ep esen
only he mesh (e.g. STL and Ma lab m- ile).
Ano he p oblem o be sol ed is he size o he model (i.e. he numbe o aces in he mesh). When ans e -
ing he segmen ed mask in o he mesh, an unnecessa ily la ge numbe o aces can be c ea ed. Compa ison o a
di e en numbe o aces and co esponding size can be ound in Fig.4. Su p isingly, educing by ou imes he
numbe o aces does no signi ican ly comp omise de ails in he 3D models. Simple shapes such as he eyeball do
no show no able e o s besides he sligh de o ma ion o he sphe e. Howe e , u he simpli ica ion o complex
shapes, such as he chond oc anium and ex aocula muscles (EOM), educes he model quali y and some de ails
a e los (Fig.4, ed a ows). Thus, depending on he s uc u es analysed and he esolu ion needed, di e en ace
educ ions should be es ed and alida ed be o e u he analysis. The e o e, he comp ession o he da a should
be cus omised acco ding o he complexi y o he model and he esolu ion o omog aphic slices.
Among he 3D p in ing so wa e, some ee so wa e enable colou modi ica ions o mesh simpli ica ion.
He e, we made use o he Meshlab47 and Blende 48 packages o his pu pose. No ably, he la e allows uni ying
di e en sub-models in o one mesh p ese ing he indi iduali y o each objec (see Supplemen a y Ma e ial1 o
mo e de ails).
C ea ion o an In e ac i e 3D PDF
Once he models a e colou -labelled and simpli ied, hey a e hen con e ed in o an in e ac i e ile. We exploi ed
3D PDF Make S andalone49 aking ad an age o he p e-p epa ed model ha p o ides an in e ac i e window
in he PDF ile by clicking on Add 3D bu on. Be o e embedding he model in o an in e ac i e ile, he use can
p epa e an o dina y PDF ile using s anda d so wa e (e.g. Mic oso O ice, Apache OpenO ice e c.). This ile
is used as a empla e showing, o example, indi idual s uc u es o p ede ined iews. These a eas (i.e. images o
signs) a e aken in o li e in 3D PDF Make by assigning wha e e a ea in he page o speci ic iew/s uc u e (see
o mo e de ails Supplemen a y Ma e ials1 and 2).
The model documen p esen ed he e (Supplemen a y Ma e ial3) consis s o ou p ede ined iews ( en al,
do sal, la e al and medial) and a possibili y o showing indi idual c anio acial s uc u es (b ain, ca ilage, bone
and ee h). I helps o manipula e he 3D model and can be se acco ding o he a eas/ iews o in e es ha a e
impo an o show. Fo example, o be e isualisa ion, some s uc u es can be se o he semi- anspa en mode,
o s uc u es can be swi ched on/o indi idually in he model ee. The in e ac i e 3D PDF also allows he use o
o a e, u n and pan he model, change he p e-de ined 3D- ende ing, change he ligh s and backg ound colou
o add a i ual c oss-sec ion on he model.
Figu e 3. Su ace ende ing o segmen ed s uc u es in a mouse emb yo 15 days pos - e ilisa ion. S uc u es o
in e es a e colou -coded.
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Discussion and Conclusion
Compa a i e mo phological s udies in he ield o de elopmen al biology ha e been challenging, bu
con as -enhanced X- ay compu ed omog aphy has b ough new possibili ies o high- esolu ion 3D isualis-
a ion15,16,50. In his s udy, we p esen a de ailed, use - iendly p o ocol o he su ace ende ing o c anio acial
s uc u es including so issues (muscles, eyeballs, cen al ne ous sys em) as well as o ha d issues (ca ilages,
bones and ee h), wi h a s ep-by-s ep p ocedu e om sample collec ion o he c ea ion o an in e ac i e 3D PDF.
The inal PDF is eadable on all ope a ing sys ems wi h he ee s anda d AdobeReade ®/Ac oba Reade DC
(Adobe Inc., Cali o nia USA).
Ou 3D econs uc ion me hodology has al eady shown i s u ili y and s eng h o isualisa ion and pheno-
ypic analysis o complex s uc u es such as he neck musculoskele al sys em50 and nasal capsules o con ol and
mu an mouse emb yos16. The e o e, a use - iendly me hod o c ea ing such iles will be o g ea u ili y o biol-
ogis s. In addi ion, ou p ocedu e can be used as a amewo k o analyse olume ic da a o any ield o esea ch
ha elies on 3D ende ing, e.g. o isualisa ion o olume ic in o ma ion o geological samples by lase -induced
b eakdown spec oscopy51.
The use o in e ac i e 3D PDF iles has a g ea po en ial o da a sha ing, communica ion and publica ions21,
bu is s ill spa sely used. We belie e ha ou wo k will inspi e esea che s wo king wi h 3D imaging o p esen
hei da a in such a use - iendly o ma .
Me hods
Use o expe imen al animals. All animal wo k was app o ed and pe mi ed by he Local E hical
Commi ee on Animal Expe imen s (No h S ockholm Animal E hics Commi ee) and conduc ed acco ding o
The Swedish Animal Agency’s P o isions and Guidelines o Animal Expe imen a ion ecommenda ions. Mice
we e sac i iced wi h iso lu ane (Bax e KDG9623) o e dose o ce ical disloca ion, and emb yos we e collec ed
in o ice-cold PBS. Subsequen ly, issue samples we e ixed in o eshly p epa ed 4% pa a o maldehyde (PFA) in
PBS solu ion o 24 hou s a +4 °C wi h slow o a ion and washed in PBS.
Figu e 4. The numbe o aces a ec s he de ec ion o de ails in chond oc anium, ex aocula muscles (EOM)
and eyeball models. Red a ows indica e de ails in he 3D model ha disappea wi h model simpli ica ion.

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issue con as ing. S aining p o ocol has been adap ed and modi ied om he o iginal p o ocol de eloped
by B ian Me sche labo a o y33,34. A e ixa ion, he samples we e dehyd a ed in inc easing concen a ion o e h-
anol se ies (30%, 50%, 70%, 90%), one day in each concen a ion o minimise he sh inkage o issues. Fo issue
con as ing, samples we e hen ans e ed in o 1% PTA (phospho ungs ic acid) in 90% me hanol o h ee weeks.
The PTA-me hanol solu ion was changed e e y wo days. Subsequen ly, he samples we e ehyd a ed by e hanol
se ies (90%, 70%, 50% and 30%) and shipped o he CT-labo a o y o scanning.
X- ay mic oc measu emen s. A e ixa ion and con as ing ea men s, samples we e ully ehyd a ed
in dis illed wa e , embedded in 1.0% aga ose gel and placed in a polyp opylene ube o a oid mo emen a e ac s
du ing omog aphy scanning. The polyp opylene ube was ixed on a plas ic od by a silicone gun. The od was
pu in he cen e o he o a ion s age axis. The mic oCT scanning was pe o med using he labo a o y sys-
em GE Phoenix | ome|x L 240 (GE Sensing & Inspec ion Technologies GmbH, Ge many) wi h a 180 kV/15 W
maximum powe nano ocus X- ay ube and la panel dynamic 41|100 wi h 4000 × 4000 px and a pixel size o
100 × 100 μm. The exposu e ime was 600 ms in each o he 2200 p ojec ions acqui ed o e a o al angle o 360°.
Th ee p ojec ions we e acqui ed and a e aged in e e y posi ion o educe he noise in he omog aphic da a.
Thus, he scanning ime was 73 minu es. The accele a ion ol age and cu en o he X- ay ube we e 60 kV and
200 μA, espec i ely. The beam was il e ed by a 0.2 mm- hick aluminium il e . The linea oxel esolu ion o he
measu emen was se o 4.2 μm in all dimensions. The omog aphic econs uc ion was conduc ed using he so -
wa e GE phoenix da os|x 2.0 (GE Sensing & Inspec ion Technologies GmbH, Ge many).
Da a p ocessing and analysis. Segmen a ion o c anio acial s uc u es was pe o med semi-manually
in A izo (The mo Fishe Scien i ic, USA). We used some au oma ic ools using a egion g owing me hod and
h esholding on 2D slices. Segmen a ions we e done on one o h ee o i e slices, and he mask was in e pola ed.
Subsequen ly, he segmen ed models we e smoo hed in VG S udio MAX 3.2 (Volume G aphics GmbH, Ge many)
acco ding o45. The segmen ed mask was hen expo ed in o a mesh (*.OBJ o ma ) and was colou -labelled and
adjus ed in Meshlab47 and Blende so wa e48. The colou -coded models we e ans e ed in o a p e-p epa ed PDF
ile in 3D PDF Make S andalone49. The de ailed manual can be ound in Supplemen a y Ma e ial1. The use o he
in e ac i e 3D PDF is a ailable in Supplemen a y Ma e ial2.
Da a a ailabili y
The da ase s gene a ed du ing and/o analysed du ing he cu en s udy a e a ailable om he co esponding
au ho on easonable eques .
Recei ed: 20 May 2019; Accep ed: 25 Sep embe 2019;
Published: xx xx xxxx
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Acknowledgemen s
This esea ch was ca ied ou unde he p ojec CEITEC 2020 (LQ1601) wi h inancial suppo om he Minis y
o Educa ion, You h and Spo s o he Czech Republic unde he Na ional Sus ainabili y P og amme II, CEITEC
Nano Resea ch In as uc u e (MEYS CR, 2016–2019) and) and Cei ec Nano+ p ojec , CZ.02.01/0.0./.0.0./16
_013/0001728 unde he p og am OP RDE. We acknowledge suppo by g an AFM Tele hon 21853. J.K. and
M.T. acknowledge he suppo o he B no Uni e si y o Technology h ough g an FSI-S-17-4506. M.T. was
inancially suppo ed by g an CEITEC VUT-J-19-5764 and by he B no Ci y Municipali y as a B no Ph.D. Talen
Schola ship Holde .
Au ho con ibu ions
M.T., E.H. and G.C. wo ked on he da a analysis and me hodology. M.K. p epa ed he samples and s ained hem
o CT measu emen . M.T. and T.Z. pe o med CT measu emen s. I.A., S.T. and J.K. designed he expe imen s.
M.T., T.Z. and J.K. w o e he manusc ip . All au ho s e iewed he manusc ip .
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