pls-07-01965 Decembe 27, 2016 Time: 12:11 # 1
PROTOCOLS
published: 04 Janua y 2017
doi: 10.3389/ pls.2016.01965
Edi ed by:
Roge Deal,
Emo y Uni e si y, USA
Re iewed by:
Michael J. Scanlon,
Co nell Uni e si y, USA
Pascal Gamas,
Cen e Na ional de la Reche che
Scien i ique, F ance
*Co espondence:
Ku Fage s ed
[email p o ec ed]
To e Nii ylä
[email p o ec ed]
†P esen add ess:
Anna Kä könen,
Na u al Resou ces Ins i u e Finland
(Luke), G een Technology,
La oka anonkaa i 7, Helsinki, Finland
‡These au ho s ha e con ibu ed
equally o his wo k.
Special y sec ion:
This a icle was submi ed o
Technical Ad ances in Plan Science,
a sec ion o he jou nal
F on ie s in Plan Science
Recei ed: 24 Oc obe 2016
Accep ed: 12 Decembe 2016
Published: 04 Janua y 2017
Ci a ion:
Blokhina O, Vale io C,
Sokołowska K, Zhao L, Kä könen A,
Nii ylä T and Fage s ed K (2017)
Lase Cap u e Mic odissec ion
P o ocol o Xylem Tissues o Woody
Plan s. F on . Plan Sci. 7:1965.
doi: 10.3389/ pls.2016.01965
Lase Cap u e Mic odissec ion
P o ocol o Xylem Tissues o Woody
Plan s
Olga Blokhina1‡, Conce a Vale io2,3‡, Ka a zyna Sokołowska3,4, Lei Zhao1,
Anna Kä könen5†, To e Nii ylä3*and Ku Fage s ed 1*
1Viikki Plan Science Cen e, Depa men o Biosciences, Uni e si y o Helsinki, Helsinki, Finland, 2Plan S ess Signaling,
Ins i u o Gulbenkian de Ciência, Oei as, Po ugal, 3Umeå Plan Science Cen e, Depa men o Fo es Gene ics and Plan
Physiology, Swedish Uni e si y o Ag icul u al Sciences, Umeå, Sweden, 4Depa men o Plan De elopmen al Biology,
Ins i u e o Expe imen al Biology, Uni e si y o W ocław, W ocław, Poland, 5Viikki Plan Science Cen e, Depa men o
Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland
Lase cap u e mic odissec ion (LCM) enables p ecise dissec ion and collec ion o
indi idual cell ypes om complex issues. When applied o plan cells, and especially
o woody issues, LCM equi es ex ensi e op imiza ion o o e come such ac o s as
igid cell walls, la ge cen al acuoles, in e cellula spaces, and echnical issues wi h
hickness and la ness o he sec ions. He e we p esen an op imized p o ocol o he
lase -assis ed mic odissec ion o de eloping xylem om ma u e ees: a gymnospe m
(No way sp uce, Picea abies) and an angiospe m (aspen, Populus emula) ee. Di e en
cell ypes o sp uce and aspen wood (i.e., ay cells, achea y elemen s, and ibe s)
we e success ully mic odissec ed om angen ial, c oss and adial c yosec ions o he
cu en yea ’s g ow h ing. Two app oaches we e applied o achie e sa is ac o y la ness
and ana omical in eg i y o he sp uce and aspen specimens. The commonly used
memb ane slides we e ine ec i e as a moun ing su ace o he wood c yosec ions.
Ins ead, in he p esen p o ocol we use glass slides, and in oduce a glass slide
sandwich assembly o he p epa a ion o aspen sec ions. To asce ain ha no only
he ana omical in eg i y o he plan issue, bu also he molecula ea u es we e no
comp omised du ing he whole LCM p ocedu e, good quali y o al RNA could be
ex ac ed om he mic odissec ed cells. This showed he e iciency o he p o ocol
and es ablished ha ou me hodology can be in eg a ed in ansc ip ome analyses o
elucida e cell-speci ic molecula e en s egula ing wood o ma ion in ees.
Keywo ds: c yosec ion, lase cap u e mic odissec ion, ay cells, RNA in eg i y, acheids, xylem ibe s
INTRODUCTION
Lase cap u e mic odissec ion (LCM) was o iginally de eloped o assis semi-au oma ic collec ion
o indi idual cells and issues om complex biological specimens o subsequen genomic and
ansc ip omic analyses (Ho ne e e al., 2007). The me hod allows p ecise analysis o a ge
cells, and is con ac - and con amina ion- ee (Gau am and Sa ka , 2015). LCM is a mic oscope-
based echnique and in ol es se e al c ucial s eps: sample p epa a ion (sample embedding and
sec ioning, o c yosec ioning), isualiza ion o in ac cells o issues o be isola ed, ou lining and
lase -cu ing o he selec ed a eas and, inally, collec ion o dissec ed cells ei he by g a i y in o
a ube, o by ca apul ing he a ge cells agains g a i y in o an adhesi e cap o a collec ion ube
(Ho ne e e al., 2007).
F on ie s in Plan Science | www. on ie sin.o g 1Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 2
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
De elopmen o new echniques o high- h oughpu da a
acquisi ion, concu en de elopmen o bioin o ma ic analyses
and inc easing a ailabili y o sequenced genomes ha e made
LCM a me hod o choice o elucida ing he cell ype-
speci ic molecula ea u es. Downs eam applica ions can co e
i ually e e y aspec o cell biology. Abo e all, gene exp ession
analyses a e, nowadays, o en pe o med h ough genome-wide
exp ession p o iling based on mic oa ay o nex gene a ion
sequencing (NGS) echnologies (whole genome sequencing,
a ge ed genome sequencing, RNA sequencing and ibosome
p o iling). The combina ion o LCM wi h NGS echniques is a
powe ul app oach o s udy cell- and issue-speci ic p ocesses
(Abbo e al., 2010;Jyske e al., 2015).
Mic odissec ion o plan issues and indi idual cells poses
a challenge due o he p esence o a cell wall, a la ge cen al
acuole in mos ully di e en ia ed cells and, in some issues, la ge
in e cellula spaces (Nakazono e al., 2003). The LCM echnique,
which was o iginally de eloped o animal issues, has equi ed
subs an ial op imiza ion o plan s udies. Asano e al. (2002)
we e he i s o apply LCM on plan issues in a s udy o phloem-
speci ic gene exp ession in ice (O yza sa i a). Ex ensi e wo k
was la e pe o med o es ablish p o ocols on plan issues, mainly
on he baceous species and by using pa a in-embedded ma e ial
(Ke k e al., 2003;Inada and Wilde mu h, 2005;Rajhi e al.,
2011;Shiono e al., 2014). Fo example, p e- ixed pa a in sec ions
o maize (Zea mays) oo s we e analyzed o di e en ial gene
exp ession be ween epide mal cells and ascula issues using
a mic oa ay (Nakazono e al., 2003). The same expe imen al
app oach has been applied in s udies on ae enchyma o ma ion
in he co ex o maize oo s (pa a in sec ions on memb ane
slides) (Rajhi e al., 2011), and on sube iniza ion o oo s du ing
o ma ion o a ba ie o adial oxygen loss in ice (Shiono e al.,
2014). The indings o he Nakazono g oup on LCM o pa a in-
embedded issues ha e been summa ized in a me hodological
a icle by Takahashi e al. (2010).
Non-pa a in embedding p ocedu es ha e been in oduced
ecen ly o LCM o plan issues. An al e na i e s a egy o sample
p epa a ion which gua an eed RNA quali y and mo phological
in eg i y, eplaces pa a in wi h S eedman’s wax. This polyes e
wax has a lowe mel ing poin han pa a in (38−40◦C s
60−65◦C), and can be washed ou by e hanol, an o e all milde
sample ea men han he pa a in emo al by xylene. The
S eedman’s wax-embedding has been pe o med, o example,
in s udies on a buscula myco hiza and oo co ical cells
o ba el medic (Medicago unca ula) (Gomez e al., 2009;
Hogekamp e al., 2011). Good esul s in e ms o RNA quali y
and RNA sequencing ha e been obse ed also a e incuba ion
o plan ma e ial in polye hylene glycol (PEG)-e hanol mix u e
and subsequen inclusion in PEG-modi ied S eedman’s wax and
polye hylene naph ala e (PEN)-coa ed slides o LCM (Roux
e al., 2014). This p o ocol was de eloped u he and 100%
PEG was used as an embedding medium wi h sec ions moun ed
on PEN slides (Ja dinaud e al., 2016). Much hicke longi udal
oo sec ions (35 µm) o ba el medic ha e been p epa ed o
LCM using embedding in issue- eezing medium and collec ed
myco hizal ma e ial was used o gene exp ession p o iling
(Gaude e al., 2012).
LCM has also been used o s udy issue- and cell-speci ic
p ocesses o non-xylem issues in woody plan s. Abbo and
co-wo ke s success ully isola ed esin duc s and cambial issues
om angen ial c yosec ions o 2-yea -old whi e sp uce (Picea
glauca) plan le s. This allowed he cha ac e iza ion o RNA,
de e mina ion o enzyme ac i i y and e penoid me aboli es
(Abbo e al., 2010). S e eo mic oscope-assis ed dissec ion o
c yosec ions ollowed by mic ogenomics has been used in a
unc ional s udy o neighbo ing me is ema ic cells in he ascula
cambium o Populus species (Goué e al., 2008, 2012). Jyske and
co-wo ke s (Jyske e al., 2015) ha e applied he LCM echnology
o assess he seasonal a ia ion in he phloem s uc u e and i s
cons i uen s in ma u e No way sp uce (Picea abies) ees. LCM
has been used also o in es iga e gene exp ession in phloem o
No way sp uce (Nagy e al., 2014). In all hese s udies, he so
phloem, cambial o co ex issues appea ed o be sui able o
p epa a ion o samples h ough ei he c yosec ioning o pa a in-
embedding o LCM.
Xylem issue wi h i s highly ligni ied cell walls has p o en
challenging o LCM applica ions. La isch e al. (2012) used LCM
o sepa a e de eloping and ma u e wood om wigs o popla
(Populus ×canescens), and subsequen ly analyzed he ma u e
wood ansc ip ome using whole-genome mic oa ays (La isch
e al., 2012). The au ho s a gued ha ay cells, as he main li ing
cells in ma u e wood, con ibu ed mos o he isola ed RNA.
Howe e , he me hod did no allow ay-cell-speci ic isola ion
o RNA due o p oblems wi h RNA deg ada ion and yield.
Recen ly, LCM was also used o p epa e ay cell-wall-en iched
ma e ial o lignin composi ion analysis om he wood o Amu
co k ee (Phellodend on amu ense) (Zheng e al., 2016). This
analysis p o ided a cell speci ic lignin p o ile, bu no RNA was
isola ed. The s udies o La isch e al. (2012) and Zheng e al.
(2016) illus a e he po en ial o LCM me hod, while a he same
ime highligh ing he need o a obus xylem-cell-speci ic LCM
p o ocol which does no comp omise RNA quali y.
He e we de eloped a p o ocol o LCM o de eloping xylem
issues om 40-yea -old No way sp uce (Picea abies) and aspen
(Populus emula) ees, ep esen a i es o gymnospe ms and
angiospe ms. The wo k wi h he gymnospe m ee was done
a he Viikki Plan Science Cen e in Finland, and wi h he
angiospe m ee a he Umeå Plan Science Cen e in Sweden.
The main goal was o achie e high quali y RNA om di e en
xylem cell ypes ( ay pa enchymal cells, xylem ibe s and xylem
achea y elemen s (i.e., acheids in sp uce) sui able o high-
h oughpu analyses. Special emphasis was placed on he isola ion
o ay pa enchymal cells, while a oiding collec ing ay acheids
ypical o sp uce. Sligh ly di e en app oaches we e ound
o be op imal o he p epa a ion and handling o sp uce
and aspen samples p io o LCM. Du ing p epa a ion o he
sec ions we es ed a ying sample hicknesses and o ien a ions.
We used PEN-coa ed slides and plain glass slides, choosing
he la e as he bes moun ing su ace o ix he c yosec ions.
We es ablished di e en dehyd a ion p ocedu es o No way
sp uce and aspen. Co ec handling o he samples p io o
c yosec ioning was c ucial o he ana omical and RNA in eg i y.
Se e al me hodological p oblems we e o e come du ing he
me hod de elopmen . These included: di icul y in achie ing
F on ie s in Plan Science | www. on ie sin.o g 2Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 3
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
su icien la ness o he sec ions on slides, s a ic elec ici y, e y
low RNA yield, and op imiza ion o lase powe o cu ing
specimens in cell wall- ich a eas in compa ison wi h he cy osolic
pa o he cells. These me hodological issues oge he wi h some
use ul ips a e add essed in he ‘c i ical no es’ sec ion and in he
oubleshoo ing able.
P o ocol O e iew
We de eloped p o ocols o he LCM o wood sec ions o
ex ac ion o cell ype-speci ic o al RNA om de eloping xylem
o No way sp uce and aspen. Disks o wood s ems o 40-yea -
old sp uce and aspen ees we e collec ed du ing he ac i e
seconda y g ow h and s o ed a −80◦C. Fo an easie handling
o he samples du ing p epa a ion o he c yosec ions, he
s em disks we e u he sawn in o smalle blocks. The blocks
we e hen used o p epa e 30/40 µm hick angen ial (sp uce)
o c oss and adial (aspen) sec ions using a c yomic o ome.
The c yosec ions, moun ed on mic oscope glass slides, we e
ixed wi h e hanol. Sp uce and aspen wood equi ed di e en
p ocedu es o dehyd a ion and la ening o he sec ions. Sp uce
angen ial sec ions we e la ened wi h a pipe e ip du ing he
las dehyd a ion s ep in 100% e hanol. Aspen c oss and adial
sec ions we e ixed in 75% e hanol and hen la ened using
a mic oscope slide sandwich du ing dehyd a ion in a eeze-
d ye chambe . In insic di e ences in wood ana omy be ween
gymnospe ms and angiospe ms de e mined he sec ioning plane
and, subsequen ly, he la ening me hod o he c yosec ions.
Fo example, he p esence o la ge essels in aspen p e en ed
he use o angen ial sec ions, because o he high p obabili y
o sec ion b eakage along he essel. Indi idual cell ypes ( ay
pa enchymal cells, acheids, and ibe s) we e isola ed by LCM
using a PALM Mic o-Beam mic oscope a oom empe a u e,
and collec ed in o adhesi e caps o collec ing ubes. Dissec ed
ma e ial was snap- ozen in liquid ni ogen and s o ed a −80◦C
un il RNA isola ion. Speci ic cell ypes collec ed o e se e al LCM
sessions we e pooled oge he o RNA ex ac ion.
Ad ances o he P o ocol
– A comp ehensi e LCM p o ocol o he de eloping xylem
o bo h angio- and gymnospe ms, applicable o angen ial,
adial and c oss sec ions.
– Less labo ious p o ocol, a oiding long embedding
p ocedu es common in se e al published p o ocols.
– Replaces memb ane-coa ed slides wi h glass mic oscope
slides, and in oduces he easy assembly o a “glass
sandwich” o keep ana omical in eg i y and la ness o he
xylem sec ions.
– Enables isola ion o eache y elemen s, ibe s and ay cells
o RNA ex ac ion.
– Enables isola ion o cell- ype speci ic, good quali y o al
RNA o high- h oughpu analyses.
STEPWISE PROCEDURES
The me hod is di ided in o i e s ages om A o E: (A) issue
sampling, handling and p epa a ion o he ozen specimens; (B)
c yosec ioning; (C) ixa ion and dehyd a ion; (D) LCM; (E) RNA
ex ac ion and quali y assessmen (Figu e 1). We adjus ed some
o he s eps o he p o ocol aking in o accoun di e en ea u es
o he wo woody ma e ials, in o de o es ablish he easies and
as es echnical p ac ice. The goal was o ex ac good quali y
RNA om he LCM-dissec ed cells, and hence, special ca e was
aken o educe he isk o RNA deg ada ion du ing each s ep o
he p o ocol.
S age A. Tissue Sampling, Handling, and
P epa a ion o he F ozen Specimens
Time: 30 min
Tempe a u e: oom empe a u e, o in liquid ni ogen
No way Sp uce
A 40-yea -old sp uce ee was elled du ing ac i e seconda y
g ow h. Disks o he unk we e sawn and immedia ely ozen in
d y ice, and hen s o ed in plas ic bags a −80◦C.
In o de o a oid hawing and o acili a e he subsequen
p epa a ion o c yosec ions, small wood blocks we e cu om
he ozen s em disks wi h a jigsaw. Fo con enience, he
s em disks we e i s sawn in hal es. Fu he sawing was
conduc ed in wo pa s: i s , a long s ip o wood, a ound
10.0 cm ×1.2 cm ×1.2 cm (H ×W×D), was sawn
angen ially om he hal s em disk (Figu e 2A). The s ip and
he emaining s em piece we e immedia ely e u ned o liquid
ni ogen. Du ing his i s s ep he ba k was easily emo ed
along he expanding zone o he de eloping xylem, and hus,
de eloping xylem wi h seconda y cell wall o ma ion on op
o he ma u e xylem was al eady exposed o c yosec ioning.
Du ing he second s ep, he ozen s ip o wood was immobilized
in a ice and cubic pieces o ca. 1.2 cm ×1.2 cm ×1.2 cm
we e sawn, placed in o 50 ml ubes in liquid ni ogen. The
i s and he las cubes om he s ip we e disca ded, since
a likely hawing in hese ou e mos zones o he s ip could
comp omise he ana omy and he RNA in eg i y. The cubes
we e s o ed a −80◦C, and used o c yosec ioning wi hin
7 days.
De eloping xylem could be iden i ied on he angen ial side o
he woody cube as a whi e, almos anslucen laye , as opposed
o he yellowish, p e ious yea ’s g ow h ing. This acili a ed he
o ien a ion o he cube du ing c yosec ioning. Al e na i ely, he
side o he ma u e xylem in he cube was ma ked ed by a quick
dipping in 0.5% Sa anin O (in 50% e hanol), be o e s o age
a −80◦C.
Aspen
Aspen wood specimens we e collec ed om a 40-yea -old ee
acco ding o he scheme illus a ed in Figu e 2B. The samples (ca.
30 cm ×20 cm ×5 cm) con aining ba k, cu en yea ’s g ow h
and se e al olde annual g ow h ings, we e cu om he unk
wi h a jigsaw, snap- ozen in liquid ni ogen and s o ed a −80◦C.
Smalle wood blocks, app oxima ely o 3 cm ×1 cm ×3 cm, we e
cu om he ozen wood ma e ial wi h a jigsaw, and kep in
liquid ni ogen be o e immedia e c yosec ioning. Al e na i ely,
hey we e s o ed a −80◦C in aluminum oil, o a longe pe iod.
In aspen, he ba k was no emo ed, since i p o ec ed he
F on ie s in Plan Science | www. on ie sin.o g 3Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 4
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
FIGURE 1 | Flow cha o he c yosec ioning and mic odissec ion p o ocol. All he s ages o he p o ocol a e indica ed wi h he ela i e empe a u e and ime
o ealiza ion. S eps pe o med di e en ly o No way sp uce and aspen a e, espec i ely, included in g een and magen a boxes. RT, oom empe a u e; o/n,
o e nigh .
de eloping xylem du ing he s o age. The ba k also helped o
o ien cu ing du ing sec ioning.
Key no es o s age A
Fo collec ion o wood samples, di e en s a egies o cu ing
can be applied (Figu e 2). The samples should be placed o
liquid ni ogen as quickly as possible a e cu ing o smalle
pieces o a oid hawing. We also ecommend o s o e he
ozen wood specimen a −80◦C in small pieces sui able o
a single expe imen . This minimizes he isk o hawing, and
consequen ly limi s he isk o RNA deg ada ion.
Du ing he p epa a ion o he small wood blocks, we ad ise
o de ine a e e ence o easily iden i y he de eloping xylem zone,
o example, by s aining as in he case o sp uce, o by keeping he
ba k in place as we did o aspen.
Equipmen and eagen s
Jigsaw; plas ic bags; aluminum oil; 50 ml ubes; d y ice; liquid
ni ogen; 0.5% Sa anin O in 50% e hanol).
S age B. C yosec ioning
Time: 1.5 h
Tempe a u e:−20◦/−24◦C
De ining he Di ec ion o Sec ioning
The o ien a ion o he block de e mines he di ec ion o
sec ioning (Figu e 3A). Table 1 summa izes ad an ages and
disad an ages o p epa ing angen ial, c oss o adial sec ions.
Tangen ial sec ions o e he bes iew o isola e di e en cell
ypes, in pa icula ay cells (Figu e 3). Howe e , a c oss sec ion
is necessa y o de ine he hickness o he de eloping xylem. In
he case o aspen, he angen ial sec ions end o c ack along he
longi udinal cell walls be ween ibe s and essels, and hence, c oss
and adial sec ions we e p e e ed.
P epa a ion o Tools o C yosec ioning
Glass slides we e acid-washed in 1 M HCl o 16 h a c.
55◦C. A e se e al washing s eps in dH2O, he slides we e
soaked in 100% e hanol, ai -d ied on a ack and s e ilized
F on ie s in Plan Science | www. on ie sin.o g 4Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 5
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
FIGURE 2 | Scheme o sampling o sp uce and aspen wood specimens. (A) Sp uce unk disk was sawn in hal es and hen, o con enience, in angen ial
s ips o ca. 10.0 cm ×1.2 cm ×1.2 cm (1). Small blocks (2) we e cu om he s ips and used o c yosec ioning. (B) Aspen wood samples (1, 2) o ca
30 cm ×20 cm ×5 cm con aining ba k, cu en yea ’s g ow h and se e al olde annual g ow h ings, we e sawn o wood disks. (3) A small aspen wood block used
o c yosec ioning. The g een dashed line ma ks he cu en yea ’s g ow h zone used o lase cap u e mic odissec ion.
FIGURE 3 | Ana omy o a No way sp uce s em wi h c oss, adial and angen ial sec ions. (A) Schema ic ep esen a ion o he h ee sec ioning planes in a
sp uce wood block. (B) Tangen ial sec ion s ained wi h Alcian blue and Sa anin O. CR, c oss sec ion; RA, adial sec ion; TA, angen ial sec ion. R, ay cell; B, ba k;
P, phloem; C, ascula cambium; RD, esin duc ; TR, acheid. Scale ba 150 µm.
a 180◦C o 4 h. Al e na i ely, he slides we e i s washed
wi h RNase decon amina ion solu ion o 0.1% DEPC (die hyl
py oca bona e)- ea ed wa e , hen wi h 100% e hanol and inally
au ocla ed. As a hi d al e na i e ea men o emo e RNase
con amina ion, he glass slides can also be incuba ed in a s e ile
Pe i dish unde UV adia ion o 30 min.
P io o c yosec ioning all he ools, including weeze s and
azo blades, we e cleaned wi h he RNase decon amina ion
solu ion. We do no ecommend he use o his solu ion o
decon amina e he c yomic o ome, which can be cleaned wi h
100% e hanol ins ead. The empe a u e o he c yomic o ome
was se o −20◦C, a leas 1 h be o e s a ing sec ioning. Du ing
he i s hou o p e-cooling, moun ing disks and glass slides
we e kep in s e ile Pe i dishes, and oge he wi h weeze s, azo
blades, and e hanol solu ions cooled inside he c yomic o ome
chambe .
Memb ane-coa ed slides used in o he published p o ocols
(Abbo e al., 2010;La isch e al., 2012;Zi hao e al., 2014), we e
F on ie s in Plan Science | www. on ie sin.o g 5Janua y 2017 | Volume 7 | A icle 1965

pls-07-01965 Decembe 27, 2016 Time: 12:11 # 6
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
TABLE 1 | Choice o he di ec ion o sec ioning.
Sec ion o ien a ion Ad an ages Disad an ages
Tangen ial Easy isola ion o pu e ay cell lines.
Easy o p epa e c yosec ions, as ibe s/ acheids go along
he cu ing plane.
Di icul o iden i y he bounda ies be ween he annual
g ow h ings du ing sec ioning. A p elimina y es ima ion o
he hickness o de eloping xylem is equi ed.
In ha dwoods, he ligni ied cell walls o ibe s/ essels cause
longi udinal c acks in he sec ion, and hampe he adequa e
la ness o he sec ions on a glass slide.
Radial Easy o iden i y ibe s and ay cells, and isola e hem in
la ge amoun s.
I he sec ion is pe ec ly adial (no oblique), he o ien a ion
o ibe s/ acheids/ essels helps o keep he sec ion la .
I he sec ion is no pe ec ly adial, he di ec ion o
ibe s/ acheids/ essels makes i di icul o ix he sample
on he slide and keep i la .
Con amina ion om he su ounding ibe / acheid cells is
no excluded.
C oss Easy o dis inguish he de eloping xylem om he ma u e
xylem, and o localize di e en ypes o cells.
The po ous appea ance o he sec ion acili a es he lase
cap u e mic odissec ion cu .
When isola ing ay cells, con amina ion om he
su ounding ibe / acheid cells canno be excluded.
o iginally de eloped o issue sec ions o animals, and ha e been
success ully adap ed o so plan issues. We disco e ed ha
glass slides wo ked be e o de eloping xylem as discussed in
mo e de ail unde “c i ical no es”.
Es ima ion o he Thickness o De eloping Xylem in
Sp uce
The hickness o de eloping xylem in sp uce was de e mined
using an ocula mic ome e om 20 µm hick c oss sec ions
s ained wi h Sa anin O-Alcian blue solu ion (Figu e 4). The
de eloping xylem laye used in ou s udy was app oxima ely
600 µm hick. Unligni ied cell walls s ain blue wi h Alcian blue,
and pa ially ligni ied cell walls show some ed s aining wi h
Sa anin O. Fully ligni ied cell walls om he p e ious yea ’s
g ow h ing appea ed unde a b igh - ield mic oscope.
P epa a ion o Tangen ial Sec ions o Sp uce
The block o de eloping xylem was moun ed wi h weeze s
on a cooled moun ing disk wi h he issue eezing medium.
We i s pe o med a imming s ep, cu ing a se ies o
20 µm hick sec ions, o emo e abou 100 µm o he
ou e mos laye o de eloping xylem. Then, 40 µm hick
sec ions we e cu and placed on p e-cooled, RNase- ee glass
slides. Each slide ca ied 3–4 sec ions. Acco ding o he
p e ious es ima ion o he hickness o he de eloping xylem,
he cu ing dep h did no exceed 600 µm. To acili a e he
a achmen o he sec ions o he slide, he slide was sligh ly
wa med om he back wi h a glo ed inge a he si e o
he sec ions o ca. 10 s. This allowed collec ion o he
eshly cu sec ions di ec ly om he c yomic o ome s age, and
a oided any u he handling o damaging o he sec ions wi h
weeze s.
P epa a ion o C oss and Radial Sec ions o Aspen
The ozen wood block was moun ed as desc ibed abo e o
sp uce. Ba k and he oldes laye s o seconda y phloem we e
emo ed by cu ing se ial, 20 µm hick angen ial sec ions. The
younges laye s o he seconda y phloem, 500–700 µm om
he cambium, we e le in ac o acili a e he c oss- and adial-
sec ioning, and o p o ide a e e ence o he o ien a ion o he
sec ions (Figu e 5). The measu emen o he hickness o he ba k
and he de eloping xylem was done on c oss sec ions as desc ibed
abo e o sp uce samples. C oss sec ions we e cu manually wi h
a azo blade om he uppe side o he wood block du ing he
imming s ep be o e changing he di ec ion o sec ioning. To
check he co ec o ien a ion and posi ion o he wood sample
on he c yo ome s age, he i s ew c yosec ions we e s ained
wi h Sa anin O-Alcian blue, and iewed unde a b igh ield
mic oscope (Figu e 5).
Th ee o ou 40 µm hick c oss o adial sec ions (a ound
1cm×1 cm/1 cm ×1.5 cm) we e cu and delica ely posi ioned
using s e ile weeze s in 50 µl o 75% ice-cold e hanol on a p e-
cooled, RNase- ee mic oscope slide. The addi ion o e hanol
helped he sec ions o adhe e o he slide. The glass slide was
kep unco e ed and ans e ed in o a 50 ml ube, which was
p e iously cooled in liquid ni ogen. The ube ca ying he slide
was closed igh ly, snap- ozen in liquid ni ogen and kep on d y
ice un il he eeze-d ying s ep.
Key no es o s age B
This s age o he p o ocol implies he longes handling ime
o he samples be o e he ixa ion. Fo his eason, app op ia e
measu es should be aken o limi he isk o RNA deg ada ion.
Tools and equipmen need o be s e ile o ea ed wi h a
RNase decon amina ion solu ion o , i no possible, wi h e hanol.
Mo eo e , he whole p ocedu e has o be done quickly and a
low empe a u e. We ecommend o keep all he ools, e.g.,
azo blades, weeze s, moun ing slides, in he c yomic o ome
chambe .
Equipmen and eagen s
C yomic o ome (Leica CM3050 S); UV C osslinke
(Spec olinke XL-1000); moun ing disks; eezing moun ing
medium (Leica Tissue F eezing Medium, Re . 14020108926);
mic oscope glass slides; glass Pe i dishes; 50 ml ubes; weeze s;
azo blades; liquid ni ogen; RNaseZAPR
decon amina ion
F on ie s in Plan Science | www. on ie sin.o g 6Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 7
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
FIGURE 4 | C oss sec ion o No way sp uce xylem o he es ima ion
o he de eloping xylem hickness. A 20 µm hick c yosec ion was s ained
wi h Sa anin O-Alcian Blue solu ion. Non-ligni ied cell walls in de eloping
xylem s ain blue wi h Alcian blue ( he ou e pa o X2). Ligni ying cells ( he
inne pa o X2) and he p e ious yea ’s g ow h ing (X1) wi h ully ligni ied cell
walls s ain ed wi h Sa anin O. No e na ow iles o ay cells (a ows). P,
phloem; C, cambium; X1, ma u e, p e ious yea ’s xylem; X2, cu en yea ’s
xylem. Scale ba 250 µm.
solu ion (AMBION AM9780); 100% e hanol; 1 M HCl; 0.1%
DEPC (die hyl py oca bona e; Sigma–Ald ichR
, 1609-47-
8)- ea ed wa e ; 75% e hanol solu ion in RNase- ee wa e
(AMBION AM9930); solu ion (1:1, / ) o 0.5% Sa anin
O(Me ck) in 50% e hanol and 0.5% Alcian blue (Na ional
diagnos ics) in 50% e hanol.
S age C. E hanol Fixa ion and
Dehyd a ion o he C yosec ions
Time: 20 min (sp uce); 30 min and o e nigh (aspen)
Tempe a u e: oom empe a u e (sp uce); oom empe a u e
and −50◦C (aspen)
Fixa ion and D ying o he Sp uce Tangen ial
Sec ions wi h E hanol
Two minu es a e he las sec ion was placed on o he slide, ice-
cold 70% e hanol was pipe ed in excess on op o he sec ions
o ully co e he whole a ea. The slide was hen ans e ed in
a s e ile glass Pe i dish in o a lamina low cabine , whe e 70%
e hanol was eplaced wi h ice-cold 100% e hanol. The subsequen
ixa ion s ep was pe o med a oom empe a u e in he lamina
low cabine . Du ing he 2-min incuba ion in 100% e hanol, he
sec ions we e gen ly s e ched and la ened wi h a pipe e ip (see
c i ical no es). A e he emo al o e hanol by pipe ing, sec ions
we e ai -d ied o 15 min. Slides wi h d y sec ions we e collec ed
in o s e ile 50 ml ubes. The ubes we e igh ly closed and s o ed
a −80◦C o no longe han 7 days.
Fixa ion and Dehyd a ion by F eeze-D ying o Aspen
Samples
The eeze-d ye chambe was i s cleaned wi h he RNase
decon amina ion solu ion and p e-cooled o −50◦C. The
c yosec ions we e d ied as ollows: (i) in a closed 50 ml ube o
20 min. The ube cap was kep loose bu no o ally unsc ewed;
(ii) in he o ally opened ube o addi ional 5–10 min; (iii) in
a closed glass sandwich kep in he 50 ml ube, o e nigh . The
glass sandwich was p epa ed by co e ing he glass mic oscope
slide ca ying he sec ions wi h a second one and closing hem
on hei sho sides wi h a pai o clips (Figu es 6A,B).
Key no es o s age C
This s age is pa icula ly impo an o ob ain la and d y sec ions
equi ed o success ul LCM and good-quali y RNA ex ac ion
(see c i ical no es). The glass sandwich keeps he specimens la
du ing and a e d ying. Howe e , we obse ed ha he sec ions
we e op imal o LCM i hey we e i s kep 30 min unco e ed
in he eeze-d ye . Du ing his incuba ion, sec ions cu l and
become pa ially de ached om he glass slide, bu a e s ill
a ached all along he phloem and he cambium. A e his ini ial
d ying s ep he sec ions can be la ened wi hou c acks by closing
he glass sandwich o he ollowing o e nigh dehyd a ion.
Equipmen and eagen s
Sp uce: ice-cold 70% e hanol in RNase- ee wa e (QIAGEN);
100% e hanol; glass slides; pipe e ips; 50 ml ubes; lamina low
cabine ; Aspen: eeze-d ye (Scan ac CoolSa eTM), RNaseZAPR

decon amina ion solu ion (AMBION AM9780); 50 ml ubes;
mic oscope glass slides; o ice clips.
S age D. Lase Cap u e Mic odissec ion
Time: 1.5 h pe slide
Tempe a u e: oom empe a u e
Lase cap u e mic odissec ion s ep was pe o med simila ly
o sp uce and aspen sec ions. The only di e ence was ha
he aspen samples equi ed an addi ional p epa a o y s ep o
immobilize he sec ions on glass slides. A he end o he
o e nigh eeze-d ying, he glass sandwich was emo ed om
he eeze-d ye and delica ely opened in he lamina low
cabine . The d ied sec ions we e s ably a ached o he glass
wi h s ips o ape on hei longe sides (Figu e 6C). The glass
sandwich was closed again, using now only one clip o a oid
F on ie s in Plan Science | www. on ie sin.o g 7Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 8
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
FIGURE 5 | C oss sec ion o aspen xylem. A manual c oss sec ion o aspen wood was cu du ing he ini ial angen ial imming s ep o c yosec ioning. A ound
500–700 µm o he phloem (P) was le as a p o ec ion and o ien a ion e e ence du ing he p epa a ion o c oss/ adial aspen sec ions. C, cambial zone; X, cu en
yea ’s xylem. Scale ba 200 µm.
dis u bance and damage o he sec ions. Samples we e kep in
s e ile Pe i dishes in a po able desicca o chambe , a oom
empe a u e un il LCM.
The sp uce samples we e anspo ed o he LCM appa a us
in a 50 ml ube kep in liquid ni ogen. P io o LCM, he ube
con aining he sp uce c yosec ions was kep a oom empe a u e
o 20 min o p e en condensa ion on o he slide.
The specimens we e dissec ed using he PALMR
Mic oBeam
Sys em (Ca l Zeiss AB). Op imal lase dissec ion was achie ed
wi h bo h ypes o wood acco ding o he ollowing se ings: cu
speed 20–30; cu ene gy 60–65; lase powe ca apul ing ene gy
25–30. The bes ocus was achie ed in he ange o 65–70 wi h
del a ocus a –2. The op imal magni ica ion o ou lining and
cu ing was 20×(sp uce) and 10×(aspen). This is simila o he
mos sui able magni ica ion o ca apul ing (20× o sp uce and
10× o aspen) (Figu es 7 and 8).
The dissec ed pieces we e collec ed by ca apul ing in o he
adhesi e cap o a 500 µl collec ing ube. Each ube and each slide
we e used du ing a single LMC session o no longe han 1.5 h.
In his ime, we we e able o ca apul a ound 15–25 and 200–300
mic odissec ed pieces o aspen and No way sp uce, espec i ely,
which gene ally co esponded o a o al a ea o 0.5–0.75 mm2
and 0.2–0.4 mm2o aspen and sp uce wood issue, espec i ely,
collec ed in a single adhesi e cap. Specimens, dissec ed du ing
se e al LCM sessions, we e combined o ob ain a de ec able
amoun o ex ac ed RNA. Fo example, he o al a ea o sp uce
LCM-isola ed ay pa enchymal cells was o ca. 12 mm2. A he
end o e e y session (usually 1.5 h long), he collec ing ube was
snap- ozen in liquid ni ogen. All he dissec ed samples we e
s o ed a −80◦C un il RNA isola ion.
Key No es
We sugges o always p epa e mo e han one slide wi h 3–4
sec ions o one LCM session in o de o ha e enough sec ions
in case one appea s unsui able o LCM. The use o RNase- ee
ools ( weeze s and needles) du ing addi ion o ape o he eeze-
d ied sec ions is ecommended al hough he sec ions a e now
ixed, and hus, mo e p o ec ed agains RNA deg ada ion. In a
oubleshoo ing Table 2, we lis he mos ele an and common
p oblems aced du ing LCM.
Equipmen and Reagen s
Lase mic odissec ion mic oscope (PALM Mic oBeam,
Zeiss); adhesi e cap ubes 500 µl opaque (Ca l Zeiss AB,
415190−9201−000); liquid ni ogen; 50 ml ubes; addi ionally
o aspen: weeze s; needles; o ice anspa en ape (MagicTM
in isible 3M, Sco chR
); scisso s; desicca o .
S age E. RNA Isola ion and Assessmen
o RNA Quali y
Time: 2 h, o acco ding o he RNA ex ac ion ki manu ac u e ’s
ins uc ions
Tempe a u e: oom empe a u e and acco ding o he RNA
ex ac ion ki manu ac u e ’s ins uc ions
F on ie s in Plan Science | www. on ie sin.o g 8Janua y 2017 | Volume 7 | A icle 1965
pls-07-01965 Decembe 27, 2016 Time: 12:11 # 9
Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem
FIGURE 6 | Glass sandwich assembled o aspen sample p epa a ion. (A) The glass sandwich holding he woody c yosec ions is closed wi h a pai o o ice
clips (B) o he o e nigh eeze-d ying p ocedu e. (C) The eeze-d ied sec ions a e a ached wi h ape on he moun ing slide and p o ec ed in a e-assembled glass
sandwich closed by a single clip. In he close-up box, an example o d ied c yosec ion con aining an in ac xylem zone sui able o lase cap u e mic odissec ion.
P, phloem; X1, old xylem; X2, cu en yea ’s xylem.
FIGURE 7 | Lase cap u e mic odissec ion o ay cells om a angen ial sec ion o No way sp uce. (A) Ray pa enchymal cells a e ou lined (blue line) o
lase cu ing. No e ha ay acheids (RT ) we e no collec ed. One cu and one uncu ile o ay cells a e seen. (B) Ray cells we e success ully cu and ca apul ed.
Do s in he middle o he cu egions a e he a eas whe e he ca apul ing pulse was applied. T , acheid. Scale ba 150 µm.
RNA was ex ac ed om he whole c yosec ions and dissec ed
cells pooled om se e al LCM sessions. To al RNA quali y was
assessed using he RNA Pico Assay o 2100 Bioanalyze .
Aspen sec ions equi ed a longe p epa a ion ime han hose
o sp uce, and we e consequen ly mo e suscep ible o RNA
in eg i y impai men , especially du ing he las p e-LCM s age a
oom empe a u e. To al RNA quali y was he e o e measu ed a
e e y s ep o he p o ocol, i.e., in a single in ac c yosec ion, in a
single in ac c yosec ion ixed and le a oom empe a u e in he
desicca o o wo days, in mic odissec ed cells, and in esidual
F on ie s in Plan Science | www. on ie sin.o g 9Janua y 2017 | Volume 7 | A icle 1965