Page 371 - konishipaper

whichIFcouldbec1earlysen.TheIFbehaviorsintheselected celswerethenconvertedintovalues，andtheaverageofthe10 valueswasca1culated.Thesemedianvalueswerethencompared using one-way analysis ofvariance (ANOVA) folowed by the Tukey-Kramer method.
3.Results
3.1. Pα t ternc/as sificationoficecrystalformationatintracel lular frezing
Examplesofthehigh-speedcameraimagesthatwereobtained from the start until the completion of intracel lular fre ezing at colingratesof-1oCfminand-100oC/minareprovidedinFigs.2 and3，respectively.TheseimagesarealsosupportedbySupple- mentaryVideos1and2，andshowthatourcryomicroscopicsystem couldcapturec1earimagesoftheIFproces，alowingustoobserve theformationofintracelularicecrystalgrains.
Supplementaryvideorelatedtothisartic1ecanbefoundat htp:/dx.do.iorg/l0.1016/j.cryobio.I2016.06.003.
TheprogresinIFfolowingtheappearanceofcrystalsinside thecelshowninFig.2isshowninFig.4A-0.Intracelularfrezing began with the sudden appearance of ice crystals inside the cel l， withintracelularicecrystalsfirstbeingobservedatthecelwal (Fig.4B).Atalcolingrates，over96%oficecrystalsappearedatthe celwal，butsomecrystalsalsoappearedatthecenterofthecel1as the coling rate increased (Fig. 4E; Supplementary Video 3). However，itwasnotposibletodeterminewhethertheseintra- cel1ularicecrystalsstartedtoappearintheinerpartofthecelor atthecelwalonthec10sestoropositesideofthefieldofview becausethecryomicroscopicsystemcouldonlyobtainimagesin twodimensions.
Supplementaryvideorelatedtothisartic1ecanbefoundat htp:/dx.doi.org/l0.l016/j.cryobio.I2016.06.003.
(A)
SchematicilustrationsofthepaternsofIFovertimearepro- videdinFig.5Aーに Thesepaternscouldbec1asifiedintothre types:formationpaternA(Fig.5A)，whereintracelularicecrystals formedaroundtheperipheryofacelafterappearingatthecel wal l; formation pa仕 ern B (Fig. 5B)，where intracel lular ice crystals formedacrosthecenterofacelafterappearingatthecelwal; andformationpatern仁 (Fig.5C)， whereintracelularicecrystals expandedtotheedgesofacelafterappearingatthecenter.Ata co olingrateof-1oCfmin，approximately53%offormationpat terns felintocategoryA，whiletheremaining47%felintocategoryB (Fig.50).However，asthecolingrateincreased，theproportionof celsexhibitingformationpaternAdecreasedandtheproportion exhibitingpaternsBand仁 increased，sothatat-100oCfmin，the proportionsofformationpaternsA andBweremoreorles reversed.
3.2.Evaluationsofceldeformationandicecrystalformationαt intracelularfrezing
Therelationbetweenthecolingrate.andthedegreeofsupeト colingandceldeformationduetoIFwasexamined.Compared withitsinitialshape(Fig.6A)，thecelareawasdeformedfolowing intracel lular fre ezing (Fig. 6B). The degree of superco oling at -10oC/minwastwicethatat-1 oC/min，whilethedegreeof supercolingat-100oC/minwasthretimesthatat-1oCfmin (Fig.6C).Inadition，theeatacolingrateof-10oC/mindecreased toapproximately68克 ofthatat-1oCfmin，whiletheeat-100oC/ mindecreasedtoapproximately35%ofthatat-1oCfmin(Fig.6C). Thus，thedegreeofsupercolingandceldeformationwasafected bythecolingrate.
Thegrowingrateofintracelularicecrystalswasevaluatedusing theareavelocityofintracelularicecrystals.Theareasofintracel- lularicecrystalsimmediatelyaftertheirappearanceinsideacel werethesameacrosalcolingrates(Fig.7).However，therelation
主Nin口'gawaet口1./CりIObiology73ρ016)20-29 25
5
O0 -0.1 -10 -10 -10 Coolingrate[OC/min]
Fig.6.Efectofcolingrateonthedegreofα1deformationandsupercolingintheepidermaltissuesofstrawberygeranium(S日xi[rag日， stolonifera仁urtis)leaves.(A)Celarea imediatelybeforeintracelularfrezing・Brokenline:celshapebeforeintracelularfrezing;whitebar:251!m.(B)Celareaimediatelyafterintracelularfrezing.Thearea outsidetheceloriginalshapeindicatesthedegreofceldeformation.(C)Relationbetwenthecolingrateandboththedegreofsupercoling(Ts)andthedegreofcel deformation(e).Bars:SE(n= 10).
(B)
Deformation ofacel
0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01
3νP3
ゆm
LUAU s=
E且・ ， 且・
zJnuqJnU
L2111 eI 4L 司
C剖
〆‘，、
Z白
勾因。Z盟国』£ω唱 =81。明ωZMω白
qc
。一回国固 。。ど邑zmh32r
咽-EA