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もd E明
thebrightnesdiferencerelatestothegrainsizeofintracelularice crystalbecausethevariationinthatreduceswithanincreasein colingrate.Thus，thestandarddeviationofthebrightnesdifer- encewasnoticedandexaminedassumingasacharacterofthe grainsizeofintracelularicecrysta.lAsthecolingrateincreased， the standard deviations of the brightnes s dif ferences decreased (Fig.9E).Itisindicatedthatthestandarddeviationofthebrightnes diferencedecreaseswithadecreaseinicecrystalgrainsize.
Toobtainamoreacurateassessmentofthegrainsizeatthe edgesofintracelularicecrystals，thebrightnesdiferencewas attemptedtousetocalculatethecharacteristicgrainsizeofthe formingintracelularicecrystals.First，adistancefromthepoint wherethebrightnesdiferencemarkedlyincreasedtothepoint whereitdroppedwastraced，andwasusedtoestimatethegrain sizeattheedgeofanintracelularicecrysta.lThiswasrepeatedat least10timesineachselectedcelandthencalculatedtheaverage valuetoobtainthecharacteristicgrainsize.Thecharacteristicgrain sizeat-10oC/mindecreasedto84克 ofthatat-1oCfmin，whilethe characteristicgrainsizeat-100oC/mindecreasedto47%ofthatat -1oCfmin(Fig.10).Thus，thegrainsizeattheedgesofforming intracelularicecrystalssignificantlydecreasedasthecolingrate increased.
4. Discussion
Cryo-scanning electron microscopy (cryo-SEM) is often a preferedtechniqueforobservingandmeasuringmicroscopicob-
jectsatlowtemperatures[4，7，101.However，althoughcryo-SEMcan measuremicroscopicobjectsextremelyacurately，itisdificultto obtainaseriesofimagesduetothemethodologyused.Bycontrast， high-speedcameraimagescanprovideinformationaboutkinetic behaviorandthemicroscopicobjectitself.Therefore，itisbeneficial tocapturec1earimagesofintracelularfrezingusingahigh-speed cameratoobtainacomprehensiveunderstandingofthemecha- nismofintracelularfrezing.
Karlson，whoisaleaderintheuseofhigh-spedcamerasto investigate intracel lular fre ezing，recommended a frame rate of 8000fpsandaresolutionof128 x 128pixelsforhigh-speed camerasincryomicroscopicsystems[14，] andStotandKarlson previouslyusedacamerawithaframerateof8000fpsandares- olutionof256x 128pixelstocaptureintracelularfrezing[32]. Thehigh-speedcamerausedinthepresentstudyhadamuchlower framerate(1000fps)thanthis，neverthelesthesamecomple- mentarymetal-oxidesemiconductor(CMOS)imagesensor.How- ever，a resolution was higher (512 x 512 pixels) than this.W h e n consideringtheperformancesofhigh-speedcameras，anincreased
(C)
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2 10 Time[ms)
Fig.7.Intracelularicecrystalgrowingrateatdiferentcolingratesintheepidermal tisuesofstrawberygeranium(S似 ifr口Iga5ωlon俳raCurtis)leaves.Bars:SE(n=10). Diferentlower-日 記 letersindicatestatisticalysignificantdiferencesbetwen treatmentsusingone-wayANOVAfolowedbytheTukey-I<ramermethod(叩 く0.05， .P<0.01).
betweentheareaofintracelularicecrystalsandthecolingrate becamestatisticalysignificantwithincreasedtimesincetheir白rst apearance.
Thegrainsizeofintracelularicecrystalswasalsoa仕 emptedto evaluate.Fig.8Ashowsanimagethatwasobtainedbeforeintra- celularfrezing，whileFig.8Bwasobtainedwhenintracelularice crystalsoccupiedhalfofthecel.lAscanbeseen，thecelappeared darkintheareaswhereicecrystalswerelocatedafterintracelular frezing(Fig.8B)，buttherewasalsoadarkareainsidethecel beforeintracelularfrezing(Fig.8A).Therefore，imagesinwhich thebrightnesoftheimageobtainedwhentheintracelularice crystalsoccupiedhalfofthecelwassubtractedfromthebrightnes oftheimageobtainedbeforeintracelularfrezing(Fig.8C)were preparedtoalowustodistinguishtheformationofintracelularice crystals.Thismodifiedimagewasusedtoevaluatethesizeofgrains at the edges of the intracel lular ice crystals (white line a- b in Fig.8C).
Thegrainsattheedgesoftheformingintracelularicecrystals became finer with an increase in coling rate (Fig.9A-C). Furthermore，as the coling rate increased，the peaks in the brightnesdiferencesbecamelowerandmotenumerous，andhad narrowerintervalsbetweenthem(Fig.9D).Itissupposedthatthe tethattheedgesoftheformingintracelularicecrystalsbecome finerwithanincreaseincolingrate.Moreover，itissugestedthat
(A)
Fig.8.Methodusedtoevaluatethesizeofintracelularicecrystalgrainsintheepidermaltisuesofstrawberygeranium(Saxifrag日 stolon俳raCurtis)leaves.(A)Celarea imediatelybeforeintracelularfrezing.Whitebar:251m.(B)Celareawhenintracelularicecrystalsocupiedhalfofthecelatacolingrateof-1oC/min.(C)Imagethathas benadjustedbysubtractingthebrightnesofimage(B)fromthatofimage(A).Thegrainsizethatwasevaluatedontheedgeofanintracelularicecrystalisindicatedbyawhite line(a-b).
、.Fノ B