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Fig.9.Brightnesdiferencesattheedgesofintracelularicecrystalsintheepidermaltisuesofstrawberygeranium(Saxifragastolonifer日， Curtis)leavesatdiferentcolingrates. (A)Theedgeofanintracelularicecrystalatacolingrateof-1oCfmin.(B)Theedgeofanintracelularicecrystalatacolingrateof-10oCfmin.(C)Theedgeofanintracelularice crystalatacolingrateof-100oCfmin.(0)BrightnesdiferencesattheedgesoftheintracelularicecrystalsshowninimagesA一仁 (E)Averagevaluesofthe50softhebrightnes dif ferences at the edges of intracel lular ice crystals at dif ferent co oling rates.Bars:5E (n = 10). Oi仔'erent lower一 日 se let ters indicate statistical ly significant dif ferences between treatmentsusingone-wayANOVAfolowedbytheTukey-Kramermethod(Pく 0.05).
frameratecancompensateforadecreasedresolution，andvice versa.TheframerateisimportantforobservingIFthroughtime， buttheresolutionisalsoimportantforobservingmoresubtle changesthatocurduringthattime.Therefore，webelievethatthe higherresolutionandlowerframerateofourcamerawasapro- priategiventhespeci白cfocusofourstudy.
Themostuniqueaspectofourcryomicroscopicsystemwasthe useofaninvertedmicroscope.Themovabledistancebetweenthe specimen'andtheobjectivelensisshorterwithaninvertedmi- croscopethanwithanordinary，uprightmicroscope.However，an invertedmicroscopecanalsoobservethecelsatthebottomofthe culturesolutioninacontaineroradishduringfrezingandpost- thawingbecausetheobjectivelenspointsupward，asaresultof whichbiosystemlaboratoriesoftenprefertouseinvertedmicro- scopes.Inadition，thisorientationalowedthemicroscopetobe refocusedonacelmorequicklythananuprightmicroscopeinthe
shorttimebetweenthedistiledwaterinsidethecoverglases frezingandtheocurenceofintracelularfrezing.Thisisbecause thedownwardexpansionofwaterduringfrezingwasrestricted duetothepresenceofthecolingstage.
Theinitialstagesofintracelularfrezingwerefirstexamined usingthemovingimagesobtainedwithourmicroscopicsystem. Intracelularicecrystalsapearedatthecelwalinover96%of celsatalcolingrates(Fig.4).However，IFprogresednotonlyin theverticaldirectionrelativetothecelwal，butalsoalongthecel outline.Plantcelsareseparatedfromtheextracelularenviron- mentbyaplasmamembrane，whichisextremelythin.Therefore， althoughtheintracelularicecrystalswerethoughttoapearatthe celwalinmostcases，itmaybethattheysometimesapearedat theplasmamembrane.Theseimagesalsoshowedthatintracelular icecrystalsapearedsucesivelyaroundtheceloutline.Thus，itis posiblethatwhenintracelularicecrystalsapearedattheplasma
