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主 Ninag口， waet口.1/Cryobio1ogy73(2016)30-39
Specimen
口
controler Fig.1.Schematicilustrationofthecryomicroscopicsystemusedinthisstudy.}.ex:excitationwavelength，}.em:emisionwavelength.Aneutraldensityfilterwasatransmisionrate
of25%andwasusedat}.em=440nm. (A)
1. Then nocol lple
2. Fiberopticthenometer 3. IRcamera
(B)
(C)
Fig.2.Methodofaplyinganelectriccurentloadtoaspecimenbetwencoverglases.(A)Schematicofthemethodofelectriccurentloadingonaspecimenbetwencover glases.Thetemperatureinsidethecoverglases，demarcatedwithaboldsquare，wasmeasuredusingathermocouple，afiberopticthermometer，andaninfrared(IR)臼 mera.(B) Coverglaseswiththespecimenplacedindistiledwater.Twoelectricalwireswerelockedontothealuminumsealsadheredtotheedgeofthecoverglas.(C)Thewaveformof electriccurentloading.Electriccurentwasloadedfor01.msecevery0.5msecwiththepolarityreversedevery60s.
withoutelectriccurentloadingatacolingrateof-1 oCfmin. Thus，intracelularfrezingwithorwithoutelectriccurentloading wascapturedat1000fpsfor2sbythehigh-spedcamera.
Finaly，thepHitransitioninjerusalemartichoketubertisues andonionbulbscaleepidermaltisueswasmeasuredwithor withoutelectriccurentloadingwhilecoling.First，thepHitran- sitioninjerusalemartichoketubertisueswithoutelectriccurent loadingwhilecolingwasmeasuredatacolingratethatvaried between -1 O(jmin and -100 O(jmin from room temperature
(aproximately220仁)untilintracelularfrezingocured.pHiwas measuredatevery4O(lowerfrom200仁 Usingcolingratesof -10oCfminand-100O(jmin，thetemperaturewasmaintainedfor 1minperevery4O(lowerfrom200仁 tocapturefluorescence images.Afterthat，thepHitransitionsinjerusalemartichoketuber tisuesandonionbulbscaleepidermaltisueswithelectriccurent loadingweremeasuredatevery4O(lowerfrom20O(atacoling rateof-1O(jmin.
AlimageswereanalyzedusingImagej(v1.46)[2，23].
60sec
Time
Nitrogen- gas
+自 凶532百ωじ200Eoω日