Page 379 - konishipaper

2.5. Evaluationofintracelularicecrystalformationbehaviors
IFbehaviorswereevaluatedusingthedegreofsupercoling， thedegreofceldeformation，andthecharacteristicgrainsizeand growingrateofintracelularicecrystals[21].
Thetemperaturesatwhichthedistiledwaterbetweenthe coverglaseswasfrozenandintracelularfrezingocured，were convertedrespectivelyintothedegreofsupercoling(Ts).Tswas ca1culatedbysubtractingthetemperatureatthereleaseofsupeト co olingfromthesolidificationpointofwater(00 C).Inad dition，the temperatureatwhichintracelularfrezingocuredwasdefined usingthetemperaturewhenintracelularfrezingocuredinhalf ofthecels(aproximately7cels)withintheareaunderobser- vationbythehigh-spedcameraorthecoledCCDcamerato reducevariabilityinthetemperatureatwhichintracelularfrezing ocuredinaspecimen.
Thedegreofceldeformation(e)indicateshowmuchacelhas beendeformedasaresultofintracelularicecrystals，andwas measuredbyca1culatingtheareaofcelsbefore(Ab)andimmedi- atelyafter(Ac)intracelularfrezing.Theewasthenca1culatedas:
Thegrainsizeofintracelularicecrystalswasevaluatedusing thechara仁teristicgrainsizeattheedgeofintracelularicecrystals. Thischaracteristicgrainsizewasobtainedusingasubtractionim- agethatwaspreparedfromanimagecapturedbeforeintracelular frezingandanimagecapturedatthetimewhentheintracelular icecrystalsocupiedhalfofthecelinside.Grainsizewasestimated bytracingthedistancefromthepointwherethebrightnesdif- ferenceincreasedsharplytothepointwhereitdecreasedbecauseit wasconsideredthattheedgeofanintracelularicecrystalexisted wherethebrightnessubstantialychanged.Thesizesofleastwise tengrainswereobtainedfromeachselectedcelusingthismethod， andtheaveragevalueofthegrainsizeswasdefinedasthechar- actenstlcgramSlze.
Thegrowingrateofintracelularicecrystalswasevaluated basedontheareavelocityofintracelularicecrystals，bymeasuring theareaofanintracelularicecrystalevery2msfromthetimeofits apearanceuntil10mshadelapsed.
2.6.pHimeasurement
ThepHiduringcolingwasmeasuredusingtheratiooffluo- rescenceintensity.FDA[33]orBCECF-AMwasusedasafluorescent reagen.tAnexcitationwavelengthwasused440nmand500nmto obtaintheratiooffluorescenceintensityandanemisionwave length by their excitation wave lengths was 530 nm. A neutral densityfilterwithatransmisionrateof25%wasusedwithan excitationwaveof440nm.Theplantspecimenswereiradiated withlightfromamercurylamp(USH-1030L，Ushio)，andfluores- cenceimageswerephotographedbythecoledCCDcamerausing ACT-1CforDXM1200Csoftwarepacl<age(Version1.1.0.25)through anobjectivelens(UPlanFllOx，Olympus).
Forfluorescencelabeling，tisuewascutintoaproximately 10m m2squaresandthenspecimenswereimmersedinadistiled- water-basedsolutioncontainingFDAorBCECF-AM.Todye，Jeru- salemartichoketubertisuesweredyedin3μMand10μMcon- centrationsofBCECF-AMandFDA，respectively.Onionbulbscale epidermaltisuesweredyedin1.5μMand5μMconcentrationsof BCECF-AMandFDA，respectively.TisueswereincubatedinBCECF- AMat270 仁 for15minandinFDAatroomtemperature(aprox- imately22OC)for10min.
2.7. Calculαtionofluorescenceintensityratio
Animageofeachfluorescenceintensityratiowaspreparedfrom twofluorescenceimagesobtainedwithexcitationat440nmand 500nm.Fluorescenceintensityratiovalue(Vr)wasobtainedfrom theaveragevalueoffluorescenceintensityratiointhetargetarea ontheimage.
2.8.pHcalibrationcurve
pHcalibrationcurveswerepreparedfromtherelationbetween pHandthefluorescenceintensityratio.Afterspecimensweredyed withafluorescentreagent，thepHiwasadjustedfrom5.5to8.5 usingpHstandardsolutionsandnigericin.ThepHstandardsolu- tionsusedinthisstudywerepreparedby仁ompoundingthepH standardsolutionsof4.01(potasiumhydrogenphthalate，50mM)， 6.86 (potasium dihydrogen phosphate， 25 m M; disodium hydrogenphosphate，25mM)，and9.18(sodiumtetraboratedeca- hydrate，10mM)，whichwerepurchasedcomercialy(DKK-TOA Corp).Specimensdyedwithafluorescencereagentweresoakedin thepHstandardsolutionspreparedfrom5.5to8，5. whichcon- tained1μMnigericinatroomtemperature(aproximately22OC) for15minUerusalemartichoke)or10min(onion).Then，fluores- cenceimageswerecapturedthreormoretimesateachpH，and thevaluesofthefluorescenceintensityratiowereobtainedfrom threormorecelsperfluorescenceimage.Thus，thefluorescence intensityratioaveragevalueswereobtainedfromnineormore samplespotsperpH.Finaly，theequationsforpHcalibrationwere obtainedasfolows:
B C E C F - A M : p H i = 0 .8 1 6 1 x V r + 4 .8 2 3 9 FDA: pHi = 0.9041 x V r + 4.4158
3. Results 3.1.Inspectionofheatgenerαtionduetoelectriccurentloading
，
Inthisresearch，athermocouplewasusedformeasuringthe temperatureofthespecimenbetweencoverglasesconsidering theadvantageofsensorsize，sensitivityandresponse.However， because thermocouple takes use ofelectromotive force，it is consideredthatelectriccurentloadmaygivetheerorofits temperaturemeasurement.Toconfirmitseror，thetemperatureat thesurfaceofspecimenonthecoverglasinsideoftheopen colingstagewithelectriccurentloadingwasmeasuredbyafiber optic thermometer， and an IR camera that are not af fected in temperaturemeasurementdirect1ybyelectriccurentloadingin aditiontoathermocouple(Fig.2A).
Fig.3Ashowsthetemperaturetransitionsonthespecimenon thecoverglaswithanelectriccurentloadingof100μAatroom temperature (26 0 C) using a thermocouple， a fiber optic ther- mometer，andanIRcamera.Themaximumdiferenceintempeト
Moreover，thetemperatureofthespecimenonthecoverglas withelectriccurentloadingwasmeasuredwiththeIRcamera equipedwithamicroscopelenstoconfirmtheinfluenceofthe heatgeneratedduetoelectriccurentloadingonthespecimen.The temperaturedistributiononaspecimenonthecoverglaswithan electriccurentloadof10μAisshowninFig.3B.Theleftimage showsanIRcameraimagewiththepositionofthespecimenonthe
主 Nin口E口waetロ1./Cryobio1ogy73(2016)30-39 33
atures measured using these thre methods was 0.5 o temperaturemeasuredusingthethermocouplewasmostlyunaf- fectedbyelectriccurentloadingandwasthereforehighlyreliable.
c.The
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