Page 367 - konishipaper

vitrification preservation can only be ap plied to micro-objects， making it dif ficult to prevent the oc cur rence of intracel lular frezingwhenlivingmaterialsarefrozen.
Frozenpreservationisusedforfodstorage，whereitisun- necesarytokeepthelivingmaterialalive.Rapidfrezingcan preservefishandmeatforalongtimeperiodwithalesdec1ineof thequality[12，18]，andsoisused，forexample，tostoretuna. However，itisdificulttoaplycryopreservationtechniquesto plantfods，suchasvegetablesandfruits，asintracelularicefor- mationleadstoadec1ineintheirtexturebecauseofmolificationof thetisue[5.13].Plantcelsaresuroundedbycelwals，anditis believedthatthemolificationofthetisueiscausedbyinjuryto thecelwalwhenicecrystalsformbothoutsideandwithinacel [9].
Basedonthisinformation，theauthorsconsiderthattheability tovisualizeintracelularicecrystalformation(IF)behaviortoun- derstandthepointatwhichintracelularfrezingbeginsandhow IFprogreses，isthekeytodevelopingimprovedcryopreservation techniques.
5tudieshavecapturedimagesofintracelularfrezinginanimal and plant cel ls and tis sues since the 1930s [6].Most of these studieshaveinvestigatedtheefectofintracelularfrezingonthe survivalrateorlevelofinjurytothecels[2，17，19，21.29]，orrelateto vitrificationpreservation[30，33，]withfewerstudiesexaminingIF behavior.However，ModilibowskaandRogers[2]filmedintra- celularfrezinginplanttisues，andreportedthebehavioroutside andinsidethecelsuponintracelularfrezing;Asahina[3]pro- videdadetailedreportonthefrezingprocesinplantcels，and recently，5totandKarlson[32]investigatedIFusingahigh-sped camera.Alofthesepreviousstudiesusedimagesthatcaptured thephenomenonknownas“flashing，"wherebytheinsideofthe celdarkensuponfrezing.However，theauthorsconsiderthatitis neces sary to capture the progres s of I IF and to investigate its behaviorinevenmoredetai.lItisknownthatintracelularfrezing beginswiththeinvasionofanicenuc1eusfromoutsidethecel [24.31].However，itisthoughtthaticenuc1eationmayocurnot onlyasaresultofextracelularfactors，butalsointracelularfac- tors，withheterogeneousorhomogeneousnuc1eationinsidethe cel.l
Togainabeterunderstandingofth
2. Materials and methods 2.1.Cryomicroscopicsystem
Thecryomicroscopicsystemusedinthisstudyconsistedofan invertedmicroscope(IX7，1Olympus)equipedwithacolingstage (HCD30，1 IN5TEC)andahigh-spedcamera(PhantomV4.2，Vision Research)(Fig.1).
Theplantspecimenswereiradiatedwithlightfroma100-W halogen lamp (12V100WHAL-L，Olympus) pased through a condenser(IX2-LWUCD，NA:0.55，Olympus).Intracelularfrezing in the plant specimens was then captured by the high-spe ed camerathroughanobjectivelens(5LCPlanFl40xOlympus).The high-spedcameracouldcaptureimagesatamaximumframerate of2000fpsandaresolutionof512x512pixelsusingPhantom CameraControlsoftwarepackage(Version9.0.640.0-CPhCon:640).
Colingwasperformedbypouringcombinationairusingliquid nitrogenandaheater(5T仁200，Instec)ontothecolingst宇ge.The colingratewascontroledbyacomputerandvariedfrom-1oCI minto-100oCfmin.Nitrogengaswasalsoflowedontothecoling stagetopreventtheobservationwindowfromc1ouding.
2.2. Plantmaterials
Epidermaltisuesontheabaxialsurfaceoftheleavesofstraw- berygeranium(SaxifragastoloniferaCurtis)plantsobtainedfrom thecampusofKanagawaInstituteofTechnology，Atsugi，japan，were usedasspecimens.Theleaveswereremovedfromtheirleafstalk andplacedinadishfiledwithwatertopreventthemfromdrying.
Initialtrialstoinvestigatetheusefulnesofourcryomicrosopic systemforobservingintracelularfrezingbehaviorwerecaried outusingspecimensobtainedinearlysummer2007(Figs.2and3.)
5pecimenstoinvestigateintracelularfrezingbehaviorsinthe tis sues were obtained between late December 2014 and mid- january2015，andinearlyDecember2015.Thestrawberygera- niumplantsgrewatthecampusofKanagawaInstituteofTech-
nology，which is located in a humid subtropical region. The meterologicalstationatneighboringEbinacityrecordedanaverage airtemperatureof7oCandanaveragedaylengthof172h.
2.3. Experimentalmethod
Eachspecimenwascutintoa10m m2sectionandplacedontoa coverglaswithdistiledwatertopreventitfromdrying.Asecond coverglaswithsilicongreaseapliedaroundtheedgeswasthen placedoverthespecimen.Thecoverglaseswereplacedonthe colingstageandcoledfromroomtemperature(aproximately 22OC)tothetempe日 tureatwhichintracelularfrezingocured atcolingratesthatrangedfrom-1oC/minto-100oC/min.
Theshuterofthehigh-spedcamerawaspresedwhenhalfof thecels(aproximatelysevencels)withintheobservationarea hadexperiencedintracelularfrezing.Thus，intracelularfrezing ineachspecimenwascapturedat1000fpsfor2sbeforeandafter presingtheshuter.Theexperimentswererepeatedatleast10 timesateachcolingrate.
AlimageswereanalyzedusingImagej(v1.46)[26，28.] 2.4. Definitionofthedegre eofsuperco oling
Thetemperatureatwhichintracelularfrezingocured(百) wasdefinedusingthetemperaturewhentheshuterofthehigh- spedcamerawaspresed.Tiwasthenconvertedintothedegre ofsupercoling(Ts)，bysubtractingthetemperatureattherelease ofsupercolingfromthesolidificationpointofwater(0OC).Thus， theTswasdefinedasfolows:
主 N i n a g 口 W 口 e t 口 .1 / C r y o b i o 1 o g y 7 3 ρ 0 1 旬 2 0 - 2 9 2 1