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increaseinthedegreofsupercoling(Fig.6仁)，asdidthebright- nesdiferenceattheedgesoftheintracelularicecrystals(Fig.9E). Thephenomenonwherebytheinsideofaceldarkensduring intracelularfrezingisknownas“flashing"andiscausedbytheice crystalsinsidethecelinterferingwiththetransmisionoflight fromthelightsource[3，17].Therefore，itisconsideredthatagreater brightnesdiferenceindicatesgreaterinterferenceinthe'light transmision.Alternatively，itisalsoposiblethatadecreaseinthe brightnesdiferencewithanincreaseincolingrateindicatesthat theedgesoftheintracelularicecrystalsarebecomingthiner.To beterunderstandtheprocesesocuring，thecharacteristicgrain sizeattheedgesoftheintracelularicecrystalswasevaluated， whichwasfoundtodecreasewithanincreaseinthedegreof supercoling(Fig.10).1thaspreviouslybeenshownthatspendinga shortertimeinthemaximumicecrystalgenerationtemperature zone leads to the formation of finer ice crystals in fo od [8，15]. Althoughthepresentstudyexaminedgrainsizeattheedgesof intracelularicecrystals，thismayafectthesizeofintracelularice crystalsjustaftertheirformation.Anincreaseinthedegreof supercolingcausedtheintracelularicecrystalgrainstobefiner， leadingtoanincreaseddensityoficecrystalsinsideacel.lThus，itis consideredthatthedegreofceldeformationdecreasedwith increasedcolingratesduetotheformationof白nerintracelular icecrystals.
1tisknownthatrapidfrezingcanreducethefodquality deteriorationduetofrezing[12，18.]Thepresentstudyshowsthat anincreaseincolingratecandecreaseicecrystalsizeformed insidenotonlyfodbutalsocelsandsugeststhatadecreasein thedegreofceldeformationduetothiscontributestothefod qualitywithrapidfrezing.Thus，high-spedcameraimagespre- sentthenewknowledgeofIFbehaviortous.
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Ourfindingsalsosugestedthatthedeformationofaceldueto intracelularfrezingmayreleaseintracelularsupercolinginthe adjacentarea.SupplementaryVideo4showsanexamplewhere intracelularfrezingapearedtobecausedbyanindirectefect from the outside to the inside of the cel l through the plasma membrane.1nthisvideo，theicecrystalinsidetheceltotheright developsinaverticaldirectiontowardtheformationdirectionof theicecrystalinsidetheceltotheleft.Whensupercoledwaterin a plastic bot tle is given a mechanical shock from the outside， superco oling is released and the water fre ezes instantly. 1t is generalyknownthataninvasionoficenuc1eifromoutsidethecel toinsidecausesicenucleationinsideacel[24，31].Moreover，the view point of thermodynamics can predict that intracelular supercolingmaybereleasedduetoheterogeneousnuc1eationin thevicinityofaplasmamembraneororganele，orhomogeneous nuc1eationoftheintracelularliquid.However，thisstudyshowed thatanewmechanismofthereleaseofintracelularsupercoling thathadnotbeensupposedevermightbepresented.
Supplementaryvideorelatedtothisartic1ecanbefoundat htp:/dx.do.iorg/10.1016/j.cryobio.I2016.06.03.
Next，IFbehaviorinrandomlyselectedcelswasexamined. Althoughthesamplesizewassmal，thisaproachalowedusto examine the degre e of cel l deformation， the growing rate of intracelularicecrystals，andthecharacteristicgrainsizeatthe edgesofintracelularicecrystalsatdiferentcolingrates.
Thedegreofsupercolingincreasedwithanincreaseincoling rate，asdidtheintracelularicecrystalgrowingrate(Fig.7).1thas previouslybeenshownthataswaterfrezes，thegrowingrateat theedgeofadendriticicecrystalincreaseswithanincreaseinthe degreofsupercoling[1].Therefore，itislikelythattheincrease inthedegreofsupercolingthataccompaniedtheincreasein colingratecausedtheintracelularicecrystalgrowingrateto increase. Furthermore，itwas found that as the coling rate increased，therewasadecreaseintheproportionofcelsexhibiting formationpaternAandanincreaseinthoseexhibitingformation paternB，inwhichintracelularicecrystalsformedacrosthe centerofacel(Fig.
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