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The25thlnternational秒mposiumonTran司portPhenomenα 5-7November2014，Krabi，Thailand
(Phantom V4.2，Vision Research) and the coled CCD came1'a(DXM1200C，Nikon)，thenormalCCDcame1'a.The colingwaspe1'fOlmedbyflowingliquidnitrogenintothe co oling stage，and co oling rate was made by compute1'- controlingthecombinationofliquidnitrogenandheater. Coling rate was va1'ied 企om lOC/min to 10oC/min. Nitrogengaswasflowedintothecolingstagetop1'event fogy on the observation window ofthe coling stage. Figure3showstheschematicofcryomicroscopicsystemin thisresearch.
PF
0.5
I A.IO .50 く>100
discusedtherelationshipbetwenthechangeofpHiand intracel lular fre ezing behaviors. Plant tis sues of Jerusalem artichoke(Helianthustuberosus)andonionbulb(Aliumcψα L.)wereobservedusingthecryomicroscopyequipedwith the high-spe ed camera and the co oled C C D camera. The co oling was can匂 d out 合 o m about 200 C to the temperature when intracel lular fre ezing oc culTed，varying co oling rate :fi'omlOC/minto10oC/min.ThevalueofpHiwasevaluated using fluo1'escence measurement. A pH-sensitive fluorescent dye ofBCECF-AM，which poseses the pH dependent fluorescenceattheexcitationwavelengthof500nmandno pHdependentfluorescenceat440nm(6)，wasused.ThepH value was calculated from the ratio ofthe fluorescence intensityateachwavelength.
Asaresult，independentofthecolingrate，pHishowed aproximately8.0befo1'ethestmiofcolingandd1'opped gradualywiththeprogresofcoling.Asthecolingrateis lower，thedegreofloweringinpHiwasmoredistinguished. ThisresultsugestedthathighcolingratecankeppHi :fi'omloweringandsubstantiatesthatrapidcolingleadsto thehighqualityp1'eservationforalivingplan.t
NOMENCLATURE
pHi CytoplasmicpH
Aex ExcitationWavelength(nm)
T Temperatu1'e(OC)
Specime.Q
Highsped ca汀lera
Liquid Nitrogen NitrogenGas
Ts
DegreeofSupercoling(OC)=Itempe1'atureatthe realeaseofsupercolingl Timeelapsedfromthestmiofcoling(s) Timeelapsedfromthestmiofintracelular frezing(s)
OLY1VIPUS，Model;IX71 PF:polarizingfilter，EF:excitationfilter，AF:analyzingfi1ter
Figu1'e3.Schematicofthecryomicroscopicsystem.
EXPERIMENTALPROCEDURES Tisue
Tube1' tisue of Jerusalem michoke (Helianthus tuberosus)andepidermaltisueofthebulbscaleofonion (AliumcepaL.)we1'eusedfo1'thisresea1'ch.
pHiMeasurement
For measurements of pHi，we used a pH四 sensitive
fluorescent dye of BCECF-AM，which have the most distinguished p H dependent fluorescence at the excitation wavelengthof500nmandnopHdependentfluorescenceat 440 nm as an isosbestic point. Figure 4 shows the relationship betwe en excited wavelength and fluorescence intensity.Takinguseofthischaracte1'istic，thepHvaluewas calculated:fi'omtheratioofthefluo1'escenceintensityateach wavelengthtop1'eventthefadingoffluorescenceintensity duetolapsedtime.
Themethodofdyeingspecimensusingthesefluo1'escence reagentsinthisresearchisasfolow;thespecimenwhichwas cut in 10 m m square was put in distil led water containing B C E C F - A M ( H e l i a n t h u s t u b e r o s u s : 3 μ M c o n c e n t 1' a t i o n
Vc Colingrate(OC/min)
ε DeformationDegreeoftheCel
EXPERIMENTALAPPARATUS
In this research，the cryomicroscopic system that
consistedoftheinvertedmicroscope(IX71，OLYMPUS) andthecolingstage(STC200，INSTEC)we1'eused.This c1'yomicroscopywasequipedwiththehighspedcamera