1A183   & 1,.)0 # 

2%+ '45(-" /!$*6t  ¯T(+¦­ 0CH6G3 Ë¯Tvu!Ë¯Tsd.0CH6G
3+(Ë¦­Y²'¦­¾ ("a l Zr+¦­V º.µ+.¢.°+1)¯Tvu!¯TYn u']¼Tvu
Po+ rË¯Tsd!T½®']¼TvuPo %vup#pÀz j\Ë®ecf ik_.Ê{uX +« ),+$ÃYµp )¦­V T]¼90<A27.}´+  Ë”¯Tsd”=DCH8H )Ë`I¦­V º.) +  +« +¢!Ë©®¯T8G=2»(GFP).HeLa¦­Ë¦­MGFP ¯Tsd ¨k_·¦­MGFP ¯Tsd!k_Ë .) +
%ËGFP ¯Tsd l¿Æh^ p.° q É ¯Tsd0CH6G3!Ë?1B;FH5DAHUÈyÁ.Ë@:>G3 a B

A

Lifetime (ns)CB

CTime (min)

E

Time

Pixel numbersTn¶~X(+¯T±ª p.°|¯T±ª.4Y[ËY[,R )¯Tsd.£X+Ã4H;.+1)ÃYµÆh¯Tp!Ël¿Æh og+Tn¶~
X(+¯T±ªÆh ª.302)AELKpË,,.¡£  §  ¦­M¯Tsd0CH6G3 k_IO.Fig. 1BC¸!HeLa¦­tudor-GFP.& .+tudor-GFP!Ë`ÀN!ËGFP WÅN.b/Ç¤.&w|Ë¯TvuS!ËÇ¤¿Y +´,+(Fig.
1A)¸¹x!ËGFP ¯Tsd!Ç¤¿YÄQ¦­VI*Ë¯Tsd 2.0 ns ¦­2.5 ns ¦­-), ¦­M¯T
sd j\ËpÂm )¥100Yx!ËJ¬&¥3.2 ns$ sdj\.´+  )Ç¤¿Y ¯Tsd!¥2.0 nsIp*Ë¦­x ¯TsdS!ËÇ¤¿Y.³Y+

Lifetime (ns)(A) Intensity image(B) 10 min(C) 100 min

0 ns

2 ns

4 ns

6 ns

Fig. 1. Fluorescence intensity image of the GFP-fusion tudor-protein
in HeLa cells (A) and the corresponding fluorescence lifetime
images at 10 min and 100 min. Excitation wavelength was 450nm, and fluorescence in the 515–565 nm region was detected.

Fig. 2. Time course of the fluorescence
lifetime of the GFP-fusion tudor-protein
in HeLa cells in the region of the granule(A) and other cellular areas (B and C).
The positions at which the lifetimes areplotted are given in the fluorescenceintensity image of (D). The time courseof the distribution of the fluorescence
lifetime is shown in (E).

 )Fig. 2A–CHeLa¯·)wÚl )¼Szd²q^,¦aÚlFig. 2D¼S¡R¦)AÛ­ÚlÝBCÄ£¼Szd 2.0 ns
¯·U2.5 ns¯·U**y)BCÚl Ý¯·M¼Szd´% p[ÝwÔt '100XÝG¶$3 nsLEQ,¦) +)DÝÛ­ÏX¼Szd(Fig. 2A)Dw)Fig. 2EÝ256256¡RU)GFP¼SzdX(8350@>)²q^)Y#2.0 ns2.5 nsÞ9C/,¼SzdX Ý¯·MÓzdb§]Ý3.3 ns9C/,
Xq^)u + )LE&(ÝÛ­LrÏXGFP¼Szd Ý¯·Mp[) ±Å*)
Â*¼Szdq^Ý¯·M GFP H)¢ºhci o q^Çg)GFP Ý1C5<A;:46ÒÀ+*V¬UÏÝ¼S,¢)¢ºhXu(Fig. 3A) vj)Ì,¢ºhXuI¿É{´e°ÈÝXuUØ»X× ,HÕ )Ìq^)Ìq^,1C5 N)«U\¹ ½  #ÝGFP ¼S,¢) ¤'*)'«UÝci,_(i"-=7Ðm&Ýs }Øn vjÝ¢ºhXuÉ{´eÍ¨s Ù,F)µ'*)Ø»X×ËrÏØn&s Ù,`) ' 3,4)Ý¯·M¢ºhci}Ønq^ ÝÉ{´eËq^,ÃÇA)µ'*)
 GFP , PVA ÜXu¸Hk#Ê!Ý
B
GFP ¼SzdrÏØn\ÝxÖw,½Z(Fig. 3B–D)PVA ¸H GFP
¼Szd® 300 ps (Fig. 3B)rÏØn&¼Szd|Ý0.5 MV cm-1 rÏØnyCÝ¼Szd ® 0.3 %|)ÝrÏØn vj) vj ¼S¾³~(Fig. 3C)(Fig. 3D)G,W) D¯·Í¨Ý¼SzdQs® 50 %q^)¼Szdq^rÏØn 2 JO) 'Ý«UrÏØn s® 6 Pq^)Á©)  )GFP ¢ºhÉ{´eË ÝrÏØn&p[) + ±Ý¯·M¼Szdp[ Ý«UrÏØn ¯·M|Çg),¦f)¯·M« + Ìq^,Ç (ÝrÏØn | µ'*)¼Szd.
?C2B0Ý¯·UÆ,Ä )Ñ¿ÎT)µ)
1) HKÝXS¥ª 55 (2006) 31. 2) M. Tsushima et al., Rev.
Sci. Instrum. 75 (2004) 479. 3) T. Nakabayashi et al., J. Am.
Chem. Soc. 127 (2005) 7041. 4) N. Ohta, Bull. Chem. Soc. Jpn.
75 (2002) 1637.

Fig. 3. (A) Schematic structure of thechromophore of GFP. (B) Fluorescencedecay (solid line) of GFP in a PVA film atzero field, together with the simulated
curve (dotted line).
Excitation andmonitoring wavelengths were 440 and 515
nm, respectively. (C) The difference(solid line) between the decays observed
at 0.5 MV cm–1 and at zero field, together
with the simulated one (dotted line). (D)The ratio of the decay observed at 0.5 MV
cm–1 relative to that at zero field (solidline), together with the simulated one(dotted line).