Lec10_FRAP_FCS_ML - A r e s p e c ific p r o te in s fix e...

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Unformatted text preview: A r e s p e c ific p r o te in s fix e d o r m o b ile in th e c e ll? F R A P – F lu o r e s c e n c e R e c o v e r y A fte r P h o to b le a c h in g S T U D Y IN G P R O T E IN D Y N A M IC S IN L IV IN G C E L L S ( 2 0 0 1 ) J e n n ife r L ip p in c o tt- S c h w a r tz , E r ik S n a p p & A n n e K e n w o r th y N a tu r e R e v ie w s M o le c u la r C e ll B io lo g y 2 , 4 4 4 - 4 5 6 1 A r e s p e c ific p r o te in s fix e d o r m o b ile in th e c e ll? S T U D Y IN G P R O T E IN D Y N A M IC S IN L IV IN G C E L L S ( 2 0 0 1 ) J e n n ife r L ip p in c o tt- S c h w a r tz , E r ik S n a p p & A n n e K e n w o r th y N a tu r e R e v ie w s M o le c u la r C e ll B io lo g y 2 , 4 4 4 - 4 5 6 2 1 Fluorescence Recovery After Photobleaching (FRAP) What does it tell us? O n a m e m b r a n e a w e ll d e fin e d r e g io n * is b le a c h e d a n d th e r e c o v e r y m o n ito r e d . T h e d a ta is fit to : Fo Im m e d ia te ly a fte r b le a c h F (t ) # ( $ ! ) n =+ F0 n! n =0 1 % 4 Dt & 1 + n '1 + 2 ( "r * ) a n d th e d iffu s io n c o e ffic ie n t is o b ta in e d . *a Gaussian intensity profile on the membrane is assumed (of course). “The paper” on single photon FPR: Axelrod, et al., (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 16:1055-1069, 1976. 3 Do we need the photobleaching or can we learn about diffusion without it? 4 2 We previously considered the noise in photon detection Light detection is the detection of photons, which is a statistical process. If for a given intensity a detector registers on average let’s say 100 photons in the detection time interval at a given intensity it will in most measurements not be e xactly 100 but may be 93, 106, 102, 89. How large are these statistical fluctuations? We found that the variance is equal to the mean photon count. 2 2 "N = m #"n = m # p = N ! M ic r o s c o p y - 2 B io p h y s ic a l M e th o d s S lid e 5 Fluorescence Correlation Spectroscopy (FCS) I ( r, z) = I0e" 2 r 2 2 2 2 # r " 2z # z e ! 6 3 Simplest cases– a single diffusion coefficient (Normal diffusion) 2D FC S 3D FC S $# &% 1 &% 2 % & % # "r $ ! &%1+ % & ( ' ' "z ( ! D $ & & & & & ( 1/ 2 " 1$ 1 G (! ) = $ Nv $ 1 + ! $! D & # % % % % ' # 1% 1 G (! ) = % Nv % 1 + ! %! D ' "D = !r2 4D τD = lateral diffusion time D = diffusion coefficient G(0) = 1 1 = NV Veff C Veff = " 3 2# r2# z ! 7 T h e a u to c o r r e la tio n fu n c tio n is Autocorrelation Function ( ACF ) G (" ) = #F ( t )#F ( t + " ) (#F (t )) 2 , #F ( t ) = F ( t ) $ Fmean , " = lag time This is calculated from digital data (counts/bin, where the fundamental “binsize” c a n b e a s s h o r t a s 1 2 n s ) . n i i s th e n u m b e r o f p h o to n s c o lle c te d in th e i th b in a n d ! j* b in s iz e i s th e la g tim e ( τ ) . N is th e to ta l n u m b e r o f b in s . N+ j G( j ) = "n i"n i + j N '2 1$ & # "n i ) N % i=1 ( N * # "n i * "n i + j , G( j ) = i= 1 $N '2 (N + j )&#"ni ) % i=1 ( , "n i = n i + n mean ! 8 4 F C S fittin g fu n c tio n s – in te r m s o f s h a p e , th e y a ll lo o k p r e tty m u c h th e s a m e a n d it c a n b e d iffic u lt to d is c r im in a te b e tw e e n m o d e ls . 1 0.8 0.6 0.4 1 0.8 0.6 0.4 0.2 0 0.001 0.01 0.1 1 10 1-comp. diff. 1-comp. diff. 0.2 0 0.001 0.01 0.1 1 10 0.5 1.8 1.6 0.4 0.3 0.2 0.1 0.01 0.1 1 triplet only (NO diffusion) 1.4 1.2 1 0.001 exponential 0.01 0.1 1 10 0 0.001 1 1.5 1 0.5 0 0.001 0.8 0.6 0.4 0.2 1-comp. diff. /1-comp. diff. + exponential 1-comp diff. + triplet 0.01 0.1 1 10 0 0.001 0.01 0.1 1 10 1 0.8 0.6 0.4 0.2 0 0.001 1 0.8 0.6 0.4 0.2 0 0.001 0.1 10 1000 1-comp. diff. / triplet 2-comp. diff. 0.01 0.1 1 10 9 F lu o r e s c e n c e c r o s s - c o r r e la tio n s p e c tr o s c o p y ( F C C S ) : 2 fo r m s : 1 . T w o - c o lo r c r o s s c o r r e la tio n – u s e s tw o d iffe r e n t d e te c to r s th a t c o lle c t th e e m is s io n fr o m tw o d iffe r e n t c o lo r flu o r o p h o r e s a n d th e n c r o s s - c o r r e la te s th e s ig n a l. G (" ) = ! FA (t ) ! FB (t + " ) FA (t ) FB (t ) 2 . S in g le c o lo r F C C S – tw o d e te c to r s a r e u s e d b u t o n ly o n e c o lo r flu o r o p h o r e . T h e e m is s io n is s p lit ( 5 0 /5 0 ) a n d s e n d to th e tw o d e te c to r s a n d c r o s s - c o r r e la te d . T h is c a n r e m o v e d e te c to r a r tifa c ts a r is in g fr o m a fte r - p u ls in g . 10 5 Dual Color Cross-Correlation: prospects for observing protein-protein interactions If the two labeled species (red and green) are not bound they move independently and do not generate a cross-correlation signal (black trace). If some fraction of the labeled species are bound together that fraction produces a crosscorrelation signal. From the G(0) values of the cross-correlation and autocorrelation the fraction of the total bound can be calculated for either species. a u to c o r re la tio n F lu o re s c e n c e B u rs t A n a ly s is (F B A ) It is also possible to record continuous streams of photon counting data which is analyzed to locate the photon bursts that occur as a molecule diffuses through the illumination volume or passes through a focused beam in a nanochannel. You can obtain: 1. The molecular brightness - number of photons per burst. 2. The burst width distribution (diffusional information). 3. The "waiting time distribution" (WTD) - the times between bursts (additional diffusional information). The molecular brightness is a more sensitive indicator of dimerization than diffusion alone. A factor of 2 increase the molecular weight changes the diffusion coefficient 21/3 = 1.2, but doubles the brightness. (t) G ab delay (ms) c ro s s -c o r re la tio n (t) G ab delay (ms) 11 12 6 ...
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This note was uploaded on 03/29/2009 for the course A&EP 470 taught by Professor Lindau during the Fall '08 term at Cornell University (Engineering School).

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Lec10_FRAP_FCS_ML - A r e s p e c ific p r o te in s fix e...

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