Raster Image Correlation Spectroscopy Seminario di relazione per la partecipazione a: 3rd European Workshop on Advanced Fluorescence Imaging and Dynamics (8-12 Ottobre 2012) Di Paolo Pozzi
Feb 22, 2016
Raster Image Correlation Spectroscopy
Seminario di relazione per la partecipazione a:3rd European Workshop on Advanced Fluorescence Imaging and Dynamics(8-12 Ottobre 2012)
Di Paolo Pozzi
FCS
1E-4 1E-3 0,01 0,1 1
0,0
0,2
0,4
0,6
0,8
1,0
Cor
rela
tion
func
tion
(ms)
πΊ (π )=β¨πΏπΌ (π‘ )πΏ πΌ (π‘+π ) β©
β¨ πΌ (π‘ ) β©2
Raster Imaging
I
t
How to do RICSπΊ
π
πΊπ (π ,π )=β¨ πΌ (π₯ , π¦ ) πΌ (π₯+π , π¦+π ) β©π₯ , π¦
β¨ πΌ (π₯ , π¦ ) β©π₯, π¦2
Spatial autocorrelation:
πΊπ (π ,π )=π (π ,π )ΓπΊ (π ,π )
π (π ,π )=ππ₯π(β12 [( 2 ππΏπ
π0 )2]
(1+4π· (π ππ+π ππ )
π02 ))
Fitting Function
Diffusive Component:
Scanning Component:
π
π
πΊ(π
)πΊ
(π)
What could go wrong
Scan too slow
Undersampling
Phototube shot noise is time correlated!!!
What could go wrongImmobile population:
Uniform concentration and uniform diffusion coefficient are needed for rics!!!
RICS profile
Immobile removal
Subtracting averange value Obtaining negative value
An arbitrary constant can be added, but the information is partially lost
Number of images and image area100 nM EGFP