raster image correlation spectroscopy
DESCRIPTION
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. Raster Imaging. I. t. How to do RICS. Spatial autocorrelation:. Fitting Function. - PowerPoint PPT PresentationTRANSCRIPT
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
Co
rre
latio
n fu
nct
ion
(ms)
𝐺 (𝜏 )=⟨𝛿𝐼 (𝑡 )𝛿 𝐼 (𝑡+𝜏 ) ⟩
⟨ 𝐼 (𝑡 ) ⟩2
Raster Imaging
I
t
How to do RICS
𝜺
𝝋
𝐺𝑆 (𝜀 ,𝜑 )=⟨ 𝐼 (𝑥 , 𝑦 ) 𝐼 (𝑥+𝜀 , 𝑦+𝜑 ) ⟩𝑥 ,𝑦
⟨ 𝐼 (𝑥 , 𝑦 ) ⟩𝑥, 𝑦2
Spatial autocorrelation:
𝐺𝑆 (𝜀 ,𝜑 )=𝑆 (𝜀 ,𝜑 )×𝐺 (𝜀 ,𝜑 )
𝑆 (𝜀 ,𝜑 )=𝑒𝑥𝑝(−12 [( 2 𝜀𝛿𝑟
𝜔0)
2](1+
4𝐷 (𝜏 𝑝𝜀+𝜏 𝑙𝜑 )𝜔0
2 ))
Fitting Function
Diffusive Component:
Scanning Component:
𝜀
𝜑
𝐺(𝜀
)𝐺
(𝜑)
What could go wrong
Scan too slow
Undersampling
Phototube shot noise is time correlated!!!
What could go wrong
Immobile 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 area
100 nM EGFP