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
𝛿𝐼 𝑡 𝛿𝐼 𝑡 + 𝜏
𝐺 𝜏 =
𝐼 𝑡 2
1,0
Correlation function
0,8
0,6
0,4
0,2
0,0
1E-4
1E-3
0,01
 (ms)
0,1
1
Raster Imaging
I
t
How to do RICS
𝜺
Spatial autocorrelation:
𝐺𝑆 𝜀, 𝜑 =
𝝋
𝐼 𝑥, 𝑦 𝐼 𝑥 + 𝜀, 𝑦 + 𝜑
𝐼 𝑥, 𝑦
𝑥,𝑦
2
𝑥,𝑦
Fitting Function
𝐺 𝜀
𝐺𝑆 𝜀, 𝜑 = 𝑆 𝜀, 𝜑 × 𝐺 𝜀, 𝜑
Diffusive Component:
−1
4𝐷 𝜏𝑝 𝜀 + 𝜏𝑙 𝜑
𝛾
𝐺 𝜀, 𝜑 =
1+
𝑁
𝜔0 2
4𝐷 𝜏𝑝 𝜀 + 𝜏𝑙 𝜑
× 1+
𝜔𝑧 2
−1/2
𝜀
𝑆 𝜀, 𝜑 = 𝑒𝑥𝑝 −
1
2
1+
2 𝜀 𝛿𝑟
𝜔0
2
𝐺 𝜑
Scanning Component:
4𝐷 𝜏𝑝 𝜀 + 𝜏𝑙 𝜑
𝜔0 2
𝜑
What could go wrong
Scan too slow
Undersampling
Phototube shot noise is time correlated!!!
What could go wrong
Immobile population:
RICS profile
Uniform concentration and uniform diffusion coefficient are needed for rics!!!
Immobile removal
Subtracting averange value
Obtaining negative value
An arbitrary constant can be added, but the 𝐺0 information is partially lost
Number of images and image area
100 nM EGFP