raster image correlation spectroscopy

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