Systems Biology (Methods in Molecular Biology)

depends on the dynamics of the molecules moving in the collected

image series. In particular, the correlation function is defined as

GðÞ¼ξ;χ;τ

hiIxðÞ;y;tIxðÞþξ;yþχ;tþτ

hiIxðÞ;y;t

 1 ð 1 Þ

whereI(x,y,t) represents the measured fluorescence intensity in

the positionx,yand at timet,ξandχrepresent the distance in the

xandydirections respectively,τrepresents the time lag, andh...i

represents the average. This function can be transformed into

GðÞ¼ξ;χ;τ

1

N

pðÞξ;χ;τ

O

WðÞξ;χ, ð 2 Þ

where “N” represents the average number of molecules in the

observation area,

N

represents the convolution operation in

space, andW(ξ,χ) represents the autocorrelation of the instrumen-

tal waist, the so-called Point Spread Function (PSF) generally well

approximated by a Gaussian function. Finally,p(ξ,χ,τ) represents

the probability to find a molecule at a distanceξandχafter a time

delayτ. If we consider a diffusive dynamics, in which molecules

move randomly in all directions and net fluxes are not present,p

(ξ,χ,τ) is also well approximated by a Gaussian function

GðÞ¼χ;ξ;τ gðÞτ exp 

χ^2 þξ^2

σ^2 ðÞτ



, ð 3 Þ

where the variance (σ^2 (τ)) can be identified as the average molecular

Mean Square Displacement directly derived from imaging, with no

need for molecular trajectories (iMSD).

2 Materials

2.1 Solutions and Gel 1. Agarose gel at 3% density (prepared from Agar) in TBE buffer.

2. Latex beads, yellow-green fluorescent, 30 nm size dissolved
   1:10 (v:v) in distilled water prior to the measurement.

2.2 Cell Manipulation 1. Cell line: Chinese Hamster Ovary (CHO)-K1 cells.

2. Humidified and thermostated incubator for cell maintenance.


3. Medium for cell maintenance: DMEM-F12 supplemented with
   10% Fetal Bovine Serum (FBS).


4. Medium for cell transfection: DMEM-F12 with no FBS.


5. 100 mm Petri dish for cell maintenance and 35 mm Glass-
   bottom Petri Dishes for optical microscopy.


6. Lipofectamine 2000 Transfection Reagent. Store at 4C.


7. Trypsin from bovine pancreas.

280 Francesco Cardarelli