FRAP / FDAP in a dendrite
FRAP and FDAP
To track diffusion of proteins in a cell, one can tag proteins in a particular region in the cell, and see how they spread from the region. One way to "tag" proteins in a area of interest is to label protein with fluorophores and photo-bleach the fluorophores in the area using a high power laser. From the time course of the fluorescence recovery after the photo-bleaching (FRAP), one can measure the diffusion constant of the protein.One problem of this method is photo-bleaching of fluorophore produces free-radicals in the cell, causing some damages to the cell.
Another way is to tag proteins is photo-activation of photo-activatable fluorophores such as photo-activatable GFP (Fluorescence decay after photoactivation = FDAP). This method should cause less damages to the cell, and give higher signal-to-noise ratio.
FRAP / FDAP in a thin dendrite
When FDAP/FRAP methods are applied in a thin dendrite and whole section of the dendrite is photo-activated (or bleached), the fluorescence change (Δ F) can be expressed as a convolution of 1D-diffusion
Δ F(t, x) ~ t-1/2 exp(-x2/4dt)
with a square function with the length of the photo-bleaching area (-b/2 < x < b/2). This results the following equation:
Δ F(t, x) ~ 1/2 [erf{(x+b/2)/(4dt)1/2} - erf{(x-b/2)/(4dt)1/2}]
Thus, the time course of the fluorescence at the center of the bleaching region is given by
Δ F(t)/ F(0) = erf(b/(16dt)1/2)
Note that the shape of the curve is very differen from exponential curve.
FRAP/FDAP in spines
The single compartment model can be used when FRAP/FDAP methods are applied in spines. Because the diffusion inside a spine reaches equilibrium quickly and diffusion between spine and dendrite is much slower, the fluorescence change can be expressed as
dF / dt = - 1/τ F
or
Δ F ~ exp(-t/τ)
where τ is the diffusion coupling time constant between a spine and the parent dendrite. τ should be proportional to volume of spine (V), length of the neck and inverse the neck diameter.
To track diffusion of proteins in a cell, one can tag proteins in a particular region in the cell, and see how they spread from the region. One way to "tag" proteins in a area of interest is to label protein with fluorophores and photo-bleach the fluorophores in the area using a high power laser. From the time course of the fluorescence recovery after the photo-bleaching (FRAP), one can measure the diffusion constant of the protein.One problem of this method is photo-bleaching of fluorophore produces free-radicals in the cell, causing some damages to the cell.
Another way is to tag proteins is photo-activation of photo-activatable fluorophores such as photo-activatable GFP (Fluorescence decay after photoactivation = FDAP). This method should cause less damages to the cell, and give higher signal-to-noise ratio.
FRAP / FDAP in a thin dendrite
When FDAP/FRAP methods are applied in a thin dendrite and whole section of the dendrite is photo-activated (or bleached), the fluorescence change (Δ F) can be expressed as a convolution of 1D-diffusion
Δ F(t, x) ~ t-1/2 exp(-x2/4dt)
with a square function with the length of the photo-bleaching area (-b/2 < x < b/2). This results the following equation:
Δ F(t, x) ~ 1/2 [erf{(x+b/2)/(4dt)1/2} - erf{(x-b/2)/(4dt)1/2}]
Thus, the time course of the fluorescence at the center of the bleaching region is given by
Δ F(t)/ F(0) = erf(b/(16dt)1/2)
Note that the shape of the curve is very differen from exponential curve.
FRAP/FDAP in spines
The single compartment model can be used when FRAP/FDAP methods are applied in spines. Because the diffusion inside a spine reaches equilibrium quickly and diffusion between spine and dendrite is much slower, the fluorescence change can be expressed as
dF / dt = - 1/τ F
or
Δ F ~ exp(-t/τ)
where τ is the diffusion coupling time constant between a spine and the parent dendrite. τ should be proportional to volume of spine (V), length of the neck and inverse the neck diameter.
posted by Ryohei at 9:46 PM
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