GFP bleaching rate as a function of laser power and pixel time
EGFP has a molar extinction coefficient (ε) of 55 000 L/(mole * cm) = 9.13 x 10-21 m2/molecule, which is also called its optical cross section (Am).
The percentage of molecules in the excited state at steady-state is ka / (ka * kf), where ka is the number of incoming photons per molecule and second from the laser, and kf is the rate of return to the ground state, which is 1 / τf where τf is the average seconds in the excited state.
For GFP, τf is 3,3 ns, making kf 3,03 * 108 molecules/s.
ka is the optical cross section times the number of photons per square meter and second. The number of photons per square meter and second is the the number of photons per second divided by the pixel area Ap. The number of photons per second is the laser power (p) in Watts (=J/s) divided by the photon energy in J. The photon energy is h c / λ, where h is Planck's constant, c is the speed of light, and λ is the light wavelength. So, ka = Am * p * λ / (Ap * h * c).
For a pixel spot with radius 0,25 µm, a laser wavelength of 488 nm, and a laser power of 0,3 mW, ka = 34 million photons / molecule and second, and x = 10%. At this rate there should not be much bleaching, especially considering that only about 22 photons will reach a molecule during a pixel time of 0,64 µs (the fastest on our confocal mic).
Same calculation for Venus (YFP variant): Extinction coefficient = 92 200 L / (mole cm) = 1,53 x 10-20 m2/molecule. λ= 515, τf = ?, ...
The percentage of molecules in the excited state at steady-state is ka / (ka * kf), where ka is the number of incoming photons per molecule and second from the laser, and kf is the rate of return to the ground state, which is 1 / τf where τf is the average seconds in the excited state.
For GFP, τf is 3,3 ns, making kf 3,03 * 108 molecules/s.
ka is the optical cross section times the number of photons per square meter and second. The number of photons per square meter and second is the the number of photons per second divided by the pixel area Ap. The number of photons per second is the laser power (p) in Watts (=J/s) divided by the photon energy in J. The photon energy is h c / λ, where h is Planck's constant, c is the speed of light, and λ is the light wavelength. So, ka = Am * p * λ / (Ap * h * c).
For a pixel spot with radius 0,25 µm, a laser wavelength of 488 nm, and a laser power of 0,3 mW, ka = 34 million photons / molecule and second, and x = 10%. At this rate there should not be much bleaching, especially considering that only about 22 photons will reach a molecule during a pixel time of 0,64 µs (the fastest on our confocal mic).
Same calculation for Venus (YFP variant): Extinction coefficient = 92 200 L / (mole cm) = 1,53 x 10-20 m2/molecule. λ= 515, τf = ?, ...
Labels: bleaching, confocal, EGFP, GFP, laser, photons, venus
posted by Erland Lewin at 16:36
2 Comments:
I recently came accross your blog and have been reading along. I thought I would leave my first comment. I dont know what to say except that I have enjoyed reading. Nice blog. I will keep visiting this blog very often.
Joannah
http://2gbmemory.net
Hm.. can we say that GFP bleaching of regions in a cell follows the diffusion equation (dp/dt = D grad^2 p) where p is the density? I'm looking at my physical chemistry notes on this topic and it looks like a connection.
http://www.diyeconomist.net
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