### GFP bleaching rate as a function of laser power and pixel time

EGFP has a molar extinction coefficient (

The percentage of molecules in the excited state at steady-state is k

For GFP, τ

k

For a pixel spot with radius 0,25 µm, a laser wavelength of 488 nm, and a laser power of 0,3 mW, k

Same calculation for Venus (YFP variant): Extinction coefficient = 92 200 L / (mole cm) = 1,53 x 10

*ε*) of 55 000 L/(mole * cm) = 9.13 x 10^{-21}m^{2}/molecule, which is also called its optical cross section (A_{m}).The percentage of molecules in the excited state at steady-state is k

_{a}/ (k_{a}* k_{f}), where k_{a}is the number of incoming photons per molecule and second from the laser, and k_{f}is the rate of return to the ground state, which is 1 / τ_{f}where τ_{f}is the average seconds in the excited state.For GFP, τ

_{f}is 3,3 ns, making k_{f}3,03 * 10^{8}molecules/s.k

_{a}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 A_{p}. 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, k_{a}= A_{m}* p * λ / (A_{p}* 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, k

_{a}= 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}m^{2}/molecule. λ= 515, τ_{f}= ?, ...Labels: bleaching, confocal, EGFP, GFP, laser, photons, venus