4. The Foerster energy transfer rate, kr, between donor and acceptor fluorophores was determined to be the following in 1946: 6 Ro -- ()* TD where to is the fluorescence decay time of the donor fluorophore, R is the actual distance from the donor absorption and acceptor emission electronic dipoles and R, is the critical distance (or Foerster Radius) at which the probability is that half of the excited state donor electrons will transfer their energy to the excited state of the acceptor. The critical distance (or Foerster Radius) can be calculated by 2108 [x²yn-47]1/0 Ro=0.2108 where is the orientational alignment factor between the donor absorption and acceptor emission electronic dipoles, D, is the fluorescence quantum yield of the donor, n is the refractive index of the media and J is the overlap integral or sum given by J = - FD(X)A(X)X²dX where Fo(A) is the wavelength dependent donor fluorescence, E(A) is the wavelength dependent acceptor absorption coefficients and A is the wavelength. For this exercise, please calculate the Foerster Radii for the following combinations of fluorophores for FRET pairs: Donor Cerulean (CFP) Acceptor mCitrine (Y pet) (YFP) To do this you will need to download the absorbance (or excitation) spectra and emission spectra of the fluorophores. You may find your own source, but it is suggested that you go to the Roger Tslen group website to download the fluorophore spectra. You will also need to obtain the fluorescence quantum yields of the donor fluorophores and the maximum absorption coefficients of the acceptor fluorophores. It will be fine to assume that the orientation factor will be random, which sets the = 2/3=0.6667, and that the refractive index will be that of water. You may use Mathematica, MATLAB, or even an MS Excel spreadsheet (heaven forbid!) to calculate these values. Please also note that the integration of Fo(A)dA is supposed to yield one. Thus, it might be necessary for you to determine the sum or integration of Fo(A)d over the spectral range and normalize the overlap integral J to this. It is important that you submit your work of your answers and notebook/workbook files/papers to show how you determined your answer.

Fig: 1