Introduction The EosFP probe is a photo-activateable protein with two distinct states: the "inactive" green state where the molecule fluoresces green light (516nm) upon illumination of 488nm light, and the "active" red state where the molecule fluoresces red light (581nm) upon illumination of 556nm light. The molecule switches from the green to red state upon illumination of near UV light (~405nm) which breaks the Phe61 structure off the chromophore which is contained within the "can" shown in figure A below. The green state is shown in figure B and the red state is shown in figure C.   The two Figures below are important for choosing the various optics for successful detection of both colors of the EosFP probe.   The two color FPALM apparatus will be used to calculate quantum yeilds for the probe. A schematic of the apparatus is shown below  Purpose The purpose of the experiment is to calculate probabilities: given an incident 405nm photon on the EosFP protein, with what probability the protein will photoswitch from the green to red state. Also given an incident 488nm photon with what probability will the protein photobleach and stop fluorescing. Finally given an incident 556nm photon with what probability will the protein photobleach and stop fluorescing. Also analysis will search for a red state protein reversing to the green state. http://www.physics.umaine.edu/ Link to the Umaine Biophysics Website: http://www.physics.umaine.edu/research/BiophysicsGrp.htm Link to the FPALM Website http://www.physics.umaine.edu/FPALM_SFS/index.htm Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP Karin Nienhaus, G. Ulrich Nienhaus, Jorg Wiedenmann, and Herbert Nar. 9156–9159 PNAS June 28, 2005 vol. 102 no. 26 Photoconvertible Fluorescent Protein EosFP: Biophysical Properties and Cell Biology Applications G. Ulrich Nienhaus, Karin Nienhaus, Angela Holzle, Sergey Ivanchenko, Fabiana Renzi, Franz Oswald, Michael Wolff, Florian Schmitt, Carlheinz Rocker, Beatrice Vallone, Wolfgang Weidemann, Ralf Heilker, Herbert Nar and Jorg Wiedenmann Photochemistry and Photobiology, 2006, 82: 351–358 Below are the links to my proposal and progress report, please note the correction to the proposal in the progress report! |