生物单分子技术-荧光.pdf

生物单分子技术 Single-molecule techniques in biology －单分子三维定向 Single molecule 3D orientation Polarization of light /articles/polarized/polarizedintro.html 2 Molecular orientation Molecular dipole moment Fluorophore Water Each fluorophore has fixed within its molecular framework an absorption and emission transition dipole moment. 3 Polarization of light for fluorophores Illumination of randomly oriented fluorophores with plane-polarized light will preferentially excite only those molecules whose absorption transition dipole moments are aligned parallel (or nearly so) to the polarization electric vector plane 4 Polarization and Anisotropy Polarization P Anisotropy r Depolarization Local Motion Absorption dipole Emission dipole (t=0) as a probe attached covalently to a protein Emission dipole (t 0) 5 Orientation imaging by wide-field epifluorescence microscopy • Defocused Orientation and Position Imaging (DOPI) • Polarized Total Internal Reflection Fluorescence Microscopy (PolTIRFM) 6 Defocused orientation and position imaging (DOPI) The defocusing method proposed and experimentally demonstrated for an immersion mirror objective used for imaging within a cryostat at low temperature. (Chem. Phys. Lett. 273, 439–443 (1997); Chem. Phys. Lett.