Fluorescence Anisotropy Imaging

We constructed optical system and developed the software to create and analyze the fluorescence anisotropy images of cells, single molecules and nanoparticles. The system includes the inverted microscope (Olympus IX71), two calcite prisms (CPs) inserted in the excitation and emission optical paths, and a CCD camera (Retiga-SRV, Q-imaging Co.). The excitation light was passed through the pinhole diaphragm, split by CP into two orthogonally polarized beams and focused by objective into two spots. The emission CP splits the fluorescence image of each spot into two images formed by horizontally and vertically polarized emission lights. The CCD camera simultaneously records four images so the fluctuation in intensity of excitation light does not affect anisotropy values and, most importantly, that two polarized components corresponds exactly to the same state of a sample. We are using this system to study the “blinking” effect of quantum dots, binding of fluorescently labeled molecules to proteins, DNA, RNA in cells.

alt Fluorescence Anisotropy Imaging 

Design of the optical system for anisotropy fluorescence imaging