Three-dimensional Fluorescence Polarization Excitation via Multiview Imaging

This technology includes a method that extends fluorescence polarization imaging so that the dipole moment of a fluorescent dye may be excited regardless of its 3D orientation. By exciting the dipole from multiple directions, we ensure that excitation may occur even if the dipole is unfavorably oriented along the axial (propagation) axis. If the dye can be rigidly attached to the structure of interest, our method also enables the 3D orientation of the structure to be estimated accurately. This method can be advantageously applied in light-sheet microscopy, where two perpendicular objectives are used to alternately excite and detect fluorescence; each objective optimally excites dipoles oriented in their focal plane, but poorly along the optical axis. By using perpendicular objectives, the excitation may be polarized along the third spatial dimension that is normally poorly accessed with a single objective. Integrating the method into light sheet microscopy also opens the door to 4D polarization analysis with minimal photobleaching and photodamage.