A CROSS BEAM EXCITATION GEOMETRY FOR LOCALIZATION MICROSCOPY.

Fluorescence microscopy is popular for its noninvasive properties and its use in imaging multiple species simultaneously (1). Furthermore, superresolution fluorescence localization microscopy methods such as fluorescence photoactivation localization microscopy (FPALM) utilize photoactivatable, photoswitchable, and photoconvertible fluorescent proteins to improve the lateral resolution of conventional fluorescence microscopy by an order of magnitude (2-4). However, our understanding of the effects of excitation light polarization coupled to the transition dipole orientation of fluorescent labels within the sample is incomplete. Specifically, the excitation rate of a given label is proportional to cos2θ, where θ is the angle between the excitation illumination polarization and the transition dipole moment of the label(1). Because of this relationship between the laser polarization and transition dipole orientation, using widefield circularly-polarized illumination, one cannot excite all possible fluorophore orientations simultaneously with equal efficiency. Since thresholds are commonly used in localization microscopy during the stage of identification of fluorophores, the number of localized molecules can also be reduced by such polarization effects. Here, we briefly present an experimental excitation geometry called cross-beam, which uses two lasers with similar wavelength to excite labels with any transition dipole orientation.