Three-dimensional localization microscopy with increased axial precision through TIRF angle modulation

• DOI: 10.1016/j.optcom.2023.129548
• Publication Date: 2023-05-09T00:46:49Z
• Authors: Daniel Fan; Jelmer Cnossen; Shih-Te Hung; Dimitri Kromm; Nynke H. Dekker; Gerard J. Verbiest; Carlas S. Smith
• Keywords: Point spread function; Reflection; Modulation (music); Fluorescence-lifetime imaging microscopy

To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial of microscopes however, limiting factor fluorescence imaging, correspondingly based single molecule localization microscopy (SMLM). Here, we present method to improve precision SMLM by combining point-spread-function engineering with total internal reflection (TIRF) fields decay lengths that vary within on-time fluorophore. Such time-varying illumination field intensity allows one extract additional location information from emitted photons. With this time varying approach, show improved two-fold over TIRF-based using astigmatic PSFs. We calculate theoretical gains for various imaging conditions via Cramér Rao Lower Bound (CRLB), commonly used metric compute best attainable SMLM.