Three-dimensional localization microscopy with increased axial precision through TIRF angle modulation
Daniel Fan (TU Delft - Team Carlas Smith)
Jelmer Cnossen (TU Delft - Team Carlas Smith)
S. Hung (TU Delft - Team Carlas Smith)
Dimitri Kromm (TU Delft - Support Delft Center for Systems and Control)
N.H. Dekker (TU Delft - BN/Nynke Dekker Lab)
G.J. Verbiest (TU Delft - Dynamics of Micro and Nano Systems)
C.S. Smith (TU Delft - Team Carlas Smith)
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Abstract
To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present a method to improve the axial localization precision in SMLM by combining point-spread-function engineering with total internal reflection fluorescence (TIRF) fields with decay lengths that vary within the on-time of a fluorophore. Such time-varying illumination field intensity allows one to extract additional axial location information from the emitted photons. With this time varying illumination approach, we show that axial localization is improved two-fold over TIRF-based SMLM using astigmatic PSFs. We calculate theoretical resolution gains for various imaging conditions via the Cramér Rao Lower Bound (CRLB), a commonly used metric to compute the best attainable localization precision in SMLM.
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