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

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

Fan, D. (TU Delft Team Carlas Smith)
Cnossen, J.P. (TU Delft Team Carlas Smith)
Hung, S. (TU Delft Team Carlas Smith)
Kromm, D. (TU Delft Support Delft Center for Systems and Control)
Dekker, N.H. (TU Delft BN/Nynke Dekker Lab)
Verbiest, G.J. (TU Delft Dynamics of Micro and Nano Systems)
Smith, C.S. (TU Delft Team Carlas Smith)

2023

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.

PSF engineering
Single molecule localization microscopy
Super-resolution microscopy
TIRF microscopy
Total internal reflection fluorescence

http://resolver.tudelft.nl/uuid:68d24b7f-ad4a-49b8-abff-108f576a1db7

https://doi.org/10.1016/j.optcom.2023.129548

0030-4018

Optics Communications, 542

Institutional Repository

journal article

© 2023 D. Fan, J.P. Cnossen, S. Hung, D. Kromm, N.H. Dekker, G.J. Verbiest, C.S. Smith