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