isoFLUX: three-dimensional modulation-enhanced localization microscopy with spatially invariant isotropic resolution

Single-molecule localization microscopy (SMLM) has pushed resolution to sub-40 nm.Combined with structured illumination, lateral resolution can be doubled or the axial resolution can be improved fourfold.However, current techniques are challenging in balancing the lateral and axial resolutions.Here, we report a new modulated illumination single-molecule localization modality, isoFLUX.Utilizing two objective lenses to form interference patterns along the x-z and y-z directions, the lateral and axial resolutions can be improved simultaneously.Compared to SMLM, isoFLUX maintains a twofold average enhancement in both lateral and axial resolutions under an astigmatic point spread function (PSF), 1.5-fold in the lateral resolution and 2.5-fold in the axial resolution under the saddle-point PSF.