Powell lens-driven flat-field TIRF illumination for quantitative super resolution imaging.

Single-molecule localization microscopy (SMLM) provides nanometer-scale resolution but suffers from inhomogeneous excitation under conventional Gaussian total internal reflection fluorescence (TIRF) illumination. Spatial intensity variations reduce photon counts, distort fluorophore blinking kinetics, and degrade localization precision, particularly at the periphery of the field of view, thereby limiting quantitative and large-field SMLM imaging. Here, we present a simple orthogonal Powell (OP) lens configuration that converts a Gaussian beam into a uniform two-dimensional flat-top profile while remaining confined within the supercritical angular domain for TIRF excitation. Compared with Gaussian illumination, OP illumination expands the effective imaging area by 18-fold and achieves a penetration depth of ~300 nm, consistent with near-critical angle TIRF theory. Using nuclear pore complexes as biological rulers, we demonstrate uniform photon counts and consistent localization precision (<3.4% variation) across peripheral regions. Furthermore, 3D reconstructions of microtubules confirm spatially uniform high-quality imaging across the entire field of view. The OP system is compact, low-cost, and easily assembled from off-the-shelf components, while its square-like illumination geometry improves intensity utilization. This approach provides a practical solution for achieving uniform, large-field TIRF-SMLM, thereby combining high performance with broad accessibility.