Computational TIRF enables optical sectioning beyond the evanescent field for widefield fluorescence microscopy

Abstract

The resolving ability of widefield fluorescence microscopy is fundamentally limited by out-of-focus background owing to its low axial resolution, particularly for densely labeled biological samples. Although total internal reflection fluorescence (TIRF) microscopy provides strong near-surface sectioning, they are intrinsically restricted to shallow imaging depths. Here we present computational TIRF (cTIRF), a deep learning-based imaging modality that generates TIRF-like sectioned images directly from conventional widefield epifluorescence measurements without any optical modification. By integrating a physics-informed forward model into network training, cTIRF achieves effective background suppression and axial resolution enhancement while maintaining consistency with the measured widefield data. We demonstrate that cTIRF recovers near-surface structures with performance comparable to experimental TIRF, and further enables both single-frame and volumetric sectioned reconstruction in densely labeled samples where conventional TIRF fails. This work establishes cTIRF as a practical and deployable alternative to hardware-based optical sectioning in fluorescence microscopy, enabled by rapid adaptation to new imaging systems with minimal calibration data.