Widefield-excitation two-photon single-pixel microscopy without temporal-focusing

Abstract

Two-photon microscopy (2PM) constitutes a key-enabling technology for imaging in weakly scattering specimens, delivering high-contrast signals through the quadratic dependence of fluorescence on excitation intensity. Extending 2PM to widefield or scan-less formats is attractive for rapid functional imaging. To maintain axially localized excitation while illuminating large areas, many scan-less widefield-excitation 2PM implementations rely on temporal focusing (TF) schemes re-compressing ultrashort pulses only on the sample plane. Introducing significant optical and dispersive complexity, typically demanding sub-200-fs sources, careful pre-compensation, and high-numerical-aperture objectives to efficiently deliver pulsed-shaped excitation. In this work, the realization of a widefield-excitation two-photon single-pixel microscope operating without TF while employing standard focusing optics is shown. A digital micromirror device is used to project sequential patterns, and images are reconstructed from a single bucket detector. A scrambled Hadamard-based intensity-correlation pipeline is implemented. Excitation is provided by a 1064nm industrial-grade laser delivering 300-fs pulses, demonstrating robust image formation with pulse durations above those commonly used in scan-less widefield-excitation 2PM. High-contrast images are recovered up to 200 ×200 µm field of view in weakly turbid samples using moderate-numerical aperture optics. Partial depth-dependent rejection is demonstrated by taking advantage of the scrambled pattern’s diffractive effect. Overall, we enable TF-free, cost-effective, scalable single-pixel implementation for widefield 2PM.