Speckle illumination temporal focusing two-photon excited fluorescence microscopy

Two-photon microscopy is a widely used technique for three-dimensional deep-tissue imaging, relying on pixel-by-pixel scanning of a tightly focused laser beam and fluorescence detection with a bucket detector such as a photomultiplier tube. Wide-field two-photon microscopy implementations using cameras have the potential to outperform conventional point-scanning approaches in terms of field of view, imaging speed, and reduced phototoxicity. However, wide-field two-photon methods have faced major limitations, including poor axial sectioning, restricted field of view, and the risk of damaging objective lenses due to the high laser powers required for wide-field nonlinear excitation. Here, we introduce a wide-field two-photon microscope that overcomes these limitations by combining temporal focusing with speckle illumination and galvo-based beam scanning. In our approach, temporally focused speckle illumination is confined to a 50 µm elementary field of view that is rapidly scanned across the sample while fluorescence is recorded on a camera. The microscope achieves a 5 µm axial resolution and operates at 10 frames per second over a 300×300µm 2 field of view. We demonstrate multicolor two-photon imaging on a variety of samples, including fluorescent beads, cultured cells, Drosophila larval epidermis, and organoids. We show that speckle illumination not only protects the objective lens from damage but also provides superior axial sectioning in scattering media. Furthermore, it enables super-resolution operation in the random illumination microscopy (RIM) mode, in which temporally focused speckle images are acquired at successive translation steps of a static speckle pattern across a region of interest. By leveraging temporal focusing for efficient rejection of out-of-focus fluorescence, we demonstrate that temporally focused RIM in thick scattering samples yields higher contrast images than conventional two-photon RIM and achieves spatial resolution beyond the diffraction limit.