Scanning stereomicroscopy with two-photon excitation and scanned Bessel beams

Bessel beams have been used in many applications due to their unique optical properties of maintaining their intensity profiles unchanged during propagation. In imaging applications, Bessel beams have been successfully used to provide extended focuses for volumetric imaging and uniformed illumination plane in light-sheet microscopy. Coupled with two-photon excitation, Bessel beams have been successfully used in realizing fluorescence projected volumetric imaging. We demonstrated previously a stereoscopic solution-two-photon fluorescence stereomicroscopy (TPFSM)-for recovering the depth information in volumetric imaging with Bessel beams. In TPFSM, tilted Bessel beams were used to generate stereoscopic images on a laser scanning two-photon fluorescence microscope; upon post image processing we could successfully provide 3D perception of acquired volume images by wearing anaglyph 3D glasses. However, tilted Bessel beams were generated by shifting either an axicon or an objective laterally; the slow imaging speed and severe aberrations made it hard to use in real-time volume imaging. In this article, we report recent improvements of TPFSM with newly designed scanner and imaging software, which allows 3D stereoscopic imaging without moving any of the optical components on the setup. This improvement has dramatically improved focusing qualities and imaging speed so that the TPFSM can be performed potentially in real-time to provide 3D visualization in scattering media without post image processing.