The small correction volume for conventional wavefront shaping methods limits their application in biological imaging through scattering media. In this paper, we take advantage of conjugate adaptive optics (CAO) and remote focusing (CAORF) to achieve three-dimensional (3D) scanning through a scattering layer with a single correction. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We demonstrate two-photon imaging with CAORF through mouse skull. The fluorescent microspheres embedded under the scattering layers can be clearly observed after applying the correction.

We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

Author(s): Tao, Xiaodong; Lu, Ju; Lam, Tuwin; Rodriguez, Ramiro; Zuo, Yi; Kubby, Joel | Abstract: We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

We demonstrate non-invasive structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins in the living Drosophila brain at cellular and subcellular resolution using three-photon microscopy and wavefront correction.

The small correction volume for conventional wavefront shaping methods limits their applications in biological imaging through scattering media. We demonstrate large volume wavefront shaping through a scattering layer with a single correction by conjugate adaptive optics and remote focusing (CAORF). The remote focusing module can keep the conjugation between the AO and scattering layer during three-dimensional scanning. This new configuration provides a wider correction volume by the best utilization of the memory effect in a fast three-dimensional laser scanning microscope. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We also demonstrated three-dimensional fluorescence imaging and multi-spot patterning through a scattering layer.

We demonstrate transcutical structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins and calcium indicators in the living Drosophila brain with cellular and subcellular resolution.