Abstract
We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.
Highlights
Multiphoton microscopy has become an important technique for imaging deep within biological tissue because of its selectivity to ballistic excitation photons in comparison to those that are scattered [1]
We have demonstrated the feasibility of conjugate Adaptive optics (AO) in a two-photon microscope configuration
As demonstrated previously in widefield microscopy, the compensated fields of view (FOV) achieved with conjugate AO in two-photon scanning microscopy is significantly larger than the corresponding compensated FOV achieved with pupil AO
Summary
Multiphoton microscopy has become an important technique for imaging deep within biological tissue because of its selectivity to ballistic excitation photons in comparison to those that are scattered [1]. Aberrations at the tissue interface or within the tissue itself lead to reduced confinement of the focused excitation spot This in turn diminishes signal intensity and limits achievable imaging depth. The FOV advantage of conjugate AO in microscopy applications has been studied using numerical simulations [27,29,30]. Our demonstration is restricted to the simplified geometry of 2D sample and well-defined interface aberrations located at a plane of known separation from the sample. As such, it is a proof of principle demonstration intended to explore some limitations of conjugate AO. We examine the axial range of conjugate AO correction, as a step toward generalization of its application to volumetric samples with axially distributed aberrations
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