This work describes design, construction and use of a two-photon scanning laser fluorescence microscope (TPSLFM) improved by adaptive optics (AO) compensation to enable deeper subsurface imaging of mouse bone marrow. AO is found to be useful to compensate for degradation of image quality, particularly in deep tissue imaging where optical aberrations degrade TPSLFM resolution and contrast. An adaptive optics (AO) TPSLFM was developed to compensate the optical aberrations in the beam path to improve contrast and resolution in the mouse bone marrow subsurface imaging. The AO system relies on a deformable mirror (DM), which is controlled by using a stochastic parallel gradient descent (SPGD) algorithm optimized by feedback from a fluorescence sensor. It was demonstrated that AO allows 80% increase in fluorescence signal intensity from bone cavities 145mm below the surface. The AO-enhanced microscope provides cellular level images of mouse bone marrow at depths exceeding those achievable without AO.

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