Adaptive optics two-photon scanning laser fluorescence microscopy.

摘要

This dissertation describes design, characterization 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 145mum below the surface. The AO-enhanced microscope provides cellular level images of mouse bone marrow at depths exceeding those achievable without AO. This dissertation describes the AOTPSLFM that was designed, constructed and tested. A novel Zernike polynomial based SPGD algorithm is introduced, and it is demonstrated that this approach can be used to overcome signal to noise limitations that are observed with conventional SPGD algorithms applied to AOTPSLFM. The optical performance of the microscope is evaluated quantitatively, both with and without AO. Electromechanical models developed as part of this project are shown to improve the controllability of the DM. Finally, the improvement in microscope performance during deep tissue imaging is demonstrated and evaluated quantitatively.