Non-linear imaging is widely used in biological imaging, primarily because of its ability to image
through tissue to depth of a few hundred micrometers. Because two photons need to be
absorbed to excite a fluorophore in this instrument, the probability of fluorescence emission of a
detectable photon scales with the intensity squared of the beam. As a result, aberrations in the
beam path that reduce the peak intensity of the focused, scanned laser spot have a significant
effect on the instrument performance. Methods for reducing those aberrations should allow higher
resolution and detection sensitivity, and deeper tissue imaging.
In this paper, I will describe a non-linear imaging microscope that has an adaptive optics (AO)
subsystem to compensate for beam path aberrations. The AO system relies on a 140 actuator
deformable mirror, controlled using a stochastic gradient descent algorithm with feedback from a
fluorescence sensor. The controlled instrument will be used for in vivo imaging of mouse skin,
lymph nodes, and skull bone marrow at depths up to 500 &mgr;m.