Adaptive optics two-photon fluorescence microscopy

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 mu m.