We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We
demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red
fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.
Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images
in deep tissue imaging. We introduce a direct wavefront sensing method using cellular structures labeled with fluorescent
proteins in tissues as guide-stars. As a non-invasive and high-speed method, it generalizes the direct wavefront sensing
method for adaptive optics microscopy. An adaptive optics confocal microscope using this method is demonstrated for
imaging of mouse brain tissue. The confocal images with and without correction are collected. The results show
increased image contrast and 3X improvement in the signal intensity for fixed mouse tissues at a depth of 70 μm. The
images of the dendrite and spines are much clearer after correction with improved contrast. The Strehl ratio is improved
from 0.29 to 0.96, a significant 3.3X improvement.
Recently, there has been a growing interest in deep tissue imaging for the study of neurons. Unfortunately, because of the
inhomogeneous refractive index of the tissue, the aberrations degrade the resolution and brightness of the final image.
In this paper, we describe an adaptive optics confocal fluorescence microscope (AOCFM) which can correct aberrations
based on direct wavefront measurements using a point source reference beacon and a Shack-Hartmann Wavefront Sensor
(SHWS). Mouse brain tissues with different thicknesses are tested. After correction, both the signal intensity and contrast
of the image are improved.