Estrada, Gerardo

In vivo two photon microscopy generally requires a cranial window implant to aid in stabilization of the
brain. These windows introduce aberration into the optical excitation, due to the use of glass coverslips
and other media with different refractive index than that for which the microscope objective is designed.
These mismatches introduce spherical aberration which can have dramatic effects on the axial pointspread
function psf. We have developed a framework for computational simulations of typically used
configurations including up to five media interfaces, as a means to understand the impact of spherical
aberration, and to search for experimental solutions to correct for it. Our simulations suggest that
spherical aberration even from a single glass coverslip ~170 microns can introduce a substantial loss of
z-axis resolution, and blurring of signals from neighboring neural elements. We propose the use
of opposite refractive index mismatch to correct for spherical aberration. Our simulations suggest that
an appropriate magnitude of opposite refractive index can almost fully recover the ideal psf, and our
experimental tests support these simulations.

Two photon microscopy is one of the fastest growing methods of in-vivo imaging of the brain. It has the capability of imaging structures on the scale of 1μm. At this scale the wavelength of the imaging field (usually near infra-red), is comparable to the size of the structures being imaged, which makes the use of ray optics invalid. A better understanding is needed to predict the result of introducing different media into the light path. We use Wolf's integral, which is capable of fulfilling these needs without the shortcomings of ray optics. We predict the effects of aberrating media introduced into the light path like glass cover-slips and then correct the aberration using the same method. We also create a method to predict aberrations when the interfaces of the media in the light-path are not aligned with the propagation direction of the wavefront.