Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator

We demonstrate fluorescence imaging with high fluorescence intensity and depth resolution in which depth-induced spherical aberration (SA) caused by refractive-index mismatch between the medium and biological sample is corrected. To reduce the impact of SA, we incorporate a spatial light modulator into a two-photon excitation fluorescence microscope. Consequently, when fluorescent beads in epoxy resin were observed with this method of SA correction, the fluorescence signal of the observed images was similar to 27 times higher and extension in the direction of the optical axes was similar to 6.5 times shorter at a depth of similar to 890 mu m. Thus, the proposed method increases the depth observable at high resolution. Further, our results show that the method improved the fluorescence intensity of images of the fluorescent beads and the structure of a biological sample. (C) 2015 Optical Society of America