Correction of Spherical Aberration in Multi-focal Multiphoton Microscopy with Spatial Light Modulator

We demonstrate that high-quality images of the deep regions of a thick sample can be obtained from its surface by multi-focal multiphoton microscopy (MMM). The MMM system incorporates a spatial light modulator to separate the excitation beam into a multi-focal excitation beam and modulate the pre-distortion wavefront to correct spherical aberration (SA) caused by a refractive index mismatch between the immersion medium and the biological sample. When fluorescent beads in transparent epoxy resin were observed using four SA-corrected focal beams, the fluorescence signal of the obtained images was ~52 times higher than that obtained without SA correction until a depth of ~1100 μm, similar to the result for single-focal multiphoton microscopy (SMM). The MMM scanning time was four times less than that for SMM, and MMM showed an improved fluorescence intensity and depth resolution for an image of blood vessels in the brain of a mouse stained with a fluorescent dye.