Reduction of spherical-aberration impact in microscopy by wavefront coding

In modern high-NA optical scanning instruments, like scanning microscopes, the refractive-index mismatch between the sample and the immersion medium introduces a significant amount of spherical aberration when imaging deep inside the specimen, spreading out the impulse response. Since such aberration depends on the focalization depth, it is not possible to achieve a static global compensation for the whole 3D sample in scanning microscopy. Therefore a depth-variant impulse response is generated. Consequently, the design of pupil elements that increase the tolerance to this aberration is of great interest. In this paper we report a hybrid technique that provides a focal spot that remains almost invariant in the depth-scanning processing of thick samples. This invariance allows the application of 3D deconvolution techniques to that provide an improved recovery of the specimen structure when imaging thick samples.