Abstract
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.
Highlights
The acquisition of high-resolution images of 3D biological samples by optical microscopes requires the production of tightly focused spots, which are usually achieved by high numerical aperture (NA) immersion objectives
In our calculations we have considered a light microscope equipped with an immersion objective of NA = 1.4, and a specimen whose refractive index is close to n2 = 1.33, which is the index of the water solution where the specimen is embedded
We have presented a hybrid technique for the reduction of impact of spherical aberration induced in the axial scanning inherent to microscopy observations
Summary
The acquisition of high-resolution images of 3D biological samples by optical microscopes requires the production of tightly focused spots, which are usually achieved by high numerical aperture (NA) immersion objectives. In this paper we take advantage of the fact that the axial values of the intensity spot obtained after focusing, through a plane interface, the light proceeding from a high-NA objective is proportional to the Fresnel transform of a properly mapped version of the objective aperture stop This fact allows us to adapt the wavefront coding technique to the aim of reducing the impact of the SA induced during the axial scanning of the sample. The hybrid technique we report here 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 with the purpose of obtaining an improved recovery of the specimen structure when imaging thick samples
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