Aberration on excitation focal spot caused by oblique interface with refractive indices discontinuous and its correction with pure-phase compensation for laser scanning microscopy.

The interface of mediums with refractive indices discontinuous, for example air-glass and glass-water, are inevitable in microscopic imaging. In this work, the aberration of oblique interface with refractive index discontinuous on the laser scanning microscope was investigated theoretically with numerical simulations. It was found that the position, shape and FWHM of focal spots, were all significantly affected by the aberration due to oblique interface. The aberration can cause serious shifting of focal spots in the axial direction of beam during z -scanning and lead to an inaccurate reconstruction of three-dimensional (3D) targets. The aberration can also lead to a decreasing spatial resolution. To correct the influence of the aberration, a pure-phase modulation method has been proposed. By applying a phase compensation map into a spatial light modulator (SLM), the oblique interface aberration had been corrected experimentally in a laser scanning microscope. We hope this research can attract the attention of researchers when using scanning microscope, especially for reconstructing 3D biological and material structures.
(© 2021 Royal Microscopical Society.)