Aberration correction for confocal imaging in refractive‐index‐mismatched media

TLDR

Confocal microscopy of thick biological tissue suffers dramatic loss of signal and resolution when imaging deep into refractive‑index‑mismatched specimens. The study aims to overcome this limitation by measuring wavefront aberrations and pre‑shaping the input beam to cancel their effects. The authors image planar and point objects in brightfield, single‑photon, and two‑photon fluorescence using an oil‑immersion objective through varying water thicknesses, and correct the measured aberrations to pre‑shape the beam. They find that correcting only the first two or three orders of spherical aberration restores adequate signal and resolution up to 50–100 wavelengths deep, making finite‑sized pinholes sufficient.

Abstract

Summary A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive‐index‐mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre‐shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single‐photon fluorescence and two‐photon fluorescence imaging. In all cases, the specimens are imaged using an oil‐immersion objective through various thicknesses of water. The question of finite‐sized pinhole is addressed and it is found, in general, that it is sufficient to correct only the first two or three orders of spherical aberration to restore adequate image signal level and optical resolution, at imaging depths of up to 50‐100 wavelengths.