Aberration correction for confocal imaging in refractive-index-mismatched media
Article first published online: 2 JUL 2004
Journal of Microscopy
Volume 192, Issue 2, pages 90–98, November 1998
How to Cite
Booth, M.J., Neil, M.A.A. and Wilson, T. (1998), Aberration correction for confocal imaging in refractive-index-mismatched media. Journal of Microscopy, 192: 90–98. doi: 10.1111/j.1365-2818.1998.99999.x
- Issue published online: 2 JUL 2004
- Article first published online: 2 JUL 2004
- Cited By
- Aberration correction;
- Confocal microscopy;
- Single photon fluorescence;
- Two photon fluorescence
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.