A microscope is a Complicated Lens n The microscope can be represented by a single convex lens.
And a sample slide n Most biological samples are aqueous, that is, they consist mostly of water.
Inside the Microscope
Lenses are not Perfect n Microscope lenses have imperfections. n These imperfections cause Coma, Spherical aberration and Chromatic aberration. n A little bit of aberration doesn’t matter much with normal microscopy but it is extremely important to a confocal.
Coma n When light passing obliquely through a lens has a different focus to that which is along the lens axis, Coma results. n It causes distortions.
Spherical Aberration n When light passing near the centre of a lens has a different focal plane to that of light passing through its edge, Spherical Aberration results.
Chromatic Aberration n When a lens has different focal planes for light of different colours, Chromatic Aberration results.
So what ? n Confocals can optically section a sample and reconstruct it in a computer. n So where is the problem ?
Aberration and Coma will Shift the Focus n Aberration and coma shift the apparent position of the focus n With the result that....
In a Confocal..... n The image vanishes or distorts.
The Lens isn’t the only Culprit n Refractive index mismatches between the immersion medium and the sample will cause aberrations.
Consider this.... n For example, deep imaging into an aqueous sample with an oil medium will cause spherical aberration.
Compare that to..... n Water medium and an aqueous sample where there is no aberration.
Or Even..... n Focus near the surface of the sample with an oil medium and an aqueous sample where there is very little aberration.
Refractive Index Mismatches n Are a major cause of poor image quality. n Cause poor resolution. n Cause secondary reflections at the sample.
And that’s bad ! n Not even the best Confocal can compensate for that !!
The Solution is n Use the correct immersion medium for the lens. n Don’t mix products from different manufacturers. n Keep the lens clean