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