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A microscope is a Complicated Lens

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

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  1. A microscope is a Complicated Lens • The microscope can be represented by a single convex lens.

  2. ........And a sample slide • Most biological samples are aqueous, that is, they consist mostly of water.

  3. Inside theMicroscope

  4. 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.

  5. 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.

  6. 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.

  7. Chromatic Aberration • When a lens has different focal planes for light of different colours, Chromatic Aberration results.

  8. So what ? • Confocals can optically section a sample and reconstruct it in a computer. • So where is the problem ?

  9. Aberration and Coma will Shift the Focus • Aberration and coma shift the apparent position of the focus • With the result that ....

  10. In a Confocal..... • The image vanishes or distorts.

  11. The Lens isn’t the only Culprit • Refractive index mismatches between the immersion medium and the sample will cause aberrations.

  12. Consider this.... • For example, deep imaging into an aqueous sample with an oil medium will cause spherical aberration.

  13. Compare that to..... • Water medium and an aqueous sample where there is no aberration.

  14. Or Even..... • Focus near the surface of the sample with an oil medium and an aqueous sample where there is very little aberration.

  15. Refractive Index Mismatches • Are a major cause of poor image quality. • Cause poor resolution. • Cause secondary reflections at the sample.

  16. And that’s bad ! • Not even the best Confocal can compensate for that !!

  17. The Solution is...... • Use the correct immersion medium for the lens. • Don’t mix products from different manufacturers. • Keep the lens clean

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