Lecture02-Principles of Microscopy and Microscope Anatomy

Lecture02-Principles of Microscopy and Microscope Anatomy -...

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Unformatted text preview: Microscope anatomy, image formation and resolution Ian Dobbie Buy this book for your lab: D.B. Murphy, "Fundamentals of light microscopy and electronic imaging", ISBN 0-471-25391-X Visit these websites: http://www.microscopyu.com http://www.olympusmicro.com Key points Basic understanding of refraction and diffraction, and properties of lenses Understanding of two different sets of conjugate planes, especially importance of objective back-focal plane Understanding of factors affecting image resolution What a microscope needs to do Magnify things Resolve points which are close together Collect as much light as possible (esp. for fluorescence) Do all of the above while introducing as little distortion as possible We need to understand the nature of light Image formation in the light microscope depends exclusively on the interactions of light with matter Diffraction: scattering of the incident illuminating light by the detailed substructure with the specimen Refraction: "bending" of light, by a lens, which causes scattered light to converge, to form an image Light as electromagnetic radiation How lenses work Refraction--the "bending", or change in the direction , of light Explaining refraction doesn't require the "wave" formalism, just the rays The speed of light depends on the medium through which light is propagating Refraction occurs when light rays travelling through one type of medium meet an interface with another type of medium The extent of refraction depends on the angle of incidence (Snell's law) air glass air glass Here the light ray is orthogonal to the interface Here the light ray is oblique to the interface light travels faster light travels slower shortest (distance) path is not taken path of least time is taken More dense materials have higher refractive indices: Air 1.0003 Water 1.33 Glycerin 1.47 Immerson Oil 1.515 (e.g.) Glass 1.52 Flint 1.66 Zircon 1.92 Diamond 2.42 Lead Sulfide 3.91 1 2 n 1 sin 1 = n 2 sin 2 n i = c/v i Lensing occurs when the interface is curved Positive (convex) lenses converge light rays. Light rays that would otherwise never meet (e.g. because they are parallel, or diverging) can now do so. Negative lenses (concave) diverge light rays Laser light passing through negative and positive lenses 1 2 1 2 n 1 sin 1 = n 2 sin 2 Image position and magnification depend on lens curvature (focal length) and on the physical distance from the object to the lens All simple lenses have associated aberrations Still may encounter: chromatic aberration on cheap microscopes (prism effect--but can be reduced by using monochromatic light), spherical aberration when imaging deep into samples (e.g. embryos, even when the objective is "corrected"), field curvature when using bright lenses for fluorescence (but this is not a problem if you're imaging cells only in the center of the field) More lens elements = better correction, but also possibly less light throughput...
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Lecture02-Principles of Microscopy and Microscope Anatomy -...

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