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BLG 151 - Chapter 2 - Microscopy.docx

BLG 151 - Chapter 2 - Microscopy.docx - Chapter 2...

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Chapter 2 Microscopy 1.2 Optics and Properties of light Lenses focus light rays at a specific place called the focal point, F . The distance between the center of the lens and the focal point is the focal length, f . The strength of the lens is determined by the focal length, whereby, the shorter the focal length, the greater the magnification Lenses are used to observe a slide with a specimen. The distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus is the working distance The working distance affects the Numerical Aperture – ½ the angle of the cone of light = θ. The larger the working distance the smaller the angle, the smaller the working distance the bigger the angle o The smaller the working distance, the greater the magnification o The larger the angle the greater the magnification Note that magnification is not always better if it there is no resolution o Magnification – enlargement of an image o Resolution – ability of a lens to separate or distinguish small objects that are close together. Without resolution, it is pointless to have magnification Resolution depends on the wavelength of light used and the numerical aperture (the ability of the lens to gather light) The shorter (amplitude) the wavelength, the greater the resolution o Resolution limit : is reached when additional magnification does not separate further detail. The result is simply an enlarged blurry image. When two waves interfere, they can either be in sync which transmits bright light or they can overlap (not be in sync) and transmit dim light The movement of waves cannot be controlled so when waves that meet overlap the dim light enables resolution. Hence why a resolution limit exists. Immersion oil allows light rays that did not enter the objective due to reflection and refraction at the surfaces of the objective lens and slide to now enter o The result is an increase in numerical aperture and therefore resolution 1.3 Light microscopes (stains) There are 4 different types of light microscopes: 1. Bright field microscopy: simple white light illumination Produces a dark image against a brighter background Commonly used to observe stained preparations Parfocal microscope – remain in focus when objectives are changed 2. Phase-contrast microscopy: phase-shifting of wavelengths due to differences in relative thickness and density of the organelles of the cell Converts differences in refractive index/cell density into detected variations in light intensity Excellent way to observe living cells (no need to stain) 3. Differential-contrast interference (DIC or Normanki imaging): phase-shifting and interference of polarized light 4. Dark-field microscopy: light is emitted at 90° to the lens and scattered by cell part
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Image is formed by light reflected or refracted by specimen Produces a bright image of the object against a dark background
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