Lect2-KR - Observing and Counting Bacteria PMB/MCB112...

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Observing and Counting Bacteria PMB/MCB112 August 30, 2010
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Bacteria are small. Typical bacteria are 1-10 μm long and 0.3-1 μm wide. Eukaryotic cells have a wide range of sizes: 10 μm in diameter (yeast) 10 μm wide and >100 μm long (skeletal muscle) up to 1 m long (neuronal axons)
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Bright-field microscopy Magnification is theoretically infinite, but resolution is limited by the wavelength of light used to capture the image, and by the light-gathering ability of the lenses. For a good microscope using visible light, the resolution limit is ~0.2 μm or 200 nm.
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Total magnification = product of magnification by objective lens (10-100x) and oculars (10x) Resolving power (resolution) is the smallest distance between two objects that allows them to be seen as distinct objects Resolution is a function of the wavelength of light used to create the image and the light-gathering ability of the objective, or its numerical aperture (NA) R=λ/2NA If we use green light (540 nm) to illuminate a sample with a high-resolution objective (NA=1.4), the resolution limit is ~200 nm.
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Air Oil Coverslip Specimen in liquid Light bends (is refracted) when it exits the glass coverslip and enters the air, because air has a lower index of refraction than glass or water. Immersion oil is has an index of refraction similar to glass, so more light rays pass into the objective, rather than being bent out of the field of view. In mathematical terms, this increases the NA of the lens, so that R is smaller. Oil-immersion lens
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Lect2-KR - Observing and Counting Bacteria PMB/MCB112...

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