BLG 311 - Chapter 9 - Visualizing cells.docx

BLG 311 - Chapter 9 - Visualizing cells.docx - Chapter 9...

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Chapter 9 Visualizing cells 9.0 Imaging Tools Needed to Observe Cells Objective lens – collects a cone of light rays to create an image Condenser lens – focuses a cone of light rays onto each point of the specimen Detection: the ability for a microscope to visualize light signals from objects much smaller than 0.2 mm, including a single molecule. However, objects appear like they are 0.2 mm in size (due to diffraction). Magnification : visual enlargement of an object. There is no limit to magnification. Resolution : “visual” separation of the individual components of an object, which previously appeared as one; quality of the image. Resolution limit : is reached when additional magnification does not separate further detail. The result is simply an enlarged blurry image. resolutionlimit = 0.61 λ nsinθ o where ,NA = n sinθ λ = light wavelength in nm n = refractive index of medium between objective and sample (air or oil) θ = half angular width of cone of light collected by objective lens Resolution depends on wavelength: the shorter the wavelength, the greater the resolution Resolution depends on NA: the higher the NA, the greater the resolution o Air objectives n =1 and Oil objectives n>1.3 Magnification depends on NA: the higher the NA, the smaller the distance between the lens and sample and thus, the higher the magnification 9.1 Wave Properties of Light 1. Interference Wave interference is a phenomenon that occurs when two waves meet while traveling along the same medium. The interference of waves causes the medium to take on a shape that results from the net effect of the two individual waves upon the particles of the medium. Wave interference can be constructive or destructive in nature. Constructive interference occurs at any location along the medium where the two interfering waves have a displacement in the same direction (do not overlap; reinforce the effects of each other resulting in bright light). Destructive interference occurs at any location along the medium where the two interfering waves have a displacement in the opposite direction (overlap and cancel the effects of each other resulting in no light) o If two molecules are closer than 200 nm these two molecules appear to be 1 object 2. Diffraction – plays a role in the existence of a resolution limit. Diffraction involves a change in direction of waves as they pass through an opening or around a barrier in their path . This makes a single particle look like a blob
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3. Phase-shifting – phase-shifting of wavelengths due to differences in relative thickness and density of the organelles of the cell 4. Refraction involves a change in the direction of waves as they pass from one medium to another 5. Colour Specific organic dyes bind to specific biological molecules. This dye indicates presence and amount of biological molecule 9.2 Light Microscope There are 4 different types of light microscopes: 1.
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