Module 3 Microscopy.docx - 3.1 Microscopy Microscope is...

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3.1 MicroscopyMicroscope is perhaps most important resource for studying biology at microscopic levelMicrometer (µm) is one-millionth of a meter and is commonly designated as 10-6mNanometer (nm) equals 10-9m or one-billionth of a meter Unaided eye can resolve (see clearly) objects typically >100µmoHowever, cellular components, organelles, etc. can be as small as 0.2µm in diameterTwo critical factors influence ability to see an object: the resolution and the contrastoResolution refers to distance between two objects at which objects still can be seen as separateThus, the closer two objects are to each other, the greater the resolution requirement will be to keep viewing two objects as separateoContrast is difference in light absorbance between two areas (objects)The lower the contrast between an object and its background, the harder it will be to see that objectThe greater the contrast between two areas, the easier it will be to visualizeType of microscope largely influences the power of resolution and the degree of contrastBright field MicroscopeBright field microscopesare simplest form of light, or optical, microscopesoLight, most often emitted from standard halogen bulb, enters microscope from base (bottom) and is reflected via mirrors towards sampleBefore light reaches sample, it first passes through a condenser converging light beams into a focused area on sampleoIris diaphragm controls amount of light that passes through sample and into objective lensObjective lens closest to sample and yields greatest amount of magnificationNote: degree of magnification is directly proportional to amount of light needed therefore to image samples clearly at higher magnifications, more light is neededoOnce light passes through sample and objective lens, it is directed through ocularlens, or eyepiece, to your eyesMost common power of ocular lens is 10xFor microscope using two lenses (objective and ocular), total magnification of specimen is multiplicativeoTherefore, 40x objective and 10x ocular result in total magnification of 400x
Figure 3.1Knowing the Microscope.The primary components of a compound microscope, often used for simple bright field microscopy are labeled accordingly.To visualize cells, samples are often stained with specific combinations of dyes that are taken up by celloStaining is often needed due to limitation of resolution on unstained cells because flat and transparent regions of cell may appear invisible under bright field conditionsoBy staining cell with various dyes, these regions can become labeled and therefore visualizedHowever, staining typically requires fixing the cells by heat or chemical methods before adding dyeCell fixation process produces its own challenges as it kills and may even distort sample
Figure 3.2 Brightfield Imaging.

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