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Adjusting a Microscope

resolution  contrast. brightness. Adjusting a Microscope. Center components on optic axis Focus objective Focus condenser Adjust illumination lamp voltage (intensity) iris diaphragm. Sample Preparation.  fixation organic solvents (acetone, alcohols) formaldehyde glutaraldehyde

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Adjusting a Microscope

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  1. resolution  contrast brightness Adjusting a Microscope • Center components on optic axis • Focus objective • Focus condenser • Adjust illumination • lamp voltage (intensity) • iris diaphragm

  2. Sample Preparation •  fixation • organic solvents (acetone, alcohols) • formaldehyde • glutaraldehyde •  sectioning • for tissues or thick samples • embed in parrafin or resin • cut with microtome •  staining • dyes differentially bind to DNA, RNA and protein • provides more contrast

  3. Light Microscopy Modifications • Phase Contrast • Differential Interference Contrast (Normarski) • Confocal Scanning • Fluorescence • Dark Field (diffracted light) • Image Enhancement

  4. Phase Contrast and Differential Interference Contrast • requires special objective and condenser lens • phase differences are converted into intensity differences • distinguish objects that only differ slightly in refractive index or thickness

  5. Light Microscopy Modifications • Phase Contrast • Differential Interference Contrast (Normarski) • Confocal Scanning • Fluorescence • Dark Field (diffracted light) • Image Enhancement

  6. resolution  contrast brightness Image Enhancement • video cameras + computers used to enhance images • correct imperfections in optical systems • overcome limitations of human eye • seeing image in dim light • seeing small intensity differences against bright background • does not increase actual resolution

  7. Limit of Resolution • distance at which two objects can be resolved • resolution limit = 0.61/numerical aperture • (NA is a lens property) •  of visible light = 0.4-0.8 mm • Electron Microscopy • samples are analyzed with electrons • particles traveling near the speed of light behave as a wave • wavelength  with  velocity • resolutions of 2 nm or less

  8. Sample Preparation • Fixation • glutaraldehyde • osmium tetroxide • Dehydration • ethanol (step-wise) • Embedding • plastic resins • Sectioning • ultramicrotome (50-100 nm thick) • Staining • heavy metals

  9. Variations of Electron Microscopy • Transmission (TEM) • Scanning (SEM) • Shadow-casting • Freeze-fracture • Freeze-etching • CryoEM • Negative Staining

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