Adjusting a Microscope

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Adjusting a Microscope

• Center components on optic axis
• Focus objective
• Focus condenser
• Adjust illumination
• lamp voltage (intensity)
• iris diaphragm

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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

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Light Microscopy Modifications

• Phase Contrast
• Differential Interference Contrast (Normarski)
• Confocal Scanning
• Fluorescence
• Dark Field (diffracted light)
• Image Enhancement

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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

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Light Microscopy Modifications

• Phase Contrast
• Differential Interference Contrast (Normarski)
• Confocal Scanning
• Fluorescence
• Dark Field (diffracted light)
• Image Enhancement

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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

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• 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

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Sample Preparation

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

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Variations of Electron Microscopy

• Transmission (TEM)
• Scanning (SEM)
• Shadow-casting
• Freeze-fracture
• Freeze-etching
• CryoEM
• Negative Staining

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