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Magnification
How much larger an object appears, compared to it's real size
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Resolving power/Resolution
Minimum separation at which two points can be distinguished as separate
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Equation for determining resolving power
d (Resolving power) = 0.5λ/(Numerical Aperture)
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Which power of ten is pico-
10^-12
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Maximum useful magnification and resolution of light microscopy
- 1000-2000X
- 0.2μm (Large organelles and bacteria)
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Purpose of staining
Provide contrast, increase visibility, accentuate features, preservation
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Fixation
- Preserving the internal and external structures of cells (and keeping them in place)
- Denatures enzymes and toughens structuers
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Types of fixation
Heat fixation, Chemical (Formaldehyde, alcohols i.e methanol etc)
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When is chemical fixation used?
- On larger, more complicated cells
- Eukaryotes
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Two parts of a staining dye
- Chromophore - Conjugated double bonds provide colour
- Binding - Bind to cell components; Ionic, covalent or hydrophobic
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Difference between positive and negative staining
- Positive; Stains the cell directly
- Negative; Stains the background
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Groups of Ionic dyes
- Basic - Pos Chomophore. E.g Crystal violet, Safranin, Malachite green and Methylene blue
- Acidic - Neg Chromophore. E.g Eosin, Acid Fuchsin
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Developer of the Gram Stain
Christian Gram (1884)
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Types of staining
Gram Stain, Endospore Stain
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Microscopy methods requiring staining
Genreally, only brightfield
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Principle of phase contrast microscopy
Difference in refractive indices alter light intensities
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Uses of phase contrast microscopy
- No staining needed
- Studying motility
- Studying living cell characteristics (shape)
- Studying internal structures (endospores, inclusion bodies)
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How does Darkfield microscopy work (simply speaking)
Only light that is passed through the sample is observed. The background remains black/dark
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Uses of Darkfield
- Unstained cells
- Allows alteration of lighting to achieve better imaging
- Internal structures of large eukaryotes and motility
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What does DIC microscopy stand for?
- Differetial interference microscopy
- AKA. Nomarsky
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How does DIC microscopy work? (Simply)
- Uses polarized light to detect different refractive indices and thickness
- Brightly coloures and 3D-ish images
- Similar to phase contrast
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Uses of DIC microscopy
Ornate cell walls, endospores, granules, vacuoles, nuclei (eukaryotes)
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How does fluorescence microscopy work? (Simply)
- Single wavelength of UV excites molecules in the specimen
- Re-emitted, lower energy light is obseved
- Often uses fluorochromes
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DAPI
- 4' 6-diamino-2-phenylindole
- Fluorochrome
- Antibodies and DNA hybridization
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CSLM
Confocal scanning laser microscopy
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How does CSLM work? (Simply)
- Laser illuminates small part of specimen
- Pinhole collects light from v. small region
- Reduces interference problems of larger lenses
- Sharp images
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Advantages of CSLM
- Improves resolution to 1um
- Can measure distributions and concentrations of molecules
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Two photon microscopy
- Uses two red-light photons
- Greater penetration
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Resolution and magnification of Electron Microscopy
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Transmission Electron Microscopy
- Low penetration
- Study cell ultrastructurePreparation is lethal to specimens
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TEM Preparation
- Chemical Fixation
- Physical prep. - Thin sectioning
- Staining (Positive, Negative and Staining) - Osmic acid, permanganate, uranium salts
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Scanning electron microscopy
- Uses electron scattering
- Specimen coated in heavy metal
- 3D Image/Depth of field
- Resolution 7nm/15X - 10,000X
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Scanned probe microscopy
- Sharp probe used to poke surface
- Often uses single atom at tip
- Sample prep not needed
- 1,000,000,000X Magnification
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