CH2 Flashcards
objective X ocular
total magnification
ability to distinguish between two objects
limited resolving resolution
why use blue light (400-500 nm)?
- shortest wavelength
- to optimize resolution
Abbe’s equation for resolution
d= 0.5 λ / s sin Θ
light must pass between two objects for them to be seen as separate
λ
refractive index (light bending properties)
n
n of air and oil
air = 1.0 oil= 1.5
-visible light, glass lenses
-magnification: 1000x to 2000x
-resolving power: use immersion oil and blue light
=0.2μm (200nm)
light microscope
- e- beam, electromagnetic lenses (
-magnification: over 100,000x
-resolving power: e- beam (λ =.005nm)
=0.5nm
electron microscope
- dark image against brighter background
- cheap and easy to use
- poor, contrast, MOs transparent, staining skills
bright field- light microscope
- light image and background dark
- useful for motile living organisms
- cheap and fairly easy to use
- good contrast without staining
- anything living or not will appear (debris)
dark field- light microscope
- slight difference in thickness/density converted to variations in light intensity
- useful for living MOs, some internal structures
- more expensive/ more difficult to use
phase contrast- light microscope
Which would have better resolution red (620nm) or green (550nm)?
green bc shorter λ
- stain specific cell/components with fluorescent dye
- expose specimen to short λ light (UV, violet)
- view the longer λ fluorescent light
- background dark, MOs fluorescent
- specificity with antibodies linked dyes
fluorescent- light microscope
- e- beam passes through specimen
- view internal cell structures
- requires very thin sections/slices, cut with glass or diamond knife
- staining (heavy metals)
transmission e- microscope TEM
- e- beam scans the surface of microbe (scattered e- collected)
- provide 3-D view of surface (internal not shown)
- easier prep
scanning e- microscope SEM
preserve/stabilize, kill, adhere, take up stain
fixation (heat/chemical)
two types of dyes
acidic and basic
colored ion
chromophore
chromophore +ive charge
basic dye
charge of most MOs
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