Microscopy 1
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Transcript of Microscopy 1
Magnification and Resolution
• Magnification provides no additional information
• Resolution often requires magnification
Resolution is a measure of distance• Resolution = d = (.61λ)/N.A• d = distance between 2
pts.• λ = wavelength of light• N.A. = Numerical
Aperture• N.A. = n sin α• n = refractive index• α = half-angle of cone of
light
Light travels in waves
• White light is a mixture of several wavelengths
• ROYGBIV Red---Violet• Red- 700nm• Violet- 400 nm• λ = wavelength of light
Refractive index• Refractive indices:• Air-• Vacuum 1 (exactly) • Air @ STP 1.0002926 • Gases @ 0 °C and 1 atm• Air 1.000293
[1]
• Helium 1.000036 • Water 1.333 • Ethyl alcohol (ethanol) 1.361 • Diamond 2.419• Amber 1.55• Sodium chloride 1.50 • Other materials • Pyrex (a borosilicate glass) 1.470 [
• Ruby 1.760• Glycerol 1.4729 • Cubic zirconia 2.15 - 2.18• Diamond 2.419 • Gallium(III) arsenide 3.927• Silicon 4.01
Field of View
• Actual diameter of microscope image at a certain mag.
• As magnification increases, field of view _______.
Depth of field
• A measure of the thickness of the focal plane of an image
• As magnification increases, depth of field _______________.
Depth of field in Photography
• Shallow depth of field prevents an entire object from being in focus
Quiz Wednesday
• Microscope care and maintenance (how to keep from breaking them)
• Microscpe anatomy (labelling of parts)
• Microscope principles (wavelength, magnification, etc.)
Microscope anatomyParts
1. Headpiece
2. Eyepieces
3. Nosepiece/turret
4. Objective leses
5. Stage clips
6. Stage
7. Condenser
8. Substage filter holder/iris diapragm lever
9. Mirror
10. Base
11. Condenser adjustment knob
12. Fine focus knob
13. Coarse focus knob
14. Arm
Electron Microscopes• Use electrons instead of light• Use magnets instead of glass
lenses• electron wavelengths are
much shorter than those of light
• TEM- sends electrons through a specimen
• SEM- specimen spraypainted with gold
Green Fluorescent Protein• discovered in 1960s by Dr. Frank
Johnson and colleagues
• closely related to jellyfish aequorin
• absorption max = 470nm
• emission max = 508nm
• 238 amino acids, 27kDa
• “beta can” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore
• amino acid substitutions result in several variants, including YFP, BFP, and CFP
40 Å
30 Å
• If you reach your fingers into the area under the microscope stage without looking, which of the following is most likely to be damaged?
• A. The base of the microscope• B. The coarse adjustment knob.• C. The fine focus adjustment knob.• D. The blue frosted glass filter.• E. The mirror
• When carrying a microscope, your principal grip should be on its:
• A. tube. • B. nosepiece. • C. stage.• D. arm. • E. coarse adjustment knob.