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Microscopy 1

Microscopy 1. Biology 101A. Announcements. Quiz- Wed, not today- 8:10am-8:25am. Magnification and Resolution. Magnification provides no additional information Resolution often requires magnification. Magnification without resolution. Magnification without resolution.

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Microscopy 1

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  1. Microscopy 1 Biology 101A

  2. Announcements • Quiz- Wed, not today- 8:10am-8:25am

  3. Magnification and Resolution • Magnification provides no additional information • Resolution often requires magnification

  4. Magnification without resolution

  5. Magnification without resolution

  6. Magnification without resolution

  7. Magnification without resolution

  8. Magnification without resolution

  9. Magnification without resolution

  10. Contrast

  11. 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

  12. Light travels in waves • White light is a mixture of several wavelengths • ROYGBIV Red---Violet • Red- 700nm • Violet- 400 nm • λ = wavelength of light

  13. 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

  14. Field of View • Actual diameter of microscope image at a certain mag. • As magnification increases, field of view _______.

  15. Depth of field • A measure of the thickness of the focal plane of an image • As magnification increases, depth of field _______________.

  16. Depth of field in Photography • Shallow depth of field prevents an entire object from being in focus

  17. Depth of field • Can be exploited for identifying layers in a substance

  18. Quiz Wednesday • Microscope care and maintenance (how to keep from breaking them) • Microscpe anatomy (labelling of parts) • Microscope principles (wavelength, magnification, etc.)

  19. Microscope anatomy

  20. Phase-contrast

  21. 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

  22. TEM

  23. SEM • Only looks at surfaces • Generates 3-D image • Often color-retouched

  24. Fluorescence microscopes look at a single wavelength of light at a time

  25. 40 Å 30 Å 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

  26. More fluorescence

  27. 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

  28. When carrying a microscope, your principal grip should be on its: • A. tube. • B. nosepiece. • C. stage. • D. arm. • E. coarse adjustment knob.

  29. Iris diaphragms are most easily damaged by: A. being touched by someone’s fingers. B. too high a light intensity. C. having too little oil on their leaves. D. too low a light intensity. E. Not being wiped clean before every use.

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