Transcript ppt - Fog.ccsf.edu

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
Magnification without resolution
Magnification without resolution
Magnification without resolution
Magnification without resolution
Contrast
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
• Red700nm
• Violet400 nm
• λ = wavelength of light
Refractive index
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Refractive indices:
AirVacuum
Air @ STP
Gases @ 0 °C and 1 atm
Air
Helium
Water
Ethyl alcohol (ethanol)
Diamond
Amber
Sodium chloride
Other materials
Pyrex (a borosilicate glass)
Ruby
Glycerol
Cubic zirconia
Diamond
Gallium(III) arsenide
Silicon
1 (exactly)
1.0002926
1.000293 [1]
1.000036
1.333
1.361
2.419
1.55
1.50
1.470 [
1.760
1.4729
2.15 - 2.18
2.419
3.927
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
Depth of field
• Can be exploited for
identifying layers in a
substance
Quiz Wednesday
• Microscope care and
maintenance (how to
keep from breaking
them)
• Microscpe anatomy
(labelling of parts)
• Microscope principles
(wavelength,
magnification, etc.)
Microscope anatomy
Parts
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
Phase-contrast
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
TEM
SEM
• Only looks at surfaces
• Generates 3-D image
• Often color-retouched
Fluorescence microscopes look at a
single wavelength of light at a time
Green Fluorescent Protein
•
discovered in 1960s by Dr. Frank
Johnson and colleagues
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closely related to jellyfish aequorin
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absorption max = 470nm
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emission max = 508nm
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238 amino acids, 27kDa
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“beta can” conformation: 11
antiparallel beta sheets, 4 alpha
helices, and a centered chromophore
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amino acid substitutions result in
several variants, including YFP, BFP,
and CFP
40 Å
30 Å
More fluorescence
• 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.
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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