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• 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

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 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

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

• 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

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.•