microscopy 1 biology 101a january 29, 2008. magnification and resolution magnification provides no...
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Microscopy 1
Biology 101AJanuary 29, 2008
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
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
Phase-contrast
Electron Microscopes
• Use electrons instead of light
• 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
Visualizing Fluorescence
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
Lab Report
• Titles:• Which do you think is best?
– Superpurple– Permeability permutations of purple anion
membrane penetration– Properties of Nonliving Membranes
Introduction• Some questions to consider for your lab report
introduction:– What was learned in the previous lab that was
pertinent to this one?– What structures exist in a normal cell membrane to
regulate passage of things into and out of the cell? (this can be revisited in the discussion/conclusion sections)
– What do the processes of diffusion, osmosis and active transport have to do with the lab at hand?
• You do not need to answer all these questions
Quiz Thursday!
• Microscope care and maintenance (how to keep from breaking them)
• Microscpe anatomy (labelling of parts)
• Microscope principles (wavelength, magnification, etc.)