Symbols

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Symbols Such

• i.e. Latin for that is
• e.g. Latin for for example
• a.k.a. also known as
• ? therefore
• ? then, leads to
• inhibits

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Symbols Such

• Same as
• ? Different from
• ? Something that can flow either way
• ? Increase
• ? Decrease
• D Change
• n.b. Latin for note well

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I have no snappy title for this lecture.

• September 29, 2008
• Overview of Microscopy
• Dr. Behonick

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Topics for today

• Components of light microscopes
• Light path
• Upright vs. inverted light microscopes
• Illumination
• Köehler Illumination
• Optics
• Image formation

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Components of Light Microscopes
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Optical Components

• light source
• condenser
• stage
• objective
• oculars

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http//micro.magnet.fsu.edu/primer/anatomy/compone
nthome.html
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Light Path
light source ? condenser ? specimen ? objective
? oculars
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http//micro.magnet.fsu.edu/primer/anatomy/compone
nthome.html
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Light Path

• transmitted light
• light source ? condenser ? specimen ? objective ?
  ocular
• epifluorescence
• light source ? objective ? specimen ? objective
  again ? ocular

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http//micro.magnet.fsu.edu/primer/anatomy/compone
nthome.html
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Light Path Resources

• Interactive Tutorial
• http//micro.magnet.fsu.edu/primer/java/component
  s/bx51lightpaths/index.html

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Light Sources

• transmitted light
• incandescent lights (heat-driven light emission)
• emit full spectrum of visible light
• widefield fluorescence
• arc lamps (xenon, mercury)
• much brighter
• full spectrum of visible light ? filters required
• first on/last off rule due to power surge
• confocal
• lasers
• emit selected wavelengths
• more expensive

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Condenser

• lens
• collects light directs to specimen
• very important to correctly align in transmitted
  light techniques (part of what is known as
  Köehlering a microscope)
• diaphragm regulates amount of light (also part of
  Köehlering)
• on fluorescence scopes, objective acts as
  condenser

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Stage

• moves in X, Y Z planes
• when motorized, can be controlled by computer
• we usually move the stage to focus image of
  specimen (but we could, theoretically, move
  oculars instead)

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Objective

• series of lenses
• magnifies image
• much info inscribed on it
• common magnifications
• 4, 10, 20, 40, 63, 100x
• N.A. (numerical aperture) affects contrast
  resolution
• called objective because its close to object
  youre viewing

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Objectives
http//micro.magnet.fsu.edu/index.html
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Objectives
http//micro.magnet.fsu.edu/index.html
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Objectives
inside a Zeiss objective
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Chromatic Aberration

• image imperfection due to differing refractive
  indices of different wavelengths of light
• ? different colors focus _at_ different distances
  from lens
• achromatic apochromatic lenses designed to
  correct for this

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Chromatic Aberration
corrected for (higher quality lens used)
not corrected for
http//upload.wikimedia.org/wikipedia/commons/6/66
/Chromatic_aberration_(comparison).jpg
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Spherical Aberration
ideal lens (doesnt exist)

• image imperfection due to increased refraction of
  light rays that strike edge of lens as compared
  to those that strike center of lens
• ? all rays dont focus _at_ same distance from lens
• achromatic apochromatic lenses designed to
  correct this

actual lens
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Achromatic Lens

• corrected to bring 2 wavelengths into focus in
  same plane
• usually red blue
• corrected for spherical aberration _at_ 1 wavelength
• lens doublet - dispersion of 1 counterbalanced by
  other

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Apochromatic Lens

• brings 3 wavelengths into focus in same plane
• usually red/green/blue
• better color correction than achromatic lens
• corrected for spherical aberration _at_ 2 wavelengths

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Oculars

• a.k.a. eyepieces
• from latin for eye
• lenses for further magnification
• often 10x
• adjust to eye comfort - look into them w/ relaxed
  eyes (as if looking far away)

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Oculars
http//micro.magnet.fsu.edu/index.html
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Oculars
http//micro.magnet.fsu.edu/index.html
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Lenses
http//micro.magnet.fsu.edu/primer/anatomy/magnifi
cation.html
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Upright vs. Inverted Microscopes

• upright
• objectives above stage
• light input condenser below stage
• inverted
• objectives below stage
• light input condenser above stage

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Inverted
Upright
images from Nikon promotional materials
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Upright vs. Inverted Microscopes
light path for BOTH Upright Inverted light
source ? condenser ? specimen ? objective ?
oculars
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Upright vs. Inverted Microscopes
Upright light ? condenser ? specimen ?
objective ? ocular
stage
Inverted light ? condenser ? specimen ?
objective ? ocular
stage
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Upright Microscope
Light paths Fluorescence A1 ? B Transmitted A2
? B
modifications by GG
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Inverted Microscope
Light paths Fluorescence A1 ? B Transmitted A2
? B
modifications by GG
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Typical Inverted Microscope
These days we use modern Digital cameras not 35
mm !!
images from Nikon promotional materials
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Major Brands

• research grade microscope manufacturers
• Leica
• Nikon
• Olympus
• Zeiss

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Illumination
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Its actually important

• illumination thought to be most important
  variable in obtaining high quality microscopic
  images

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Proper illumination of a diatom
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Köehler Illumination

• aka - Köehlering
• method for achieving optimal microscopy sample
  illumination
• introduced by August Köehler (Zeiss) in 1893

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Köehler Illumination

• why?
• proper alignment of illumination source for
  transmitted light microscopy
• aligning condenser lens to ensure
• optimal resolution
• even lighting of image (consistently lit
  throughout)
• no contrast artifacts

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Whats an artifact?
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Whats an artifact?

• artifact error in data caused by tool used for
  observation
• examples in microscopy
• anything that happens to a tissue while its
  being prepared for slides
• folds
• tears/holes
• tissue damage that affects staining results

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References

• Dykstra, M.J., Reuss, L.E. (2003) Biological
  Electron Microscopy Theory, Techniques, and
  Troubleshooting. Springer.
• Giorgi, G. Lecture 2. Merritt College Biology
  035, 24 January 2008.
• Molecular Expressions lthttp//micro.magnet.fsu.edu
  gt
• Robinson, J.P. The Principles of Microscopy,
  Part 1. Purdue University Cytometry
  Laboratories. 24 September 2008.
  lthttp//www.cyto.purdue.edu/flowcyt/educate/pptsli
  de.htmgt