Bright-field microscope

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?????????????????????

• ???????????????????????????
• Bright-field microscope
• Dark-field microscope
• Phase-contrast microscope
• Fluorescence microscopes

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???????????????????????

• ?????????????????? 1000 2000 ????
• ???????????????????????? ?????????????
• ???????????????????????????????
• parfocal microscopes remain in focus when
  objectives are changed
• ???????????????
• ??????????????????????? x
  ??????????????????????????

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???????????????? (Resolution power)

• ????????????????????? 2 ????????????
• ???????????????
• shorter wavelength ? greater resolution

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??????????????
Eyepiece
Binocular tube
Revolving nosepiece
Arm
Objective
Mechanical stage
Fine adjustment
Stage
Course adjustment
Condenser
Illuminator
Base
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???????????????????????????????????????

• Resolution power
• ??????????????????????????????????????????????????
  ????????
• R 0.6 x ?/NA
• Numerical aperture
• ??????????????????????????????????????????????????
  ?????????????
• NA n sin ?

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Working distance
Low power
High power
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Working distance
Oil Immersion
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• ??????????????????????????

• working distance
• - ????????????????????????? ?????????????
  ???????????????? ???? ????????

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Mechanical Lengths
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Objective Specifications
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The 3 Classes of Objectives
Chromatic and Mono-Chromatic Corrections
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Plan Objectives
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???????????????? (Dark-Field Microscope)

• ?????????????????????????????
• ???????????????????????????????
  ??????????????????? ns

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Dark field microscope
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Phase-contrast microscope

• ??????????????????????????????????
  ????????????????????????????????????????????????
• ????????????????????????????
• ???????????????????????
• ????? annual diaphragm ??? condenser ??? phase
  plate ?? objective lens
• wave length 1/4 ???????

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Phase contrast microscope
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Figure 2.10
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Fluorescence Microscope

• ?????? ultraviolet, violet ???? blue light
• ??????????????????? ?? fluorochromes
• ?????????? ???????????? ??????????????????????????
  ???? ????????????????

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Fluorescence Microscope
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?????????????????? Fluorescence Microscope
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?????????????????????????????

• ????????????????????????????????????????????????
• ????????????????????????????????????????????????
• ?????????????????

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Fixation

• ???????????????? ??????????
• ??????????????????????????????????????????????????
  ??????????????????????
• ??????????????????????????????????????????????
• ?????????????????
• ??????????????????????????????????????????????????
  ??
• ????????????????
• ?????????????????????????? ??????? ????
  Formaldehyde, Odmium tetroxide (Os4) , potassium
  permanganate, Glutaraldehyde

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???????????? ???????????????? (dehydration and
simple staining)

• ?? (stain)
• ??????????????????????????????????????????????????
  ???????? ?????????????????????
• ????????????????
• ?????????????????
• ???? ??????? (basic dyes)

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Electron Microscopy

• ???????????????????????????????
• ??????????????????????????????????????????????????
  ??????????????

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Transmission Electron Microscope

• electrons scatter when they pass through thin
  sections of a specimen
• transmitted electrons (those that do not scatter)
  are used to produce image
• denser regions in specimen, scatter more
  electrons and appear darker

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EM
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Ebola
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The Scanning Electron Microscope

• uses electrons reflected from the surface of a
  specimen to create image
• produces a 3-dimensional image of specimens
  surface features

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Fly head
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Confocal microscopy

• have extremely high resolution
• can be used to observe individual atoms

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Confocal Microscopy

• confocal scanning laser microscope
• laser beam used to illuminate spots on specimen
• computer compiles images created from each point
  to generate a 3-dimensional image

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Figure 2.30
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Biochemical Techniques

• ????????????
• ???????????????????????????????????????????????
  ????????
• Molecule

Small
Assembly of macromolecule
Large
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???????

• ??? ???????????????????????????? (Cell
  fractination) ?????????????????????????????? ???
• 1. ?????????????????????????
  (Homogenization) ??????????????????????
• 1. ???????? (Pestle /Moltar)
• 2. ???????????????? (Ultrasonic vibration)
• 3. ??????????????? (Wareing bleden/ Potter
  homogenizers)
• 4. ??????????????????????? (Freeze and Thaw)
• 5. ???????????????????? (Deactivation)
• 6. ????????????? (Chemical)
• 7. ????????????? (Enzyme)

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• Homogenizer

http//www.freewebs.com/ltaing/chpt7.3Cellfraction
ation.gif
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Ultrasonic vibration
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Deep freezer
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• Gel Filtration

http//fig.cox.miami.edu/cmallery/150/protein/pro
teinsb.htm
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• 2. ????????????????????? ?????????
• (Separation of the homogenate)
• 1. ?????????????? (Centrifugation)
• - Gravity (g)
• - Relative centrifugal force (RFC)
• - Rate of sedimentation of components
• (size, shape, density of particle
  and solvent, speed) - Differential centrifugation
• - Rate-zonal or density-gradient centrifugation

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• Differential Centrifigation

http//www.freewebs.com/ltaing/chpt7.3Cellfraction
ation.gif
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• Differential Centrifigation

http//www.freewebs.com/ltaing/chpt7.3Cellfraction
ation.gif
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• 3. ?????????????????????? (Separation of
  biomolecules)
• 1. ???????????????????? (Solubility)
• 2. ???? (size)
• - Centrifugation
• - Dialysis
• - Ultrafiltration
• - Gel filtration
• - SDS -PAGE (Sodium Dodecyl Sulphate
  Polyacrylamide Gel Electrophoresis)

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Ultrafiltration
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• 3. ?????????? (Charge)
• Ion exchange chromatography
• Isoelectric focusing
• Electrophoresis
• 4. Biological properties
• Affinity of chromatography
• Immunoelectrophoresis
• Blotting - Southern blot (DNA)
• - Northern blot (RNA)
• - Western blot (Protein)
• Chromatography ????? (paper. Gas ,
  Thin-layer)
• PCR (Polymerase chain reaction)

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• Ion Exchange

http//fig.cox.miami.edu/cmallery/150/protein/ion
_exchange.jpg
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• Immunoelectrophoresis

http//www.haps.nsw.gov.au/edrsrch/edimages/myelom
a2.jpg
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• Southern blot
• ?????????????????? DNA ??????????????????
  agarose gel electrophoresis

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• Northern blot
• ?????????????????? RNA

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Western blot
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• PCR (Polymerase Chain Reaction)
• ?????????????????? DNA ???????????????
  ??????????????????????????? DNA
  ?????????????????
• 1. Denaturation ????????????? DNA
  ????????????????????????? ?????????????? ??????
  90-95oC
• 2. Annealing ???????????????????????????
  Primer ??????????? DNA ?????? ??????????????
  ?????? 50-55oC
• 3. Extension (primer extension, synthesis)
  ?????????????? ?????? 70-75oC

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Buffer Optimalization

• 10 mM Tris HCl, pH 8.3
• 50 mM KCl
• 1.5 - 2.5 mM MgCl2
• 50 - 200 mM dNTP
• DNA template concentration
• Optional compounds

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Primer Considerations

• Primer lengths
• GC contents
• Melting temperature
• Tm 2(TA)4(GC)
• Tm difference
• 3 and 5 end considerations

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PCR Principle
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