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Visualizing Prokaryote Cells

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					Visualizing Prokaryote
          Cells
   Chapter 3 - Black
Light
Key Words -

   Visible light
   Ultraviolet light
   Reflection
   Transmission
   Refraction
   Absorption
Light Microscopy
Resolution

   Resolution is the ability to see two
    images as separate and discrete.
   The wavelengths of visible light
    from 420 to 620 prevent resolution
    of two points closer than 220 nm
   By using the light emitted from an
    electron it is possible to resolve two
    points that are .2nm apart
Specialized Microscopy

   Dark field
   Phase Contrast
   Differential interference
   Fluorescence
   Confocal
   Digital
Digital photomicrosopy

   Camera can be
    used to
    photograph images
   Specimen can be
    viewed on TV
    screen as well as
    on computer
    screen
Classroom Microscopy
Phase Contrast Microscopy
   Accentuate small              micro.magnet.fsu.ed
    differences in the             u/.../dicphasecompar
    refractive index of            ison.html
    the specimen
   More detail is
    apparent in living cells
   Assist in the
    visualization of cell
    structure in
    transparent cells
Differential interference
microscopy
   Produces higher
    resolution
   Depends on a
    gradient
   It can produce
    almost a three
    dimensional image
Comparison of Phase Contrast and
Differential Interference Contrast
Fluorescent Microscopy
   Organisms such as
    Mycobacterium tubercuolosis
    and Treponema pallidum are
    treated with a fluorochrome
    dye
   Ultra violet light is used to
    excite the fluorochrome
    molecules and produce a
    glowing image
   Used in clinical work
   Also used with antigens and
    antibodies to identify the
    presence of molecules on the
    surface of a cell
Fluorescent antibody
staining
   Used in immunology
   Fluorescent
    antibodies are used to
    detect antigens on
    the surface of cells
   In the picture to the
    right are Bacillus
    anthracis cells tagged
    with a fluorescent
    antibody
Confocal Microscopy

   Utilizes beams of ultraviolet
    light to excite fluorescent
    dye molecules.
   The exciting light is focused
    on the specimen with a thin
    optical fiber
   Resulting fluorescence is
    focused through a narrow
    aperture
   The light is detected and
    analyzed by a computer
   Very sharp focus
   For thick specimens an image
    is constructed in layers
Confocal images

   http://www.microscopyu.com/galleri
    es/confocal/applecedarrustaeciaand
    pycnia.html
   http://www.microscopyu.com/galleri
    es/confocal/chlamydomonas.html
Electron Microscopy

   Transmission electron microscopy
   Scanning electron microscopy
Transmission Electron
Microscopy
   Electrons are used
    as the source of
    light
   Produced by a high
    voltage current
    running through a
    tungsten filament
   www.steve.gb.com
    /science/electron
    _microscopy.html
Transmission Electron
Microscope
   The lenses are
    electromagnetic
   They act on the
    negatively charged
    electrons to focus
    them in a
    concentrated path
    through the specimen
   The image is
    magnified by
    additional lenses and
    visualized on a screen
TEM images
   Images produced
    display high resolution
   Staining with heavy
    metals that interact
    with the electrons
   Gradations of black,
    gray, and white
    contrast areas of
    greater density that
    absorb the stain
Scanning Electron Microscopy


   Electrons are
    reflected and
    collected off of
    the surface of a
    cell
SEM

   Images show
    surface contours
   Three dimensional
    image
Freeze Fracture
   Cells are quickly frozen in liquid nitrogen (196C), which
    immobilizes cell components instantly.
    2. Block of frozen cells is fractured. This fracture is
    irregular and occures along lines of weakness like the
    plasma membrane or surfaces of organelles.
    3. Surface ice is removed by a vacuum (freeze etching)
    4. A thin layer of carbon is evaporated vertically onto the
    surface to produce a carbon replica.
    5. Surface is shadowed with a platinum vapor.
    6. Organic material is digested away by acid, leaving a
    replica.
    7. Carbon-metal replica is put on a grid and examined by a
    transmission electron microscope.
Freeze Fracture and
Etching
Freeze Fracture
References

   http://micro.magnet.fsu.edu/primer
   http://www.mos.org/sln/SEM/

				
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posted:11/29/2011
language:English
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