Visualizing Prokaryote
Cells
Chapter 3 - Black
Light
Key Words 
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Visible light
Ultraviolet light
Reflection
Transmission
Refraction
Absorption
Light Microscopy
Resolution
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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
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Dark field
Phase Contrast
Differential interference
Fluorescence
Confocal
Digital
Digital photomicrosopy
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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
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Accentuate small
differences in the
refractive index of
the specimen
More detail is
apparent in living cells
Assist in the
visualization of cell
structure in
transparent cells
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micro.magnet.fsu.ed
u/.../dicphasecompar
ison.html
Differential interference
microscopy
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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
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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
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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
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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
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http://www.microscopyu.com/galleri
es/confocal/applecedarrustaeciaand
pycnia.html
http://www.microscopyu.com/galleri
es/confocal/chlamydomonas.html
Electron Microscopy
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Transmission electron microscopy
Scanning electron microscopy
Transmission Electron
Microscopy
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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
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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
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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
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Electrons are
reflected and
collected off of
the surface of a
cell
SEM
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Images show
surface contours
Three dimensional
image
Freeze Fracture
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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
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http://micro.magnet.fsu.edu/primer
http://www.mos.org/sln/SEM/