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Microscopy
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UNITS OF MEASUREMENT
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1000 mm (millimeters) in 1 meter (m)
1000 µm (microns) in 1 mm
Bacteria are about 1µm or smaller
1000 nm (nanometers) in 1 µm
Viruses are about 1nm
 1000 viruses can fit into one bacterium
Protozoa are fairly large single-celled animals. You can see them
with the naked eye. That is called being “macroscopic”
Bacteria are so small, they are measured in µm. They are the
smallest things you can see under a microscope with the oil
immersion lens.
Viruses are even smaller, so they are measured in nm.
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Limits of Resolution
Figure 4.3 33
VOCABULARY
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Immersion oil: keeps light from bending and allows lens to be refracted.
Parfocal: focused in all lenses.
Depth of field: how much of the background is in focus at the same time
that the foreground is in focus.
Resolution: ability of two lenses to distinguish two points.
RESOLUTION: the ability of the lenses to distinguish two points. A
microscope with a resolving power of 0.4 nm can distinguish between two
points greater than or equal to 0.4 nm. When you go to the eye doctor, you
look at the chart (Snellen chart) and read it from 20 feet away. If you can read
what a normal sighted person can read from 20 feet away, it is called 20/20
vision. If you can’t read it well, your eyesight has less resolution than normal.
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TYPES OF MICROSCOPES
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SIMPLE MICROSCOPE: Has only one lens,
like an ocular (eyepiece)
COMPOUND MICROSCOPE: More than
one lens, like an ocular and an objective. An
example is the Brightfield microscope.
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There are two main types of compound
microscopes: Light Microscopes and Electron
Microscopes.
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SIMPLE MICROSCOPE
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COMPOUND MICROSCOPE:
One Eyepiece
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COMPOUND MICROSCOPE:
Two Eyepieces
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Types of Compound Microscopes
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Dissecting
Brightfield
Darkfield
Phase-contrast
Differential Interference contrast
Fluorescence
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Dissecting Microscope:
For looking at large objects
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BRIGHTFIELD ILLUMINATION:
For live organisms with no stain
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BRIGHTFIELD ILLUMINATION:
Can also use for stained tissues
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DARKFIELD ILLUMINATION
For live organisms with no stain
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DARKFIELD ILLUMINATION
For fluorescent organisms
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Brightfield vs Darkfield
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PHASE CONTRAST MICROSCOPY
For seeing organelles in live organisms
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DIFFERENTIAL INTERFERENCE CONTRAST
For seeing organelles in live organisms in three
dimensions
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DIFFERENTIAL INTERFERENCE
CONTRAST
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DIFFERENTIAL INTERFERENCE
CONTRAST
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Fluorescence Microscopy
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Cells are stained
with fluorescent
dyes called
fluorochromes.
UV Light is shined
on the specimen.
Fluorescent
substances absorb
UV light and emit
visible light.
203.6b
Figure
FLUORESCENCE MICROSCOPY
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FLUORESCENCE MICROSCOPY
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Transmission Electron Microscope
Much higher
resolution than
microscopes.
Can only be
used on dead
cells
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Transmission Electron Microscope
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Transmission Electron Microscope:
Inside of a Plant Cell
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Scanning Electron Microscope
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Very high resolution like a Transmission
Electron Microscope except makes images in
three dimensions.
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Scanning Electron Microscope
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Scanning Electron Microscope:
Flea
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Scanning Electron Microscope:
Pollen
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Scanning Probe Microscope
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A scan is passed over the specimen, line by line.
The surface dimensions are recorded and sent to
a computer, which shows the image in false
color.
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Scanning Probe Microscope:
Red Blood Cells
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Scanning Probe Microscope:
Chromosomes
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COMPARISON OF MICROSCOPES
BRIGHTFIELD
Dark objects are visible against a bright background.
Light reflected off the specimen does not enter the objective lens
Not for looking at live cells
Maximum resolution is 0.2µm and maximum magnification is
2000x
Stains are used on specimens
DARKFIELD
Light objects are visible against dark background
Used for live cells, cilia, flagella
Especially good for spirochetes
Uses special condenser with an opaque disc that eliminates all light
in the center
PHASECONTRAST
No staying required
Accentuates diffraction of the light that passes through a specimen
Good for live cells; good contrast
Most sensitive; cilia shows up
Not three-dimensional
DIFFERENTIAL Uses two beams of light
INTERFERENCE Shows three dimensions
CONTRAST
Has a prism to get different colors
Good for live cells (unstained)
Best resolution
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COMPARISON OF MICROSCOPES
FLUORESCENCE
Uses ultraviolet light
Stained cells with fluorescent dye; energizes electrons and creates
visible light
No live cells
Quick diagnosis of TB and syphilis
TRANSMISSION
ELECTRON
Get flat images
Have vacuum pumps to allow electrons to float better
Stain with heavy metal salts
Shows sections of cell, revealing organelles
Requires an ultramicrotome
Best resolution of all microscopes
SCANNING
ELECTRON
Surface view only
Needs a vacuum
No live cells
Three-dimensional view
SCANNING
PROBE
Physical probe scans the specimen
Raster scan: image is cut up into pixels and transmitted to computer
Not limited by diffraction
Slower in acquiring images
Maximum image size is smaller
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For the next 20 minutes….
• Everyone who does not have a lab group,
stand to the side of the room.
• Decide what people to join with to form a lab
group.
• Discuss ideas for your group project.
• How will you conduct it? What supplies will
you need, and how will you measure the
results? Will you count how many of each
different colony you get?
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