Microscopy
UNITS OF MEASUREMENT
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1mm = 1000µm
1µm = 10-3mm (convert mm to µm by multiplying by 1000 = 3 zeros)
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1nm = 10-6mm (convert nm to mm by dividing by 1000000 = six zeros)
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Bacteria are about 1µm or smaller
Viruses are about 1nm
1000nm = 1µm
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1000 viruses can fit into one bacterium
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0.001µm = 1 nm
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Bacteria are so small, they are measured in µm.
Viruses are even smaller, so they are measured in nm.
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VOCABULARY
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Immersion oil: keeps light from bending and allows lens to be
refracted.
Resolution: ability of two lenses to distinguish two points.
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.
Refractive Index: a measure of the light-bending ability of a
medium
Numerical aperture: numerical aperture increases as depth of
field decreases.
Resolution power: limits the useful magnification of the
microscope resolving power.
RESOLUTION
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The ability of the lenses to distinguish two
points.
RESOLVING POWER
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The distance between two closely adjacent
objects where the objects still appear separate
and distinct. The shorter the distance (the
smaller the number), the better the resolving
power (the sharper the image).
RESOLVING POWER
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To calculate the resolving power (to see how
close two objects can be so you can still see
them):
D = distance (smaller number is better)
 0.61 = a constant number, does not change
 NAobj = numerical aperture of the objective (larger
number causes D to decrease, which is better)
 λ = (lambda): the wavelength (nm) of the light going
through the microscope. Convert nm to mm by dividing
by 1000000 (six zeros)
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D = 0.61 λ / NAobj
Resolving Power
2 objects
resolved
2 objects not
resolved
Same 2 objects resolved with
better optical instrument
Resolving power limits for several
optical instruments:
Optical Instrument
Human eye
Light microscope
Scanning electron microscope
(SEM)
Transmission electron microscope
(TEM)
Resolving Power
0.2 millimeters
(mm)
0.25 micrometers
(µm)
5-10 nanometers
(nm)
0.5 nanometers
(nm)
PRISM
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This is a triangular device that breaks up light
into its various wavelengths so you can see all
the colors of the rainbow (the visible spectrum).
The visible spectrum of colors starts with violet
(350nm), and goes on to indigo, blue, green
(550nm), yellow, orange, red (700nm).
Sample Problem
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When we want to observe the color green
(550nm) under an oil-immersion objective lens
of a microscope, where the NAobj is 1.25, the
resolution is as follows:
D = (0.61)(0.000550) / 1.25
D = 0.0002684 mm  convert this to
microns (µm) by multiplying by 1000
D = 0.27 µm
Sample Problem
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The NAobj for the high-dry (400x) lens is 0.65
What is the resolving power (D) of this
objective when viewing a wavelength of 550nm?
D = 0.61 λ / NAobj
D = (0.61)(0.000550) / 0.65
D = 5.1615mm
D = 0.52µm
Conclusions
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Therefore, we can see an organism such as E. coli,
which is 2µm long and 1µm wide because it is larger
than the resolving power. However, we could not see
Haemophilus influenza, which is 0.2µm long because it is
smaller than our resolving power.
Therefore, the resolving power limits the useful
magnification of the microscope.
Resolution determines the magnification.
REFRACTION
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Refraction is the bending of
light caused by the
surrounding medium.
N = Refraction Index of the
medium surrounding the lens
Air: N= 1
Glass: N = 1.5
Immersion Oil: N = 1.51
(about the same as glass)
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.
SIMPLE MICROSCOPE
COMPOUND MICROSCOPE:
One Eyepiece
COMPOUND MICROSCOPE:
Two Eyepieces
Types of Compound Microscopes
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Dissecting
Brightfield
Darkfield
Phase-contrast
Differential Interference contrast
Fluorescence
Dissecting Microscope
BRIGHTFIELD ILLUMINATION:
No Stain
BRIGHTFIELD ILLUMINATION:
With Stain
DARKFIELD ILLUMINATION
DARKFIELD ILLUMINATION
Brightfield vs Darkfield
PHASE CONTRAST MICROSCOPY
DIFFERENTIAL INTERFERENCE
CONTRAST
DIFFERENTIAL INTERFERENCE
CONTRAST
DIFFERENTIAL INTERFERENCE
CONTRAST
Fluorescence Microscopy
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Uses UV light.
Fluorescent
substances absorb
UV light and emit
visible light.
Cells may be stained
with fluorescent
dyes
(fluorochromes).
Figure 3.6b
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
Transmission Electron Microscope
Transmission Electron Microscope
Transmission Electron Microscope:
Inside of a Plant Cell
Scanning Electron Microscope
Scanning Electron Microscope:
Flea
Scanning Electron Microscope:
Pollen
Scanning Probe Microscope:
Red Blood Cells
Scanning Probe Microscope:
Chromosomes
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
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