Lab 3: Microscopes and Microscopy

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Lab 3 Microscopes and Microscopy
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I. Background Reading

• From Textbook (Biology Concepts and Connections
  by Campbell et al., 2006)
• p 52-54 Microscopes and cell sizes
• p 55-67 Cell structure

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II. Lab Three Objectives

• To illustrate the parts of the microscope and
  their functions.
• 2. to outline good microscopical technique
• 3. to illustrate optical properties of the
  microscope, including image orientation and plane
  of focus
• 4. to discuss other forms of microscopy,
  including scanning and electron microscopy.
• 5. to learn how to measure specimens with
  compound light microscope and in electron
  micrographs.

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Microscope quality depends on the capacity to
Resolve, not magnify objects.

• Resolving power ability to distinguish detail,
  (or the ability to separate two dots).
• ex. A human eye can separate dots 0.25 mm apart.
• A Compound light microscope can separate
  dots 0.1 µm apart.
• A transmission electron microscope can
  separate dots less than 5 angstroms apart.
• Magnification enlargement of the current image.

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• Contrast is based on the differential
  absorption of light or electrons by parts of the
  specimen.
• Thick structures absorb light or electrons more
  than thin structures.
• Structures having natural pigments (e.g.
  chlorophyll, carotenoids) absorb light more than
  opaque structures.
• Contrast can be improved by using stains or dyes
  that bind to cellular structures absorb enough
  light or electrons to provide contrast.

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The ocular lenses are located at the top of the
body tube. These lenses have a magnification
power equal to 10x.
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The objective lenses are mounted on the
nosepiece. The three objectives include
scanning (power 4x) low power (power 10x)
high power (power 40x)
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The stage is where you place your slide with your
specimen.
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The condenser is a system of lenses that collect
light rays and converge them to focus on a
specimen. The condenser is adjusted using the
condenser adjustment knob.
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The diaphragm is an adjustable light barrier that
controls the angle of the cone of light
pre-presented to the specimen. The diaphragm
controls light intensity, and is adjusted by
sliding the bar across the front of the
condenser.
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The light source is located in the base of the
microscope.
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The coarse adjustment is for large corrections in
focus. The coarse adjustment should only be
used in the scanning or low power objectives.
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The fine adjustment is for small corrections in
focus. The fine adjustment knob is the smaller
knob attached to the coarse adjustment knob.
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Procedure (page 6) Making a slide.
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• Total magnification is the product of ....

Ocular Lens Magnification
X
Objective Lens Magnification

• Ocular mag. x Obj. mag. Total mag.

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Measurement using the ocular micrometer in a
compound microscope

• Use the Ocular micrometer glass disc in the
  right ocular lens on hich are etched uniformly
  spaced lines.

1. Superimpose the scale on the image of the
specimen or part of specimen being observed. 2.
Determine the magnification of the objective lens
being used. 3. Count the number of divisions
(small or large) covered by specimen.
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• Using calibration chart provided, determine the
  value (in micrometers) of each division.

Example 3 large units at 40x 75 µm
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Chloroplast (2)
Chloroplast (1)
http//www.biol.tsukuba.ac.jp/inouye/ino/g/char/C
losterium.GIF
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The Stereoscopic dissecting microscope has 2
objective lenses for each magnification whereas
compound has only one
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Anatomy of a planarian
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Electron Microscopes
I. Characteristics
A. Specimens must be killed and fixed with
chemicals.
B. Illumination source is a beam of electrons
having wavelength much smaller than light
greater resolution.
C. Beam of electrons enclosed in a vacuum so
their path not interfered with by air
molecules.
D. Electromagnets instead of glass lenses in
light microscopes used to focus electron
beam on specimen.
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Two types
2. Scanning Electron Microsopes (SEM)
1. Transmission Electron Microscopes (TEM)
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Comparison of light, TEM, SEM
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Specimen preparation for TEM