Honors Biology
Microscopes
Important tool for all biologists
Honors Bio: Microscopes
Use light or electrons to magnify
Enable us to see the shape and structure of
very small objects
• Cells and cell parts
• Tissues
• Molecules (only with electron microscopes)
• Small and microscopic organisms
Value of Magnification
Real size
Magnified 400 X
Cell walls
Elodea canadensis
Pond weed
chloroplasts
cytoplasm
central vacuole
Magnification
Magnification = object size ~ image size
Total magnification = ocular lens X objective lens
chloroplasts
flagellum
nucleus
food vacuole
Euglena, a one-celled organism 1000X
Resolution or Resolving Power
Resolution = sharpness, clarity of focused image
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•
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“Ability to show two close points as separate”
Depends on shape and perfection of lenses
Human eye can see objects as small as 0.2 mm
A light microscope can resolve objects as small as 0.2 m
high resolution lens
lower resolution lens
Comparing Resolutions
RP in Angstroms
Optical Instrument
Resolving Power
Human eye
0.2 millimeters (mm)
2,000,000 A
Light microscope
0.20 micrometers (µm)
2000 A
Scanning electron
microscope (SEM)
5-10 nanometers (nm)
50-100 A
(TEM) Transmission
0.5 nanometers (nm)
electron microscope
5A
Depth of Field
• Thickness or layer in focus
• Higher magnification  thinner layer
Light Microscopes
Send LIGHT through a thin specimen
an early microscope
binocular light
microscope
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Light Microscopes (LM)
• Light waves pass through a thin specimen
• Lenses bend light to magnify image
– Simple microscope – one lens
– Compound microscope – two lenses
• Magnifies image twice
Leeuwenhoek’s Microscope
• Anton von Leeuwenhoek, 1600s
• First powerful scope with high resolution
– Single lens
– Magnify ~ 300 X
Leeuwenhoek’s microscope
LE 4-1a
Eyepiece
Ocular
lens
Objective lens
Specimen
Condenser
lens
Light
source
BINOCULAR MICROSCOPE – has ocular lens for each eye
How two lenses magnifies
Epithelial cell
Photosynthetic
cells
Chloroplast
(dots inside cell)
Leaf cross-section (LM)
Stoma
(leaf opening)
Advantages of light microscopes
- Can magnify up to 2000 times
- Shows shape and structure of cells and
tiny organisms
- Specimens can be alive
Disadvantages
- Specimens must be thin enough for light to
pass through
- Image appears inverted and backwards
- Often need stain to see image
Cheek cells with stain
Light microscope
LM “dark field”
Common stains: methylene blue, Lugol’s iodine
“Vital stains” - stain without killing cells
Phase-Contrast Microscope
“Differential Interference Microscope”
Increases contrast between tissue densities
– don’t need stain; good for living organisms
Cheek cells without stain
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Compound Microscope
cheek cells – stained
nucleus
cytoplasm
cell membrane
Phase-Contrast
Microscope
cheek cells –unstained
nucleus
cytoplasm
cell membrane
Amoeba, one-celled organism
preserved, stained
Compound scope
alive, moving
Phase-Contrast scope
Cell cycle, under phase contrast
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Phase-contrast micrograph of a
roundworm 630X
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Stereomicroscope
“Dissecting microscope”
Has ocular lens and
objective lens for
each eye 
Stereoscopic vision,
3-D
Image NOT inverted
Magnifies 10-50X
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Advantages of stereoscopes
• Image NOT inverted or backwards
• Makes manipulation easy
• Specimens can be solid, living
• Disadvantage: magnifies up to ~50 X
Stereomicroscope – whole specimens
chick embryo
soil worm
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Fluorescent Microscopy
• Uses lasers on thin slices; confocal scope
• Fluorescent dyes show different molecules
Cancer cells tagged with 3 fluorescent dyes shows cell microtubules
(blue), microfilaments (yellow), DNA (green)
Fluorescent – shows different cell parts
as different colors
• Details in a single layer
Green – microtubules in cytoplasm
Red -DNA
http://www.microscopyu.com/tutorials/java/virtual/conf
ocal/index.html
Fruit fly embryo –
developmental layers
Confocal Microscopy
E. Coli bacteria
Specialized Cells in the Ear
Electron Microscope
• Uses electrons instead of light
• Magnets focus the beam
• Image shows on monitor
• Magnify up to 1 million times
• Show cell details, interior
- “ultrastructure
• Invented 1930’s
• Nobel for Ruska 1986
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Electron Microscope
• How does it work?
– Specimen is coated with a metal
film
– Electron beam hits metal, ejects
electrons from metal atoms
– These electrons make the image
Advantages
of electron microscopy
• Electron are much smaller than the
wavelength of light – show things that light
cannot show
• Very high magnification – up to 1,000,000X
• Very high resolution - up to 1 nanometer
• DISADVANTAGE – specimen must be
dead, dried, coated, in vacuum chamber
Scanning Electron Microscope
SEM
• Electron beam skims across specimen
surface
• Shows tiny surface structures in great detail
• Magnifies up to 50,000 times
• DISADVANTAGE: shows surface, but not
interior
Compare LM and SEM
Blood cells (LM)
Blood cells (SEM)
SEM micrographs
Euglena (protist)
SEM
Ant head, SEM
Scanning Electron Microscope (SEM)
shows surface details
Electrons scan across surface of specimen
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SEM of DNA
Image made with special scanning “tunneling” microscope
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Transmission Electron Microscope
(TEM) shows inside cells
• Electrons pass through thin specimen
• Shows great detail of internal structure
• Magnifies up to 1,000,000 times!!
Rough ER
Mitochondria
Nucleus
Transmission Electron Microscope
Bacterium dividing
Muscle fibers
Phage virus
Liver cells
Cilia and basal bodies
Chloroplast
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Comparing microscopes
Euglena SEM
Euglena LM
Euglena TEM
Which type of microscope
produced these micrographs?
Amoeba, preserved and stained
Vacuole inside a cell
Amoeba, alive and unstained
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Which type of microscope made
these micrographs?
Female and male fruit fly
Closterium Unicellular green alga
40
Name the microscope
Leaf cross-section 400X
chloroplast 5,000 X
Name the microscope
Iridescent beetle
Eye of a housefly
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Which microscope?
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