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Concept: Biologists use microscopes and the tools
of biochemistry to study cells
• first compound microscope – Zacharias
Jansen in 1590
• three important parameters of microscopy
– Magnification: the ratio of an
object’s image size to its real size
– Resolution: the measure of the
clarity of the image, or the minimum
distance of two distinguishable
points
• inversely related to the wavelength of
the radiation a microscope uses
– Contrast: visible differences in
parts of the sample
Human height
1m
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Chicken egg
1 cm
Frog egg
1 mm
100 m
in compound microscopes – more than one lens
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–
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e.g. magnifying lens
0.1 m
ocular and objective lenses
improves resolution and allows for more than one
magnification
LMs can magnify effectively to about 1,000 times
the size of the actual specimen
this allows for individual cells within a tissue to be
visualized
Human egg
Most plant and
animal cells
10 m
1 m
100 nm
Nucleus
Most bacteria
Mitochondrion
Smallest bacteria
Viruses
Ribosomes
10 nm
Proteins
Lipids
1 nm
0.1 nm
Small molecules
Atoms
Superresolution
microscopy
Electron microscopy
•
in a light microscope (LM) - visible light is passed
through a specimen and then through glass lenses
the lenses refract (bend) the light - so that the
image is magnified
in a simple microscope - there is one lens for
magnification
Light microscopy
•
Length of some
nerve and
muscle cells
Unaided eye
Studying Cells: Microscopy
10 m
Light Microscopy (LM)
• various techniques enhance contrast of a
LM and enable cell components to be
stained or labeled
• BUT - most subcellular structures,
including organelles, are too small to be
resolved by an LM
• LMs cannot resolve detail finer than 0.2um
- regardless of magnification
50 m
Brightfield
(unstained specimen)
Brightfield
(stained specimen)
Phase-contrast
Differential-interferencecontrast (Nomarski)
Fluorescence
10 m
• to improve resolution and magnification – allowing for
imaging of subcellular structure - development of two
other microscopes in the 1950s
• called electron microscopes (EMs)- use a focused beam
of electrons rather than light
• resolution increase – due to the shorter wavelength of
the electron beam
• two types:
– 1. Transmission Electron Microscope (TEM)
– 2. Scanning Electron Microscope (SEM)
• 1. Scanning electron microscopes (SEMs) –
electron beam is focused onto the surface of a
subject
Blood cells
– providing images that look 3D
– SEM electron beam excites the electrons of the gold
on the subject’s surface
– several kinds of electrons are produced
– these electrons are detected by the scope and
projected onto a video screen as a magnified image
that appears 3D
– can be colorized
Pollen grains
•
2. Transmission electron microscopes (TEMs)
focus a beam of electrons through a specimen
–
–
–
–
–
–
subject is sliced into a very thin layer
so TEMs are used mainly to study the internal
structure of cells
subject is stained with heavy metals that adhere to
the internal structures of the cell
so some parts of the cell become more electron
dense than others
the electron beam passes through those less dense
and scattered/reflected by the more dense regions
the electrons that pass through hit a piece of film
negative or hit a detector for displaying the image