Microscopy
Learning Objectives
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Identify cells that can be seen with a
light microscope
Use of light microscope to view
prepared slides and own slides
State the meaning of magnification
and resolution
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Work on units of length and
magnification calculations
In groups list the different
components of an animal cell
Which components are visible
with a light microscope?
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Cell membrane
Nucleus
Cytoplasm
Components not visible
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Ribosomes
Lysosome
Mitochondrion
RER
Golgi body
Nucleolus
Nuclear membrane or pore
How small is a cell?
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Magnification
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This is the increase in the apparent size of
an object
The total magnification of a light
microscope is worked out by:
Magnification of objective lens X magnification of eyepiece (ocular) lens
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There is no limit to magnification produced
by a light microscope. However above a
certain magnification the image becomes
blurred.
This limit is known as the resolving power
or resolution of the microscope
Resolution
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Resolution is the ability to show two
objects as separate
A light microscope’s resolution is limited
by the wavelength of light
No lens no matter how strong it can
magnify can resolve 2 dots that are
separated by less than 200nm (half the
wavelength of light)
Advantages of a light
microscope
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Specimen can be living or dead
Relatively quick and cheap to
prepare
Relatively cheap
Coloured images
No vacuum needed
Less training needed
Disadvantages of light
microscope
Low resolution
Smaller image
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Units of Measurement
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Standard SI units used in microscopy
are
Metre
m
= 1m
Millimetre
mm
= 10-3m
Micrometre
μm
= 10-6m
Nanometre
nm
=10-9m
Picometre
pm
=10-12m
Units of measurement
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Millimetre (mm) 1/1000th of a m
(10-3 m)
Micrometre (μm) 1/1000th of a mm
(10-6 m)
Nanometre (nm) 1/1000th or a μm
(10-9 m)
Summary of light Microscopes
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Specimens illuminated by light
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Focussed by glass lenses and viewed using photographic
film or eyes
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Specimens can be living or dead
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Maximum resolution of 200nm (good enough to see cells
but not details of organelles)
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Maximum magnification of 1500x
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Images are coloured
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Cheap and easy to prepare
Calculating magnification
I = IMAGE SIZE
A = ACTUACL SIZE
M = MAGNIFICATON
I
A
M
Virtual microscope
Mini Quiz
1.One advantage of using a light microscope
2.One disadvantage of using a light
microscope
3.What does resolution mean?
4.How do you calculate magnification?
5. Convert 3mm into μm
6. Is 10-6m a micrometre or nanometre?
Homework gridsmicroscopy
Learning Objectives
1.Understand the principles by
which TEM and SEM work.
2.State the differences between
TEM and SEM
3.State limitations of TEM and
SEM
What differences do you notice?
1
3
2
The Electron microscope
What is the main limitation of the light
microscope?
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Poor resolution due to the
wavelength of light
Main advantages of electron
microscopes?
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Uses a beam of electrons which has a
shorter wavelength than light so has a
high resolving power (1nm)
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Magnification up to 500,000x
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Revealed the detailed ultra structure of
cells
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2 main types of electron microscope:
transmission and scanning
Transmission electron microscope
(TEM)
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TEM essentially looks ‘through’ a thin
slice of a specimen
Resolution of 0.1nm
Scanning electron microscopes (SEM)
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SEM looks at the surface of a solid object
SEM has a lower resolving power than
TEM (20nm)
Advantageous as has ability to image a
large area of specimen
Images are often easier to interpret than
TEM
Transmission electron microscope (TEM)
Parts of the
specimen absorb
electrons and
appear dark, and
parts allow
electrons to pass
through and appear
bright.
Principles of TEM
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Specimens have to be extremely thin to allow electrons to
penetrate
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Specimen placed in chamber (under vacuum), as electrons
are absorbed by the molecules in air.
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Beam of electrons focused onto the specimen by condenser
electromagnet
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Beam passes through small section of specimen
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Parts of specimen absorb electrons (dark) or allow electrons
to pass through it (bright)
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Image produced on a screen which can be photographed to
give photomicrograph ( a 2D image)
Scanning electron microscope (SEM)
Principles of SEM
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Similar technique to TEM, but directs beam of electrons
onto surface of specimen from above
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Beam passes through pairs of scanning coils which deflect
the beam horizontally and vertically
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Beam scans across portion of specimen in a regular pattern
resulting in scattering of electrons and emission of
secondary electrons from surface of specimen
Pattern of scattering depends on contours of specimen
surface
A 3D image is created from computer analysis
Resolving power of 20nm, (less than the TEM)
Limitations of electron microscopes
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Vacuum used to prevent scattering of electrons
so only dead specimens can be viewed
TEM sample must be extremely thin
Complex preparation and staining of specimens is
required. Image only in black and white.
Image (photomicrograph) may contain ‘artefacts’
which result from the way specimens are
prepared e.g. dehydration of specimen may
result in distortion of key features
Learning Objectives
1.Understand the principles by
which TEM and SEM work.
2.State the differences between
TEM and SEM
3.State limitations of TEM and
SEM
For each image write down a
fact