Microscopy
What is the limitation of a light
microscope?
How can an electron
microscope overcome this?
In terms of magnification,
define ‘object’.
It cannot distinguish between two objects less than 0.2 m apart.
Define ‘image’.
The way the material appears when viewed through the microscope.
Define ‘magnification’.
How many times bigger the image of an object is compared to its actual size.
How do you find out the
magnification of an image?
How do you find out the size of
the object?
Refer to drawing g, Figure 1,
p. 41, showing a ribosome.
What is the actual size of the
ribosome? (Take size of image
as between X and Y)
Size of image/size of object
Define ‘resolution’.
Beams of electrons have a shorter wavelength.
The material put under the microscope
Size of image/magnification
21mm
1,000,0000
 2.1 x10 5  21nm
The minimum distance apart two objects must be in order to be distinguished from each other.
Increasing magnification always
False
increases resolution. True or
false?
Cell
fractionation
What is cell fractionation?
Where cells are lysed and the contents separated.
Why must the solution the
tissue is placed in before cell
fractionation be...
1. Cold?
To minimise the activity of enzymes that could
digest organelles
2. Isotonic?
To prevent damage to the organelles by
osmosis
To keep pH constant
3. Buffered?
Cell
fractionation
(continued)
Introduction
TEM
What is used to break up cells?
Homogeniser (or blender)
The fluid obtained is called:
Homogenate
How are the organelles in the
homogenate separated?
By using an ultracentrifuge.
Which organelles are heaviest?
Nuclei
Where in the centrifuged tube
are the heaviest cell
components found?
What is the fluid at the top of
the tube called?
Study Figure 2 and Table 2 on
p. 42. Which of the organelles
in Table 2 would still be left in
Supernatant 3?
What are the two main
advantages of an electron
microscope?
What special condition must
an electron microscope
operate in?
Two types of electron
microscope:
How is the beam focused in a
TEM?
Does the beam pass through
the specimen?
At the bottom, in the sediment.
Supernatant
Ribosomes
1. Short wavelength
A near-vacuum
2. Beam can be focused using electromagnets
Why is this?
1. Transmission electron microscope (TEM)
A condenser electromagnet
Yes
Molecules in the air can
absorb electrons
2. Scanning electron microscope (SEM)
Why do some parts of the
specimen appear dark?
Why do some parts of the
specimen appear bright?
What is the photographed
image produced called?
They absorb electrons
The electrons pass straight through
Photomicrograph
1. Vacuum
Limitations:
2. Process is complex and image is not a colour image
3. Specimen must be very thin
4. Artefacts
SEM
How can a 3D image be
produced from TEM images?
In a SEM, does the electron
beam come from above or
below?
How is a 3D image produced
using an SEM?
By taking cross sections through a specimen and studying the sections in order.
What is the resolution of:
Light microscopes: 0.2 m
Below
Electrons scatter as the beam is passed back and forth over the sample; the pattern of scattering
is analysed and used to make an image.
TEM: 0.1 nm
SEM: 0.2 nm