2.1.1 Cells and microscopy
Resolution - the ability to distinguish between two separate
points
Light Magnification - the number of times an image is enlarged
microscope compared to the actual object
s o Total magnification = eyepiece magnification x objective
magnification
Light microscope
Magnification up to x1500
Resolving power up to 200 nm
o Resolution can be improved by using shorter wavelength
radiation (eg. UV, beam of electrons)
How it works:
Visible light passes from a bulb under the stage, through a
condenser lens and up through the specimen. The beam of light
is then focused through the objective lens and through the
eyepiece lens.
Advantages & disadvantages
You can view a large range of specimens, including living cells
Inexpensive
Easy to use
Limited resolution means most internal structures cannot be
seen
Low max. magnification
Two main types of electron microscope: transmission electron
microscope (TEM) and scanning electron microscope (SEM).
Electron
microscope Advantages & disadvantages
s Much greater resolving power than light microscopes
Therefore smaller objects and higher detail can be seen
Large and expensive
Specimen must be dead as it must be placed in a vacuum
Require special training and location
Requires a complex staining process
General about EMs:
Specimens must be much thinner than when using light
microscopes because electrons cannot penetrate materials as
well as light rays can.
Heavy metal stains are used to stain the specimen, because the
atoms of these heavy metals have large, positively charged
nuclei that scatter the electrons.
The specimens must be dead as they need to be placed in a
vacuum, because air molecules absorb the electrons.
, Transmission electron microscope
Magnification up to x500000
Resolving power of 0.5 nm
How it works:
Specimen is placed in a vacuum and must be very thin in order
to allow electrons to pass through the specimen. The structures
in the cell that take up the heavy metal stain appear as dark
images.
Scanning electron microscope
Magnification up to x100000
Resolving power of 3-10 nm
How it works:
Specimen is placed in a vacuum where electrons don’t pass
through the specimen, instead they are reflected off its surface.
The beam of electrons is passed forwards and backwards over
the surface of the specimen, and the pattern of reflected
electrons is used to produce a 3D image of the surface.
Confocal laser scanning microscope
Developed in the 1980’s
Magnification up to x400
Resolution depends on light source
Can produce high resolution images of thick specimens
How it works:
Source of light is directed at the object plane
Point of reflected light is detected through a pinhole
Object is scanned point by point to produce very thin optical
sections
Optical sections are combined and a 3D image of the object is
produced.
Stains are used to reveal different structures within tissues and cells.
Differential
The stages in the preparation of a temporary slide are:
staining and Fixation, staining, and mounting.
blood
smears Differential staining can be carried out on a variety of plant tissues,
and is used to differentiate between things inside the cell, by making
some structures appear darker or a different colour from other
structures.
Differential staining of blood cells
Erythrocytes can be seen clearly under a microscope
Resolution - the ability to distinguish between two separate
points
Light Magnification - the number of times an image is enlarged
microscope compared to the actual object
s o Total magnification = eyepiece magnification x objective
magnification
Light microscope
Magnification up to x1500
Resolving power up to 200 nm
o Resolution can be improved by using shorter wavelength
radiation (eg. UV, beam of electrons)
How it works:
Visible light passes from a bulb under the stage, through a
condenser lens and up through the specimen. The beam of light
is then focused through the objective lens and through the
eyepiece lens.
Advantages & disadvantages
You can view a large range of specimens, including living cells
Inexpensive
Easy to use
Limited resolution means most internal structures cannot be
seen
Low max. magnification
Two main types of electron microscope: transmission electron
microscope (TEM) and scanning electron microscope (SEM).
Electron
microscope Advantages & disadvantages
s Much greater resolving power than light microscopes
Therefore smaller objects and higher detail can be seen
Large and expensive
Specimen must be dead as it must be placed in a vacuum
Require special training and location
Requires a complex staining process
General about EMs:
Specimens must be much thinner than when using light
microscopes because electrons cannot penetrate materials as
well as light rays can.
Heavy metal stains are used to stain the specimen, because the
atoms of these heavy metals have large, positively charged
nuclei that scatter the electrons.
The specimens must be dead as they need to be placed in a
vacuum, because air molecules absorb the electrons.
, Transmission electron microscope
Magnification up to x500000
Resolving power of 0.5 nm
How it works:
Specimen is placed in a vacuum and must be very thin in order
to allow electrons to pass through the specimen. The structures
in the cell that take up the heavy metal stain appear as dark
images.
Scanning electron microscope
Magnification up to x100000
Resolving power of 3-10 nm
How it works:
Specimen is placed in a vacuum where electrons don’t pass
through the specimen, instead they are reflected off its surface.
The beam of electrons is passed forwards and backwards over
the surface of the specimen, and the pattern of reflected
electrons is used to produce a 3D image of the surface.
Confocal laser scanning microscope
Developed in the 1980’s
Magnification up to x400
Resolution depends on light source
Can produce high resolution images of thick specimens
How it works:
Source of light is directed at the object plane
Point of reflected light is detected through a pinhole
Object is scanned point by point to produce very thin optical
sections
Optical sections are combined and a 3D image of the object is
produced.
Stains are used to reveal different structures within tissues and cells.
Differential
The stages in the preparation of a temporary slide are:
staining and Fixation, staining, and mounting.
blood
smears Differential staining can be carried out on a variety of plant tissues,
and is used to differentiate between things inside the cell, by making
some structures appear darker or a different colour from other
structures.
Differential staining of blood cells
Erythrocytes can be seen clearly under a microscope
