Cell Structure
• Magnification: how many times bigger the image is produced
• Resolution: ability to distinguish between 2 points that are close together as
separate structures
Optical microscopes:
• Use light to form an image- limits the resolution of the microscope
• Produce a colour image
• Maximum resolution: 200nm
• Can be used to observe eukaryotic cells (living or dead), their nuclei
and maybe mitochondria and chloroplast
• Cant be used to observe smaller organelles i.e. ribosomes, ER or
lysosomes
• Maximum magnification: x1500
Electron microscopes:
• Uses electrons to form an image
• Vacuum needed for electron microscopes
• Maximum resolution: 0.5nm
• Can be used to observe small organelles such as ribosomes, ER or lysosomes, viruses
and DNA
• Magnification: x500,000
• 2 types:
o TEM: electrons transmitted through the specimen (denser parts of the
specimen absorb more electrons)
▪ Advantages:
• resolution 0.5nm
• Allows internal structures within cells to be seen
▪ Disadvantages:
• Can only be used with very thin specimens
• Cannot be used to observe live specimens
• Lengthy treatment to prepare specimens
• Don’t produce a colour image
o SEM: electrons scanned across the surface of the specimen. This beam
bounces off the surface of the specimen and the electrons are detected,
forming an image- 3D image produced (resolution: 3-10nm)
▪ Advantages:
• Thick or 3D specimens
• Allow the external 3D structure of specimens to be observed
▪ Disadvantages:
• Lower resolution images than TEM
• Cannot be used to observe live specimens
, • Don’t produce a colour image
Laser scanning confocal microscope:
• The cells being viewed must be stained with
fluorescent dyes
• A thick section of tissue or small living organisms are
scanned with a laser beam to produce an image
• The laser beam is reflected by the fluorescent dyes
• Fluorescence: absorption and re-radiation of light.
• A laser scanning confocal microscope moves a single
spot of focused light across a specimen (point
illumination)- causes fluorescence from the components
with dye
• Emitted light from the specimen is filtered through a
pinhole aperture.
• Only light radiated from very close to the focal plane
(distance that gives the sharpest focus) is detected
• Advantages:
o Can be used for thick/ 3D specimens
o Very clear images are produced- laser beam is focused at a very specific depth
o Can see the structure of the cytoskeleton in cells
• Disadvantages:
o Slow process- takes a long time to obtain an image
o Laser has the potential to cause photodamage to the cells
Preparation of microscope slides
• Light is directed through the thin layer of biological material that is supported on a
glass slide
• The light is focused through several lenses so that the image is visible through the
eyepiece
• The magnifying power of the microscope can be increased by rotating the higher
power objective lens into place
• Coarse focus knob: the big one- used to focus the low and medium power objective
lenses
• Fine focus knob: the small one- used to focus the high power objective lens
Sample preparation
1. Dry mount: specimens viewed whole or cut into very thin slices with a sharp blade-
sectioning
Specimen is placed on the centre of the slide and a cover slip is placed over the
sample
2. Wet mount: sample suspended in a liquid
Coverslip placed on from an angle
• Magnification: how many times bigger the image is produced
• Resolution: ability to distinguish between 2 points that are close together as
separate structures
Optical microscopes:
• Use light to form an image- limits the resolution of the microscope
• Produce a colour image
• Maximum resolution: 200nm
• Can be used to observe eukaryotic cells (living or dead), their nuclei
and maybe mitochondria and chloroplast
• Cant be used to observe smaller organelles i.e. ribosomes, ER or
lysosomes
• Maximum magnification: x1500
Electron microscopes:
• Uses electrons to form an image
• Vacuum needed for electron microscopes
• Maximum resolution: 0.5nm
• Can be used to observe small organelles such as ribosomes, ER or lysosomes, viruses
and DNA
• Magnification: x500,000
• 2 types:
o TEM: electrons transmitted through the specimen (denser parts of the
specimen absorb more electrons)
▪ Advantages:
• resolution 0.5nm
• Allows internal structures within cells to be seen
▪ Disadvantages:
• Can only be used with very thin specimens
• Cannot be used to observe live specimens
• Lengthy treatment to prepare specimens
• Don’t produce a colour image
o SEM: electrons scanned across the surface of the specimen. This beam
bounces off the surface of the specimen and the electrons are detected,
forming an image- 3D image produced (resolution: 3-10nm)
▪ Advantages:
• Thick or 3D specimens
• Allow the external 3D structure of specimens to be observed
▪ Disadvantages:
• Lower resolution images than TEM
• Cannot be used to observe live specimens
, • Don’t produce a colour image
Laser scanning confocal microscope:
• The cells being viewed must be stained with
fluorescent dyes
• A thick section of tissue or small living organisms are
scanned with a laser beam to produce an image
• The laser beam is reflected by the fluorescent dyes
• Fluorescence: absorption and re-radiation of light.
• A laser scanning confocal microscope moves a single
spot of focused light across a specimen (point
illumination)- causes fluorescence from the components
with dye
• Emitted light from the specimen is filtered through a
pinhole aperture.
• Only light radiated from very close to the focal plane
(distance that gives the sharpest focus) is detected
• Advantages:
o Can be used for thick/ 3D specimens
o Very clear images are produced- laser beam is focused at a very specific depth
o Can see the structure of the cytoskeleton in cells
• Disadvantages:
o Slow process- takes a long time to obtain an image
o Laser has the potential to cause photodamage to the cells
Preparation of microscope slides
• Light is directed through the thin layer of biological material that is supported on a
glass slide
• The light is focused through several lenses so that the image is visible through the
eyepiece
• The magnifying power of the microscope can be increased by rotating the higher
power objective lens into place
• Coarse focus knob: the big one- used to focus the low and medium power objective
lenses
• Fine focus knob: the small one- used to focus the high power objective lens
Sample preparation
1. Dry mount: specimens viewed whole or cut into very thin slices with a sharp blade-
sectioning
Specimen is placed on the centre of the slide and a cover slip is placed over the
sample
2. Wet mount: sample suspended in a liquid
Coverslip placed on from an angle
