3.2.1 Studying cells Flashcards
What is magnification?
How much bigger the image is than the specimen
Magnification = image size / actual size
What is resolution?
The shortest distance between two points on a specimen that can still be distinguished by the microscope camera as separate entities
What is resolution limited by?
Wavelength
How does an optical light microscope produce an image?
Using light rays that are transmitted through the specimen and two lenses
What is the maximum magnification of an optical light microscope?
x 1 500
What are the limitations of an optical microscope?
Low resolution - maximum about 0.2µm (long wavelength of light)
Cannot see cell ultrastructure smaller than 0.2µm: lysosomes, ribosomes, endoplasmic reticulum
How does an electron microscope produce an image?
Using electrons to produce black and white images
What is the maximum magnification of an electron microscope?
x 1 500 000
What is the resolution of an electron microscope and why?
High resolution - maximum about 0.0002µm (x1000 light microscope) - electrons have very short wavelength
Can see more organelles
What are the limitations of an electron microscope?
Process involves specimen preparation: staining with toxic metal, viewing in a vacuum, specimen cannot be alive
Colour is only added artificially
Specimen must be very thin - cut with microtome (TEM)
Artefacts could be present in photomicrograph from method of preparation
What are the two types of electron microscope?
Transmission EM
Scanning EM
How does a TEM work?
Electromagnets are used to focus a beam of electrons, which is then transmitted through the specimen
Denser parts of the specimen absorb more electrons - darker images
This helps to identify where organelles are located
Its high resolution allows you to see internal structure of organelles e.g. chloroplasts
What are the limitations of TEM?
Specimen must be view in a vacuum, so movement living organisms could not be observed
They can only be used on very thin specimens (cut using a microtome) so that electrons could pass through them
How does a SEM work?
A beam of electrons is scanned across the specimen, focused using magnetic condensers
Electrons are reflected off from the specimen and are gathered in a cathode ray tube to form an image
They produce 3D images
What is cell fractionation?
The process of breaking cells and separating out different organelles within
What is done before cell fractioning?
Homogenisation and fractionisation
What happens in homogenisation?
Tissue is cut up and stored in cold, isotonic buffered solution
They are further cut up using a homogeniser
This releases organelles from the cell
What happens after homogenisation?
Filtration
The resultant liquid from homogenisation is called the homogenate
It will then be filtered to remove any complete cells or large pieces of cell/tissue debris
What happens after filtration?
Ultracentrifugation
Solution is poured into a tube and placed into a centrifuge
It will be spun first at a slower speed, which forces the heaviest organelles (nuclei) to the bottom of the tube - forming a pellet. The rest of the organelles stay suspended in the fluid above called the supernatant
The supernatant will be drained off and placed into another tube. This process is repeated at a higher speed and a new pellet and supernatant will form.
This process is repeated until the desired organelles are spun out with the centrifuge
