Methods of studying cells Flashcards
What is magnification?
the number of times greater an image is from the actual size
What is resolution?
the minimum distance apart 2 objects can be to distinguish them as separate objects
What are the principles of optical microscopes?
- light focused using glass lenses
- light passes through specimen where different structures absorb different amounts
- can view living organisms
- simple preparation
- shows colour image
What are the limitations of optical microscopes?
- low resolution due to long wavelength of light
- internal structures of organelles aren’t visible
- low magnification (x1500)
- generates 2D image of cross-section
What are the principles of transmission electron microscopes (TEM)?
- electrons focused using electromagnets
electrons pass through specimen where denser parts absorb more and appear darker - high resolution due to short wavelength of electrons
- high magnification (x1000000)
- internal structures of organelles are visible
What are the limitations of transmission electron microscopes (TEM)?
-generates 2D image of cross-section
- specimen must be very thin
- only able to view dead/dehydrated specimens viewed since vacuum used
- complex prep so artefacts often present
- image not colour
What are the principles of scanning electron microscopes (SEM)?
- electrons focused using electromagnets
- electrons deflected off specimen surface
- generates 3D image of surface
- high resolution due to short wavelength of electrons
- specimen doesn’t need to be thin
- high magnification (x100000)
What are the limitations of scanning electron microscopes (SEM)?
- can’t see internal structures
- only dead/dehydrated specimens viewed since vacuum used
- complex prep so artefacts often present
- image not colour
How is the difference between artefacts & organelles distinguished in a cell?
preparing specimens in different ways and if object was only seen with one technique it is likely to be an artefact
What is the formula to calculate magnification?
M = image size/ actual size
How can the size of an object viewed with an optical microscope be measured?
- line up eyepiece graticule with stage micrometre
- calibrate eyepiece graticule by using stage micrometre to calculate size of divisions on eyepiece graticule
- take micrometre away & use graticule to measure how many divisions make up object
- recalibrate eyepiece graticule at different magnifications
What is cell fractionation?
method used to separate cell components which involves homogenisation & ultracentrifugation
What are the principles of cell fractionation?
- Homogenise tissue - breaks open cells & releases organelles
- Place in cold, buffered isotonic solution - cold to reduce enzyme activity, buffered to keep constant pH & isotonic so water doesn’t move in the cell and burst it
- Filter homogenate - removes large unwanted debris
- Ultracentrifugation - separates organelles in order of density by spinning the sample at increasing high speeds where the heaviest organelles sink first
