methods of studying cells Flashcards
magnification
number of times greater an image is than size of the real (actual) object
resolution
minimum distance apart 2 objects can be to be distinguished as separate objects
optical microscope features
- light focused using glass lenses
- light passes through specimen, different structures absorb different amounts and wavelengths
- generates 2D image of a cross-section
- low resolution due to long wavelength of light
- can’t see internal structure of organelles or ribosomes
- specimen = thin
- low magnification (x1500)
- can view living organisms
- simple preparation
- can show colour
transmission electron microscope (TEM)
- electron focused using electromagnets
- electron pass through specimen, denser parts absorb more and appear darker
- generates 2D image of a cross-section
- very high resolution due to short wavelength of electrons
- can see internal structure of organelles and ribosomes
- specimen = very thin
- high magnification (x1,000,000)
- can onlly view dead/dehydrated specimens as uses a vacuum
- complex preparation so artefacts are often present
- doesn’t show colour
scanning electron microscope (SEM)
- electron focused using electromagnets
- electron deflected/bounce off specimen surface
- generates 3D image of surface
- high resolution due to short wavelength of electrons
- can’t see internal structures
- specimen doesn’t need to be thin
- high magnification (x1,000,000)
- can onlly view dead/dehydrated specimens as uses a vacuum
- complex preparation so artefacts are often present
- doesn’t show colour
how does the scientific community distinguish between artefacts (e.g. dust, air bubbles etc.)
- scientists prepared specimens in different ways
- if an object was seen with one technique but not another, it was more likely to be an artefact than an organelle
what are the steps in calculations involving magnification, real size + image size
- note formula/rearrange if necessary (I = AM)
- convert units if necessary - image and actual size must be the same unit
- calculate the answer and check units required or if standard form etc. is required
how do you convert between different units
metre - x1000 - millimetre - x1000 - micrometre - x1000 - nanometre
describe how the size of an object viewed with an optical microscope can be measured
- line up (scale of) eyepiece graticule with (scale of) stage micrometre
- calibrate eyepiece graticule - use stage micrometre to calculate size of divisions on eyepiece graticule
- take micrometre away and use graticule to measure how many divisions make up the object
- calculate size of object by multiplying number of divisions by size of division
- recalibrate eyepiece graticule at different magnifications
steps of cell fractionation
- homogenise
- place in cold, isotonic, cold, buffered solution
- filter homogenate
- ultracentrifugation
what is step 1. homogenise and why do we do it?
- use a blender
- this disrupts the cell membrane, breaking open cells to release contents/organelles
what is step 2. place in a cold, isotonic, buffered solution and why do we do it?
- cold to reduce enzyme activity - so organelles aren’t broken down/damaged
- isotonic so water doesn’t move in or out of organelles by osmosis - so they don’t burst
- buffered to keep pH constant so enzymes don’t denature
what is step 3. filter homogenate and why do we do it?
- remove large, unwanted debris e.g. whole cells, connective tissue
what is step 4. ultracentrifugation and why do we do it?
- centrifuge homogenate in a tube at low speed
- remove pellet of heaviest organelle and re-spin supernatant at a higher speed
- repeated at increasing speeds until separated out, each time the pellet is made of lighter organelles (nuclei then chloroplasts/mitochondria then lysosomes then ER then ribosomes)
