3. analysis of cell components Flashcards
what is magnification?
how much bigger the image is than the specimen
how is magnification calculated
magnification = size of image / size of real object
triangle formula for calculating magnification / size of real object (actual)
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what is resolution?
how detailed the image is
how well a microscope distinguishes between two points that are close together
what do optical (light) microscopes use to form an image
light
- what is the maximum resolution of an optical microscope?
2. what can you see and what cant you see?
- O.2um (micrometres)
- can see - nucleus
can’t see ribosomes, endoplasmic reticulum and lysosomes.
what is the maximum useful magnification of a light microscope
x1500
what do electron microscopes use to form an image
electrons
what is the maximum resolution of an electron microscope?
0.0002 um
higher resolution than optical = more detailed image
what is the maximum useful magnification of an electron microscope
x1,500,000
converting units
micrometres (um) -> millimeters (mm) / 1000
how do Transmission electron microscopes work (TEM)
1, use electromagnets to focus beam of electrons which is then transmitted through specimen
2. denser part of specimen absorbs more electrons which makes them look darker on the image
advantage of TEM
give high resolution images
see internal structure of organelles
disadvantage of TEM
can only be used on thin specimens
how do Scanning electron microscopes work (SEM)
- scan a beam of electrons across specimen.
this knocks off electrons from specimen, which are gathered in a cathode ray tube to form an image
advantage of SEM
images show surface of specimen and can be 3D
can be used on thick specimen
disadvantage of SEM
lower resolution images than TEM
how to prepare a temporary mount of a specimen on a slide
4 steps
- pipette a small drop of water onto slide
- use tweezers to place thin section of specimen on top of water drop
- add drop of stain (highlights objects in a cell)
- add cover slip
examples of stains used on temporary mounts
eosin makes what show up?
cytoplasm
examples of stains used on temporary mounts
iodine in potassium iodide solution stains what?
starch grains in plant cells
why do you have to CAREFULLY tilt and lower cover slip onto specimen/slide
avoid air bubbles (obstruct view of specimen)
how to separate organelles
cell fractionation
- homogenisation - break up cells
- filtration - getting rid of big bits
- ultracentrifugation (separating organelles)
cell fractionation
1. homogenisation (break up cells)
grind up cells in blende
breaks up plasma membrane and releases organelles into solution
must be kept ice cold to reduce enzyme activity that break down organelles
solution should be isotonic (same conc of chemicals as cells being broke down)
prevents damage of cells through osmosis
buffer solution added to maintain PH
cell fractionation
2. filtration (getting rid of big bits)
homogenised cell solution filtered through gauze to separate any large cell debris or tissue debris from organelles (organelles much smaller so pass through gauze)
cell fractionation
3. ultracentrifugation (separating the organelles)
- cell fragments poured into tube. tube put in centrifuge which is spun at a low speed. heaviest organelles nuclei form pellet at bottom. rest of the organelles remain suspended in fluid above (the supernatant)
- supernatant drained off poured in another tube and spun in centrifuge at a higher speed. heaviest organelles form pellet (this time mitochondria) supernatant drained off and spun again at a higher speed
- process repeated at higher and higher speeds until organelles are separated out
order of separation in ultracentrifuge
nuclei mitochondria lysosomes endoplasmic reticulum ribosomes
