3.1 Methods of studying cells Flashcards
(16 cards)
Describe the difference between magnification and resolution
● Magnification = number of times greater image is than size of the real (actual) object
○ Magnification = size of image / size of real object (Or I=A x M)
● Resolution = minimum distance apart 2 objects can be to be distinguished as separate objects
What are the characteristics of the optical microscope
(10)
- Light is focused using glass lenses
- Light passes through specimen differnet structures absorb different amounts of light and wavelengths
- Generates a 2D image of a cross section
- Low resolution due to long wavelength of light
- Cant see internal structures of organelles or ribosomes
- The specimen is thin
- Low magnification (x1500)
- Can View living organisms
- Simple preparations
- Can show colour
What are the characteristics of the Transmission electron microscope
(10)
- Electrons focused using electromagnets
- Electrons pass through specimen, Denser parts absorb more and appear darker
- Generates a 2D image of a cross section
- Very high resolution due to short wavelength of electrons
- Can see internal strctures of organelles and ribosomes
- Specimen= very thin
- High magnification (1,000,000)
- Can onlu view dead/dehydrated specimens as a vacuum
- Complex preparations so artefact often present
- Does not show colour
What are the characteristics of the scanning electron microscope
(10)
- Electrons focused using electromagnets
- Electrons deflected/ bounce off specimen surface
- Generates a 3D image
- High resolution due to short wavelength of electrons
- Cant see internal strcutres
- Specimen does not need to be thin
- High magnification (x1,000,000)
- Can only view dead/dehydrated specimens as it uses a vacuum
- Complex preparations so artefacts are present
- Does not show colour
What is an artefact
Artefacts are visible details that aren’t part of the specimen being observed, such as air bubbles or fingerprints
Suggest how the scientific community distinguished between artefacts (eg. dust, air bubbles occurring during preparation) and cell organelles
● 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
List the steps in calculations involving magnification, real size & image size (3)
1 Note formula / rearrange if necessary (I = AM)
2 Convert units if necessary - image and actual size
must be in same unit
3 Calculate answer and check units required or if
standard form etc. is required
Describe how the size of an object viewed with an optical microscope can be measured (5)
Stage micrometer stuff
- 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
What is cell fractionation
A method used in cell biology to separate the different parts of a cell (specifically organelles) based on their size and density.
Describe and explain the principles of cell fractionation and
ultracentrifugation as used to separate cell components (only frist two steps)
- Homogenise tissue /use a blender
● Disrupts the cell membrane, breaking open cells to release contents/organelles
FILTER - Place in a cold, isotonic, buffered solution
● Cold to reduce enzyme activity
○ So organelles not 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
Describe and explain the principles of cell fractionation and
ultracentrifugation as used to separate cell components (steps 3-4)
- Filter homogenate
● Remove large, unwanted debris eg. whole cells, connective tissue - Ultracentrifugation -separates organelles in order of density/mass
● Centrifuge homogenate in a tube at a low speed
● Remove pellet of heaviest organelle and respin supernatant at a higher speed
● Repeat at increasing speeds until separated out, each time the
pellet is made of lighter organelles (nuclei→chloroplasts/mitochondria→lysosomes→ER→ribosomes)
What is a homogeniser
A blender
What is ultracentrifugation
the process used to separate organelles based on their density.
What is the process of ultracentrifugation (6)
- The filtrate is placed (ina tube) in the centrifuge and spun at low speeds
- The heaviest organelles,nuclei, are forced to the bottom (of the tube) where they form a thin sediment or pellet
- The fluid at the top of the test tube (supernatant) is removed
- The supernatant is transferred to another tube and spun in the centrifuge at a faster speed
- The next heaviest organelle is forced to the bottom of the tube
- Repeat this process and the speed is increased every time
Although the resolving power of the TEM is 0.1nm why can this not always be achieveid
There could be diffuclties preparing the speicmen
A higher energy electron beam is required and this may destroy the specimen
Why are some areas brighter in the TEM
Some parts of the specimen allow the electron to pass through
Low density
