Cell Fractionation + microscopy Flashcards
define cell fractionation
a technique which allows scientists to study the functions of organelles
Separating out all of the different organelles/components
define homogenisation
Homogenise means to break up the tissue and break open the cells
what is ultracentrifugation
ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge
what does the centrifuge do
spins tubes of homogenate at very high speeds to produce a centrifugal force
this force causes homogenate to separate
describe homogenisation
In the first stage, take a sample of tissue containing the cells that we are interested in e.g. heart muscle tissue
Next, homogenise the tissue
Homogenise means to break up the tissue and break open the cells
note: the ER is a very large organelle - but this tends to get broken up during homogenisation
This can be done in a blender
Or we can use a homogeniser ( a glass tube containing a plunger)
Place the tissue sample into the glass tube
Cover with a buffer solution
Buffers keep the pH constant
This is important because if the pH changes, enzymes in the cells organelles could denature ||(COULD ALTER STRUCTURE OF ORGANELLES OR AFFECT THE FUNCTIONING OF ENZYMES)
The water potential of the buffer is the same as inside the cell
This prevents water from moving into the organelles by osmosis and causing them to burst
PREVENT ORGANELLES BURSTING OR SHRINKING AS A RESULT OF OSMOTIC GAIN OR LOSS OF WATER
The homogeniser is then placed on ice
Cooling the sample means that enzymes work more slowly, preventing any destructive enzymes from damaging the organelles REDUCE ENZYME ACTIVITY THAT MIGHT BREAK DOWN THE ORGANELLES
Now push the plunger up and down, to disrupt the tissue and break open the cells
This produces a cell homogenate
The cell homogenate contains all the organelles that we find in the cell
what does the cell homogenate contain
The cell homogenate contains all the organelles that we find in the cell
contains all of the organelles which we would find in a cell
what needs to be done to find out what the organelles in the cell homogenate do
In order to find out what these organelles do, we need to separate them
describe cell fractionation
This produces a cell homogenate
The cell homogenate contains all the organelles that we find in the cell
In order to find out what these organelles do, we need to separate them (in order to understand this, we need to look at relative sizes of different organelles - nucleus, mitochondria, lysosomes, ribosomes)
Separating out all of the different organelles is called fractionation
ultracentrifugation
This is carried out using a machine called a centrifuge
1) We place the tubes containing the filtered cell homogenate into the sample holder of the centrifuge
2)The centrifuge now spins the sample
The organelles are flung , forced towards the bottom of the tube by the forces generated
Larger organelles…
3)First we start with a relatively low speed spin.
the tube looks like this (at the end of the spin)
https://cdn.savemyexams.com/cdn-cgi/image/w=1920,f=auto/uploads/2021/02/Cell-fractionation-stages-3.png - first picture
4) As the centrifuge spins, the larger organelles/heaviest such as the nuclei are flung, forced to the bottom of the tube by the forces generated forming a pellet (pellet containing nuclei)
5) Larger organelles (with a greater mass) such as the nucleus experience a greater force and move towards the bottom of the tube faster than the smaller organelles
6) The remaining organelles stay suspended in the liquid. This liquid is called the supernatant.
Now transfer the supernatant into a new tube, and centrifuge this at a higher speed
After the higher speed spin, the heaviest organelles (which are now mitochondria) now are forced to the bottom of the test tube and form pellet now contains mitochondria
process is repeated at higher and higher speeds untill all organelles are separated out. each time, the pellet at the bottom of the tube is made up of lighter and lighter organelles
Once again, transfer the supernatant to a new tube and centrifuge again at a higher speed. After this spin, the pellet now contains lysosomes
Take the supernatant one more time and transfer this to another tube for a final very high speed spin.
After this spin, the pellet now contains ribosomes
At this point, all of the organelles have been separated by size
Now we can test each fraction to determine how the organelles work
note: in plant cells the chloroplasts come after the nuclei but before the mitochondria
the order in which organelles are separated out - they are separated out in order of mass -from heaviest to lightest: nuclei, mitochondria, lysosomes, ER, ribosomes
Additional points about cell fractionation
Additional - we need to keep the pellets on ice until we use them. this is to slow down enzymes which might damage the organelles
it is extremely difficult to separate the organelles fully e.g. the mitochondrial fraction might contain a very small number of nuclei and lysosomes
Also there are other organelles such as the endoplasmic reticulum and Golgi apparatus which might be present in your fractions
How are the organelles separated in ultracentrifugation
In ultracentrifugation, the organelles are separated in order of mass - from lightest to heaviest
describe what was happening after each spin
Each time, the pellet at the bottom is made from lighter and lighter organelles
what is a micrometre
1 x 10-6 metre
1 mm is how many micrometres
1 mm = 1000 micrometre
nanometre size
1 x 10-9 metre
1 micrometre = how many nanometres
1 micrometre = 1000 nanometres
Advantage of using light microscopes compared to other microscopes
Light microscopes can be used on living cells - we can look at living cells
This means that we can explore processes such as cell division or movement of cells / chromosomes moving during mitosis
Disadvantage of using light microscopes compared to other microscopes
Sometimes a stain needs to be used (before viewing the cell - to help see the cell) - and these can kill cells
poor Resolution - this is due to the nature of light
the wavelength of visible light is around 400nm to 700 nm
if two objects are closer than 200nm (limit of resolution for a standard light microscope) then we cannot see them
resolution of light microscope 200nm—
because… wavelength of visible light is simply too large to allow us to resolve objects closer than that
In conventional light microscopy, light passes through the sample
when trying to view certain parts of the cell which are further away, the light that we see will already have passed through the parts of the cell before reaching what you’re actually trying to view - makes the image blurred and resolution even worse than it could be
define resolution
The minimum distance between two objects where they can still be seen as two separate objects
the ability to distinguish between two separate objects
describe how laser scanning confocal microscopy works
In laser scanning confocal microscopy, a laser is used to scan the object that we are interested in
This allows us to view a very precise layer of the object
This means that the image produced by laser scanning confocal microscopy has a higher resolution than conventional light microscopy
using laser scanning confocal microscopy we can produce
describe the advantages of laser scanning confocal microscopy over conventional light microscopy
In laser scanning confocal microscopy, a laser is used to scan the object that we are interested in
This allows us to view a very precise layer of the object
________________________
This means that the image produced by laser scanning confocal microscopy has a higher resolution than conventional light microscopy
using laser scanning confocal microscopy we can produce three dimensional images of cells
we can visualise specific proteins and structures within the cell and watch them move.
_________________
this technique also allows us to tag specific proteins and structures within cells by using special dyes and antibodies
with laser scanning confocal microscopy we can watch proteins moving around living cells
This helps us to work out the functions of those proteins
formula for magnification
magnification = size of image / size of real object
scale bar = 10mm which represents 1 micrometre
nucleus = 63 mm
work out actual size of nucleus
scale bar = 10mm
nucleus = 63mm
63/10 = 6.3
means that the nucleus is 6.3 times the length of the scale bar
1 micrometre x 6.3 = 6.3 micrometres
always do organelle / scale bar
scale bar = 37mm
mitochondrion = 21 mm
scale bar represents 2 micrometres
work out actual size of mitochondria
scale bar = 37 mm
mitochondrion = 21mm
21/37 = 0.57
means that the mitochondrion is 0.57 times the length of the scale bar
0.57 x 2 micrometres = 1.14
object size of mitochondrion = 0.8 micrometres
length = 30mm
work out magnification
magnification = size of image / size of real object
= 30,000 / 0.8 = 37,500 x
describe how electron microscopy works
This microscope uses electrons instead of light
Electrons have a very short wavelength (so the resolution is 2000x better than a light microscope)
An electron gun produces a beam of electrons
These electrons pass down the microscope
The inside of an electron microscope contains a vacuum, so the electrons can pass through without bouncing off the molecules in air
Electrons are negatively charged. This means that we can focus the electron beam using electromagnets called electromagnetic lenses
The specimen is placed in the path of the electron beam
Electrons can pass through some parts of the specimen more easily than other parts
The final image is produced on a fluorescent screen
how can particle have a wavelength
electrons have properties of both particles and waves - so they can have a wavelength
draw a diagram of electron microscopy
https://www.researchgate.net/profile/Lakmal-Jayarathna/publication/299750087/figure/fig8/AS:614003847987255@1523401333840/Image-formation-method-on-in-a-one-lens-transmission-electron-microscope.png
electron gun
electron beam
electromagnetic lens
specimen
image on fluorescent screen
describe the advantages of electron microscopy over a light microscope
the resolution of an electron microscope is around 2000x better than a light microscope -
resolve up to 0.1nm under good conditions - means we can achieve a far greater level of magnification before the image becomes blurred
because of this higher resolution, the electron microscope has been used to make some major discoveries in biology e.g. ribosomes and structure of cell membrane
describe disadvantages of electron microscopy
The interior of an electron microscope is a vacuum. This means that we cannot view living specimens using an electron microscope
Electron microscopy requires very careful staining of the specimen and the specimen often has to be very thin
Electron microscopy can lead to artefacts (false images)
(with electron microscopy we can get artefacts)
Artefacts are false images created by the staining process or the conditions inside the electron microscope
biologists have to be careful to check that what they are seeing with an electron microscope is actually real and not created by the conditions in the microscope or by the stain
describe how transmission electron microscopy works
This microscope uses electrons instead of light
Electrons have a very short wavelength (so the resolution is 2000x better than a light microscope)
An electron gun produces a beam of electrons
These electrons pass down the microscope
The inside of an electron microscope contains a vacuum, so the electrons can pass through without bouncing off/ colliding the molecules in air
Electrons are negatively charged. This means that we can focus the electron beam using electromagnets called electromagnetic lenses
The specimen is placed/positioned in the path of the electron beam
Electrons can pass through some parts of the specimen more easily than other parts
The final image is produced on a fluorescent screen
how can particle have a wavelength
electrons have properties of both particles and waves - so they can have a wavelength
what are the two types of electron microscopes
transmission electron microscopes
scanning electron microscopes
describe how a scanning electron microscope works
The electron beam does not pass through the specimen.
Instead electrons are scattered from the surface of the specimen and detected
draw a scanning electron microscope
electron gum
electron beam
electromagnetic lenses
electron detector - one either side
specimen
compare transmission electron microscopy with scanning electron microscopy
In a transmission electron microscope, the electron beam passes through the specimen
The transmission electron microscope produces flat 2-dimensional images
The transmission electron microscope only works if the specimen is very thinly sliced
Transmission electron microscope has a very high resolution
VS
The scanning electron microscope produces 3-dimensional images
The scanning electron microscope does not require the specimen to be thinly sliced
The scanning electron microscope has a lower resolution than the transmission electron microscope
The transmission electron microscope also requires that the specimen is coated with metal such as gold
This can lead to artefacts
describe the difference of the two types of electron microscopes terms of function
In a transmission electron microscope, the electron beam passes through the specimen
In a scanning electron microscope,
The electron beam does not pass through the specimen.
Instead electrons are scattered from the surface of the specimen and detected
1 order of magnitude meaning
10 x bigger
apple 10cm
tree 100cm
tree is 1 order of magnitude bigger than the apple
