Methods Of Studying Cells Flashcards
What is material that is put under a microscope referred to as?
An object
What is the appearance of the object when viewed under a microscope referred to as?
The image
What is the magnifications equation?
Magnification= image size/actual size
What is a key thing to remember when using the magnification equation?
The units.
How do you get from m to mm?
X 1000
How do you get from nm to mm?
/1000 then /1000
What is meant by resolution?
This is the minimum distance apart that two objects can be in order for them to appear as separate items.
What does the resolving power depend on?
The wavelength or form of radiation used.
What is the resolving power of a light microscope?
0.2 micromètres
Is a high resolution good or bad?
This is good as the image is more clear.
What does every microscope have?
A limit of resolution.
What is meant by the limit of resolution?
This is where increasing the magnification will create a larger but more blurred image.
In what process are cells broken up to separate their organelles?
Cell fractionation
What type of solution is the tissue placed in before being broken up?
Cold, isotonic bigger solution.
Why is the solution cold?
To reduce enzyme activity that may break down the organelles.
Why is the solution isotonic?
To keep the water potential the same.
Why is it important that the water potential of the solution is the same as the tissue?
To prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Why is the solution buffered?
To stop the pH from fluctuating.
How could a change in pH affect cell fractionation?
Any change in pH could alter the structure of the organelles or affect the functioning of enzymes.
What are the two stages of cell fractionation?
Homogenation
Ultracentrifugation
What is homogenation?
This is where cells are broken up in a homogeniser to release the organelles from the cell.
What is a homogeniser?
A blender
What is the resultant fluid of homogenation called?
Homogenate
What happens to the homogenate after being broken up by a homogeniser?
It is filtered to remove any complete cells and large pieces of debris.
What is ultracentrifugation?
This is the process by which the fragments in the filtered homogenate are separated in a machine known as a centrifuge.
What does a centrifuge do?
This spins the tubes of homogenate at very high speeds in order to create a centrifugal force.
Explain how the ultracentrifugation of animal cells works.
The tube of filtrate is placed in a centrifuge and spun at a slow speed.
The heaviest organelles are forced to the bottom of the tube to form a pellet.
The supernatant is removed to leave just the pellet.
The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before and the process repeats at faster and faster speeds to remove lighter and lighter organelles.
What speed should be used to separate nuclei?
1000 revolutions/min
What speed should be used to separate the mitochondria?
3,500 revolutions/min
What speed should the lysosomes be centrifuged at?
16,500 revolutions/min
Why do light microscopes have a low resolution?
This is because light has a long wavelength.
What are two advantages of electron microscopes?
The electron beam has a short wavelength and therefore has a high resolving power.
As electrons are negatively charged, the electrons can be focused using electromagnets.
What are the two types of electron microscope?
TEM
SEM
What does TEM stand for?
Transmission Electron Microscope
What does SEM stand for?
Scanning Electron Microscope
Explain how TEM works.
An electron beam is focused by electromagnets onto the thin specimen. The electrons then penetrate the specimen and are either allowed to pass through or are absorbed by the specimen. An image of the specimen can be seen on a screen this can then be photographed to make a photomicrograph.
Why is the resolving power of a TEM sometimes limited?
Difficulties preparing the specimen limit the resolution that can be achieved.
A higher energy electron beam is required which may damage the specimen.
What do areas which absorb electrons show up as on a TEM?
Areas where electrons are absorbed show up dark.
What do areas that allow electrons to pass through show up as on TEMs?
They appear as bright.
What are the main limitations of TEM?
The whole system must be a vacuum so living specimens cannot be absorbed.
A complex “staining” process is required however, the image is still not in colour.
The specimen needs to be thin.
Photomicrographs contain artefacts.
A 2D image is produced unless cross sections are used to build up a 3D image but this is slow and complicated.
What is an advantage of TEM?
Smaller samples can be viewed.
Higher resolving power than light microscopes.
How does SEM work?
A beam of electrons is passed over a specimen back and forth in a regular pattern. The electrons are then scattered depending on the contours of the surface. This can then be used to build up a 3D image on a computer.
What are advantages of SEM over TEM?
The specimens do not need to be thin.
In false colour.
3D image
How can a 3D image be produced from SEM?
The computer can analyse the pattern of scattered electrons and secondary electrons.
What is an advantage of TEM over SEM?
Higher resolving power.
What is an eyepiece graticule?
This is a glass disc that is placed in the eyepiece of a microscope.
In what type of microscope can an eyepiece graticule be found?
A light microscope.
What is the scale that is etched on the glass disc of an eyepiece graticule?
A scale that is typically 10mm long and is divided into 100 sub-divisions.
When is the graticule scale visible?
When looking down the eyepiece of a microscope.
Why can the scale on the eyepiece graticule not be used to directly measure the size of objects under a microscope’s objective lens?
Each objective lens magnifies to a different degree.
What should you do if you want to use the graticule to measure the size of an object?
Calibrate it.
What type of microscope slide needs to be used to calibrate an eyepiece graticule?
A stage micrometer.
What does a stage micrometer slide have etched into it?
A scale
What do the eyepiece graticule and stage micrometer need to be in order to calibrate the graticule?
They need to be lined up.
