Studying Cells Flashcards
What is the equation for calculating magnification
Magnification= image size/actual size
Define resolution/resolving power
The resolution of a microscope is the minimum distance apart that two objects can be in order for them to appear as separate items.
What effect does a high resolution have on an image
The greater the resolution, the greater the clarity and precision of the image.
What is a microscopes limit of resolution?
The limit of resolution is the point where before reaching it, increasing the microscopes magnification will reveal more detail in an image but after it, increasing magnification will increase the size of the image but will not reveal more detail- the image will just become more blurred.
What is cell fractionation
The process where cells are broken up and the different organelles that they contain are separated out
What is magnification
The scale/number of times an image has been enlarged from its actual size to the size of the image.
What are the two stages in cell fractionation
Homogenation
Ultracentrifugation
What must happen to the tissue before cell fractionation can begin
It must be placed in a cold, isotonic buffer solution.
Why must the solution that the tissue is placed in before cell fractionation be cold
To reduce enzyme activity that might break down the organelles.
Why must the solution that the tissue is placed in before cell fractionation be buffered
It must be buffered to ensure that the PH does not fluctuate as a change in PH could alter the structure of organelles and affect how enzymes function.
Why must the solution that the tissue is placed in before cell fractionation be isotonic
The solution must have the same water potential as the tissue to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.
Describe the first stage of cell fractionation- homogenation
The cells are broken up by a homogeniser (blender). The resultant fluid (homogenate) is filtered to remove any complete cells and large pieces of debris.
Briefly describe the second stage of cell fractionation- ultracentrifugation
Ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge which spins the solution at different speeds to separate the different organelles.
Describe the process of ultracentrifugation
- The tube of filtrate is placed in the centrifuge and spun at a slow speed
- The heaviest organelles- the nuclei are forced to the bottom of the tube where they form a thin layer of sediment or a pellet.
- The fluid at the top of the tube (supernatant) is removed,leaving just the sediment of nuclei.
- the supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before.
- The next heaviest organelles (the mitochondria) are forced to the bottom of the tube to form a pellet or thin layer of sediment.
- This process is continued so that at each increase in speed, the next heaviest organelle is sedimented and separated out.
Briefly describe each step in the whole process of cell fractionation.
1) Tissue is cut into pieces in cold conditions in a buffered, isotonic solution
2) The tissue is homogenised to release the organelles from the cell
3) The homogenate formed is filtered
4) the filtrate is centrifuges at a low speed
5) The supernatant is decanted and re-centrifuged at higher speeds until the desired organelle is separated out.
6) the contents of the supernatant can be analysed
At roughly what speed of centrifugation (revolutions per minute) is a nuclei pellet formed
1000
At roughly what speed of centrifugation (revolutions per minute) are mitochondria and chloroplast pellets formed
3,500
At roughly what speed of centrifugation (revolutions per minute) are lysosome and endoplasmic reticulum pellets formed
16,500
At roughly what speed of centrifugation (revolutions per minute) are ribosome pellets formed
100,000
What are the two main advantages of an electron microscope
1) An electron beam has a very short wavelength and so electron microscopes can resolve objects very well-they have a high resolving power.
2) As electrons are negatively charged they can be focused using electromagnets.
Why must a near-vacuum be created within the chamber of an electron microscope
To prevent the electrons from being absorbed or deflected by molecules in the air
What are the two types of electron microscope
Scanning electron microscope
Transmission electron microscope
What are the main disadvantages/limitations of a transmission electron microscope
- The whole system must be in a vacuum and therefore living specimens cannot be observed.
- A complex ‘staining’ process is required and even then the image is not in colour.
- the specimen must be extremely thin to allow the electrons to penetrate and so the result is a 2D image.
- The image may contain artefacts which are abnormalities as a result of specimen preparation or due to the irradiation from the electron beam.
Why do the artefacts seen on an image created by an electron microscope (TEM & SEM) cause problems
Artefacts may appear on the finished photomicrograph but are not part of the natural specimen therefore it is difficult to tell if what we see on the photomicrograph really exists in that form.
What can scientists do to try to help the fact that only 2D images are produced by a TEM
Taking a series of different sections through the specimen and looking at them through a TEM then using the photomicrographs produced to build up a 3D image. However, this is slow and complicated to do.
Describe how a transmission electron microscope (TEM) works
- An electron gun produces a beam of electrons that is focused onto the specimen by a condenser electromagnet.
- The beam of electrons then passes through a thin section of the specimen.
- Parts of the specimen absorb electrons so appear dark other parts allow the electrons to pass through and so appear bright.
- The electrons that pass through the specimen are detected on a fluorescent screen on which the image is displayed.
- This image can be photographed to give a photomicrograph.
What is the resolving power of a transmission electron microscope (TEM)
0.1nm
Why can the TEMs resolving power of 0.1nm not always be reached.
- difficulties preparing the specimen can limit the resolution
- A higher energy electron beam is required and this may damage the specimen.
Describe how a scanning electron microscope (SEM) works
The SEM directs a beam of electrons onto the surface of a specimen from above. The beam is then passed back and forth across a portion of the specimen in a regular pattern. The electrons are scattered by the specimen and the pattern of this scattering depends on the contours of the specimen surface. We can build up a 3D image of the specimen by analysing the pattern of scattered electrons and secondary electrons produced.
What are the limitations of a scanning electron microscope (SEM)
- The whole system must be in a vacuum so living specimens cannot be observed
- A complex staining process must be used and even then images are not in colour
- The image may contain artefacts- abnormalities created by the preparation of the sample or due to the irradiation from the electron beam.
What is the resolving power of a scanning electron microscope
20nm
Describe the three key differences between a scanning electron microscope and a transmission electron microscope
RESOLVING POWER
A TEM has a resolving power of 0.1nm (but this is rarely achieved due to limitations preparing specimens and the higher energy electron beam destroying the specimen) whereas the SEM has a lower resolving power of 20nm
SPECIMEN
The specimen used in a TEM must be extremely thin to allow the electrons to pass through but is a SEM the specimen does not need to be thin as electrons do not pass through but bounce/scatter off the specimens surface
IMAGE
The TEM produces a 2D image whereas the SEM produces a 3D image.
What is the resolving power of a light microscope
0.2 micrometers (um)
Why do electron microscopes have a higher resolving power than light microscopes
Because electron beams have shorter wavelengths than light waves.
What are the advantages of light microscopes
Living cells can be observed
Natural colour can be seen
Movement can be observed
The specimen is rarely distorted during cell preparation.
What are the disadvantages of using a light microscope
Limited magnification and limited resolution
What is an eyepiece graticule
A glass disc that can be placed in the eyepiece of a light microscope to measure the size of a specimen being observed.
Why can an eyepiece gratitude not be used directly to measure the size of an object under a microscopes objective lense
Because each objective lense will magnify to a different degree and so the graticule must first be calibrated.
How do you calibrate an eyepiece graticule
Use a stage micrometer. Place the stage micrometer under the microscope and line it up with the eyepiece graticule. Calculate the length of the divisions of the eyepiece graticule under the objective lense being used.
