2.1.3 methods of studying cells Flashcards
What is the magnification formula with a scale bar?
Actual length of scale bar / representative length of scale bar.
What is the magnification formula without a scale bar?
Image size / actual size.
What is magnification?
The degree to which an image is bigger than the object (specimen) being viewed under the microscope.
What is resolution?
The clarity of an image; the greater the resolution, the ability to distinguish between 2 points that are closer together.
How are specimens illuminated in an optical (light) microscope?
Using light.
How is the image produced in an optical microscope?
Some parts of the specimen absorb more light than others.
What state do specimens need to be in an optical microscope?
They can be living or dead.
How can you make specimens more visible in an optical microscope?
By staining the specimen with a coloured dye.
What is the maximum useful magnification of a light microscope?
x 1500.
What is the maximum resolution of a light microscope?
0.2 micrometers.
Can you view organelles with an optical microscope?
NO - only cells.
How do transmission electron microscopes (TEM) work?
TEMs use electromagnets to focus a beam of electrons, which is transmitted through the specimen; denser parts absorb more electrons and appear darker.
What is the thickness of the specimen like in the TEM?
Must be very thin slices - 100nm or less.
Can specimens be living in a TEM?
NO - they must be dead; they are put in a vacuum, chemically fixed and dehydrated.
Is the image produced in a TEM black and white or colour?
Black and white.
Is the image produced in an optical microscope black and white or colour?
Colour.
Is a TEM expensive to use?
YES.
What is the resolution of a TEM?
1nm.
What is the magnification of a TEM?
x500,000.
What are the steps of using an electron microscope?
- Fix the specimen.
- Dehydrate the specimen.
- Cut into thin slices.
- Stain.
- Mount onto a copper grid.
- Place in a vacuum.
What is the magnification of a scanning electron microscope (SEM)?
x 250000.
What is the resolution of a SEM?
1nm.
How is a specimen prepared for SEM?
- Coated in a thin layer of gold.
- Put in a vacuum.
Can the specimens be living in a SEM?
No - they must be dead.
Is the image produced in colour in a SEM?
No - black and white.
Are SEMs cheap or expensive to use?
Expensive.
How does a SEM work?
- Beam of electrons onto surface of specimen from above, not below.
- Beam passed back and forth across portion of specimen in a regular pattern.
- Electrons scattered by specimen; then can build up 3D image by computer analysis based on pattern and secondary electrons produced.
Describe how to use a micrometer.
- Reference EPU.
- Unknown scale.
- Use micrometer.
- Known 1mm.
- Line up.
- Work out EPU equivalent to number of micrometers.
What is the role of organelles in the production, transport and release of proteins from eukaryotic cells?
- DNA in nucleus is code for protein.
- Ribosomes/rough endoplasmic reticulum produce protein.
- Mitochondria produce ATP for protein synthesis.
- Golgi apparatus package/modify.
What is one advantage of using a transmission electron microscope compared with a scanning electron microscope?
Higher resolution/view internal structures.
Describe how to make a temporary mount of a piece of plant tissue to observe starch grains.
- Add drop of water to glass slide.
- Obtain thin section of plant tissue and place on slide.
- Stain with potassium iodide.
- Lower cover slip using mounted needle.
What is the function of a mitochondrion?
Aerobic respiration.
Describe the steps of ultracentrifugation. (6 steps)
Tube of filtrate is placed in centrifuge and spun at a low speed.
The heaviest organelles (nuclei) are forced to the bottom, forming a sediment or pellet.
The fluid at the top, called the 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.
The next heaviest organelles (mitochondria) are forced to the bottom.
The process is continued with the supernatant transferred each time so that progressively less heavy organelles are separated (until only cytoplasm is left).
Explain why a homogenate is filtered before spinning at low speed in the centrifuge. (2)
removes debris / intact cells / sand;
which would contaminate the 1st sediment / interfere with the results;
An optical light microscope cannot be used to see a plasma membrane. Explain why.
Does not have the resolution / cannot distinguish between points this close together;
As light has longer wavelength;
Contrast how an optical microscope and a transmission electron microscope work and contrast the limitations of their use when studying cells.
TEM uses electrons vs light
TEM- uses a greater resolution
TEM - can see smaller organisms
LM - see living specimens
TEM - dead
TEM can’t see in colour LM can
TEM - longer process to prepare sample
LM - quick don’t need to be skilled
TEM - thinner specimens
Describe and explain how cell fractionation and ultracentrifugation can be used to isolate mitochondria from a suspension of animal cells.
Cell Homogenisation - to break open cells
Filter to remove whole cells
Isotonic solution - to prevent damage to mitochondria
Cold - prevents enzymes activity
Buffered solution - prevents enzymes denaturing
Spins - centrifugation -at low speed - separates out nuclei into pellet
Left over stiff - supernatant - re-spin to get pellet
Centrifuge again at higher speed to get mitochondria
Describe how to determine size and structure with microscope of an organisms
1- measure and divide by magnification
2- Micrometers to cm x10000
or
1- measure and divide by length of scale bar
2- Multiply by actual length of scale bar
Describe how you could make a temporary mount of a piece of plant tissue to observe the position of starch grains in the cells when using an optical light microscope. (4marks)
Add a drop of water to a glass slide
Place a section of a thin tissue sample onto a glass slide
Put onto drop of water
Add iodine solution to stain it
And add cover slip lower with mounted needle - prevents air bubbles
Describe the limitations of a light microscope.
low magnification
low resolution
2D
How can the size of an object under a light microscope be measured?
Focus lens with specimen underneath.
Remove specimen and replace with stage micrometer (without changing magnification)
Use the stage micrometer to find the value that each division on the eyepiece graticule holds
Remove the stage micrometer and replace with specimen.
Measure the size of object with eyepiece graticule divisions.
Multiply the number of divisions by the value for one division.
In the preparation for cell fractionation, why is the tissue placed in a buffered solution?
to maintain a constant pH so that structure of organelles and enzyme activity is not altered
In the preparation for cell fractionation, why is the tissue placed in a cold solution?
to reduce enzyme activity which may break down organelles
In the preparation for cell fractionation, why is the tissue placed in a isotonic solution?
to prevent organelles bursting/shrinking due to osmosis
What are the advantages of a light microscope?
specimen can be alive
easier to use (than electron microscopes)
can use stains
can see natural colour
What are the advantages of SEM?
3D image (shows contours)
very detailed
What are the advantages of TEM?
highest resolution (out of light and SEM)
highest magnification (out of light and SEM)
What are the two stages of cell fractionation?
Homogenation
Ultracentrifugation
What is the order of organelle fractionation?
Nuclei
Chloroplasts
Mitochondria
Lysosomes
Endoplasmic Reticulum
Ribosomes
What is the preparation for cell fractionation?
tissue is placed in a cold, isotonic buffer solution
Why do electron microscopes produce high resolution images?
the beam of electrons has a short wavelength (shorter wavelength than light)
Why is a buffer solution used when homogenising cells?
keep pH the same / controls pH;
prevent change to / denaturing of proteins/enzymes;
why is the Resolution in Electron microscopes higher?
shorter wavelength between electrons
longer wavelength In light rays
