Methods of Studying Cells Flashcards
1
Q
Features of Optical Microscopes:
A
- Use light to form a 2D image.
- Visible light has a longer wavelength so lower resolution 200nm.
- Lower magnification x1500.
2
Q
Features of Scanning Electron Microscope:
A
- Use electrons to from a 2D image.
- Beams of electrons scan surface, knocking off electrons from the specimen, which are gathered in a cathode ray tube to form an image.
- Electrons shorter wavelength.
- Higher resolution 0.2nm
- Higher magnification x 1 500 000
3
Q
Features of Transmission Electron Microscope:
A
- Use electrons to form a 3D image.
- Electromagnets focus beam of electrons onto specimen, transmitted, more dense- more absorbed= darker appearance.
- Electrons shorter wavelength.
- Higher resolution 0.2nm
- High magnification x1 500 000.
4
Q
Pros and Cons of Optical Microscope:
A
- 2D image
- Thin specimens
- Low resolution: cannot see internal structures of organelles or organelles smaller than 200nm like ribosomes.
- Low magnification.
- Living organisms can be seen.
5
Q
Pros and Cons of SEM:
A
- Vacuum; can’t see living organisms.
- Lower resolution than TEM.
- 3D image.
- High resolution; can see internal structures of organelles.
- High magnification.
- Used on thick specimens
6
Q
Pros and Cons of the TEM:
A
- 2D image.
- Thin specimens.
- Vacuum; can’t see living organisms.
- High resolution; see internal structures of organelles.
- High magnification
7
Q
Explain the difference between magnification and resolution
A
- Magnification - how much bigger the image of a sample is compared to the real size, measured by
- Resolution -a measure of the microscope’s ability to distinguish between two points which are close together on an object.; shows amount of detail; limited by wavelength of radiation used e.g. light
8
Q
Explain how you would measure the size of an object viewed with an optical microscope?
A
- Line up eyepiece graticule with stage micrometer
- Use stage micrometer to calculate the size of divisions on eyepiece graticule at a particular magnification
- Take the micrometer away and use the graticule to measure how many divisions
make up the object - Calculate the size of the object by multiplying the number of divisions by the size of division
- Recalibrate eyepiece graticule at different magnifications
9
Q
Explain how you would prepare a ‘temporary mount’ of a specimen on a slide?
A
- Use tweezers to place a thin section of specimen e.g. tissue on a water drop on a microscope slide
- Add a drop of a stain e.g. iodine in potassium iodide solution used to stain starch grains in plant cells
- Add a cover slip by carefully tilting and lowering it, trying not to get any air bubbles
10
Q
Explain how you would use cell fractionation to separate cell components?
A
- Homogenise tissue using a blender
- Disrupts cell membrane / break open cell
- Release contents / organelles - Place in a cold, isotonic, buffered solution
- Filter homogenate
- Remove large, unwanted debris e.g. whole cells, connective tissue
11
Q
Why is the solution cold, isotonic and buffered?
A
- Isotonic so water doesn’t move in/out of organelles by osmosis so they don’t burst / shrivel
- Buffered keeps pH constant so enzymes don’t denature
- Cold reduces enzyme activity so organelles aren’t broken down
12
Q
Explain how you would use ultracentrifugation to separate cell components?
A
- Centrifuge homogenate in a tube at a low speed
- Remove pellet of heaviest organelle and spin supernatant at a higher speed
- Repeated at higher and higher speeds until organelles separated out, each time pellet is made of lighter organelles
- Separated in order of mass/density
13
Q
What is the order of separation?
A
- Nucleus
- Chloroplasts
- Mitochondria
- Lysosomes
- Endplasmic reticulum
- Ribosomes
14
Q
Explain how the scientific community distinguished between artefacts and cell organelles?
A
- Repeatedly prepared specimens in different ways
- If an object could only be seen with one preparation technique, but not another it was more likely to be an artefact than an organelle
