3.2.1.3 Methods Of Studying Cells Flashcards
Define magnification
How many times lager the image produced by the microscope is than the actual size of the object being measured
Define resolution
The ability to distinguish between objects that are close together (the minimum distance between two objects in which they can be viewed as separate). It depends of the wavelength of the light or the type of radiation used.
How does an optical/light microscope work?
A beam of light is shone on a specimen, and passes through to our eye to produce an image.
Advantages of an optical/light microscope
Images can be seen in colour
Can be used to view both living and dead specimens
Disadvantages of an optical/light microscope
Low resolution as light has a long wavelength
Lower magnification than other microscopes
What can be seen through an optical/light microscope?
Eukaryotic cells, nuclei, cell membrane and cytoplasm, but not any smaller organelles.
How does a transmission electron microscope work?
Uses an electron gun to produce a beam of electrons that are focused by an electromagnet when passed through the specimen. Denser parts absorb more electrons so appear darker.
Advantages of a TEM
Have a high resolution (more detail)
Have a high magnification
Allows eve internal structure of organelles to be seen
Disadvantages of a TEM
Can only observe dead specimens as must be in a vacuum
Only produces images in black and white
Only works for very thin specimens so image may contain artefacts (tares or folds)
Complex and long standing process needed
Only produces 2D images
What can be seen using a TEM?
All organelles and even their internal structures
How does a scanning electron microscope work?
A beam of electrons is scanned across the surface of the specimen
Electrons bounce off the surface and detected to form an image
Advantages of a SEM
They produce a 3D image of the surface of a specimen to allow their external structure to be observed
Can view thick specimens
Higher magnification and resolution than an optical microscope
Disadvantages of a SEM
Lower resolution images than TEM (less detail)
Cannot observe live specimens
Do not produce a colour image but false colour can be added
What can be seen using an SEM?
Can see external 3D structures of specimens
How is the total magnification calculated?
Total magnification = eyepiece magnification x objective magnification
How is the image size calculated?
Image size = actual size x magnification
What is an eyepiece graticule?
An eyepiece graticule is a small disc in the eyepiece of a microscope that has a scale. It has no fixed units so must be calibrated (depending on the objective lens that is in use).
What is a stage micrometer?
A stage micrometer is a scale that is engraved on a microscope slide, that enables us to calculate the number of units of measurement that each unit on the graticule is worth.
Why are biological drawings made?
they are used to show accurately the components of individual cells that are observed under a microscope.
What are the 8 rules for biological drawings?
- The drawing must have a title
- The magnification under which the observations shown by the drawing were made must be shown
- Drawings should be done in pencil and on plain white paper
- Lines should be clear with no shading
- It should be large (take up as much of the page as possible)
- The drawing should be made using proper proportions
- The drawing should be labelled (all labels on one side)
- It should include a scale bar
What is cell fractionation?
Cell fractionation refers to the process of separating different organelles of a cell, so that their structures and functions can be studied.
What does it mean to isolate something?
Removing something from its normal location, usually to see if it behaves differently.
How is a pure sample of an organelle obtained?
Homogenisation and then differential centrifugation
What does isotonic mean?
The same water potential as the inside of the cell
What is the first step of cell fractionation?
The tissue is cut up and kept in a cold isotonic buffer solution
Why is the solution in cell fractionation ice cold?
It is ice cold to reduce the activity of enzymes that break down organelles.
Why is the solution in cell fractionation isotonic?
The solution is isotonic to prevent water moving into the cells or organelles by osmosis.
Why is the solution in cell fractionation pH buffered?
It is pH buffered to prevent organelles proteins from being denatured.
What is homogenisation?
The process by which cut up tissue is broken up in a homogeniser and filtered to remove large cell debris.
It breaks down the cell wall and the cell membrane, and is filtered to remove any fragments of the cell wall or membrane.
What is a supernatant?
A supernatant is the solution that has not been separated and so contains less dense organelles.
What is a pellet?
A pellet is the heavier organelle that has been spun out, at the bottom of the test tube.
What is differential centrifugation?
The homogenised tissue is spun in an ultracentrifuge at a low speed.
Different organelles will have different densities, so will separate out at different speeds. Heave organelles will be spun out at lower speeds and sink to the bottom of the tube.
The supernatant is transferred to another tube and is spun at a higher speed to separate the rest of the organelles.
What is the order in which the organelles are spun out in differential centrifugation?
Nucleus
Mitochondria or chloroplasts
Endoplasmic reticulum or Golgi
Ribosomes
