3.1.3 Substances are exchanged between organisms and their environment by passive or active transport across exchange surfaces. The structure of plasma membranes enables control of the passage of substances across exchange surfaces.
- The principles and limitations of transmission and scanning electron microscopes. The difference between magnification and resolution.
- Principles of cell fractionation and ultracentrifugation as used to separate cell components.
Cells are so small that they need to be seen using microscopes. We can divide microscopes into two main categories, which can be further subdivided:
- Light microscopes
- Electron microscopes
- Transmission electron microscopes
- Scanning electron microscopes
Light microscopes are the microscopes found in school laboratories for GCE AQA Biology. They use a normal light to illuminate the specimen so that we can see it magnified via a convex lens 🔍.
There are some limitations to using light, that can be overcome by using electron beams instead:
- The long wavelength of light means that microscopes have relatively low resolution, and can only see disparate objects separately if they are relatively far apart.
- In contrast, electron beams have short wavelengths, so the microscope has a higher resolving power
- Unlike light, negatively-charged electrons can be focused into a single, narrow beam using electromagnets.
1. Transmission Electron Microscope
- Electron gun fires an electron beam
- Electrons are focused onto the specimen by a condenser electromagnet
- Some parts of the specimen are more permeable to electrons than others
- The parts that absorb more electrons appear darker, and vice versa
- This produces an image that can be photographed = the final outcome is a photomicrograph
- The highest resolving power of the TEM can’t always be achieved because
- Higher energy beam needed destroys the specimen
- The specimen may not be of perfect condition
- Vacuum is required, so specimens need to be dead
- Complicated staining procedure needs to be carried out
- The image is not in colour
- The specimen needs to be very thin
- Improper preparation of the specimen leads to artefacts, making it hard for us to confirm whether the image is the actual specimen or artefacts
- The thin specimen means we can’t produce 3D images. A slow, laborious, complicated process of taking many 2D images to build up a single 3D image is required
2. Scanning Electron Microscope
- A beam of electrons passes through the specimen from above
- The beam is moved back and forth regularly across the specimen
- The specimen reflects and scatters electrons
- The pattern of scattering is determined by the 3D contours of the specimen
- The computer can then analyse the scattering and build up a 3D image
- All of the TEM limitations, except that the specimen doesn’t have to be super thin
- Lower resolving power than TEM
Magnification is how many times the image is bigger than the object.
- The greater the magnification, the greater the size of the image.
Resolution/resolving power is the minimum distance apart two objects need to be so that they appear as distinct, separate objects in the image.
- This depends on the wavelength of the radiation used.
- The greater the resolution, the clearer the image.
How to prepare cells for microscopy 🥼
In order to see the individual organelles inside cells clearly, we need to separate them.
Cell fractionation: process of breaking up cells and separating out the organelles inside the cells 🦠
1. Submerge the cells in a special solution 🧪
- The solution is cold - stop enzyme activity that breaks down organelles
- Same water potential as tissue - prevent entry or loss of water that could cause bursting or shrinking of the organelles
- pH buffered - prevent damage to the organelles or the enzymes
- The cells are broken up in a homogenizer
- This produces a fluid (homogenate)
- The homogenate contains the organelles of the cell, all mixed up
- The homogenate is put into a centrifuge
- The centrifuge spins the homogenate very quickly, separating the organelles by weight
- In animal cells:
- Slow spinning causes the heaviest organelles (nuclei) to sink and form a pellet
- The remaining fluid (supernatant) is separated and spun again more quickly
- This causes the next heaviest organelles to sink again (mitochondria)
- The process repeats, with the speed increasing each time, so that organelles are sedimented out by weight
That's all for today!
Toole, G., & Toole, S. (2015). Aqa biology A level. Oxford: Oxford University Press.