3.2.1.3 Methods of studying cells Content

This section is all about microscopes. The material we put under the microscope is the object, the appearance of this material when viewed under the microscope is the image. The magnification of an object is how many times bigger the image is when compared to the object:

magnification = size of image / size of object

NOTE: remember to keep the units of measurement the same!

Resolution is different to magnification. The resolution of a microscope is the minimum distance apart that two objects can be in order for them to appear as separate items. The resolving power depends on the wavelength/form of radiation emitted from the microscope. Increasing the magnification will increase the size of an object but not necessarily the resolution (every microscope has a limit resolution).

Okay so there are three types of microscope we use to study cells: the optical/light microscope, the transmission electron microscope, and the scanning electron microscope. We need to know the principles and limitations of using each one:

• The light microscope
• can only distinguish between objects more than 2μm apart due to the long wavelength of light.
• the transmission electron microscope
• can be focused by electromagnets as electrons are negatively charged
• can resolve objects that are just 0.1nm apart
• beams pass through a thin section of the specimen. Parts of this specimen absorb electrons and appear darker (other parts allow the electrons to pass through and so appear bright)
• an image is produced on a screen which can be photographed to produce a photomicrograph
• the resolving power (0.1nm) cannot always be achieved due to difficulties in preparing the specimen/the high energy electron beam may destroy the specimen
• the main limitations are as follows:
• whole system must be in a vacuum (living specimens cannot be observed)
• image produced is black and white
• a complex staining process is required
• specimen must be extremely thin
• image may contain artefacts
• 2D image produced
• the scanning electron microscope
• can be focused by electromagnets as electrons are negatively charged
• can resolve objects that are 20nm apart
• directs a beam of electrons on to the surface of the specimen from above (rather than penetrating from below). The beam is passed back and forth across a portion of the specimen in a regular pattern - the electrons are scattered depending on the contours of the specimen surface.
• A 3D image is produced by computer analysis of the pattern of scattered electrons and secondary electrons produced.
• the main limitations are as follows:
• whole system must be in a vacuum (living specimens cannot be observed)
• image produced is black and white
• a complex staining process is required
• image may contain artefacts

Cell fractionation
This is used to obtain large numbers of isolated organelles. It is the process whereby cells are broken up and the different organelles are separated out. Before cell fractionation can occur the tissue is placed in a cold buffered solution of the same water potential. this is because:

• cold to reduce enzyme activity that might break down the organelles
• is of the same water potential to prevent organelles bursting/shrinking as a result of osmotic gain/loss of water
• buffered so that the pH does not fluctuate.

The two stages of cell fractionation are homogenation and ultracentrifugation:

• homogenation
• cells are broken up by a homogeniser which releases the organelles from the cell. The resultant fluid is known as a homogenate and is filtered to remove any complete cells/large pieces of debris
• ultracentrifugation
• this is the process by which the fragments in the filtered homogenate are separated in a machine (a centrifuge). this spins the tubes of homogenate at very high speeds which creates a centrifugal force:
• the tube of filtrate is placed in the centrifuge and spun at slow speeds
• the heaviest organelles are forced to the bottom and form a pellet
• the supernatant is removed
• the supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
• the next heaviest organelles are forced to the bottom
• etc