methods of studying cells?

what is the magnification of an object?

Making the image appear larger or bigger to see more clearly

What is resolution?

The minimum distance apart that 2 objects can be distinguished as separate objects in an image.

• the greater the resolution, the more clear the image will be

give 4 ways a student could ensure they produce a correct biological drawing?

• no sketching

• show labels

• no shading

• show magnification/scale bar

How to calculate magnification?

Magnification = image size / actual size

What are the 2 main types of electron microscopes?

Scanning electron and transmission electron

How do electron microscopes work?

• they use electrons to form an image

• they have a higher resolution than optical microscopes and give a more detailed image

• they have a maximum resolution of 0.0002 micrometres

• the maximum useful magnification is about x1 500 000

• They send a beam of electrons that are focused by electromagnets inside a vacuum environment.

• the vacuum environment is needed so that particles in the air do not deflect the electrons out of the beam alignment.

How does the transmission electron microscope work?

• beam of electrons is transmitted through specimen

• denser parts of the specimen absorb more electrons, making them appear darker

• they give high resolution images, enabling you to see internal structures like organelles

• can only be used on thin specimens

How does the scanning electron microscope work?

• scan a beam of electrons across the specimen

• this knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image

• the images show the surface of the specimen

• SEMs can be used on thick specimens

• they give lower resolution images than TEMs

difference between TEM and SEM?

TEM:

• has a higher resolution

• has a resolution in the range 0.05nm to 2nm

• shows the cell interior

• shows the ultrastructure of the cell

SEM:

• has a resolution in the range 5nm to 50nm

• shows a 3D image

• shows the cell surface

Limitations of the electron microscopes?

• whole system must be in a vacuum so living specimens cannot be observed

• A complex staining process is required which may introduce artefacts into the image

• Specimens have to be very thin, particulary for TEM so that electrons can pass through

• SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope because electrons have a short wavelength

• image produced is not in 3D/ only 2D images produced

How does the light microscope work?

• they use light to form an image

• light has a longer wavelenght

• have a maximum resolution of 0.2 micrometres

• therefore, you cannot see ribosomes, SER and RER, lysosomes (because they are smaller than 0.2 micrometres)

• you may be able to see mitochondria

• you can see the nucleus

• the maximum useful magnification is about x1500

What is cell fractionation?

The process in which different parts and organelles of a cell are separated so that they can be studied in detail

• the most common method of cell fractionation is differential centrifugation

Process of homogenation?

• cells are blended first in a homogeniser to break open the cells

• then filtered to remove whole cells/ large debris forming the resultant fluid called the homogenate. This tube of homogenate is then placed in a centrifuge and spun at low speed

• The heaviest organelles, the nuclei, are forced to the bottom of the tube, where a thin sediment or pellet forms

• The fluid at the top, called the supernatant, is removed, which leaves just the sediment of the nuclei.

• The supernatant is then transferred to another tube and spun at a slightly faster speed. This time, the pellet that forms contains the next heaviest organelle, the mitochondria

• This process continues so that each time the speed is increased, the next heaviest organelle is sedimented and separated out.

Properties of the homogenate at the beginning?

• placed in a cold, buffered solution, of the same water potential as the cells

• This is to prevent the organelles from bursting under osmotic pressure

• , to inactivate any enzymes from breaking down organelles/ inhibit enzyme action

• and so that the pH does not fluctuate, so stopping enzymes from denaturing