Methods of studying cells

• MAGNIFICATION:

  • DEFINITION: The process of enlarging an object in appearance. Image size(mm)= how big the object appears to be (in a picture/ drawing). Actual size (μm)= how big the object is in reality

  CALCULATING MAGNIFICATION: Magnification= image size/ actual size
• RESOLUTION:

  DEFINITION: The ability to distinguish 2 adjacent structures as separate. Higher resolution= higher clarity and detail of the image
• CELL FRACTIONATION:

  • EFFECT: Separates organelles according to size
  • ORDER OF FRACTIONATION: Nucleus, chloroplasts, mitochondria, lysosomes, endoplasmic reticulum, ribosomes
• Cell fractionation

  0. Tissue kept in specific conditions before fractionation

  1. Homogenisation

  2. Filtration

  3. Ultracentrifugation

  4. Ultracentrifugation repeated
• CELL FRACTIONATION: 1) Homogenisation

  1. Tissue sample is homogenised using a blender to break the cells

  2. Resultant fluid is called homogenate
• CELL FRACTIONATION: 2)Filtration

  1. Tissue sample/homogenate is filtered into tubes through a gauze

  2. Gauze separates larger components from the small organelles

  3. Organelles are filtered into tubes to be fractionated using ultracentrifugation
• CELL FRACTIONATION: 3)Ultracentrifugation

  1. Samples are spun at low speed in a centrifuge

  2. Tubes are balanced directly opposite each other

  3. Centrifugation separates the sample into fractions

  4. Heavier organelles forced to the bottom, lighter organelles move to top of the tube

  5. Cell debris (e.g. cell walls) form a pellet at the bottom

  6. Supernatant (a liquid) above the pellet contains the organelles
• CELL FRACTIONATION: 4)Repeated Ultracentrifugation

  1. Supernatant is poured off

  2. Centrifuged at a higher speed to separate the next heaviest organelles (the nuclei)

  3. This is repeated at increasingly higher speeds to separate each fraction
• CELL FRACTIONATION: 0) Conditions

  The tissue is kept in specific conditions before fractionation: Ice cold (reduces enzyme activity that might damage the cells), isotonic solution (prevents osmosis that could shrink/burst organelles NOT CELLS!), buffered solution (keeps pH constant. Avoids damaging protein structures)
• LIGHT MICROSCOPES:

  • OPTICAL (LIGHT) MICROSCOPES: Visible light passes and is bent through the lens to enable the user to see the specimen. The specimen can be alive and individual cells are generally transparent & their components not distinguishable (unless coloured with special stains) as light has a relatively long wavelength
  • EFFECTS: Maximum resolution: 0.2 μm. The nucleus and mitochondria can be seen. Maximum magnification: x 1500
• Electron microscopes

  Use a beam of electrons instead of a beam of light, allowing higher magnification and higher resolving power (more detail can be seen) as electron wavelengths are shorter
• Types of electron microscopes

  • TEM (transmission electron microscope)
  • SEM (scanning electron microscope)
• TEM (transmission electron microscope)

  • The electron beam penetrates the cell and provides details of a cell's internal structures
  • They use electromagnets to focus the electron beam and are high resolution microscopes
  • Denser parts absorb more electrons -> appear darker
  • In thin specimens you can see the internal structures of organelles e.g. chloroplasts
  • Artefacts may appear
• SEM (scanning electron microscope)

  • A beam of electrons move back and forth across a cell's surface creating details of a cell surface characteristics
  • SEMs knock electrons off the specimen and these electrons come together to form an image, which can be 3D
  • Specimens do not have to be thin, but the resolution is lower than by a TEM
• Maximum resolution of electron microscopes: 0.0002 μm (x1000 light microscopes)
• Maximum magnification of electron microscopes: x 1,500,000
• LIGHT MICROSCOPE ADVANTAGES:

  • Objects are living
  • Images are in colour, but staining may be used (to see some structures)
  • Cheap so can be viewed in most settings eg hospitals or schools
• ELECTRON MICROSCOPE ADVANTAGES:

  -Huge power of magnification and resolution

  -Can discover new structures & biological processes
• ELECTRON MICROSCOPE DISADVANTAGES:

  -Image can be fuzzy

  -Specimens must be dead

  -Severe treatment can produce artefacts

  -Expensive, only found in research labs
• TEM

  Specimens can only be viewed in a vacuum

  (Since the specimens are in a vacuum) they have to be dead

  Specimen must be stained (even if the image produced has no colour)

  Specimen must be thin
• SEM

  Specimens can only be viewed in a vacuum

  Specimens must be dead

  Specimen must be stained (the image produced is 3D and is in colour)
• LIGHT MICROSCOPE DISADVANTAGES:

  • Preservation & staining can produce artefacts
  • Low resolution and magnification