Methods of studying cells

• Convert:

  1. millimetres to micrometres
  2. micrometres to millimetres
  3. micrometres to nanometres
  4. X1000
  5. /1000
  6. X1000
• Magnification equation:

  M= O/A
• How to calculate magnification from a scalebar:

  1. Measure scale bar in mm to get the ratio of mm : micrometres
  2. Convert the value to micrometres
  3. M= observed size of bar / actual size
• Define resolution:

  The minimum distance 2 objects can have between them where they can be seen as 2 separate objects.
• Describe the principles of a transmitting electron microscope:

  • no air inside to prevent electrons scattering
  • electrons produced by a hot metal source
  • transmitted through a thin section of a specimen
  • those absorbed appear dark and those that pass through appear bright
  • electrons are detected by projecting them onto a screen and taking a 2D, black and white photomicrograph
  • high resolution as electrons are used (short wavelength)
• Limitations of a TEM:

  1. Cannot view living specimens
  2. Specimen must be extremely thin
  3. Image is in 2D black and white
  4. Image may produce false structures / artefacts
• Describe the principles of a scanning electron microscope (SEM):

  • electrons are scanned across surface of the specimen
  • they scatter across the surface / contours of the specimen
  • a computer analyses the electrons scattered producing a 3D black and white image
  • high resolution as electrons have short wavelength but lower than TEM
• Limitations of SEM:

  1. Cannot view living specimens
  2. Black and white image
  3. Cannot see internal structures
• Describe the principles of an optical microscope:

  • uses light which is focused by glass lenses
  • light reflects from a specimen, passes back through a lens towards the eye
  • living specimens can be viewed
  • 2D images produced in black and white or colour
• How do we measure cells viewed with a light microscope?

  An eyepiece graticule - a glass disc placed into the eyepiece lens.
• What must we do to an eyepiece graticule before using it?

  Calibrate it using a stage micrometer for the given magnification.
• How is a stage micrometer used?

  It is lined up with the eyepiece graticule to allow the length of the divisions on the graticule to be worked out.
• Describe a stage micrometer:

  A microscope slide with a scale engraved onto its central circular region. The smallest sub-divisions are 0.01mm = 10 micrometres.
• How would you measure the length of a mitochondrion in a cell? (3)

  • Measure the mitochondrion using an eyepiece graticule that has been calibrated by a stage micrometer.
  • Measure many mitochondria in different fields of view.
  • Calculate a mean.
• What is cell fractionation?

  Breaking open cells to release organelles.
• The 6 basic steps to obtain a sample of mitochondria/chloroplasts:

  1. Cut up the tissue.
  2. Place it into an ice cold, isotonic buffered solution.
  3. Blend the tissue in a homogeniser to fractionate the cells.
  4. Filter to remove any debris and whole cells.
  5. Centrifuge the filtrate at a low speed to obtain a pellet of nuclei.
  6. Centrifuge the supernatant at a higher speed to obtain a pellet of mitochondria/chloroplasts.
• How are the organelles separated during ultracentrifugation?

  Based on their density (weight). The more dense organelles will form pellets first at lower speeds.
• Why is homogeniser solution ice cold?

  To reduce enzyme activity which could damage organelles.
• Why is homogeniser solution isotonic?

  To prevent damage to organelles e.g. lysis or shrivelling, due to osmotic gain or loss of water.
• Why is homogeniser solution buffered?

  To maintain a constant pH, so enzymes in the organelles are not denatured.