Microscopes

• Magnification

  The degree to which the size of an image is larger than the specimen itself (simply: how many times larger the size of the image is than the object)
• Define resolving power + what a high resolving power means when viewing specimens.

  The degree to which it is possible to distinguish between two objects that are very close together. High resolving power = better clarity and detail when viewing specimens

  
• A light microscope's eyepiece magnification is x10. The objective magnifications are x4, x10, x40 and x100. We find the overall magnification by multiplying the eyepiece and objective magnification together, e.g. 10x4 = x400.
• What is the magnification equation?

  Magnification = size of image/size of real object
• The resolving power of a microscope depends on the wavelength or type of radiation used.
• There is always a limit of resolution on a microscope.
• TEM

  Transmission electron microscope
• How does a TEM work?

  The beam of electrons is focused onto the specimen by a condenser electromagnet and passes through a thin section of a specimen. Parts of the specimen absorb electrons so it appears dark, whereas other parts allow electrons to pass through so it appears bright. The image produced is called a photomicrograph.
• Why might the resolving power of a TEM be difficult to be achieved?- DISADVANTAGE

  The resolving power of a TEM is 0.1nm. However, difficulties in preparing the specimen and the need for a high energy electron beam (which can destroy specimens) can prevent this from being achieved.
• TEM have no living specimens due to the vacuum. They require a complex staining process, and the specimens must be extremely thin. They produce flat, 2D images with no colour (we can see inside of them). DISADVANTAGE
• SEM

  Scanning electron microscope
• How does a SEM work?

  Beams of electrons reflect (bounce off) off the sample's surface (they are scattered)
• SEM have a lower resolving power than TEM. They produce 3D images with no colour. PRO/CON
• False-colour micrograph

  Micrograph with added colour (via a computer)
• Eyepiece graticule

  Scale on a glass disc within the eyepiece lens that has 100 subdivisions. It always appears the same size, and it must be calibrated for each objective lens e.g. x4, x10, x40
• Stage micrometre

  Scale on a glass disc, with each division worth 0.01mm or 10 micrometres
• What are the steps in the calibration of a microscope? (edit)

  Line up the eyepiece graticule and stage micrometre and choose a section that both scales line up perfectly with. Measure the length of that section on the eyepiece graticule to give the number of EPG divisions. Measure the length of the section on the stage micrometre to give the number of micrometre divisions. Number of micrometre divisions x value of micrometre division on the stage micrometre. Divide that answer by the number of EPG divisions.
• What is a quick way to find the calibration of similar magnifications like x40 and x400?

  x400 is 10 times bigger than x40. This means that we divide the calibration of x40 by 10 to get the calibration of x400.
• Cell fractionation

  Process where cells are broken up and the different organelles within them are separated out
• What is the first step in cell fractionation?

  The tissue is placed in a cold, isotonic, buffered solution.
• What is the second step in cell fractionation?

  Homogenisation - The process of cells being broken up by a homogeniser (blender)

  The organelles are consequently released from the cell.
• Why is a cold, isotonic, buffered solution used?

  Cold - reduces enzyme activity that breaks down organelles

  Same water potential (isotonic) - prevents osmosis which would cause the organelles to burst

  pH buffer - prevents the denaturing of proteins
• What is the third step in cell fractionation?

  Filtration - the homogenate is filtered through a gauze to remove any large pieces of debris
• Homogenate

  Solution produced from the homogeniser
• What is the fourth step in cell fractionation?

  Ultracentrifugation - the process by which organelles in the filtered homogenate are separated in a centrifuge. The centrifuge spins tubes of the homogenate at increasing speeds.
• What are the 4 points of ultracentrifugation?

  Tube of homogenate is placed in the centrifuge and spun at a slower speed.

  Heavier organelles (e.g. nuclei) are forced to the bottom to form a sediment pellet.

  Lighter organelles stay suspended in the fluid above the sediment pellet, called the supernatant.

  The supernatant is transferred to another tube and spun in the centrifuge at a higher speed.
• Heavier organelles are spun at a lower speed than the lighter organelles, which are spun at a higher speed
• Organelles are separated in order of mass. Nuclei (heaviest) -> mitochondria -> lysosomes -> endoplasmic reticulum -> ribosomes (lightest)
• TEM can see internal structures of organelles, whereas SEM cannot - SEM can only see the 3D structure of organelles. We remember this because TEM *transmits* light into the cell, whereas SEM *scans* the organelle
• Artefacts

  Things you can see down the microscope that are not part of the specimen that you're viewing. E.g. dust, air bubbles, fingerprints. Usually made during preparation of slides. Common in electron micrographs because EMs require a lot of preparation for specimens
• What are the 5 advantages of an optical microscope?

  1. Inexpensive - (THINK ABOUT MONEY)
  2. Portable - (THINK ABOUT TRAVEL)
  3. Easy to operate
  4. Small and easy to store
  5. Living and dead samples can be viewed, in their original pigments
• What are the 3 limitations of an optical microscope?

  1. Light has wavelength of 400nm -> 750nm - cannot view organelles smaller than 400nm.
  2. Limited magnification and resolving power
  3. Samples are often distorted during preparation
• What are the 2 advantages of an electron microscope?

  1. Higher resolving power
  2. Higher magnification
• What are the 6 limitations of an electron microscope?

  1. Expensive - (THINK ABOUT MONEY)
  2. Large - (THINK ABOUT SIZE)
  3. Must be controlled
  4. Specimens must be dead (due to the near-vacuum)
  5. Sample preparation is complex
  6. Black and white images are produced
• What is the difference between optical and electron microscopes in terms of resolving power?

  Optical - 0.2 micrometres is the resolving power. Light has longer wavelength.

  Electrons - 0.1 nanometres is the resolving power. Beams of electrons have shorter wavelengths.
• What is the difference between optical and electron microscopes in terms of how they work?

  Optical - uses convex glass lenses in pairs. Has no vacuum

  Electron - uses electromagnets. Has a near-vacuum so that the electrons won't be absorbed or deflected by molecules in the air (e.g. dust particles) and, ultimately, interfere