A2.2

• Cells


  The basic structural unit of all living organisms
• Deductive reasoning can be used to generate predictions from theories. Based on cell theory a newly discovered organism can be predicted to have one or more cells.
• Microscopy


  The use of microscopes to investigate cell structure
• Microscopes are devices that produce a magnified image of objects too small to be seen directly by eyes. Developments in microscopy have led to discoveries in biology.
• Microscope types


  • Light Microscope
  • Transmission Electron Microscope
  • Scanning Electron Microscope
  • Cryogenic Electron Microscope
• How to Focus a Light Microscope

  1. Use the lowest objective lens (x4)

  2. Raise stage as close to objective lens as possible

  3. While looking through the eye piece, use the coarse focus knob and lower the stage until the image is focused

  4. Using the fine focus knob make small adjustments

  5. Increase magnification and use the fine focus only to adjust
• Total Magnification


  Objective lens X ocular lens
• Making temporary mounts of cells and tissues (Wet mounts)

  1. Using a sharp scalpel, cut a small square of onion

  2. Using tweezers, peel off a thin inside layer of the onion

  3. Transfer the thin layer of onion onto a glass slide

  4. Using a pipette, add a small drop of iodine onto your specimen

  5. Starting with the coverslip at a 90° angle, gently lower the cover slip over the specimen to avoid bubbles

  6. If bubbles do occur, gently press the cover slip with the eraser end of a pencil to push out the bubble
• Stains


  Can be used to highlight certain features of the cell
• Stains


  • Iodine - plant cells stains starch
  • Methylene blue - animal cells binds to the nucleus
• Measuring Cell Size


  1. Using a graticule and a mini grid

  2. Graticule - Transparent grid mounted in the eyepiece, when the specimen is in focus so is the scale, the size of the object may then be recorded in arbitrary units

  3. Mini grid - Placed on the stage, when the mini grid and graticule are superimposed the true dimension of the cell can be estimated in micrometers
• Field of view


  Diameter of microscopes image (circle)
• Sketching what you see under the microscope

  1. Use a sharp lead pencil

  2. Draw only the lines that you see (no shading or colouring)

  3. Your diagrams should take up about a third to half a page each

  4. Record the magnification next to each diagram

  5. Add a scale bar

  6. State the name of the specimen and the date of observation

  7. A written description is also often of considerable value

  8. When you are viewing many cells at one time, it is often useful to select and draw only two or three representative cells for each observation
• Light microscopes are limited in resolution by the wavelengths of visible light (400–700 nm)
• Electrons have a much shorter wavelength (2 – 12 pm) therefore electron microscopes have a much higher resolution
• Light microscopes are usually limited to 1000x because, due to the resolution, nothing is gained by increasing the magnification
• Resolution


  The shortest distance between two points that can be distinguished
• Resolution of different microscopes

  • Human eye: 0.1mm, 100μm, 100,000nm
  • Light microscopes: 0.0002mm, 0.2μm, 200nm
  • Electron microscopes: 0.000001mm, 0.001μm, 1nm
• Transmission Electron Microscopes (TEM)

  • Have a much shorter wavelength producing greater magnification (500000x) and greater resolution
  • Useful for studying cellular components and viruses
  • Passes beams of electrons through a very thin specimen stained with metal ions, these are either absorbed by the denser parts of the specimen and scattered or pass through the less dense parts and picked up by the electron detector to form an image
  • Disadvantages: Unlike with light microscopes the specimen must to be non-living or dead, images in black and white
• Scanning Electron Microscopes (SEM)


  • The electron beam is scanned back and forth across the surface of the specimen
  • Electrons are reflected from the surface and detected to be converted into a three-dimensional image
• Light microscopes allow us to see the structure of cells, electron microscopes allow us to see the ultrastructure of cells, such as these pancreatic exocrine cells
• Ultrastructure


  All the structures of a biological specimen that are at least 0.1nm in their smallest dimension
• Cryogenic Electron Microscopy


  • Solutions of proteins or biomolecules are frozen rapidly (flash freezing) to very cold temperatures (-180ºC)
  • Specimen is then exposed to electrons to produce high resolution image of individual molecules
• Freeze fracture


  • Biological material is instantly frozen in liquid nitrogen preserving their shape
  • The solid sample is then broken in a vacuum
  • The exposed surfaces are allowed to lose some of their ice - producing an etched surface
  • The surface is then replicated in carbon and coated in metal
  • The mask is then examined with
• Electron microscopes


  Allow us to see the ultrastructure of cells, such as these pancreatic exocrine cells
• Electron microscopes can see viruses (0.1μm diameter), but light microscopes cannot
• Cryogenic Electron Microscopy


  Solutions of proteins or biomolecules are frozen rapidly (flash freezing) to very cold temperatures (-180ºC), then exposed to electrons to produce high resolution image of individual molecules
• Freeze fracture


  Biological material is instantly frozen in liquid nitrogen preserving their shape, then the solid sample is broken in a vacuum and the exposed surfaces are allowed to lose some ice - producing an etched surface, which is then replicated in carbon and coated in metal, and examined with an electron microscope
• Immunofluorescence


  Fluorescent markers are attached to antibodies which are able to bind to specific antigens, and the fluorescent markers emit light when exposed to certain light wavelengths, which is detected by a fluorescence microscope
• Fluorescent dyes


  Dye is added to a sample and will bind to target structure, and the tagged structures will appear as a brightly coloured spot when viewed through microscope
• All organisms are composed of one or more cells (cell theory)
• All cells consist of

  • Plasma membrane
  • Cytoplasm
  • DNA as their genetic material
• Plasma membrane


  A lipid layer that surrounds the cytoplasm, encloses and protects the contents from surrounding environment, allowing conditions inside the cell to differ, and controls movement of substances in and out of the cell
• Cytoplasm


  Composed mainly of water, contains ingredients for chemical reactions to take place, molecules need a solvent for reactions to proceed
• DNA


  Stores and transfers information from the parent to daughter cells, controls the production of proteins essential to cell function including enzymes which control chemical reactions
• E. Coli is a model organism used in research and teaching, some strains are toxic to humans and can cause food poisoning
• In prokaryotes, we refer to the cell parts/ultrastructure rather than use the term organelle as very few structures in prokaryotes are regarded as organelles
• Warning: only draw and label features you can clearly see - don't put structures in because you think they should be there
• Plasma membrane


  Encloses and protects the contents of the cell
• Cell wall / pili


  Provides structure and support to the cell