01 Microscope in cell studies


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01 Microscope in cell studies

  2. 2. Early days <ul><li>Hans and Zacharias Janssen of Holland in the 1590’s created the “first” compound microscope </li></ul>ALBIO9700/2006JK
  3. 3. <ul><li>1665 - Robert Hooke discovered and described the fundamental unit of all living things (cells) by examining thin slices of cork </li></ul>ALBIO9700/2006JK
  4. 4. <ul><li>1674 - The first man to witness a live cell under a microscope was Anton Van Leeuwenhoek, describing the algae Spirogyra and named the moving organisms animalcules, meaning &quot;little animals&quot; </li></ul>ALBIO9700/2006JK
  5. 6. <ul><li>1838 – Matthias Schleiden suggested that all plants are made of cells </li></ul><ul><li>1839 – Theodor Schwann suggested the same for animals </li></ul><ul><li>General cell theory emerged: </li></ul><ul><li>“ the basic unit structure and function of all living organisms is the cell” </li></ul>ALBIO9700/2006JK
  6. 7. <ul><li>1855 – Rudolph Virchow’s theory: “all cells arise from pre-existing cells by cell division” </li></ul>ALBIO9700/2006JK
  7. 8. Unit of Measurements in Cell Studies <ul><li>metre m = 1 m </li></ul><ul><li>millimetre mm = 10 -3 m </li></ul><ul><li>micrometre µm = 10 -6 m </li></ul><ul><li>nanometre nm = 10 -9 m </li></ul><ul><li>picometre pm = 10 -12 m </li></ul><ul><li>angstrom A = 10 -10 m (obsolete) </li></ul>ALBIO9700/2006JK
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  9. 10. Magnification and Resolution <ul><li>Magnification </li></ul><ul><li>- the number of times larger an image is compared with the real size of the object </li></ul><ul><li>- magnification = size of image </li></ul><ul><li>actual size of specimen </li></ul><ul><li>Resolution </li></ul><ul><li>- the ability to distinguish between two separate points </li></ul>ALBIO9700/2006JK
  10. 11. The Microscope in Cell Studies <ul><li>2 different types of microscopes: </li></ul><ul><li>i) light microscope – uses light as a source of radiation </li></ul><ul><li>ii) electron microscope – uses electrons </li></ul>ALBIO9700/2006JK
  11. 12. Light microscope ALBIO9700/2006JK
  12. 13. Carry a Microscope Correctly
  13. 14. Images Produced by Light Microscopes Amoeba Streptococcus bacteria Anthrax bacteria Human cheek cells Plant cells Yeast cells
  14. 15. Electron microscope ALBIO9700/2006JK
  15. 16. Images Produced by Electron Microscopes Cyanobacteria (TEM) Lactobacillus (SEM) Campylobacter (SEM) Deinococcus (SEM) House ant Avian influenza virus Human eyelash Yeast
  16. 17. <ul><li>Electrons – negatively charged particles which orbit the nucleus of an atom </li></ul><ul><li>When electrons gain high energy, they escape from their orbits. Free electrons behave like electromagnetic radiation </li></ul><ul><li>Short wavelength (greater energy, shorter wavelength) </li></ul><ul><li>Advantage of using electrons for microscopy: </li></ul><ul><ul><li>Wavelength extremely short </li></ul></ul><ul><ul><li>Negatively charged (can be focused easily using electromagnets) </li></ul></ul>ALBIO9700/2006JK
  17. 18. <ul><li>Electromagnetic spectrum – whole range of wavelengths </li></ul><ul><li>The longer the electromagnetic waves, the lower their frequency </li></ul><ul><li>The greater the energy, the shorter the wavelength </li></ul><ul><li>The limit of resolution is about ½ the wavelength of the radiation used to view the specimen (if an object is any smaller than half the wavelength of the radiation used to view it, it cannot be seen separately from nearby objects) </li></ul><ul><li>Best resolution using microscope that uses visible light is 200 nm, since shortest wavelength of visible light is 400 nm (violet light). Smaller objects can never be seen using light (ribosomes – 22 nm) </li></ul><ul><li>If an object is transparent it will allow light waves to pass through it and will still not be visible </li></ul>The electromagnetic spectrum ALBIO9700/2006JK
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  20. 21. <ul><li>Beam of electrons passed through the specimen before being viewed </li></ul><ul><li>Only electrons that are transmitted (pass through the specimen) are seen </li></ul><ul><li>Allows to see thin sections of specimens (inside the cell) </li></ul>Transmission Electron Microscope ALBIO9700/2006JK
  21. 22. ALBIO9700/2006JK
  22. 23. <ul><li>Electron beam is used to scan the surfaces of structures and only the reflected beam is observed </li></ul><ul><li>Surface structures can be seen </li></ul><ul><li>Great depth of field is obtained so that much of the specimen is in focus at the same time </li></ul><ul><li>Cannot achieve same resolution as a transmission electron microscope </li></ul>Scanning Electron Microscope ALBIO9700/2006JK
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  24. 25. Viewing sample with the electron microcope <ul><li>Electron beam projected onto a fluorescent screen (areas hit by electrons shine brightly giving overall a ‘black and white’ picture) </li></ul><ul><li>Stains used to improve contrast contain heavy metal atoms which stop the passage of electrons </li></ul><ul><li>Electron beam must be in vacuum to avoid collision with air molecules </li></ul><ul><li>Specimen must be dehydrated because water boils at room temperature in vacuum (only dead material can be examined) </li></ul><ul><li>Great efforts to preserve material in life-like state when preparing specimen </li></ul>ALBIO9700/2006JK
  25. 26. Comparison of advantages and disadvantages of the light and electron microscope ALBIO9700/2006JK LIGHT MICROSCOPE ELECTRON MICROSCOPE Advantages Disadvantages Cheap to purchase Expensive to purchase Cheap to operate – uses a little electricity where there is a built-in light source Expensive to operate – requires up to 100 000 volts to produce the electric beam Small and portable Very large and must be operated in special rooms Unaffected by magnetic fields Affected by magnetic fields Preparation of material is relatively quick and simple, requiring only a little expertise Preparation of material is lengthy and requires considerable expertise and sometimes complex equipment Material rarely distorted by preparation Preparation of material may distort it Living as well as dead material may be viewed A high vacuum is required and living material cannot be observed Natural colour of material can be observed All images are in black and white Disadvantages Advantages Magnifies objects up to 1500X Magnifies objects over 500 000X Can resolve objects up to 200 nm apart Has a resolving power for biological specimens of around 1 nm The depth of field is restricted It is possible to investigate a greater depth of field
  26. 27. <ul><li>Graticule </li></ul><ul><li>Stage micrometer </li></ul>ALBIO9700/2006JK