Honors Biology Microscopes

1. Honors BiologyMicroscopes Important tool for all biologists

2. Honors Bio: Microscopes Use light or electrons to magnify Enable us to see the shape and structure of very small objects • Cells and cell parts • Tissues • Molecules (only with electron microscopes) • Small and microscopic organisms

3. Value of MagnificationReal size Magnified 400 X Cell walls cytoplasm Elodea canadensis Pond weed chloroplasts central vacuole

4. Magnification Magnification = object size ~ image size Total magnification = ocular lens X objective lens chloroplasts flagellum nucleus food vacuole Euglena, a one-celled organism 1000X

5. Resolution or Resolving Power Resolution = sharpness, clarity of focused image • “Ability to show two close points as separate” • Depends on shape and perfection of lenses • Human eye can see objects as small as 0.2 mm • A light microscope can resolve objects as small as 0.2 m high resolution lens lower resolution lens

6. Comparing Resolutions

7. Depth of Field • Thickness or layer in focus • Higher magnification thinner layer

8. Light MicroscopesSend LIGHT through a thin specimen an early microscope binocular light microscope

9. Light Microscopes (LM) • Light waves pass through a thin specimen • Lenses bend light to magnify image • Simple microscope – one lens • Compound microscope – two lenses • Magnifies image twice

10. Leeuwenhoek’s Microscope • Anton von Leeuwenhoek, 1600s • First powerful scope with high resolution • Single lens • Magnify ~ 300 X

11. Leeuwenhoek’s microscope

12. Eyepiece Ocular lens LE 4-1a Objective lens Specimen Condenser lens Light source BINOCULAR MICROSCOPE – has ocular lens for each eye

13. How two lenses magnifies

14. Epithelial cell Photosynthetic cells Chloroplast (dots inside cell) Stoma (leaf opening) Leaf cross-section(LM)

15. Advantages of light microscopes • Can magnify up to 2000 times • Shows shape and structure of cells and tiny organisms • Specimens can be alive Disadvantages • Specimens must be thin enough for light to pass through • Image appears inverted and backwards • Often need stain to see image

16. Cheek cells with stain Light microscope LM “dark field” Common stains: methylene blue, Lugol’s iodine “Vital stains” - stain without killing cells

17. Phase-Contrast Microscope“Differential Interference Microscope” Increases contrast between tissue densities – don’t need stain; good for living organisms Cheek cells without stain

18. Phase-Contrast Microscope cheek cells –unstained Compound Microscope cheek cells – stained nucleus cytoplasm cell membrane nucleus cytoplasm cell membrane

19. Amoeba, one-celled organism preserved, stained alive, moving Compound scope Phase-Contrast scope

20. Cell cycle, under phase contrast

21. Phase-contrast micrograph of a roundworm 630X

22. Stereomicroscope“Dissecting microscope” Has ocular lens and objective lens for each eye Stereoscopic vision, 3-D Image NOT inverted Magnifies 10-50X

23. Advantages of stereoscopes • Image NOT inverted or backwards • Makes manipulation easy • Specimens can be solid, living • Disadvantage: magnifies up to ~50 X

24. Stereomicroscope – whole specimens chick embryo soil worm

25. Fluorescent Microscopy • Uses lasers on thin slices; confocal scope • Fluorescent dyes show different molecules Cancer cells tagged with 3 fluorescent dyes shows cell microtubules (blue), microfilaments (yellow), DNA (green)

26. Fluorescent – shows different cell parts as different colors • Details in a single layer Fruit fly embryo – developmental layers Green – microtubules in cytoplasm Red -DNA • http://www.microscopyu.com/tutorials/java/virtual/confocal/index.html

27. Confocal Microscopy Specialized Cells in the Ear E. Coli bacteria

28. Electron Microscope • Uses electrons instead of light • Magnets focus the beam • Image shows on monitor • Magnify up to 1 million times • Show cell details, interior • - “ultrastructure • Invented 1930’s • Nobel for Ruska 1986

29. Electron Microscope • How does it work? • Specimen is coated with a metal film • Electron beam hits metal, ejects electrons from metal atoms • These electrons make the image

30. Advantagesof electron microscopy • Electron are much smaller than the wavelength of light – show things that light cannot show • Very high magnification – up to 1,000,000X • Very high resolution - up to 1 nanometer • DISADVANTAGE – specimen must be dead, dried, coated, in vacuum chamber

31. Scanning Electron MicroscopeSEM • Electron beam skims across specimen surface • Shows tiny surface structures in great detail • Magnifies up to 50,000 times • DISADVANTAGE: shows surface, but not interior

32. Compare LM and SEM Blood cells (LM) Blood cells (SEM)

33. SEM micrographs Euglena(protist) SEM Ant head, SEM

34. Scanning Electron Microscope (SEM) shows surface details Electrons scan across surface of specimen

35. SEM of DNA Image made with special scanning “tunneling” microscope

36. Transmission Electron Microscope (TEM) shows inside cells • Electrons pass through thin specimen • Shows great detail of internal structure • Magnifies up to 1,000,000 times!! Rough ER Mitochondria Nucleus

37. Transmission Electron Microscope Bacterium dividing Muscle fibers Phage virus Liver cells Cilia and basal bodies Chloroplast

38. Comparing microscopes Euglena SEM Euglena LM Euglena TEM

39. Which type of microscope produced these micrographs? Amoeba, preserved and stained Vacuole inside a cell Amoeba, alive and unstained

40. Which type of microscope made these micrographs? Female and male fruit fly Closterium -Unicellular green alga

41. Name the microscope Leaf cross-section 400X chloroplast 5,000 X

42. Name the microscope Iridescent beetle Eye of a housefly

43. Which microscope?