1. Microscopy 1

2. UNITS OF MEASUREMENT 1 m = 1000 mm (millimeters) 1 m = 1000 mm (millimeters) 1000 mm = 1 µm (microns) 1000 mm = 1 µm (microns) Bacteria are about 1µm or smaller Bacteria are about 1µm or smaller 1000 µm = 1nm (nanometers) 1000 µm = 1nm (nanometers) Viruses are about 1nm Viruses are about 1nm 1000 viruses can fit into one bacterium 1000 viruses can fit into one bacterium Protozoa are fairly large single-celled animals. You can see them with the naked eye. Bacteria are so small, they are measured in µm. They are the smallest things you can see under a microscope with the oil immersion lens. Bacteria are so small, they are measured in µm. They are the smallest things you can see under a microscope with the oil immersion lens. Viruses are even smaller, so they are measured in nm. Viruses are even smaller, so they are measured in nm. 2

3. 3 Figure 4.3 Limits of Resolution 3

4. VOCABULARY Immersion oil: keeps light from bending and allows lens to be refracted. Resolution: ability of two lenses to distinguish two points. Parfocal: focused in all lenses. Depth of field: how much of the background is in focus at the same time that the foreground is in focus. RESOLUTION: the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points greater than or equal to 0.4nm. When you go to the eye doctor, you look at the chart (Snellen chart) and read it from 20 feet away. If you can read what a normal sighted person can read from 20 feet away, it is called 20/20 vision. If you can’t read it well, your eyesight has less resolution than normal. 4

5. TYPES OF MICROSCOPES SIMPLE MICROSCOPE: Has only one lens, like an ocular (eyepiece) SIMPLE MICROSCOPE: Has only one lens, like an ocular (eyepiece) COMPOUND MICROSCOPE: More than one lens, like an ocular and an objective. An example is the Brightfield microscope. COMPOUND MICROSCOPE: More than one lens, like an ocular and an objective. An example is the Brightfield microscope. There are two main types of compound microscopes: Light Microscopes and Electron Microscopes. There are two main types of compound microscopes: Light Microscopes and Electron Microscopes. 5

6. SIMPLE MICROSCOPE 6

7. COMPOUND MICROSCOPE: One Eyepiece 7

8. COMPOUND MICROSCOPE: Two Eyepieces 8

9. Types of Compound Microscopes Dissecting Dissecting Brightfield Brightfield Darkfield Darkfield Phase-contrast Phase-contrast Differential Interference contrast Differential Interference contrast Fluorescence Fluorescence 9

10. Dissecting Microscope: For looking at large objects 10

11. BRIGHTFIELD ILLUMINATION: For live organisms with no stain 11

12. BRIGHTFIELD ILLUMINATION: Can also use for stained tissues 12

13. DARKFIELD ILLUMINATION For live organisms with no stain 13

14. DARKFIELD ILLUMINATION For fluorescent organisms 14

15. Brightfield vs Darkfield 15

16. PHASE CONTRAST MICROSCOPY For seeing organelles in live organisms 16

17. DIFFERENTIAL INTERFERENCE CONTRAST For seeing organelles in live organisms in three dimensions 17

18. DIFFERENTIAL INTERFERENCE CONTRAST 18

19. DIFFERENTIAL INTERFERENCE CONTRAST 19

20. Cells are stained with fluorescent dyes called fluorochromes. Cells are stained with fluorescent dyes called fluorochromes. UV Light is shined on the specimen. Fluorescent substances absorb UV light and emit visible light. Fluorescent substances absorb UV light and emit visible light. Fluorescence Microscopy Figure 3.6b 20

21. FLUORESCENCE MICROSCOPY 21

22. FLUORESCENCE MICROSCOPY 22

23. Transmission Electron Microscope Much higher resolution than microscopes. Can only be used on dead cells 23

24. Transmission Electron Microscope 24

25. Transmission Electron Microscope: Inside of a Plant Cell 25

26. Scanning Electron Microscope Very high resolution like a Transmission Electron Microscope except makes images in three dimensions. Very high resolution like a Transmission Electron Microscope except makes images in three dimensions. 26

27. Scanning Electron Microscope 27

28. Scanning Electron Microscope: Flea 28

29. Scanning Electron Microscope: Pollen 29

30. Scanning Probe Microscope A scan is passed over the specimen, line by line. A scan is passed over the specimen, line by line. The surface dimensions are recorded and sent to a computer, which shows the image in false color. The surface dimensions are recorded and sent to a computer, which shows the image in false color. 30

31. Scanning Probe Microscope: Red Blood Cells 31

32. Scanning Probe Microscope: Chromosomes 32

33. COMPARISON OF MICROSCOPES BRIGHTFIELDDark objects are visible against a bright background. Light reflected off the specimen does not enter the objective lens Not for looking at live cells Maximum resolution is 0.2µm and maximum magnification is 2000x Stains are used on specimens DARKFIELDLight objects are visible against dark background Used for live cells, cilia, flagella Especially good for spirochetes Uses special condenser with an opaque disc that eliminates all light in the center PHASE- CONTRAST No staying required Accentuates diffraction of the light that passes through a specimen Good for live cells; good contrast Most sensitive; cilia shows up Not three-dimensional DIFFERENTIAL INTERFERENCE CONTRAST Uses two beams of light Shows three dimensions Has a prism to get different colors Good for live cells (unstained) Best resolution 33

34. COMPARISON OF MICROSCOPES FLUORESCENCEUses ultraviolet light Stained cells with fluorescent dye; energizes electrons and creates visible light No live cells Quick diagnosis of TB and syphilis TRANSMISSION ELECTRON Get flat images Have vacuum pumps to allow electrons to float better Stain with heavy metal salts Shows sections of cell, revealing organelles Requires an ultramicrotome Best resolution of all microscopes SCANNING ELECTRON Surface view only Needs a vacuum No live cells Three-dimensional view SCANNING PROBE Physical probe scans the specimen Raster scan: image is cut up into pixels and transmitted to computer Not limited by diffraction Slower in acquiring images Maximum image size is smaller 34

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