1. Microscopy

2. UNITS OF MEASUREMENT 1000 mm (millimeters) in 1 meter (m)
1000 µm (microns) in 1 mm
Bacteria are about 1µm or smaller
1000 nm (nanometers) in 1 µm
Viruses are about 1nm
1000 viruses can fit into one bacterium
Protozoa are fairly large single-celled animals. You can see them with the naked eye. That is called being “macroscopic”
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.

3. Limits of Resolution 3
Figure 4.3

4. VOCABULARY
Immersion oil: keeps light from bending and allows lens to be refracted.
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: ability of two lenses to distinguish two points.
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 0.4 nm. 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.

5. TYPES OF MICROSCOPES
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.
There are two main types of compound microscopes: Light Microscopes and Electron Microscopes.

6. SIMPLE MICROSCOPE

7. COMPOUND MICROSCOPE: One Eyepiece

8. COMPOUND MICROSCOPE: Two Eyepieces

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

10. Dissecting Microscope: For looking at large objects

11. BRIGHTFIELD ILLUMINATION: For live organisms with no stain

12. BRIGHTFIELD ILLUMINATION: Can also use for stained tissues

13. DARKFIELD ILLUMINATION For live organisms with no stain

14. DARKFIELD ILLUMINATION For fluorescent organisms

15. Brightfield vs Darkfield

16. PHASE CONTRAST MICROSCOPY For seeing organelles in live organisms

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

18. DIFFERENTIAL INTERFERENCE CONTRAST

19. DIFFERENTIAL INTERFERENCE CONTRAST

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

21. FLUORESCENCE MICROSCOPY

22. FLUORESCENCE MICROSCOPY

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

24. Transmission Electron Microscope

25. Transmission Electron Microscope: Inside of a Plant Cell

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

27. Scanning Electron Microscope

28. Scanning Electron Microscope: Flea

29. Scanning Electron Microscope: Pollen

30. Scanning Probe Microscope
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.

31. Scanning Probe Microscope: Red Blood Cells

32. Scanning Probe Microscope: Chromosomes

33. COMPARISON OF MICROSCOPES
BRIGHTFIELD
Dark 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
DARKFIELD
Light 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

34. COMPARISON OF MICROSCOPES
FLUORESCENCE
Uses 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
Surface view only
Needs a vacuum
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

36. For the next 20 minutes….
Everyone who does not have a lab group, stand to the side of the room.
Decide what people to join with to form a lab group.
Discuss ideas for your group project.
How will you conduct it? What supplies will you need, and how will you measure the results? Will you count how many of each different colony you get?