1. Microscopy

2. UNITS OF MEASUREMENT 1mm = 1000µm
1µm = 10-3mm (convert mm to µm by multiplying by 1000 = 3 zeros)
Bacteria are about 1µm or smaller
1nm = 10-6mm (convert nm to mm by dividing by = six zeros)
Viruses are about 1nm
1000nm = 1µm
1000 viruses can fit into one bacterium
0.001µm = 1 nm
Bacteria are so small, they are measured in µm.
Viruses are even smaller, so they are measured in nm.

3. 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.
Refractive Index: a measure of the light-bending ability of a medium
Numerical aperture: numerical aperture increases as depth of field decreases.
Resolution power: limits the useful magnification of the microscope resolving power.

4. RESOLUTION
The ability of the lenses to distinguish two points.

5. RESOLVING POWER
The distance between two closely adjacent objects where the objects still appear separate and distinct. The shorter the distance (the smaller the number), the better the resolving power (the sharper the image).

6. RESOLVING POWER
To calculate the resolving power (to see how close two objects can be so you can still see them):
D = distance (smaller number is better)
0.61 = a constant number, does not change
NAobj = numerical aperture of the objective (larger number causes D to decrease, which is better)
λ = (lambda): the wavelength (nm) of the light going through the microscope. Convert nm to mm by dividing by (six zeros)
D = 0.61 λ / NAobj

7. Resolving Power 2 objects resolved 2 objects not resolved
Same 2 objects resolved with
better optical instrument
                       
                  
          

8. Resolving power limits for several optical instruments:
Human eye
0.2 millimeters (mm)
Light microscope
0.25 micrometers (µm)
Scanning electron microscope (SEM)
5-10 nanometers (nm)
Transmission electron microscope (TEM)
0.5 nanometers (nm)

9. PRISM
This is a triangular device that breaks up light into its various wavelengths so you can see all the colors of the rainbow (the visible spectrum).
The visible spectrum of colors starts with violet (350nm), and goes on to indigo, blue, green (550nm), yellow, orange, red (700nm).

10. Sample Problem
When we want to observe the color green (550nm) under an oil-immersion objective lens of a microscope, where the NAobj is 1.25, the resolution is as follows:
D = (0.61)( ) / 1.25
D = mm  convert this to microns (µm) by multiplying by 1000
D = 0.27 µm

11. Sample Problem The NAobj for the high-dry (400x) lens is 0.65
What is the resolving power (D) of this objective when viewing a wavelength of 550nm?
D = 0.61 λ / NAobj
D = (0.61)( ) / 0.65
D = mm
D = 0.52µm

12. Conclusions
Therefore, we can see an organism such as E. coli, which is 2µm long and 1µm wide because it is larger than the resolving power. However, we could not see Haemophilus influenza, which is 0.2µm long because it is smaller than our resolving power.
Therefore, the resolving power limits the useful magnification of the microscope.
Resolution determines the magnification.

13. REFRACTION
Refraction is the bending of light caused by the surrounding medium.
N = Refraction Index of the medium surrounding the lens
Air: N= 1
Glass: N = 1.5
Immersion Oil: N = 1.51 (about the same as glass)

14. 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.

15. SIMPLE MICROSCOPE

16. COMPOUND MICROSCOPE: One Eyepiece

17. COMPOUND MICROSCOPE: Two Eyepieces

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

19. Dissecting Microscope

20. BRIGHTFIELD ILLUMINATION: No Stain

21. BRIGHTFIELD ILLUMINATION: With Stain

22. DARKFIELD ILLUMINATION

23. DARKFIELD ILLUMINATION

24. Brightfield vs Darkfield

25. PHASE CONTRAST MICROSCOPY

26. DIFFERENTIAL INTERFERENCE CONTRAST

27. DIFFERENTIAL INTERFERENCE CONTRAST

28. DIFFERENTIAL INTERFERENCE CONTRAST

29. Fluorescence Microscopy
Uses UV light.
Fluorescent substances absorb UV light and emit visible light.
Cells may be stained with fluorescent dyes (fluorochromes).
Figure 3.6b

30. FLUORESCENCE MICROSCOPY

31. FLUORESCENCE MICROSCOPY

32. Transmission Electron Microscope

33. Transmission Electron Microscope

34. Transmission Electron Microscope: Inside of a Plant Cell

35. Scanning Electron Microscope

36. Scanning Electron Microscope: Flea

37. Scanning Electron Microscope: Pollen

38. Scanning Probe Microscope: Red Blood Cells

39. Scanning Probe Microscope: Chromosomes

40. 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

41. 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