Biology 303: Microbiology Microscopy

BARER, R. Lecture Notes on the Use of the Microscope. 3rd ed. Oxford: Blackwell Scientific Publications, 1968. BRADBURY, SAVILE. The Optical Microscope in Biology. London: Edward Arnold, 1976. GRIMSTONE, A. V. The Electron Microscope in Biology. 2nd ed. London: Edward Arnold, 1977. RICHARDS, JAMES A., FRANCIS WESTON SEARS, M. RUSSELL WEHR, & MARK W. ZEMANSKY. Modern University Physics. Reading MA: AddisonWesley, 1960.

I.

Behavior of light as it passes from one transparent medium with one refractive index into a different medium with a different refractive index.
A. Law of reflection: light is reflected from a plane surface with an angle of reflection equal to the angle of incidence. Snell's law: When light strikes the boundary surface between two transparent media, the ratio of the sine of the angle of incidence to the sine of the angle of refraction is equal to the ratio of the velocities in the two media; thus if n, the refractive index, is defined to be c c n = and n ' = v v' then v n' = v' n sin n ' = sin ' n

B.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY and

n sin = n ' sin '

Figure 2-1. When light is reflected off a surface, the angle of incidence, , equals the angle of reflection, r. The angle of refraction, ', obeys Snell's law, n sin = n' sin '. RICHARDS et al., p. 618.

C.

Total internal reflection

Figure 2-2. When light passes from one medium of index of refraction n to a second medium with a lower index of refraction (n'), sin ' is larger than sin at all angles. At some critical angle, c, ' = 90, and at angles of incidence larger than c, all the light is internally reflected. RICHARDS et al., p. 619.

1 n' = = 0.67 n 1.5 for an air-glass interface. Thus c = 42 sin c =

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY D. The ability of a high-power lens to gather light is critical, and so oil of index of refraction close to 1.5 is used to eliminate internal reflection.

Figure 2-3. The principle of oil immersion. In the top figure four rays are shown passing from the point P in the object through the coverslip into the air space between the latter and the lens AB. Only rays 1 and 2 can enter the objective. Ray 4 is totally reflected. In the lower figure the air space is replaced by oil of the same refractive index as glass. The rays now pass straight through without deviation so that rays 1, 2, and 3 can enter the objective. BARER, p. 18.

II.

In addition to reflection and refraction, diffraction plays an important role in determining the limit of resolution of a light microscope.
A. The Airy disc

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Figure 2-4. (a) The appearance of the Airy disc. The central bright spot of light is surrounded by diffraction rings. (b) Densitometer tracing across the Airy disc in (a). (c) Two Airy discs produced by two points close together. (d) Densitometer tracing across the Airy disc in (c). The intensity drop between the two maxima is about 20% and the two points would just be resolved. BRADBURY, p. 4.

B.

The 20% criterion was first proposed by Lord Rayleigh for the resolution of spectroscopic images; it is fulfilled if the central maximum of one pattern just coincides with the first minimum of the other. It is possible to calculate the separation (dmin) of points in the object which will just satisfy this criterion:
dmin = 0.61 n sin

C.

1.

Where is the wavelength of the illuminating light, n is the refractive index of the medium through which the light passes, and is half the acceptance angle of the lens (see Figure 2-5). N sin is often defined to be the numerical aperture (N.A.) of the lens; thus 0.61 dmin = N.A.

2.

Figure 2-5. The acceptance angle of this lens is AOB. BRADBURY, p. 15.

3.

Thus the resolution of the microscope is a function of the wavelength of the light (short wavelengths are better), the refractive index of the medium through which the light passes (bigger is better), and the acceptance angle of the lens (bigger is better).

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY

III.

Behavior of the thin lens

A. A simple lens, with the object beyond the focal length: a model for the microscope objective

Q'

FO Q O

FO P'

Figure 2-6. A simple lens, with the object (PQ) beyond the focal length. Lens O produces a real image that is upside down and backwards.

1.

The earliest microscopes were single-lens microscopes

Figure 2-7. Microscope used by Antony van Leeuwenhoek (1632-1723). BRADBURY, p. 7.

2.

The modern objective lens is considerably more complicated.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY

Figure 2-8. A diagrammatic section of an oil immersion planapochromatic lens of high aperture. The large number of lenses are designed to minimize spherical and chromatic aberration. BRADBURY, p. 16.

B.

Modern compound microscopes use a second lens, the eyepiece or ocular, to produce a virtual image of the image cast by the objective

Q''

Q'

F P' E

P''
Figure 2-9. A simple lens, with the object (P'Q') within the focal length. Note that rays to the right diverge in such a way as to create the impression that they are coming from the virtual image, P''Q''.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY C. The compound microscope thus magnifies the image twice.
Q''

P'''

Q'

FO Q O

FO

F P' E

Q'''

P''
\ Figure 2-10. The optical design of the compound microscope, including the viewing eye.

IV.

Other types of light microscopy, besides brightfield.

A. Darkfield microscopy

Figure 2-11. A diagram of the ray paths producing darkfield (dark ground) illumination by insertion of a patch stop below the condenser. BRADBURY, p. 23.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY B. Phase contrast microscopy

Figure 2-12. The optical system of a phase contrast microscope. BRADBURY, p. 30.

C.

Fluorescence microscopy

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY

Figure 2-13. The optical arrangement of an incident-light fluorescence microscope using a vertical illuminator with dichroic mirror. BRADBURY, p. 36.

V.

Electron microscopy
A. Transmission 1. A simplified diagram

Figure 2-14. Diagram of an electron gun. GRIMSTONE, p. 7.

Figure 2-15. Diagram of an electron lens in section. GRIMSTONE, p. 8.

Figure 2-16. Diagram showing the layout of a simple electron microscope. GRIMSTONE, p. 9.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY 2. What it really looks like

Figure 2-17. A transmission electron microscope, Philips Model EM 200. GRIMSTONE, p. 11.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY 3. Staining techniques for viruses

Figure 2-18. Negative staining of viruses, in which the specimen is made to stand out against and electron-opaque (dark) background. GRIMSTONE, p. 19.

Figure 2-19. Shadowing technique. GRIMSTONE, p. 20.

B.

Scanning electron microscopy

Figure 2-20. The layout of a scanning electron microscope. GRIMSTONE, p. 57.

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BIOLOGY 303: MICROBIOLOGY LECTURE 02: MICROSCOPY

Larson, Gary. Hound of the Far Side. Kansas City: Andrews and McMeel, 1984, p. 56.

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