1. Chapter 8 Companion site for Light and Video Microscopy Author: Wayne

2. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 2 Interference of light reflected from a thin film in air. FIGURE 8.1

3. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 3 Interference of light reflected from a thin film when n1 < n2 < n3. This is the case for the thin films of metal oxides that are responsible for the iridescent colors of glass. FIGURE 8.2

4. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 4 Vector representation of an image point (P) produced by the nondiffracted (U) and diffracted waves (D) in a bright-field microscope. FIGURE 8.3

5. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 5 Vector representations of an image point (P¢) produced by the nondiffracted (U) and diffracted waves (D) in positive and negative phase-contrast microscopes. FIGURE 8.4

6. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 6 Vector representation of a background point (U¢) produced by the vector sum of the nondiffracted light (U) and the reference beam (R). Vector representation of an image point (P¢) produced by the vector sum of the diffracted (D), nondiffracted (U), and the reference beam (R) in an interference microscope. The vector sum of the diffracted (D) and nondiffracted (U) light is equal to the vector (p) that represents all the light that comes from a point in the specimen. FIGURE 8.5

7. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 7 By varying the phase of the reference beam (R), we can bring the background to extinction. FIGURE 8.6

8. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 8 By varying the phase of the reference beam (R), we can bring a point in the image to extinction. FIGURE 8.7

9. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 9 The phase angle of a point in the image compared to the background can be determined from the difference in the angles that bring the image point and the background to extinction. FIGURE 8.8

10. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 10 Methods to form and to recombine two coherent beams. FIGURE 8.9

11. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 11 A diagram of a Mach-Zehnder interferometer composed of fully silvered mirrors and half-silvered mirrors in the absence of a specimen. FIGURE 8.10

12. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 12 A diagram of a Mach-Zehnder interferometer in the presence of a specimen whose phase angle is 180°. The specimen appears dark. Fully-silvered mirror (M), half-silvered mirror (H). FIGURE 8.11

13. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 13 Compensation of a specimen in a Mach-Zehnder interferometer. The specimen appears bright. Fully- silvered mirror (M), half-silvered mirror (H). FIGURE 8.12

14. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 14 Diagram of a Leitz interference microscope based on a Mach-Zehnder interferometer. FIGURE 8.13

15. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 15 Diagram of a Dyson interference microscope based on a modified Mach-Zehnder interferometer. FIGURE 8.14

16. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 16 Diagram of a Zeiss interference microscope based on a Jamin-Lebedeff interferometer. FIGURE 8.15

17. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 17 A Wollaston prism splits light into two coherent waves. The waves that strike the interface between the two prisms at the midway point in the prism leave the prism in phase. An optical path difference between the ordinary wave and the extraordinary wave can be introduced by sliding the Wollaston prism. The ordinary wave emerges ahead of the extraordinary wave in the beam splitter when the prism is shifted to the left, and the extraordinary wave emerges ahead of the ordinary wave in the beam splitter when the prism is shifted to the right. FIGURE 8.16

18. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 18 A Wollaston prism can also recombine two coherent waves. An optical path difference between the recombined ordinary wave and the extraordinary wave can be introduced by sliding the Wollaston prism of the recombining prism. The beam that enters the recombining prism as the ordinary wave emerges ahead of the extraordinary wave when the prism is shifted to the right and the beam that enters the recombining prism as the extraordinary wave emerges ahead of the ordinary wave when the prism is shifted to the left. FIGURE 8.17

19. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 19 A Smith interference microscope. Note the antiparallel orientation of the cement line of the two Wollaston prisms. In most publications, the cement lines in the two Wollaston prisms are shown with a parallel orientationan arrangement that could not work. I used to tell my students that I thought that the orientation of the two prisms should be antiparallel, even though the majority of publications, including the technical report put out by Zeiss (Lang, 1968) show parallel Wollaston prisms, and since I may be crazy, they were free to go with the majority opinion. After many years of saying this, I finally called Zeiss and told them that I think that their technical publication was incorrect and that the Wollaston prisms must have an antiparallel orientation to split and recombine the beams. Ernst Keller of Zeiss graciously called me back, saying, You have the whole building upside down and indeed I was right I use this as an example for my students, to base their conclusions on first principles and not on what the majority says FIGURE 8.18

20. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 20 A diagram of a Smith-Baker interference microscope based on polarized light. FIGURE 8.19

21. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 21 Diagrams and densitometer tracings of a sarcomere before and after myosin extraction from Huxley and Hanson (1957). FIGURE 8.20

22. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 22 Diagrams of the optical arrangements designed by Andrew Fielding Huxley using various prisms made from quartz and calcite to observe interference images of muscle (Huxley, 1952, 1954). FIGURE 8.21

23. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 23 Diagram of two possible arrangements used in reflection-interference microscopes. FIGURE 8.22

24. Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 24 A ray diagram of the reflections that occur when looking at cell attachments with a reflection- interference microscope. FIGURE 8.23