Chapter 6 Companion site for Light and Video Microscopy Author: Wayne

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 2 To achieve dark-field illumination, the specimen must be illuminated with a hollow cone of light that is too wide to enter the objective lens. FIGURE 6.1

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 3 Oil placed between the condenser and the slide allows us to use an objective lens with greater numerical aperture when using dark-field illumination. FIGURE 6.2

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 4 Two kinds of dark-field condensers. FIGURE 6.3

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 5 Rheinberg illumination and Rheinberg filters. FIGURE 6.4

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 6 Two ways of producing oblique illumination. FIGURE 6.5

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 7 Comparison between axial, Köhler, and oblique illumination. The numbers on the diffracted rays indicate the diffraction order. With Köhler illuminate the zeroth-order rays that enter the left side of the objective lens, will coexist with the negative first-order diffracted rays from the axially-illuminated specimen and the negative second-order diffracted rays from the specimen illuminated with oblique rays that enter the right side of the objective lens. FIGURE 6.6

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 8 Amplitude objects reduce the amplitude of the incident wave, but phase objects influence the phase of the wave without changing the amplitude. FIGURE 6.7

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 9 Wave and vector representation of light that does not interact with the specimen (A) and light that does interact with a point on the specimen (P). FIGURE 6.8

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 10 Vector representation OA represents the undiffracted light, AP represents the diffracted light, and OP represents the vector sum of the diffracted light and undiffracted light that makes up the image point. FIGURE 6.9

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 11 Vector representations of specimens observed with positive phase-contrast microscopy. FIGURE 6.10

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 12 Vector representations of specimens observed with negative phase-contrast microscopy. FIGURE 6.11

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 13 The relationship between intensity of an image point and phase angle. The phase angles for negative phase-contrast microscopy are given in parentheses. FIGURE 6.12

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 14 Phase-contrast microscope based on axial illumination. FIGURE 6.13

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 15 Phase-contrast microscope based on annular illumination. FIGURE 6.14

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 16 Vector representation of the influence of putting an absorbing layer on the annulus of the phase plate on the contrast of the image. FIGURE 6.15

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 17 Shading-off effect in a phase-contrast microscope. FIGURE 6.16

Companion site for Light and Video Microscopy. by Wayne Copyright © 2009 by Academic Press. All rights reserved. 18 Vector representation of the generation of a pseudo-relief image by oblique illumination. Notice that the negative diffracted orders produce a dark image and the positive diffracted orders produce a bright image. The difference in refractive index between an object and the surround leads to a difference in the wavelength of Huygens wavelets passing through the object and the surround. Consequently, the diffraction pattern created by the interface of the object and surround is tilted left or right, depending on the direction of the difference in the refractive indices of the object and the surround. When using oblique illumination, the objective lens preferentially captures the positive diffraction orders from one side of the object and the negative diffraction orders from the other side of the object to produce a pseudo-relief image. FIGURE 6.17