Aeskulap-Stab
Introduction
Resolution and 
 focal depth
Principles of 
 luminance contrast
Modification of 
 lenses
Mirror objectives
Illuminating  
 apparatus
Material and  
 methods
Results
Further technical 
 developements
Discussion
Summary
Links
Contact
References
Image gallery
Principles of luminance contrast

In luminance contrast, the specimen is illuminated by a very small axial light beam that is congruent with the optical axis. Suitable light beams can be achieved, when the aperture diaphragm of a common bright field condenser and the field diaphragm for Köhler-illumination are appropriately closed. On this way, the specimen is punctually illuminated by a central beam.

Inside of the objective, a small light beam stop has to be situated, so that the illuminating axial light beam is blocked.

By this optical modification, the light path within the objective changes radically.

When the illuminating central beam is completely blocked by the light stop, it no longer contributes to the microscope image. Nevertheless, the specimen remains illuminated centrically by this beam. When the intensity of the illuminating light is adequate, the specimen is visible in a maximized homogeneous contrast, situated in a dark black background (luminance dark field). This image is a result of scattered light components bent and reflected by the specimen. The scattered beams can pass all lenses of the objective, because their optical pathway is different from the optical axis and the central illuminating beam and are not blocked by the beam stop. A simplistic diagram of the light path in luminance dark field is shown in fig. 3.

 

 

 

 

Fig. 3: Light path in luminance contrast / luminance dark field

  1 = light source
  2 = collector lens
  3 = field iris diaphragm (light diaphragm, Köhler illumination)
  4 = condenser aperture diaphragm (iris diaphragm)
  5 = special light mask (facultative, see fig. 7b)
  6 = condenser
  7 = specimen (slide and cover slip)
  8 = objective
  9 = light stop (within the objective´s back focal plane)
10 = eyepiece (field lens)
11 = eyepiece (loupe lens)
12 = eye

 


 

Now, the aperture diaphragm in the condenser can be opened in tiny steps so that a small peripheral component of the central light beam is no longer covered by the light stop and can pass the objective together with the scattered beams emitted by the specimen. A moderate brightening of the background results from this. Both light components (scattered light from the specimen and background light from the periphery of the central beam) interfere with each other. Phase differences within the specimen and its surroundings acquire contrast similar to negative phase contrast (luminance phase contrast). The brightness of the background and the intensity of the contrast can be controlled and regulated by the aperture diaphragm. Existing phase differences remain visible, as long as the brightness of the background remains lower than the brightness of the specimen. Otherwise, the illumination changes to bright field.

When the specimen occurs in luminance phase contrast, a part of the illuminating beam can be covered by a non transparent mask shifted into the condenser from one side, situated near the aperture diaphragm. In this way, oblique illumination is achievable so that the structure of the specimen is visible in a three dimensional manner comparable with interference contrast. As described above, the brightness of the background and the intensity of the resulting contrast can be regulated via the aperture diaphragm.

The effects of luminance interference contrast are also achievable without using an additional mask when the light stop is slightly uncentered in the back focal plane of the objective. In this case, an oblique illumination occurs when the aperture diaphragm is in the centered position and closed adequately.

When a phase contrast condenser is available instead of a bright field condenser, luminance dark field can also be achieved by producing the centered illuminating light beam with the suitable ring shaped mask. In this case, a small condenser annulus is necessary so that the passing light beams are completely covered by the light stop in the objective; in this technical variant, the condenser aperture diahragm remains wide open.

In very small specimen, e.g. bacterial cell walls, diffraction lines can be visible, especially when the size of the respective structure is near the optical resolution limit or less. Thus, some small structures which could not directly be detected by a light microscope can be recognized because of their diffraction pattern. In some specimen multiple diffraction lines can occur, consisting of alternating diffraction maxima and minima with decreasing intensity. Distances and width of these diffraction lines are dependent on the wavelength, the area or diameter and coherency of the illuminating beam and the size of the specimen or its particular structure. In practice, the intensity and number of visible diffraction lines can be influenced by the aperture diaphragm and the wave length, especially when monochromatic light is used.

Moreover, the area of the illuminating light beam is smaller than in corresponding conventional techniques; in luminance contrast, this area is about 10 or 20 % of the usual area necessary for correct illumination. Therefore, the illuminating light is more coherent than in conventional modes. The coherence length will increase furthermore when the specimen is illuminated in monochromatic light. An improved detection of fine details may result from this when luminance contrast is used.

Copyright: Joerg Piper, Bad Bertrich, Germany, 2007

 

[Introduction]
[Resolution and focal depth]
[Principles of luminance contrast]
[Modification of lenses]
[Mirror objectives]
[Illuminating apparatus]
[Material and methods]
[Results]
[Further technical developements]
[Discussion]
[Summary]
[Links]
[Contact]
[References]
[Image gallery]