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
Results

The 125 x magnifying water immersion leads to excellent results when small transparent specimen are examined in thin-layer preparations. This mirror objective can be successfully combined with photo oculars up to 12.5 x magnification.

The focal depth is dependent on the respective magnification (maximum here: 125 x 12.5 = 1562 x) and the numerical aperture (here: 0.90). As demonstrated in fig. 2,  the focal depth is about 0.8 µm when the magnification is 1600 x; it increases up to 1.0 µm when the magnification is reduced to 1000 x. These values for focal depth are about 66-78 % higher than the corresponding values calculated for a conventional oil immersion lens with a numerical aperture of 1.30 (3). Thus, specimen can be examined and photographed easily, because the focal depth of this mirror objective is above average.

The lateral resolution is determined by the numerical aperture (fig. 1). When the aperture is 0.9, the resolution is about 0.38 µm. When the aperture increases up to 1.30, the resolution is near 0.30 µm (4). Thus, the lateral resolution of this mirror objective is reduced by about 27 % when compared with a common oil immersion lens. In practice, this moderate reduction of the calculated resolving power is compensated by the specific illumination effects of luminance contrast (further explanations in the discussion).

The contours of cell membranes and cell walls, including their superficial structures, are seen in extraordinary clarity. Discontinuances of these structures, for example pores or perforations in nucleus membranes or cell walls can be detected accurately as well as various intracellular structures like photoreceptors, plastides, vaculoes, and ejectiosomes etc.. Also fine nuances in the cytoplasm of bacteria and protozoa are visible, especially in luminance dark field.

The 40 x magnifying mirror objective is suitable for examination of fine structures in crystallizations and other particles in preparations without coverslips. The color filters shown in fig. 7 b and c can also be used successfully for bicolor double-contrast techniques in colorless specimen when this objective is used.

All images taken with mirror objectives are characterized by a planarity and a complete absence of articifactual reflections or vignetting as well as high saturation and clarity of colors; the faithfulness of color detection is higher than in apochromatic lenses.

When modified objectives with glass lenses are used, all variants of luminance contrast can be achieved. As the respective light beam stops are situated in the back focal plane, they are not visible in microscopic images. In all variants of luminance contrast, the 10 x magnifying lens is free from any reflections or vignetting. Bicolor double-contrast can be achieved in the same manner using higher magnifying mirror objectives. All variants of luminance contrast can also be carried out in polarized light, when anisotropic materials, e.g. chitin structures are to be examined. 

When carried out using the 45 and 100 x magnifying lenses, luminance contrast leads to extraordinary visible enhancements of optical resolving power when compared with conventional illuminating techniques. For example, marginal structures in bacteria walls are visible in images taken with the 45 x lens that are smaller than the usual limit of light microscopic resolving power. Using the 100 x magnifying oil immersion, fine textures on the surfaces of epithelial cells and villous structures on their marginal cell membranes can be recognized in a similar manner than using the 125 x magnifying mirror objective.

The locally manufactured prototypes of these lenses and the mounted plane-parallel plates are not optimally coated. Therefore, some reflections occur in luminance dark field, when the field diaphragm is not appropriately closed. When this diaphragm is properly closed most of these artifacts can be greatly reduced so that coresponding photographs are free from visible artifacts.

All results mentioned above are demonstrated by exemplary images (see image gallery).


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]