Unstained transparent specimens (phase objects), e.g. native living cells, chromosomes and bacteria, are usually
examined in phase contrast, interference contrast or dark field illumination when a light
microscope is used.
These methods have several characteristic limitations. In dark field, some transparent objects, e.g. small bacteria, are barely visible; fine structures inside of them are
often not visible and the margins of such
cells can show blooming effects. In phase contrast, the discernment of fine details can be reduced by halo artifacts; the intensity of contrast is constant and
cannot be adjusted by the user. Interference contrast images are free
from halo artifacts, but their contrast may be lower than in corresponding phase contrast or dark field images. In all illumination techniques, the resolving power is dependent on the wavelength of the visible light
spectra and the numerical aperture of the lenses; the usual limit of light microscopic resolution is about 0,20 mikrometers.
Luminance contrast is a new illumination technique characterized by several advantages in comparison with the usual examination methods, mentioned above. The light path is completely different from common dark field,
phase and interference contrast. With the help of luminance contrast, phase objects can be examined in several variations of contrast effects, which are similar to dark field, phase and interference contrast.
Therefore, these variants have been named luminance dark field, luminance phase contrast and luminance interference contrast.
Differing from common dark field, the illumination of specimens is much more homogeneous; central structures inside of them are also visible in high
contrast. Differing form common phase and interference
contrast, the intensity of contrast is adjustable in tiny steps; halo artifacts are reduced or non-existent. In all variants of luminance contrast, the specimen occurs in a self-luminous, flourescent manner.
Probably because of this, even very small structures can be visible in luminance contrast which are lower than the usual resolving power of the respective optical system. Fundamental improvements
resolving power result from this.
Because of their extraordinary contrast , excellent sharpnesss and supramicroscopic resolution, the quality of luminance contrast images is visibly improved when compared with conventional illuminating modes, also in
The contours of cell membranes and cell walls including their superficial structures are contrasted
with extraordinary clarity. Interruptions of these structures, for example pores or perforations in nucleus
membranes or cell walls can be detected accurately as well as various intracellular structures. Also fine nuances in the cytoplasm of bacteria and other cells are visible, especially in luminance dark field.
Moreover, this method is suitable for examinations of fine structures in crystallizations and other particles in preparations without coverslips.
Luminance contrast can be achieved by using a modified condenser and modified objectives with glass lenses or by using microscopic mirror objectives (Cassegrain-Schwarzschild type). Images taken with mirror
objectives are characterized by a high planeness and a high saturation and clarity of colors; the
integrity of color detection is higher than in apochromatic lenses. As mirror lenses lead to excellent results in
luminance contrast imaging, their usefulness has also been evaluated with regard to other applications in light microscopy.
As specimens occur as self illuminating bodies, luminance contrast is also comparable to flourescence techniques aud suitable to be combined with them.Thus, luminance contrast might also be
able to give new impulses for improving flourescence microscopy.
For luminance contrast, a patent has been applied
for. Further information about all relevant technical details
is given at the URL: www.luminance-contrast.com.
Native dental bacillus in Luminance contrast
Luminance darkfield, white light, mirror objective 125x, horizontal field width: 80 Ám
Detail in luminance darkfield, white light, mirror objective 125x, horizontal field width: 20 Ám
Detail in luminance-phase contrast, green light, glass lens 45x, horizontal field width: 15 Ám
Visible differences in cytoplasmatic density, marginal line breadth of the bacteria wall: ca. 0,2 Ám
Native epithelial cell in luminance interference contrast, white light,
mirror objective 125x, horizontal field width: 100 Ám
Luminance-contrast - a new visible light technique for examining transparent specimens
(submitted: 07 June 2007, accepted: 08 June 2007)
(magazine owned of the Microscopy Society of America / MSA)
Piper, J.: Luminance contrast, a new illumination technique in light miroscopy: optical basics, practical evaluations, further developements
(submitted: 03.11.2007, accepted without modifications: 30.03.2008)
Opt. Int. J. Light Electron. Opt. (2008) doi: 10.1016/j.ijleo.2008.03.032, Elsevier, 2008
Opt. Int. J..Light Electron. Opt. 120, 963-975, Elsevier, 2009
Piper, J.: Mirror lenses in microscopy - a pleading for optical dinosaurs
submitted: 12.09.2009, accepted: 14.09.2009)
Piper, J.: Mirror lenses in light microscopy - theoretical considerations and practical implications
(submitted: 06.10.2009, accepted without modifications: 20.10.2009)
Microscopy Research and Technique, Wiley-Blackwell, 2010
Copyright: Joerg Piper, Bad Bertrich, Germany, 2010