Brightfield, darkfield and phase contrast are established in light microscopy as
most used standard techniques for examination of transparent specimens. Interference contrast (DIC), fluorescence and
polarization microscopy are additional techniques which are well suited for special tasks. These different techniques are all characterized by typical advantages and disadvantages and several optical limitations are
prevalent in these methods, especially when transparent specimens have to be examined which are characterized by strong marginal contours, fine internal structures, high ranges in local thickness and a complex
three-dimensional architecture such as crystallizations, casts or impressions of volatile crystals, skeletons of diatoms or foraminiferida and biological specimens consisting of high density light absorbing and low
density phase shifting components.
In usual brightfield illumination, light absorbing specimens can be visualized
as they modulate the amplitude of transmitted light. The lateral resolution is
reduced in brightfield and artifacts caused by diffraction become apparent when the aperture diaphragm is closed in order to enhance depth of focus and contrast, or accentuate fine marginal contours at barely
visible low density and colorless specimens. Thus, the marginal contours of such specimens mostly appear in a black or dark color when the width of the condenser aperture diaphragm is appropriately reduced.
Nevertheless, existing fine internal details may remain barely visible or not at all, especially
when their optical density and contrast are very low.
In standard darkfield illumination,
high density light reflecting specimens can be examined as well as very small low density specimens leading to diffraction of the incoming light so that phase boundaries are accentuated.
small structures which are not resolved by the
optical system can be detected in darkfield
illumination when the illuminating light is
scattered so that visible diffraction patterns are
generated (Tyndall effect, point spread function). On the other hand, fine
internal structures can be lost when they are not hit by the illuminating light beams which run from the condenser´s periphery in an oblique direction to the specimen. Darkfield imagery is affected with a high
contrast caused by a very high range from bright
to dark so that bright structures will often be overexposed and dark details may appear underexposed in photomicrographs and prints. Marginal contours often
appear highly irradiated by blooming and scattering so that the clarity of fine details can be reduced. The aperture of darkfield condensers cannot be reduced
under normal circumstances, and the aperture diaphragm – if
present in the respective condenser – has to be wide open. Therefore, in normal darkfield, the focal depth cannot be influenced by the aperture diaphragm and it will be lower than in corresponding brightfield
images. In contrast to other techniques, the zeroth principal order maximum does not contribute to the microscopic image so that the imaging light is only based on the secondary and higher order maxima.
For examinations of low density phase shifting specimens, so-called phase specimens, phase contrast (Zernike, 1942) is mostly used in practice as
the standard technique. Phase contrast images are
mostly affected with haloing which can be regarded as a typical artifact for this technique, especially in specimens consisting of a critical density or thickness so that the morphologic appearance of their
structures could be masked or altered. In analogy with darkfield, the condenser aperture diaphragm – when present in the condenser used - has to be wide open in phase contrast examinations also, so that the depth of
field cannot be enhanced by reducing the condenser´s aperture. Shade-off (zone-of-action effect) is another typical artifact associated with phase contrast leading to loss of homogeneity in large phase specimens
which have a constant thickness and optical density so that – in standard positive phase contrast - the peripheral zones of such specimens appear in lower brightness whereas the central parts are highlighted up to
the brightness of the surrounding medium. Reversed effects can be observed in negative phase contrast. Also by shade-off, some fine details can be lost in normal phase contrast examinations. The advantages and
disadvantages of phase contrast have been reviewed by several authors (Robertson, 1970, Slaghter
& Slaghter, 1992, Glückstad et al., 2001, Murphy, 2001, Murphy et al. 2012).
has been developed as an attractive alternative for phase contrast and brightfield illumination. However, for this technique, the specimen´s ideal thickness and density should be medium, i.e. somewhat higher than in typical phase specimens and lower than in typical brightfield specimens (Determann and Lepusch, 1981). Very thin and low density specimens mostly appear in lower contrast when compared with phase contrast, and rather thick specimens are imaged as “optical sections” when examined in interference contrast (Lichtscheidl, 2011) so that their three-dimensional (3D) architecture cannot be well presented. Interference contrast is free from blooming and haloing, but the depth of field is lower than in the other examination modes. Relief effects visible in interference contrast can be generated by regional variance of the refractive index so that they are not real but just “pseudo-reliefs”.
In birefringent colorless specimens only, fine details can be accentuated when illuminated with polarized light, and fluorescence techniques
are well suited for presenting only fine fluorescent details in high resolution. But, of course, these techniques cannot be used for examinations of specimens which are isotropic or
In order to
overcome some artifacts and optical limitations described, I succeeded in developing several new illumination techniques each promising significant advantages in examinations of so-called “problem specimens”. In
many cases, the visual information achievable will be higher than in the concurrent conventional standard techniques, in visual observations as well as in photomicrographs and
In detail, three different techniques have been developed and rigorously evaluated in practice:
- Variable Bright-Darkfield Contrast (VBDC)
- Variable Phase-Darkfield Contrast (VPDC)
- Variable Phase-Brightfield Contrast (VPBD).
In these methods, two complementary illumination techniques are combined with each other and simultaneously carried out so that a couple of different partial images is generated and superimposed. In VBDC
, a brightfield-like partial image is superimposed with a darkfield image. Phase contrast is combined with darkfield in VPDC or coincident with brightfield in VPBC. In all variants,
where darkfield is
carried out, i.e. VBDC and VPDC, the darkfield-like partial image can be generated based on concentric-peripheral light as
is usual in normal darkfield or it is achievable with axial light which has to be
covered by a light stopper within the objective (so-called axial or central darkfield).
of the coactive partial images and so the weighting of the images superimposed can be
modified by the
user. Thus, the final images can be dominated by one of the partial images, or they can be equalized so that both partial images contribute to the resulting sandwich in comparable proportions.
In VBDC and VPDC, the illuminating light components associated with the respective partial images can be differently colorized so that fine details can be accentuated still more, and in VPBC, these different
light components must be filtered at different colors for suitable results.
In special circumstances, also three partial images can be generated based
on brightfield, darkfield and phase contrast
leading to superior visual information ("triplex
All optical and technical details of my methods, current practical results and suggestions for further technical developments are reported in the following chapters.
Last Update: August 10th, 2012
Copyright: Timm Piper, 2012