Introduction

Copyright: Jugend forscht

Timm Piper´s
microcopy site
Introduction
Principles of VBDC
Principles of VPDC
Principles of VPBC
Materials and Methods
Results of VBDC
Results of VPDC
Results of VPBC
Further developments
in VBDC
Further developments
in VPDC
Further devepolments
in VPBC
Optical calculations
Discussion
Summarizing remarks
and conclusions
Supplementary
images
Video Downloads
References
List of Personal 
Publications
Patents
Acknowledgements
About me
Contact Details

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. Very 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).

Interference contrast 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 non-fluorescent.

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 prints.

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).

The intensities 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 mode").

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