Brightfield, darkfield and phase contrast are widely used as routine applications leading to complementary results in visualizations of different structures. Thus, global visual information can be significantly enhanced when these illumination techniques are combined with each other. By superimposition of different partial images being based on different optical phenomena (absorption, diffraction, reflection, phase shift, principal and secondary order maxima) new qualities of visual information can be achieved which can compete with the established standard methods.

In several variants of the techniques presented, axial resolution and focal depth (depth of field) are significantly enhanced; the lower the condenser aperture the higher the depth of field will be. Nevertheless, the lateral resolution is not visibly reduced when the numerical aperture of the objective lens is adequate. Moreover, potential loss of lateral resolution caused by a low illuminating aperture can be compensated by oblique or concentric-peripheral illumination as the lateral resolution is higher in oblique illumination than in axial (azimuthal) illumination.

The shortest distance “d” of two separate points which can be resolved in light microscopy determines the lateral resolution. It is dependent on the numerical aperture (NA) of the objective and the wavelength of the illuminating light and can be estimated as follows:
d = 0.6 λ / NA (Robertson, 1970).

This formula can be used for observations in incident light. When transmitted light is used for illumination, the aperture of the condenser is an additional factor influencing the distance “d”. In this case, “d” can be estimated as follows:
d =  λ / (NA_objective + NA_condenser) (Determann and Lepusch, 1981a).

In maximum oblique illumination, the lateral resolution can be doubled so that
d =  0.5 λ / (NA_objective + NA_condenser) (Leitz Wetzlar GmbH, 1969a).

The axial resolution is dependent on the respective aperture and the global magnification (magnification_objective x magnification_ocular) (Determann and Lepusch, 1981c).

The graph and nomogram presented in the Figures 62 and 63 show these relations calculated for standard brightfield illumination. Some representative values for the minimum distance “d” and the axial resolution according to Fig. 62 and 63, which result from the respective numerical aperture and magnification are compiled in Table 4; the corresponding graphical presentation is given in Fig. 64 . When the aperture is reduced, the axial resolution increases much more than the lateral resolution will decline. Therefore, an increase of the axial resolution can be of higher importance for the visual information than the consequent diminution of lateral resolution, especially in complex three-dimensional specimens which are characterized by a high regional thickness.

Fig. 62: Numerical aperture (NA) and lateral resolution,                         Fig. 63: Focal depth (µm), magnification and aperture,
calculated for monochromatic green light (λ = 550 nm),                         modified from Determann and Lepusch, 1981
modified from E. Leitz Wetzlar GmbH, 1969

Fig. 64: Aperture, minimum distance “d” calculated for 550 nm green light (indicator for the lateral resolution, graph 1) and focal depth (graph 2 and 3) calculated for 400x (graph 2) and 100x (graph 3) magnification

Numerical aperture	0.05	0.10	0.20	0.30	0.40	0.50	0.90
Lateral resolution [µm] for λ = 550 nm	10	4	2	1.2	0.9	0.7	0.4
Axial resolution [µm], for 100x magnification	250	100	40	25	16	10	4
Axial resolution [µm], for 400x magnification	200	60	25	10	6	5	1.6

Table 4: Numerical aperture, lateral and axial resolution


In Fig. 23 (see section: “results of VBDC"), an impressive example is given for the axial resolution achievable with VBDC. The axial resolution is circa 8 µm in VBDC when 100 fold magnifying lenses are combined with 12.5 fold magnifying eyepieces. In normal circumstances, this axial resolution can be achieved by use of 16 or 25 fold magnifying objectives instead of 100 fold magnifying lenses (see Fig. 63). This example emphasizes the utilization of VBDC for examinations of thick transparent specimens especially in medium or high magnification.

It can be regarded as a fundamental additional advantage that the angle of incidence can be continuously varied in several techniques developed so that axial (azimuthal), equatorial and concentric illumination can be carried out as well as oblique and eccentric illumination. By this feature, the illumination can be optimally adjusted to the specific properties of the specimen. Moreover, the specimen´s three -dimensionality can be accentuated in an impressive manner, because the specimen is simultaneously illuminated by separate light components at different angles of incidence.

In comparison with standard darkfield illumination, blooming and scattering are reduced in VBDC and VPDC so that fine internal structures often appear in higher clarity. Fine internal structures which might be lost in normal darkfield when not hit by the oblique light beams often appear in higher clarity, especially when partial images are generated based on axial darkfield.

In contrast to phase contrast examinations, haloing and shade-off are reduced or absent in VPDC and VPBC, and in contrast to both darkfied and phase contrast, the condenser aperture diaphragm can be used for improving the image quality in the same or a similar manner as usual in brightfield microscopy. Moreover, the background brightness and character of the resulting image can be continuously modified so that the resulting contrast between specimen and background can be manually adjusted. This feature, too, distinguishes the new methods from normal techniques.

When darkfield is carried out with axial light, some additional characteristic advantages are relevant. In this case, the darkfield-like partial image is based on a small axial light beam which is much more collimated and more coherent than in normal darkfield. In axial darkfield, the specimen is illuminated by perpendicular light so that also very fine structures sized at the optical resolution limit are hit by the illuminating light and detected in maximized resolution like self-luminous bodies. Even in very low density material affected with minimal differences in refractive index, ultra-fine marginal contours can be accentuated in high contrast. In axial darkfield images, the vertical resolution is maximized because the aperture of the illuminating apparatus is as small as possible. Nevertheless, lateral resolution is not reduced in a visible or relevant manner – at least in all practical evaluations carried out until now. The characteristic blooming and scattering of normal darkfield images are lower in axial darkfield because the illuminating light takes an axial path.

As demonstrated in several exemples of images, the methods reported are suitable for visualizing the real tree-dimensional relief in many specimens. And also in rather thick specimens, existing structures situated in different planes or heights are mostly imaged sharp and distinct. In particular, these methods are suitable for visualizing the three-dimensional architecture and relief in many specimens. These attributes differentiate my methods also from interference contrast which can lead to pseudo relief effects and which - because of its very low focal depth - usually produces “optical sections” especially in thick specimens. In the methods presented, colorless phase structures can be visualized together with surrounding light absorbing material in natural colors. Moreover, the clarity of colorless structures can be accentuated further by bicolor light filtering which can be regarded as an additional interesting feature of the new techniques.

As the intensity of the background illumination can be modulated by the user, my methods could also be well combined with fluorescence techniques if fluorescent regions have to be visualized together with surrounding non-fluorescent environmental structures.

In addition to these general aspects, some more special considerations can be made with regard to condenser-based concentric and eccentric VBDC. In these techniques, the whole cross section area of the objective can be used completely for the transfer of the imaging and illuminating light; no phase rings or light modulators are necessary in this technique. Thus, the optical performance of the respective objective lenses is not affected by such “foreign bodies”. Especially in this respect, VBDC also differs from the Hoffmann modulation contrast (HMC) (Hoffmann, 1977, Hoffmann and Gross, 1975a, b and 1976, Modulation Optics, 2011, Abramowitz and Davidson, 2011) and the integrated modulation contrast (IMC) (Kleine and Schué, 2009), which has been developed as a derivate by the Leica company in 2009; for in these techniques, about at least 10 percent of the optical relevant cross section area is lost for imaging as covered by the light modulator. The homogeneity of the background is a further matter for discussion. In HMC and IMC images, the light modulator leads to a background gradient which spans from dark to bright, whereas the background has a good homogeneity in VBDC. Moreover, most of HMC microscopes provided by manufacturers and also the Leica IMC mode are only based on oblique illumination. But, oblique illumination is not the preferable technique for examinations of all specimens. In the new techniques, however, the user can themselves decide whether to use oblique illumination or not.

Also for VPBC, some particular considerations can be added. In order to obtain satisfying results in VPBC, bicolor light filtering is obligatory, because poor image quality results from simple superpositions of phase contrast and bright-field images. Moderate mismatching of light annulus and phase ring may be the easiest way to combine both techniques. When phase ring and light annulus are not properly aligned, a small part of the illuminating light runs beside the phase ring so that bright-field illumination is added. Nevertheless, such additions of bright-field-producing light lead to lessening of the image quality, because the phase contrast image is “disturbed” by the bright field light added. For the same reason also, the bright-field and phase contrast mixing mode which has been implemented in the historical phase contrast condenser developed by Heine (E. Leitz Wetzlar) could not be widely established for a long time. In this condenser, a very narrow light annulus can be shifted in a vertical direction so that it is projected on to the objective´s back focal plane at a variable diameter. Thus, concentric bright-field illumination can be added, when the internal diameter of the light annulus is projected on to an objective somewhat smaller than the internal diameter of the phase ring, and such additional bright-field can also be generated when the external diameter of the light annulus is projected somewhat greater than the external diameter of the phase ring. In both variants the phase contrast and bright-field producing light components are not separated from each other, and they run to the specimen at the same angle of incidence. Moreover, in the Heine condenser, the breadth of the light annulus cannot be influenced by the user, and the illuminating light which corresponds with bright-field and phase contrast cannot be filtered at different colors. Lastly, this condenser is not fitted with an aperture diaphragm. Because of these technical limitations, in most specimens the Heine condenser will not lead to superior results when bright-field and phase contrast are combined. Further information about this condenser can be obtained on the internet (James, 2003).

On the other hand, VPBC is characterized by several technical attributes which can be regarded as “keys to success”: As the illuminating light associated with bright-field and phase contrast is filtered at different colors, all particular visual information which is immanently visible in both techniques remains detectable and distinguishable also in superimposed images. The different illuminating light components are separated from each other and run to the specimen at different angles. Thus, the plasticity and 3D appearance of three-dimensional specimens can be also accentuated in this method. The dominance of the partial images superimposed can be varied by the user so that the weighting of bright-field and phase contrast can be continuously modulated in high variance. Moreover, several parameters which are relevant for the appearance and quality of the final image such as contrast, depth of field and contour sharpness can be influenced by the condenser aperture diaphragm. It can be regarded as an additional advantage that the bright-field illumination added can easily be swapped from peripheral to axial by changing the light mask.

In economic and practical aspects, it can be regarded as an additional advantage that the majority of my new techniques described can be carried out with all types of standard objectives designed for normal brightfield or phase contrast. Thus, no specific modifications in the optical design of the objectives are necessary for these techniques. Owing to the fact that also low or medium magnifying objectives can be used, my methods could also be integrated in stereo microscopes when equipped for examinations in transmitted light. 

Beyond applications carried out with transmitted light, the new techniques might also be suitable for further tasks such as microscopic quality controlling of transparent materials in transmitted or non transparent probes in incident light when integrated into vertical illuminators.

All in all, my methods can be regarded as attractive and interesting complementary techniques in several fields of light microscopy leading to excellent results especially in transparent colorless specimens having a high range in density or regional thickness and complex three-dimensional morphology.   



Last Update: August 10th, 2012
Copyright: Timm Piper, 2012