Final remarks and summarizing conclusions

For hundreds of years, the microscope has been the daily tool of many scientists, and also nowadays, it is an important instrument for several users in research and routine tasks. At the beginning of their development, microscopes worked only with bright-field illumination so that specimens could be illuminated by transmitted light running in axial direction. In this technique, very small and thin, low density specimens such as living cells, native bacteria and microorganisms remain barely visible or invisible. Dark-field illumination was developed as the next “milestone”. In the year 1837, Joseph Bancroft Reade published first constructions of an illuminating apparatus modified for darkfield examinations, followed by Francis Herbert Wenham who reported several principles of darkfield illumination in the years 1852-1856. In 1905, Spirochaeta pallidum (pathogen of syphilis / lues) was  visualized for the first time by Fritz Schaudinn and Erich Hoffmann with the help of a darkfield microscope. Phase contrast was developed by Fritz Zernike and published in 1942; 11 years later, he was awarded the Nobel Prize for physics for this invention. In phase contrast, even very low density colorless specimens could now be directly observed in a new and unprecedented grade of clarity. Thus, several fundamental phenomena such as mitosis and meiosis could be visualized for the first time in living cells by use of phase contrast microscopes.

Continuing this tradition, brightfield, darkfield and phase contrast are the most widely used applications in light microscopy even today. Nevertheless, each of these illumination techniques is affected with characteristic limitations and artifacts, and each of them leads to complementary visual information. Thus, for instance, low density and thin colorless specimens cannot be well perceived in brightfield (as already mentioned), and light absorbing specimens which are predestined for brightfield are not well suited for examinations in phase contrast. In darkfield, however, fine details inside specimens can be lost when they are not hit by the oblique running illuminating light. In brightfield, the lateral resolution is limited by diffraction, and in phase contrast, fine structures can be masked by typical artifacts (haloing and shade-off). In darkfield, the clarity of details can be restricted by blooming and scattering. Moreover, in darkfield and phase contrast, the appearance of the image cannot be influenced by the aperture diaphragm, because this diaphragm has to remain wide open.

In view of these limitations, I took upon myself the task of developing several improved illumination techniques based on adequate combinations of the three basic modes described. In particular, bright-field was combined with darkfield and phase contrast, and phase contrast was also combined with dark-field. Moreover, these three illumination modes were simultaneously carried out. Thus, all potential combinations were consequently implemented into standard microscopes equipped for routine examinations and could be rigorously tested in practice.

In order to obtain best optical results, several modifications had to be made with regard to the illuminating light pathway. And so, standard condensers designed for normal bright-field, phase contrast and dark-field were modified for my new methods and fitted with several specialized hand-made light masks. In addition to this, mirror lenses were used as objectives and special inserts were made for modifying the light pathway within objectives based on glass lenses. All optical modifications were made in a particular manner so that the condenser aperture diaphragm could be used for modulations of the image´s appearance and enhancements of the final image quality.

Regarded scientifically, my project is special in that the total visual information achievable in light microscopy can be significantly enhanced especially in observations of so-called “problem specimens” which cannot be well perceived in the standard techniques. The figures presented show several impressive examples of the improvements achievable. In particular, the three-dimensional architecture of complex structured specimens can be visualized in superior clarity as can superficial textures and fine details inside transparent specimens. High density light absorbing or light reflecting structures and low density phase shifting structures can be simultaneously imaged. In specimens characterized by high ranges in regional thickness and density, all zones can be simultaneously observed in maximum image quality. The complementary information of microscopic images based on bright-field, dark-field and phase contrast is combined in the resulting images and typical artifacts such as haloing, shade-off, blooming and scattering are reduced. Thus, the images achievable with my new methods are characterized by loss of artifacts, improved precision in visualizing of fine details, enhanced focal depth (depth of field) and excellent lateral resolution.

In many scientific fields dealing with light microscopy and using brightfield, darkfield and phase contrast as standard techniques, the new methods presented promise fundamental improvements of the global image quality and visual information. Thus, my project is of relevance to those people who are engaged in light microscopy and use light microscopes for their tasks in research and routine. Moreover, also those people might benefit who do not use microscopes themselves, but profit from the scientific or practical results elaborated by use of microscopes.

The practical evaluations carried out until now are based on hand-made prototypes of several light masks and objective inserts. Following on this, universal condensers could be built up fitted with modified light masks arranged on a pivoted turret. Moreover, double iris diaphragms and several polarizers could be integrated in such condensers as suggested in my paper so that the intensities of the respective partial images superimposed and the corresponding illuminating apertures could be regulated in maximum variance. For tasks based on axial (central) darkfield, specialized phase contrast objectives could be made fitted with a phase plate carrying an additional light stop in the middle of the phase ring or designed with a miniaturized light stopping slide which can be inserted in the objective´s back focal plane on demand. For tasks, darkfield illumination is added, i.e. VBDC and VPDC, the objectives used could also be fitted with an iris diaphragm so that blooming and scattering associated with darkfield could be reduced still more. Moreover, mirror lenses could be fitted with phase rings so that their centric convex mirror could act as a light stopper necessary for axial darkfield whereas the phase ring could lead to simultaneous phase contrast illumination. Thus, the particular advantages of mirror lenses such as long working distances, great areas and high planarity of field and loss of any chromatical aberration could also be used for my new methods. Lastly, my methods could also be implemented in vertical illuminators so that material controlling and material sciences could get new impulses. All in all, manufacturers of optical equipment could embrace my ideas and create new generations of light microscopes equipped with all modifications described so that I think that my methods might have a great potential to be commercialized by such manufacturers.

I think that my methods can be implemented for low or moderate costs so that the techniques described can also be used by persons or institutions whose budget is restricted. And so, even in the developing world, my methods can be used without spending high financial resources.



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