Journal of Microscopy, Vol. 255, Issue 1 2014, pp. 30–41
doi: 10.1111/jmi.12134
Received 21 October 2013; accepted 9 April 2014
Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field T . P I P E R ∗ & J . P I P E R ∗ , †, ‡, §
∗ Department of Light Microscopy, Laboratory for Applied Microscopy Research, Marienburgstr. 23, Bullay, Rheinland-Pfalz, Germany
†Department of Internal Medicine, Clinic ‘Meduna’, Bad Bertrich, Germany ‡Faculty of Medicine and Pharmacy, University of Oradea, Romania §Faculty of Human Sciences, University of North West Europe, Kerkrade, Netherlands
Key words. Bright field, contrast tube, dark field, incident light, interference contrast, multimodal microscopy, phase contrast, transmitted light, vertical illuminator.
Summary Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark- or bright-field illumination. Thus, the particular visual information associated with interference and phase contrast, darkand bright-field illumination is joined in real-time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark-field illumination. In particular, haloing and shade-off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators.
Introduction In light microscopy, image quality and visual information can be significantly improved when different illumination techniques, which are normally used on their own, are combined with each other and simultaneously carried out. Several specimens, especially problematic objects that cannot be well perceived in standard techniques can often be observed and photographed in superior clarity with the help of such optical superimposition. Optical principles, technical realization and practical results of these techniques based on phase contrast, Correspondence to: Dr. J¨org Piper, Department of Internal Medicine, Clinic ‘Meduna’, Clara Viebig Road No. 4, D-56864 Bad Bertrich, Germany. Tel: +49-0-2674-1823184 or 182-3335; fax: +49-0-2674-182-3182; e-mail: [email protected]
bright- and dark field have been reported in several previous contributions. Up till now, we were successful with various implementations of multimodal bright–dark field microscopy (Piper & Piper, 2012a, 2013b, c), variable combinations of phase contrast and peripheral or axial dark field (Piper & Piper, 2012b, c) or phase contrast and bright field illumination carried out simultaneously (Piper & Piper, 2013a). For technical reasons, however, we did not combine interference contrast with phase contrast, dark- and bright field in the past. On the other hand, potential advantages could also be expected from such combinations. The particular visual information immanent in the respective single modes could also be fused into final images resulting from optical superimposition of differently illuminated images when interference contrast is simultaneously carried out with other illumination techniques mentioned. The presentation of fine details might be optimized further when the intensity of the coactive illuminating light components and thus the weighting of the partial images superimposed could be regulated by the user so that variable contrast effects would be achievable. In this paper, several technical implementations are presented, which can lead to variable interference-phase contrast (VIPC), variable interference-dark field contrast (VIDC) and variable interference-bright field contrast (VIBC). Moreover, some examples are given for practical use of these methods.
Optical solutions of variable multimodal microscopy based on interference contrast Interference contrast can be superimposed with phase contrast or central dark-field illumination in maximal variability when a special ‘contrast tube’ is available as shown in Figure 1. Within this tube, the image forming light is divided into two parts running parallel in two separate light corridors. This separation of light is achieved with a splitting prism C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy
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Fig. 1. Special tube for variable multimodal microscopy designed with two light corridors for interference contrast and phase contrast or central dark field (technical drawing modified from Beyer & Sch¨oppe, 1965; Carl Zeiss Jena, 1965): 1, light source; 1a, collector lens; 2, condenser (facultative zoom system); 3, specimen slide; 4, objective; 5, tube lens; 6, prism for photo tube (removable); 7, Bertrand lens (removable); 8, eyepiece; 8a, eye; 9, photo-ocular; 9a, camera; 10, light mask; 10a, light annulus; 11, convex lens (10 dioptres); 12, polarizer; 13, quarter lambda compensator; 14, lambda compensator; 15, DIC prism; 16, projection lens group 1; 17, projection lens group 2; 18.1, splitting prism; 18.2, joining prism; 19, revolving turret; 20, phase plate; 20.1, phase ring; 20.2, annular light stop; 21, DIC prism; 22, analyser; 23 and 24, double polarizers; BFP, back focal plane; IMI, intermediate image.
(18.1). One light corridor can be used for generating interference contrast and the other for achievement of phase contrast, central dark field or bright field. Moreover, the tube contains several projection lenses arranged in two groups (16 and 17) so that the objective’s back focal plane (BFP 1) is duplicated and projected within the tube in both light corridors into a secondary back focal plane (BFP 2) and the first intermediate image (IMI 1) is duplicated as well so that a secondary interme C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
diate image (IMI 2) results from this optical arrangement. In order to generate interference contrast, the first differential interference contrast (DIC) prism, Wollaston or Nomarski prism (15), is situated beneath the condenser lens (or integrated into the condenser); the second DIC prism (21) is placed within the tube and inserted into one of the two parallel light corridors; the secondary BFP 2 is the ideal place for this second DIC prism. Of course, all additional optical components that are
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Fig. 2. Special tube for variable multimodal microscopy designed with two light corridors for interference contrast and bright field, labelling as in Figure 1.
needed for handling interference contrast illumination (polarizer, lambda or quarter lambda compensators and analyser, 12, 13, 14, 22) can be inserted into the light path and used according to normal technical standards. Both DIC prisms (15 and 21) can be designed as Wollaston prisms and mounted in fixed position, whereas the polarizer (12) is pivoted and designed as a rotatable polarizer. In this arrangement, interference contrast can be achieved according to SMITH. On the other hand, interference contrast can also be carried out according to the NOMARSKI method when both DIC prisms are designed and mounted as NOMARSKI prisms so that the second DIC prism (21) can be shifted perpendicularly to the optical
axis, whereas the polarizer (12) does not need to be pivoted. The other light corridor contains a phase ring (20.1) or an annular light stopper (20.2) mounted on a phase plate (20) instead of a phase ring also placed in the BFP 2. Various light modulating components (phase rings and/or light stoppers) of different shapes and sizes can be arranged on a revolving turret (19) on demand so that they can be easily changed, removed and replaced. A light mask (10) containing an annular light outlet (10.1) is mounted beneath the condenser. This light annulus (10.1) is coupled with a convex lens (11) so that it is projected into the BFP 2. When the condenser is designed as a zoom system, the projection size of the light annulus can C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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be varied in finest steps by changing the condenser aperture. Phase contrast or central dark field can be generated when the condenser light annulus (10.1) and the corresponding phase ring or annular light stopper (20.1, 20.2) are optically congruent and conjugate so that the projection image of the light annulus is completely covered by the light modulator used (phase ring or light stopper). When light annulus and phase ring or light stopper are somewhat misaligned so that a small proportion of the illuminating light runs beside the respective light modulator, a bright-field image is added as third component (triplex mode). The higher the proportion of aberrant light the higher are the intensity and dominance of the bright-field illumination. A pure bright-field image can be added and combined with interference contrast when phase rings and light stoppers are removed from the light corridor (Fig. 2). All in all, the specimen is illuminated by a concentric light cone; interference contrast is produced in one of the light corridors, and phase contrast or central dark field and facultative additional bright field or pure bright field are generated in the other light corridor. Both parallel image forming light beams associated with interference contrast and the complementary illuminating light are reunited by an opposite joining prism (18.2) so that the coactive partial images are superimposed in the plane of the secondary IMI 2. This IMI 2 can be observed with an eyepiece in the usual manner. As interference contrast and phase contrast or dark field/bright field are generated in different light corridors, the intensities of both corresponding partial images can be regulated independent from each other over the full range of brightness and darkness so that variable transitions between interference contrast and other complementary illumination techniques can be achieved. Thus, both coactive illumination techniques contribute to the final composite image in variable proportions. For this task, each light corridor is fitted with a couple of rotatable polarizers (23 and 24) so that the intensity of light can be modulated by turning these polarizers. When the polarizers (23 or 24) are turned into a crossed position, the image forming light is completely blocked from passing these polarizers. Thus, specimens can be observed also in pure interference contrast when the other light corridor is blocked, or examinations can be made in pure phase contrast, dark-field or dark-/bright-field illumination, when the interference contrast forming light is completely reflected by the polarizers. Of course, grey filter sets could also be used for analogous regulations of these intensities instead of double polarizers. A simpler technical variant also leading to interferencephase contrast or interference-dark-field illumination is demonstrated in Figure 3. In this arrangement, the light pathway is based on a single light corridor so that interference contrast and phase contrast or dark-field illumination are generated on the same optical axis. In this variant, the second DIC prism (21) is situated in or near to the objective’s first BFP 1, whereas a phase ring or an annular light stopper is mounted within the tube in the secondary BFP 2. The C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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additional equipment needed for interference contrast (polarizer, analyser, lambda and quarter lambda compensators, 12, 13, 14, 22) is used according to technical standards. Also in this arrangement, interference contrast can be carried out according to SMITH or NOMARSKI. The condenser light annulus (10.1) situated on the light mask (10) has to be aligned with the phase ring or the annular light stopper (20.1, 20.2) as already described. Thus, specimens can be observed using interference contrast combined with phase contrast or central dark field. When the area of the condenser light annulus projected into the secondary BFP 2 is completely covered by the phase ring or the light stopper, the intensities of the phase contrast- or dark-field–based partial images superimposed with interference contrast are maximized. The proportions of phase contrast and dark field can be reduced when the light annulus is slightly turned into a misaligned (off-centred) position. Alternatively, the light annulus can be designed with a higher breadth so that its external zone is projected outside of the phase ring or the annular light stopper, whereas its internal zone is covered by the light modulator used. Now, the light annulus can remain in centred position, and the external illuminating light that runs beside the light modulator leads to a higher dominance of interference contrast. In this arrangement, the external diameter of the light outlet and thus the relative dominance of the interference contrast image can be regulated with an iris diaphragm situated near to the condenser light mask. When the external light zone projected outside of the light modulator is completely covered by the iris diaphragm, the relative dominance of phase contrast or central dark field will be maximized. When all illuminating light is covered by an annular light stopper (20.2), pure central dark-field illumination can be achieved, and phase contrast can contribute to the final composite image by approximately 50%, when the annular light stopper is replaced by a phase ring of the same size. The intensity of interference contrast illumination cannot be selectively reduced further, because interference and phase contrast are generated in the same light corridor. Specimens can be observed in pure phase contrast when at least one of the DIC prisms (15, 21) and/or polarizers and analysers (12, 22) are removed, and they can be examined in pure interference contrast when the condenser light mask (10) is removed out of the light path. Instead of the arrangement described, the breadth of the light annulus can also be reduced. In this case, the external zone of the light annulus is projected into the phase ring or the annular light stopper, and the internal zone is projected beside the light modulator used. Phase contrast or central dark field are now associated with the external zone of the light annulus and interference contrast is generated by the internal portion of illuminating light. In this variant, the dominance of the phase contrast or dark-field illumination can be regulated with an iris diaphragm as already described.
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Fig. 3. Simplified special tube for variable multimodal microscopy with interference contrast and phase contrast or central dark field based on a single light corridor, labelling form Figure 1.
Materials and methods In order to evaluate the optical effects resulting from the multimodal methods described, a ‘hybrid microscope’ was built up as a prototype consisting of the following components: A Leitz/Leica laboratory microscope (Dialux) was fitted with a Leitz interference contrast ‘T’ device (Fig. 4). In this equipment, a special condenser designed for interference contrast (ICT condenser, Fig. 4 A) contains a set of four different Wollas-
ton prisms arranged in fixed positions on a rotatable revolving turret. The second Wollaston prisms are inserted in fixed positions situated in the BFP of four special ‘ICT objectives’ (16×, 25×, 40× and oil 100×, Fig. 4B). A rotatable polarizer is mounted at the bottom of the ICT condenser followed by a quarter lambda compensator and an additional lambda compensator, which can be switched into the light path or removed as required. The lambda compensator can be used for colour contrast effects and it can be removed when interference contrast has to be carried out C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 6. Adapter ‘Zeiss to Leitz’, manufactured by Dipl. Ing. Manfred L¨ochel, Germany.
Fig. 4. ‘Interference contrast T’ device from E. Leitz Wetzlar, ICT condenser (A) and ICT objectives (B).
Fig. 7. Revolving turret of the contrast tube showing an annular light stopper.
Fig. 5. Contrast tube from C. Zeiss Jena, mounted with a Leitz/Leica ‘Dialux’ microscope: 1, sliding handle of the prism for the phototube; 2, sliding handle of the Bertrand lens; 3, focusing wheel of the Bertrand lens; BFP, back focal plane; IMI, intermediate image.
based on grey tones. An analyser, mounted on a slide can be inserted above the objective. These optical components lead to interference contrast according to SMITH. In addition to the four Wollaston prisms, the condenser’s revolving turret contains two further light masks that are not fitted with Wollaston prisms: one outlet for bright field (position ‘H’) and one light annulus for phase contrast (position ‘Phaco’). A special ‘contrast tube’ manufactured by Carl Zeiss Jena (Fig. 5) was mounted with the Leitz/Leica ‘Dialux’ microscope; a hand-made adapter (Fig. 6), manufactured by Manfred L¨ochel, Germany, was necessary for this fitting so that the different bayonets from Leitz/Leica and Zeiss could match with each other. The contrast tube used is designed according to the technical drawing in Figure 3. It contains a revolving turret (Fig. 7) fitted with various phase rings and annular light stoppers situated in the secondary BFP 2. A light C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
mask carrying a light annulus (Fig. 8A) was localized beneath the condenser. This mask could be simply put on the light outlet of the microscope’s stand. An achromatic convex lens system, 10 dioptres, manufactured by Sebastian Hess, Germany, was mounted beneath the ICT condenser so that the light annulus situated beneath the condenser was projected into the secondary BFP leading to a sharp and distinct projection image (Figs. 8A,B). In the ‘hybrid microscope’ described, interference contrast can be achieved by optical components situated beneath the tube, that is, according to the normal technical standard, and phase contrast or central dark field are simultaneously generated in the tube, based on duplications of the objective’s BFP and the IMI. Results of practical evaluations According to several practical tests carried out up till now, the quality of interference contrast images is not influenced by the light annulus needed for phase contrast and axial dark field; the visual character of interference contrast images observed remains the same regardless of whether the specimen is illuminated by a circular light cone or by axial light beams. Moreover, the image quality is not diminished by the 10 dioptres convex lens mounted beneath the condenser used for
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Fig. 8. Illuminating apparatus modified for superimposition of interference contrast and phase contrast or central dark field, light mask with light annulus put on the microscope stand (A), achromatic convex lens system (manufactured by Sebastian Hess, Germany) mounted with the base of the ICT condenser (A and B).
Fig. 9. Flat and low-density diatom, diameter: 0.07 mm, objective 25×, bright field (A), interference contrast (B), phase contrast (C) and variable interference-phase contrast (VIPC) (D); preparation: Klaus Kemp, United Kingdom.
generating a projection image of the light annulus in the secondary BFP 2. Some examples of practical use are given in Figures 9 and 10, showing a small and low-density diatom. Figure 9 was
Fig. 10. Preparation and equipment from Figure 9, interference contrast (A), VIDC (B, C) and central dark field (D).
taken with a 25× magnifying ICT objective and illuminated in bright field, interference contrast, phase contrast and VIPC. It is obvious that most details can be perceived in best sharpness, maximum contrast and enhanced plasticity when VIPC is carried out. In bright field (Fig. 9A), only a few details can be seen in very low contrast because of the low density of C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 11. Examples of light masks fitted with two separate light outlets (bottom row) and corresponding phase plates (top row), designed for phase contrast and central dark field (A and B), phase contrast and axial dark field (C), phase contrast and bright field (D and E) and dark and bright field (F and G).
this specimen even when the condenser aperture diaphragm is maximally closed. In interference contrast (Fig. 9B), typical relief effects are apparent in medium contrast, and fine details can be revealed, especially in the centre and at the margin of the frustule. In phase contrast (Fig. 9C), the diatom is significantly higher contrasted than in interference contrast, but contours and fine details appear less sharply and are surrounded by typical haloing. In VIPC (Fig. 9D), the high level of contrast, which is immanent in phase contrast, is transferred into the composite image; nevertheless, fine details which are imaged indistinctly in phase contrast are revealed with the same precision as when using pure interference contrast. Marginal contours can also be perceived in best sharpness and clarity. Figure 10 demonstrates transitions between interference contrast and central dark field. Photomicrographs were taken in pure interference contrast (Fig. 10A), in VIDC dominated by interference contrast (Fig. 10B) or dark field (Fig. 10C) and in pure dark field (Fig. 10D). It may clearly be seen that most fine details, especially the multiple fine perforations of the discoid diatom shell are apparent in VIDC, revealed in maximal resolution and contrast. Suggestions for further technical developments In the technical drawings presented in Figures 1–3, the illuminating light is formed to a circular light cone by a single light annulus. Instead of a single light annulus of this sort, light masks could also be fitted with two separate concentric light outlets so that two different illumination modes can be achieved in one optical axis and combined with interference contrast. Some technical examples are given in Figure 11. In such arrangements, the external light outlet can be partially or completely covered by an iris diaphragm so that the intensity of the partial image generated by the respective external illuminating light can be easily varied. Moreover, the light flow in both outlets can be regulated in maximum variability independent of each other, when light masks are fitted with double diaphragms or double polarizers. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 12. Suggestions for light masks fitted with double diaphragms, central iris diaphragm and fixed light annulus (A), fixed central perforation and variable light annulus (B) and double iris diaphragm system (C).
Fig. 13. Suggestions for light masks fitted with polarization filters, discoid (P1) and annular (P2) polarizers, mounted in fixed crossed position or in a facultative pivoted arrangement, additional rotatable polarizer (P3) beneath the light mask.
Examples of construction plans are given in the Figures 12 and 13. Interference contrast could also be combined with phase contrast and/or dark field in incident light when a vertical illuminator is modified according to Figure 14. In the technical drawing shown here, a special objective for epi-brightand dark-field examinations is used so that two different light corridors are available within the objective: an external light corridor for epi-dark field and an internal light corridor for interference and phase contrast. The vertical illuminator is fitted with a light mask on a slide (29), consisting of two separate light outlets: an external light annulus (29.2) for epidark-field illumination and an internal light annulus (29.1) for epi-phase and interference contrast. The objective is fitted with a phase plate (36) containing a phase ring (36.1) that has to be conjugate with the internal light annulus (29.1). Moreover, a Wollaston prism (37) is inserted near the phase plate so that interference contrast is added. The light components associated with both light corridors can be filtered at different colours (Fig. 15A). The intensities of these light components can also be regulated independent from each other by use of polarizers (examples in Fig. 15B), grey filters (Fig. 15C) or (iris) diaphragms (Fig. 15D). Thus, interference contrast could also be used together with dark field
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Fig. 14. Vertical illuminator and special objective for dark- and bright-field illumination based on incident light, modification for variable multimodal interference contrast (technical drawing modified from E. Leitz Wetzlar, 1969): 25, illuminator tube; 26, light source; 27, illuminator lenses; 28, (iris) diaphragm; 29, slide with light mask; 29.1, internal light annulus for phase and interference contrast; 29.2, external light annulus for dark field; 30, external light cone for dark-field illumination; 31, internal light cone for interference and phase contrast illumination; 32, semipermeable mirror; 33, specimen; 34, objective; 34.1, reflecting internal area; 35.1, external objective lenses for dark-field illumination; 35.2, internal objective lenses (imaging lens system) passed by illuminating light for bright field, phase and interference contrast; 36, phase plate; 36.1, phase ring; 37, DIC prism; 38, polarizer P; 39, analyser A; 40, tube lenses; 41, joined light beams running to the eyepiece.
and phase contrast in material controlling of nontransparent probes. The contrast tubes described can also be used for other variants of multimodal light microscopy when the DIC prisms are removed and replaced by other light modulating components. Thus, variable phase-dark-field contrast, variable bright-darkfield contrast and variable phase-bright-field contrast can be achieved with tubes of this sort when they are appropriately modified for the respective task. Figure 16 gives an example of a contrast tube designed for variable phase-dark-field contrast based on two separate light corridors. The tube contains two different revolving turrets equipped with several phase rings and annular light stoppers. In order to achieve variable phase-dark-field contrast, a couple of light modulators – phase ring and annular light stopper of the same size – have to be turned into the light path. The condenser light annulus, which is projected into the plane of both light modulators, has to be congruent and conjugate with both light modulators. As a result of this optical arrangement, a phase contrast image is generated in one light corridor and a central dark-field image in the other. The weighting of both partial images can be regulated with double polarizers as already described. Moreover, both images superimposed, the dark field and the phase contrast image, can be filtered at different colours on demand. When one of the revolving turrets is removed, a brightfield image can be achieved in the respective light corridor and a phase contrast or dark-field image can be added, which is generated in the other corridor. Thus, specimens can be
Fig. 15. Vertical illuminator from Figure 14, light corridors fitted with different colour filters 42.1 and 42.2 (A), double polarizers 43.1 and 43.2 (B), grey filters 44.1 and 44.2 (C) or iris diaphragms 45.1 and 45.2 (D), further explanations in the text.
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Fig. 16. Contrast tube modified for variable phase-dark-field contrast (VPDC), labelling as in Figure 1.
illuminated in variable bright-dark-field contrast or variable phase-bright-field contrast.
Discussion Bright and dark field, phase and interference contrast are used as standard techniques in many fields of light microscopy. Each method is characterized by typical well-known advantages and disadvantages including optical limitations and characteristic artefacts, which have been compiled by several authors (Robertson, 1970; Determann & Lepusch, 1981; Slaghter ¨ & Slaghter, 1992; Gluckstad et al., 2001; Murphy, 2001; C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
Oldfield, 2001; Lichtscheidl, 2011). Thus, for instance, bright field is not suited for observation of low-density specimens. Diffraction is a typical artefact in bright-field techniques, especially when the aperture of the condenser is reduced by closing the aperture diaphragm. Phase contrast images are affected with haloing and shade-off so that fine details situated in the zones of these artefacts can be masked. Dark-field illumination is characterized by ultrahigh ranges of brightness and contrast and by irradiation caused by reflection and scattering of the incoming light. In interference contrast, pseudo-relief effects can be generated by regional variation of the specimen’s refraction index, and the level of contrast may be lower in interference contrast than in phase contrast. Moreover, the depth of
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focus achievable with interference contrast is lower when compared with some other concurrent techniques so that thick specimens appear like ‘optical sections’. On the other hand, especially unstained phase specimens can be visualized with high accuracy, as interference contrast is not affected with haloing and shade-off. In view of these facts, significant improvements of the resulting image quality can be expected when various illumination techniques are combined with each other and simultaneously carried out. The particular visual information that can be obtained by use of different single techniques can be joined in composite images. Also fine details, which may be lost in one illumination technique carried out on its own, can be contributed by the other complementary technique superimposed. Additional contrast effects can be achieved when the partial images superimposed interfere with each other. Thus, the global visual information can be enhanced in the respective composite images. Until now, interference contrast, phase contrast and other illumination techniques have been carried out successively and compared with each other based on observation of the same specimen. Such comparisons have already been carried out several years and decades ago. Thus, for instance, interference and phase contrast have been compared in examination of bacterial cells (Hitchins et al., 1968), spermatozoa (Cortner et al., 1978) and organs of corti (Kuhn et al., 1971). An extensive comparison of phase contrast and DIC microscopy can be obtained from the web (Murphy et al., 2005). In interference contrast, regions characterized by very shallow optical path slopes such as extended and flat specimens produce insignificant and low contrast so that they often appear at the same intensity level as the background (Murphy et al., 2005). Moreover, in several specimens, the level of contrast achievable with DIC is influenced by the orientation of the specimen with respect to the optical axis of the microscope, whereas phase contrast leads to constant contrast levels being independent of the specimen’s rotation around the optical axis (Murphy et al., 2005). On the other hand, regions that are masked by haloing in phase contrast images can appear in superior clarity when interference contrast is used. Thus, for instance, halo artefacts can surround the periphery of cell membranes so that visual information about intercellular contacts may be lost in phase contrast. Interference contrast, however, can lead to superior clarity in imaging of cellular membranes, but internal details of cells may appear in lower contrast and clarity when compared with phase contrast (Murphy et al., 2005). According to these considerations, both methods can be regarded as complementary techniques each leading to complementary visualization of particular structures. Following this, superimposition of different images of the same specimen illuminated by use of different techniques can lead to superior global visual information.
Although computer-based digital superimpositions of various photomicrographs taken from the same specimen at different illumination can only be carried out with ‘static’ specimens (permanent slides, for instance), the optical superimpositions, which are achievable by our methods, can also be used for visual observation in real time including examination and documentation of motile native specimens showing spontaneous movement and locomotion. Moreover, interference phenomena of the imaging light associated with partial images, which are simultaneously generated and optically superimposed, can produce additional contrast effects so that additional visual information may result. Otherwise, any interference effects are absent in computerbased superimpositions. Primarily, our methods are characterized by a technical and optical value. In particular, thin and low-density biological structures appear in higher contrast than in interference contrast – the grade of contrast can be compared with phase contrast. Fine details and marginal contours appear in higher precision than achievable with phase contrast, because loss of visual information caused by haloing and shade-off can be avoided. Finally, the relief or pseudo-relief effects that are typical for interference contrast remain immanent in our technique. These characteristics are evident in the examples presented in Figures 9 and 10. When particular biological probes can be observed in higher clarity and precision compared with conventional concurrent techniques carried out on their own, this surplus of image quality can also lead to a ‘biological value’. In particular, the several types of ‘contrast tubes’ described should be predestined for development of high-end multimodal microscopes for variable combination of bright- and dark field, phase and interference contrast, because all complementary illumination techniques can be combined with each other in highest variability based on the same technical platform. Acknowledgements The authors thank Mr. Klaus Kemp, United Kingdom, and Mr. Eberhard Raap, Germany, for arranging slides of diatoms, Mr. Dipl. Ing. Manfred L¨ochel for an individual created handmade special adapter ‘Zeiss to Leica’ and Mr. Sebastian Hess, Germany, for supplying achromatic lens systems provided for his ‘magniflash’ device. We thank Mrs. Linda Tennant, United Kingdom, for revising our English manuscript.
References Beyer, H. & Sch¨oppe, G. (1965) Interference device for microscopes based on transmitted light. Jena News 10, 99–105. Carl Zeiss, Jena (1965) Interphako NF for Transmitted Light. Factory print No 30-305-1, pp. 1–11. Carl Zeiss Jena Company, Jena, Germany. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Cortner, G.V., Boudreau, M.S. & A.J. (1978) Phase contrast microscopy versus differential interference contrast microscopy as applicable to observation of spermatozoa. J. Forensic Sci. 23(4), 830– 832. Determann, H. & Lepusch, F. (1981) Darkfield microscopy, phase contrast microscopy, interference contrast microscopy. The Microscope and Its Applications (eds by H. Determann & F. Lepusch), pp. 18–24. E. Leitz Wetzlar Company, Germany. ¨ Gluckstad, J., Mogensen, P.C. & Eriksen, R.L. (2001) The generalised phase contrast method and its applications, DOPS-NYT 1, 49–54. Hitchins, A.D., Kahn, A.J. & Slepecky, R.A. (1968) Interference contrast and phase contrast microscopy of sporulation and germination of Bacillus megaterium. J. Bacteriol. 96(5), 1811–1817. Kuhn, F.A., Thalmann, R. & Marovitz, W.F. (1971) A comparison of Nomarski differential interference contrast and phase contrast microscopy of the guinea pig organ of corti. Laryngoscope. 81(7), 1090–1118. E. Leitz Wetzlar (1969) Vertical illuminators. Image Forming and Illuminating Optics of the Microscope: Objectives, Eyepieces, Condensers, Factory Print, p. 36. E. Leitz Wetzlar Company, Wetzlar, Germany. Lichtscheidl, I.(ed.). (2011) Interference contrast. Light Microscopy Online: Theory and Practical Use. Department of Cell Imaging and Submicroscopic Research, University of Vienna, Vienna. Available at: http://www.univie.ac.at/mikroskopie/2_kontraste/interferenz/1 einleitung.htm Murphy, D.B. (2001) Phase contrast microscopy. Fundamentals of Light Microscopy and Electron Imaging (ed. by D. B. Murphy), pp. 97–112. Wiley, Chicester. Murphy, B.M., Hinsch, J., Spring, K.R. & Davidson M.W. (2005) Comparison of Phase Contrast and DIC Microscopy, Molecular Expressions, Microscopy Primer. Available at: http://micro.magnet.fsu.edu/
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primer/techniques/dic/dicphasecomparison.html. Accessed May 4, 2014. Oldfield, R. (2001) Encyclopedia of Life Sciences. Differential Interference Contrast Light Microscopy (eds by A. Clarke, A.F. Agr´o, Y. Zheng et al.) pp.1–4. Wiley & Sons, Chicester. Piper, T. & Piper, J. (2012a) Variable bright-darkfield-contrast (VBDC), a new illumination technique for improved visualization of complex structured transparent specimens. Microsc. Res. Techn. 75, 537–554. Piper, T. & Piper, J. (2012b) Variable phase-darkfield-contrast (VPDC), a variant illumination technique for improved visualizations of transparent specimens. Micros. Microanal. 18, 343–352. Piper, T. & Piper, J. (2012c) Axial phase-darkfield-contrast (APDC): a new technique for variable optical contrasting in light microscopy. J. Microsc. 247, 259–268. Piper, T. & Piper, J. (2013a) Variable phase bright-field contrast (VPBC): an alternative illumination technique for improved imaging in transparent specimens. Micros. Microanal. 19, 11–21. Piper, T. & Piper, J. (2013b) Universal variable bright-darkfield contrast: a variant technique for improved imaging of problematic specimens in light microscopy. Micros. Microanal. 19, 1092–1105. Piper, T. & Piper, J. (2013c) Phase contrast without phase plates and phase rings: optical solutions for improved imaging of phase structures. Microsc. Res. Techn. 76, 1050–1056. Robertson, D. (1970) The phase contrast microscope. The World Beneath the Microscope (ed. by D. Robertson), pp. 26–30. Weidenfeld and Nicholson, London Slaghter, E.M. & Slaghter, H.S. (1992) Imaging of phase objects. Light and Electron Microscopy (eds by E. M. Slaghter & H. S. Slaghter), pp. 149–167. Cambridge University Press, Cambridge.
doi: 10.1111/jmi.12134
Received 21 October 2013; accepted 9 April 2014
Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field T . P I P E R ∗ & J . P I P E R ∗ , †, ‡, §
∗ Department of Light Microscopy, Laboratory for Applied Microscopy Research, Marienburgstr. 23, Bullay, Rheinland-Pfalz, Germany
†Department of Internal Medicine, Clinic ‘Meduna’, Bad Bertrich, Germany ‡Faculty of Medicine and Pharmacy, University of Oradea, Romania §Faculty of Human Sciences, University of North West Europe, Kerkrade, Netherlands
Key words. Bright field, contrast tube, dark field, incident light, interference contrast, multimodal microscopy, phase contrast, transmitted light, vertical illuminator.
Summary Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark- or bright-field illumination. Thus, the particular visual information associated with interference and phase contrast, darkand bright-field illumination is joined in real-time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark-field illumination. In particular, haloing and shade-off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators.
Introduction In light microscopy, image quality and visual information can be significantly improved when different illumination techniques, which are normally used on their own, are combined with each other and simultaneously carried out. Several specimens, especially problematic objects that cannot be well perceived in standard techniques can often be observed and photographed in superior clarity with the help of such optical superimposition. Optical principles, technical realization and practical results of these techniques based on phase contrast, Correspondence to: Dr. J¨org Piper, Department of Internal Medicine, Clinic ‘Meduna’, Clara Viebig Road No. 4, D-56864 Bad Bertrich, Germany. Tel: +49-0-2674-1823184 or 182-3335; fax: +49-0-2674-182-3182; e-mail: [email protected]
bright- and dark field have been reported in several previous contributions. Up till now, we were successful with various implementations of multimodal bright–dark field microscopy (Piper & Piper, 2012a, 2013b, c), variable combinations of phase contrast and peripheral or axial dark field (Piper & Piper, 2012b, c) or phase contrast and bright field illumination carried out simultaneously (Piper & Piper, 2013a). For technical reasons, however, we did not combine interference contrast with phase contrast, dark- and bright field in the past. On the other hand, potential advantages could also be expected from such combinations. The particular visual information immanent in the respective single modes could also be fused into final images resulting from optical superimposition of differently illuminated images when interference contrast is simultaneously carried out with other illumination techniques mentioned. The presentation of fine details might be optimized further when the intensity of the coactive illuminating light components and thus the weighting of the partial images superimposed could be regulated by the user so that variable contrast effects would be achievable. In this paper, several technical implementations are presented, which can lead to variable interference-phase contrast (VIPC), variable interference-dark field contrast (VIDC) and variable interference-bright field contrast (VIBC). Moreover, some examples are given for practical use of these methods.
Optical solutions of variable multimodal microscopy based on interference contrast Interference contrast can be superimposed with phase contrast or central dark-field illumination in maximal variability when a special ‘contrast tube’ is available as shown in Figure 1. Within this tube, the image forming light is divided into two parts running parallel in two separate light corridors. This separation of light is achieved with a splitting prism C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy
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Fig. 1. Special tube for variable multimodal microscopy designed with two light corridors for interference contrast and phase contrast or central dark field (technical drawing modified from Beyer & Sch¨oppe, 1965; Carl Zeiss Jena, 1965): 1, light source; 1a, collector lens; 2, condenser (facultative zoom system); 3, specimen slide; 4, objective; 5, tube lens; 6, prism for photo tube (removable); 7, Bertrand lens (removable); 8, eyepiece; 8a, eye; 9, photo-ocular; 9a, camera; 10, light mask; 10a, light annulus; 11, convex lens (10 dioptres); 12, polarizer; 13, quarter lambda compensator; 14, lambda compensator; 15, DIC prism; 16, projection lens group 1; 17, projection lens group 2; 18.1, splitting prism; 18.2, joining prism; 19, revolving turret; 20, phase plate; 20.1, phase ring; 20.2, annular light stop; 21, DIC prism; 22, analyser; 23 and 24, double polarizers; BFP, back focal plane; IMI, intermediate image.
(18.1). One light corridor can be used for generating interference contrast and the other for achievement of phase contrast, central dark field or bright field. Moreover, the tube contains several projection lenses arranged in two groups (16 and 17) so that the objective’s back focal plane (BFP 1) is duplicated and projected within the tube in both light corridors into a secondary back focal plane (BFP 2) and the first intermediate image (IMI 1) is duplicated as well so that a secondary interme C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
diate image (IMI 2) results from this optical arrangement. In order to generate interference contrast, the first differential interference contrast (DIC) prism, Wollaston or Nomarski prism (15), is situated beneath the condenser lens (or integrated into the condenser); the second DIC prism (21) is placed within the tube and inserted into one of the two parallel light corridors; the secondary BFP 2 is the ideal place for this second DIC prism. Of course, all additional optical components that are
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Fig. 2. Special tube for variable multimodal microscopy designed with two light corridors for interference contrast and bright field, labelling as in Figure 1.
needed for handling interference contrast illumination (polarizer, lambda or quarter lambda compensators and analyser, 12, 13, 14, 22) can be inserted into the light path and used according to normal technical standards. Both DIC prisms (15 and 21) can be designed as Wollaston prisms and mounted in fixed position, whereas the polarizer (12) is pivoted and designed as a rotatable polarizer. In this arrangement, interference contrast can be achieved according to SMITH. On the other hand, interference contrast can also be carried out according to the NOMARSKI method when both DIC prisms are designed and mounted as NOMARSKI prisms so that the second DIC prism (21) can be shifted perpendicularly to the optical
axis, whereas the polarizer (12) does not need to be pivoted. The other light corridor contains a phase ring (20.1) or an annular light stopper (20.2) mounted on a phase plate (20) instead of a phase ring also placed in the BFP 2. Various light modulating components (phase rings and/or light stoppers) of different shapes and sizes can be arranged on a revolving turret (19) on demand so that they can be easily changed, removed and replaced. A light mask (10) containing an annular light outlet (10.1) is mounted beneath the condenser. This light annulus (10.1) is coupled with a convex lens (11) so that it is projected into the BFP 2. When the condenser is designed as a zoom system, the projection size of the light annulus can C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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be varied in finest steps by changing the condenser aperture. Phase contrast or central dark field can be generated when the condenser light annulus (10.1) and the corresponding phase ring or annular light stopper (20.1, 20.2) are optically congruent and conjugate so that the projection image of the light annulus is completely covered by the light modulator used (phase ring or light stopper). When light annulus and phase ring or light stopper are somewhat misaligned so that a small proportion of the illuminating light runs beside the respective light modulator, a bright-field image is added as third component (triplex mode). The higher the proportion of aberrant light the higher are the intensity and dominance of the bright-field illumination. A pure bright-field image can be added and combined with interference contrast when phase rings and light stoppers are removed from the light corridor (Fig. 2). All in all, the specimen is illuminated by a concentric light cone; interference contrast is produced in one of the light corridors, and phase contrast or central dark field and facultative additional bright field or pure bright field are generated in the other light corridor. Both parallel image forming light beams associated with interference contrast and the complementary illuminating light are reunited by an opposite joining prism (18.2) so that the coactive partial images are superimposed in the plane of the secondary IMI 2. This IMI 2 can be observed with an eyepiece in the usual manner. As interference contrast and phase contrast or dark field/bright field are generated in different light corridors, the intensities of both corresponding partial images can be regulated independent from each other over the full range of brightness and darkness so that variable transitions between interference contrast and other complementary illumination techniques can be achieved. Thus, both coactive illumination techniques contribute to the final composite image in variable proportions. For this task, each light corridor is fitted with a couple of rotatable polarizers (23 and 24) so that the intensity of light can be modulated by turning these polarizers. When the polarizers (23 or 24) are turned into a crossed position, the image forming light is completely blocked from passing these polarizers. Thus, specimens can be observed also in pure interference contrast when the other light corridor is blocked, or examinations can be made in pure phase contrast, dark-field or dark-/bright-field illumination, when the interference contrast forming light is completely reflected by the polarizers. Of course, grey filter sets could also be used for analogous regulations of these intensities instead of double polarizers. A simpler technical variant also leading to interferencephase contrast or interference-dark-field illumination is demonstrated in Figure 3. In this arrangement, the light pathway is based on a single light corridor so that interference contrast and phase contrast or dark-field illumination are generated on the same optical axis. In this variant, the second DIC prism (21) is situated in or near to the objective’s first BFP 1, whereas a phase ring or an annular light stopper is mounted within the tube in the secondary BFP 2. The C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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additional equipment needed for interference contrast (polarizer, analyser, lambda and quarter lambda compensators, 12, 13, 14, 22) is used according to technical standards. Also in this arrangement, interference contrast can be carried out according to SMITH or NOMARSKI. The condenser light annulus (10.1) situated on the light mask (10) has to be aligned with the phase ring or the annular light stopper (20.1, 20.2) as already described. Thus, specimens can be observed using interference contrast combined with phase contrast or central dark field. When the area of the condenser light annulus projected into the secondary BFP 2 is completely covered by the phase ring or the light stopper, the intensities of the phase contrast- or dark-field–based partial images superimposed with interference contrast are maximized. The proportions of phase contrast and dark field can be reduced when the light annulus is slightly turned into a misaligned (off-centred) position. Alternatively, the light annulus can be designed with a higher breadth so that its external zone is projected outside of the phase ring or the annular light stopper, whereas its internal zone is covered by the light modulator used. Now, the light annulus can remain in centred position, and the external illuminating light that runs beside the light modulator leads to a higher dominance of interference contrast. In this arrangement, the external diameter of the light outlet and thus the relative dominance of the interference contrast image can be regulated with an iris diaphragm situated near to the condenser light mask. When the external light zone projected outside of the light modulator is completely covered by the iris diaphragm, the relative dominance of phase contrast or central dark field will be maximized. When all illuminating light is covered by an annular light stopper (20.2), pure central dark-field illumination can be achieved, and phase contrast can contribute to the final composite image by approximately 50%, when the annular light stopper is replaced by a phase ring of the same size. The intensity of interference contrast illumination cannot be selectively reduced further, because interference and phase contrast are generated in the same light corridor. Specimens can be observed in pure phase contrast when at least one of the DIC prisms (15, 21) and/or polarizers and analysers (12, 22) are removed, and they can be examined in pure interference contrast when the condenser light mask (10) is removed out of the light path. Instead of the arrangement described, the breadth of the light annulus can also be reduced. In this case, the external zone of the light annulus is projected into the phase ring or the annular light stopper, and the internal zone is projected beside the light modulator used. Phase contrast or central dark field are now associated with the external zone of the light annulus and interference contrast is generated by the internal portion of illuminating light. In this variant, the dominance of the phase contrast or dark-field illumination can be regulated with an iris diaphragm as already described.
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Fig. 3. Simplified special tube for variable multimodal microscopy with interference contrast and phase contrast or central dark field based on a single light corridor, labelling form Figure 1.
Materials and methods In order to evaluate the optical effects resulting from the multimodal methods described, a ‘hybrid microscope’ was built up as a prototype consisting of the following components: A Leitz/Leica laboratory microscope (Dialux) was fitted with a Leitz interference contrast ‘T’ device (Fig. 4). In this equipment, a special condenser designed for interference contrast (ICT condenser, Fig. 4 A) contains a set of four different Wollas-
ton prisms arranged in fixed positions on a rotatable revolving turret. The second Wollaston prisms are inserted in fixed positions situated in the BFP of four special ‘ICT objectives’ (16×, 25×, 40× and oil 100×, Fig. 4B). A rotatable polarizer is mounted at the bottom of the ICT condenser followed by a quarter lambda compensator and an additional lambda compensator, which can be switched into the light path or removed as required. The lambda compensator can be used for colour contrast effects and it can be removed when interference contrast has to be carried out C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 6. Adapter ‘Zeiss to Leitz’, manufactured by Dipl. Ing. Manfred L¨ochel, Germany.
Fig. 4. ‘Interference contrast T’ device from E. Leitz Wetzlar, ICT condenser (A) and ICT objectives (B).
Fig. 7. Revolving turret of the contrast tube showing an annular light stopper.
Fig. 5. Contrast tube from C. Zeiss Jena, mounted with a Leitz/Leica ‘Dialux’ microscope: 1, sliding handle of the prism for the phototube; 2, sliding handle of the Bertrand lens; 3, focusing wheel of the Bertrand lens; BFP, back focal plane; IMI, intermediate image.
based on grey tones. An analyser, mounted on a slide can be inserted above the objective. These optical components lead to interference contrast according to SMITH. In addition to the four Wollaston prisms, the condenser’s revolving turret contains two further light masks that are not fitted with Wollaston prisms: one outlet for bright field (position ‘H’) and one light annulus for phase contrast (position ‘Phaco’). A special ‘contrast tube’ manufactured by Carl Zeiss Jena (Fig. 5) was mounted with the Leitz/Leica ‘Dialux’ microscope; a hand-made adapter (Fig. 6), manufactured by Manfred L¨ochel, Germany, was necessary for this fitting so that the different bayonets from Leitz/Leica and Zeiss could match with each other. The contrast tube used is designed according to the technical drawing in Figure 3. It contains a revolving turret (Fig. 7) fitted with various phase rings and annular light stoppers situated in the secondary BFP 2. A light C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
mask carrying a light annulus (Fig. 8A) was localized beneath the condenser. This mask could be simply put on the light outlet of the microscope’s stand. An achromatic convex lens system, 10 dioptres, manufactured by Sebastian Hess, Germany, was mounted beneath the ICT condenser so that the light annulus situated beneath the condenser was projected into the secondary BFP leading to a sharp and distinct projection image (Figs. 8A,B). In the ‘hybrid microscope’ described, interference contrast can be achieved by optical components situated beneath the tube, that is, according to the normal technical standard, and phase contrast or central dark field are simultaneously generated in the tube, based on duplications of the objective’s BFP and the IMI. Results of practical evaluations According to several practical tests carried out up till now, the quality of interference contrast images is not influenced by the light annulus needed for phase contrast and axial dark field; the visual character of interference contrast images observed remains the same regardless of whether the specimen is illuminated by a circular light cone or by axial light beams. Moreover, the image quality is not diminished by the 10 dioptres convex lens mounted beneath the condenser used for
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Fig. 8. Illuminating apparatus modified for superimposition of interference contrast and phase contrast or central dark field, light mask with light annulus put on the microscope stand (A), achromatic convex lens system (manufactured by Sebastian Hess, Germany) mounted with the base of the ICT condenser (A and B).
Fig. 9. Flat and low-density diatom, diameter: 0.07 mm, objective 25×, bright field (A), interference contrast (B), phase contrast (C) and variable interference-phase contrast (VIPC) (D); preparation: Klaus Kemp, United Kingdom.
generating a projection image of the light annulus in the secondary BFP 2. Some examples of practical use are given in Figures 9 and 10, showing a small and low-density diatom. Figure 9 was
Fig. 10. Preparation and equipment from Figure 9, interference contrast (A), VIDC (B, C) and central dark field (D).
taken with a 25× magnifying ICT objective and illuminated in bright field, interference contrast, phase contrast and VIPC. It is obvious that most details can be perceived in best sharpness, maximum contrast and enhanced plasticity when VIPC is carried out. In bright field (Fig. 9A), only a few details can be seen in very low contrast because of the low density of C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 11. Examples of light masks fitted with two separate light outlets (bottom row) and corresponding phase plates (top row), designed for phase contrast and central dark field (A and B), phase contrast and axial dark field (C), phase contrast and bright field (D and E) and dark and bright field (F and G).
this specimen even when the condenser aperture diaphragm is maximally closed. In interference contrast (Fig. 9B), typical relief effects are apparent in medium contrast, and fine details can be revealed, especially in the centre and at the margin of the frustule. In phase contrast (Fig. 9C), the diatom is significantly higher contrasted than in interference contrast, but contours and fine details appear less sharply and are surrounded by typical haloing. In VIPC (Fig. 9D), the high level of contrast, which is immanent in phase contrast, is transferred into the composite image; nevertheless, fine details which are imaged indistinctly in phase contrast are revealed with the same precision as when using pure interference contrast. Marginal contours can also be perceived in best sharpness and clarity. Figure 10 demonstrates transitions between interference contrast and central dark field. Photomicrographs were taken in pure interference contrast (Fig. 10A), in VIDC dominated by interference contrast (Fig. 10B) or dark field (Fig. 10C) and in pure dark field (Fig. 10D). It may clearly be seen that most fine details, especially the multiple fine perforations of the discoid diatom shell are apparent in VIDC, revealed in maximal resolution and contrast. Suggestions for further technical developments In the technical drawings presented in Figures 1–3, the illuminating light is formed to a circular light cone by a single light annulus. Instead of a single light annulus of this sort, light masks could also be fitted with two separate concentric light outlets so that two different illumination modes can be achieved in one optical axis and combined with interference contrast. Some technical examples are given in Figure 11. In such arrangements, the external light outlet can be partially or completely covered by an iris diaphragm so that the intensity of the partial image generated by the respective external illuminating light can be easily varied. Moreover, the light flow in both outlets can be regulated in maximum variability independent of each other, when light masks are fitted with double diaphragms or double polarizers. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
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Fig. 12. Suggestions for light masks fitted with double diaphragms, central iris diaphragm and fixed light annulus (A), fixed central perforation and variable light annulus (B) and double iris diaphragm system (C).
Fig. 13. Suggestions for light masks fitted with polarization filters, discoid (P1) and annular (P2) polarizers, mounted in fixed crossed position or in a facultative pivoted arrangement, additional rotatable polarizer (P3) beneath the light mask.
Examples of construction plans are given in the Figures 12 and 13. Interference contrast could also be combined with phase contrast and/or dark field in incident light when a vertical illuminator is modified according to Figure 14. In the technical drawing shown here, a special objective for epi-brightand dark-field examinations is used so that two different light corridors are available within the objective: an external light corridor for epi-dark field and an internal light corridor for interference and phase contrast. The vertical illuminator is fitted with a light mask on a slide (29), consisting of two separate light outlets: an external light annulus (29.2) for epidark-field illumination and an internal light annulus (29.1) for epi-phase and interference contrast. The objective is fitted with a phase plate (36) containing a phase ring (36.1) that has to be conjugate with the internal light annulus (29.1). Moreover, a Wollaston prism (37) is inserted near the phase plate so that interference contrast is added. The light components associated with both light corridors can be filtered at different colours (Fig. 15A). The intensities of these light components can also be regulated independent from each other by use of polarizers (examples in Fig. 15B), grey filters (Fig. 15C) or (iris) diaphragms (Fig. 15D). Thus, interference contrast could also be used together with dark field
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Fig. 14. Vertical illuminator and special objective for dark- and bright-field illumination based on incident light, modification for variable multimodal interference contrast (technical drawing modified from E. Leitz Wetzlar, 1969): 25, illuminator tube; 26, light source; 27, illuminator lenses; 28, (iris) diaphragm; 29, slide with light mask; 29.1, internal light annulus for phase and interference contrast; 29.2, external light annulus for dark field; 30, external light cone for dark-field illumination; 31, internal light cone for interference and phase contrast illumination; 32, semipermeable mirror; 33, specimen; 34, objective; 34.1, reflecting internal area; 35.1, external objective lenses for dark-field illumination; 35.2, internal objective lenses (imaging lens system) passed by illuminating light for bright field, phase and interference contrast; 36, phase plate; 36.1, phase ring; 37, DIC prism; 38, polarizer P; 39, analyser A; 40, tube lenses; 41, joined light beams running to the eyepiece.
and phase contrast in material controlling of nontransparent probes. The contrast tubes described can also be used for other variants of multimodal light microscopy when the DIC prisms are removed and replaced by other light modulating components. Thus, variable phase-dark-field contrast, variable bright-darkfield contrast and variable phase-bright-field contrast can be achieved with tubes of this sort when they are appropriately modified for the respective task. Figure 16 gives an example of a contrast tube designed for variable phase-dark-field contrast based on two separate light corridors. The tube contains two different revolving turrets equipped with several phase rings and annular light stoppers. In order to achieve variable phase-dark-field contrast, a couple of light modulators – phase ring and annular light stopper of the same size – have to be turned into the light path. The condenser light annulus, which is projected into the plane of both light modulators, has to be congruent and conjugate with both light modulators. As a result of this optical arrangement, a phase contrast image is generated in one light corridor and a central dark-field image in the other. The weighting of both partial images can be regulated with double polarizers as already described. Moreover, both images superimposed, the dark field and the phase contrast image, can be filtered at different colours on demand. When one of the revolving turrets is removed, a brightfield image can be achieved in the respective light corridor and a phase contrast or dark-field image can be added, which is generated in the other corridor. Thus, specimens can be
Fig. 15. Vertical illuminator from Figure 14, light corridors fitted with different colour filters 42.1 and 42.2 (A), double polarizers 43.1 and 43.2 (B), grey filters 44.1 and 44.2 (C) or iris diaphragms 45.1 and 45.2 (D), further explanations in the text.
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Fig. 16. Contrast tube modified for variable phase-dark-field contrast (VPDC), labelling as in Figure 1.
illuminated in variable bright-dark-field contrast or variable phase-bright-field contrast.
Discussion Bright and dark field, phase and interference contrast are used as standard techniques in many fields of light microscopy. Each method is characterized by typical well-known advantages and disadvantages including optical limitations and characteristic artefacts, which have been compiled by several authors (Robertson, 1970; Determann & Lepusch, 1981; Slaghter ¨ & Slaghter, 1992; Gluckstad et al., 2001; Murphy, 2001; C 2014 The Authors C 2014 Royal Microscopical Society, 255, 30–41 Journal of Microscopy
Oldfield, 2001; Lichtscheidl, 2011). Thus, for instance, bright field is not suited for observation of low-density specimens. Diffraction is a typical artefact in bright-field techniques, especially when the aperture of the condenser is reduced by closing the aperture diaphragm. Phase contrast images are affected with haloing and shade-off so that fine details situated in the zones of these artefacts can be masked. Dark-field illumination is characterized by ultrahigh ranges of brightness and contrast and by irradiation caused by reflection and scattering of the incoming light. In interference contrast, pseudo-relief effects can be generated by regional variation of the specimen’s refraction index, and the level of contrast may be lower in interference contrast than in phase contrast. Moreover, the depth of
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focus achievable with interference contrast is lower when compared with some other concurrent techniques so that thick specimens appear like ‘optical sections’. On the other hand, especially unstained phase specimens can be visualized with high accuracy, as interference contrast is not affected with haloing and shade-off. In view of these facts, significant improvements of the resulting image quality can be expected when various illumination techniques are combined with each other and simultaneously carried out. The particular visual information that can be obtained by use of different single techniques can be joined in composite images. Also fine details, which may be lost in one illumination technique carried out on its own, can be contributed by the other complementary technique superimposed. Additional contrast effects can be achieved when the partial images superimposed interfere with each other. Thus, the global visual information can be enhanced in the respective composite images. Until now, interference contrast, phase contrast and other illumination techniques have been carried out successively and compared with each other based on observation of the same specimen. Such comparisons have already been carried out several years and decades ago. Thus, for instance, interference and phase contrast have been compared in examination of bacterial cells (Hitchins et al., 1968), spermatozoa (Cortner et al., 1978) and organs of corti (Kuhn et al., 1971). An extensive comparison of phase contrast and DIC microscopy can be obtained from the web (Murphy et al., 2005). In interference contrast, regions characterized by very shallow optical path slopes such as extended and flat specimens produce insignificant and low contrast so that they often appear at the same intensity level as the background (Murphy et al., 2005). Moreover, in several specimens, the level of contrast achievable with DIC is influenced by the orientation of the specimen with respect to the optical axis of the microscope, whereas phase contrast leads to constant contrast levels being independent of the specimen’s rotation around the optical axis (Murphy et al., 2005). On the other hand, regions that are masked by haloing in phase contrast images can appear in superior clarity when interference contrast is used. Thus, for instance, halo artefacts can surround the periphery of cell membranes so that visual information about intercellular contacts may be lost in phase contrast. Interference contrast, however, can lead to superior clarity in imaging of cellular membranes, but internal details of cells may appear in lower contrast and clarity when compared with phase contrast (Murphy et al., 2005). According to these considerations, both methods can be regarded as complementary techniques each leading to complementary visualization of particular structures. Following this, superimposition of different images of the same specimen illuminated by use of different techniques can lead to superior global visual information.
Although computer-based digital superimpositions of various photomicrographs taken from the same specimen at different illumination can only be carried out with ‘static’ specimens (permanent slides, for instance), the optical superimpositions, which are achievable by our methods, can also be used for visual observation in real time including examination and documentation of motile native specimens showing spontaneous movement and locomotion. Moreover, interference phenomena of the imaging light associated with partial images, which are simultaneously generated and optically superimposed, can produce additional contrast effects so that additional visual information may result. Otherwise, any interference effects are absent in computerbased superimpositions. Primarily, our methods are characterized by a technical and optical value. In particular, thin and low-density biological structures appear in higher contrast than in interference contrast – the grade of contrast can be compared with phase contrast. Fine details and marginal contours appear in higher precision than achievable with phase contrast, because loss of visual information caused by haloing and shade-off can be avoided. Finally, the relief or pseudo-relief effects that are typical for interference contrast remain immanent in our technique. These characteristics are evident in the examples presented in Figures 9 and 10. When particular biological probes can be observed in higher clarity and precision compared with conventional concurrent techniques carried out on their own, this surplus of image quality can also lead to a ‘biological value’. In particular, the several types of ‘contrast tubes’ described should be predestined for development of high-end multimodal microscopes for variable combination of bright- and dark field, phase and interference contrast, because all complementary illumination techniques can be combined with each other in highest variability based on the same technical platform. Acknowledgements The authors thank Mr. Klaus Kemp, United Kingdom, and Mr. Eberhard Raap, Germany, for arranging slides of diatoms, Mr. Dipl. Ing. Manfred L¨ochel for an individual created handmade special adapter ‘Zeiss to Leica’ and Mr. Sebastian Hess, Germany, for supplying achromatic lens systems provided for his ‘magniflash’ device. We thank Mrs. Linda Tennant, United Kingdom, for revising our English manuscript.
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