DE102013110497B4 - Method and device for generating a variable and simultaneous phase contrast image in combination with one of the images dark field image or bright field image or polarization image - Google Patents

Abstract

In a microscope for variable and simultaneous generation of phase contrast and dark/bright field/polarization imaging, the object is illuminated differently in transmitted and/or reflected light and the light beam is divided into two separate light beams after passing through or reflecting off the object. Changes in at least one of the light beams of the partial images in terms of phase position, polarization position, spectral composition and/or intensity can be documented by interference of these light beams with the summation image in the eyepiece. For this purpose, the microscope has an illumination apparatus with a condenser with a condenser diaphragm and at least one light passage for phase contrast and/or dark field, the tube of which has projection lenses (16, 17) which extend the first objective focal plane (OBE1) and the first intermediate image plane (ZBE1) to the second objective focal plane (OBE2) and second intermediate image level (ZBE2) duplicate. Beam splitting means form two partial beam paths, with a phase ring (20.1) for phase contrast being arranged in one and a ring-shaped or centric light stopper (20.2) for central/axial dark field and/or for bright field or polarization illumination being removed from the respective light passage in the other partial beam path. Means are provided for combining the partial beams of rays in the second intermediate image plane (ZBE2), the summation image being viewable by means of eyepieces (8, 9).

Description

The invention relates to a method for generating a microscopic image of an object with simultaneously executed bright, dark field, polarization, phase contrast and / or fluorescence illumination, the illumination intensities of the respectively generated and optically superimposed on bright, dark field, polarization and / or Phase contrast-based sub-images can be continuously varied independently of one another, it also relates to a microscope with illumination apparatus, object stage with object, tube with eyepiece and lens, and optionally an additional reflected-light illuminator for fluorescence or reflected-light bright and dark field, in which the illumination apparatus includes a condenser , which is provided with at least one ring-shaped diaphragm opening located outside the optical axis, optionally with an additional ring-shaped or axial (centric) light passage and an optionally rotatable polarization filter, and in which the tube is designed as a special tube for generating multi-modal illumination, provided with projection lenses for Duplication of rear lens focal plane and first intermediate image plane, further provided with a phase ring that can be removed from the beam path and/or a ring-shaped light stopper, if required also with an axially (centrally) located small-area light stopper and/or an insertable analyzer and optionally provided with a splitter prism, see above that two separate light corridors running parallel to each other are created inside the tube, so that phase contrast, polarization, bright and dark field images, if necessary, are generated simultaneously in different light corridors and superimposed with one another, with the intensities of the superimposed partial images being steplessly and independently adjustable .

The illumination of objects plays a crucial role in microscopy for resolution and image generation. Based on the well-known Köhler illumination apparatus designed to generate a bright field image, dark field illumination was introduced, which offers advantages over the bright field technique in terms of resolution and image generation. This was followed by the development of phase-contrast lighting, in which existing weak path differences in objects with low optical density are contrasted and made visible using a wave-optical process. In the usual optical design, the phase contrast method is optimized for the contrasting of thin biological objects, for example living cells in a single layer arrangement, which cause a phase shift of about a quarter wavelength. Halo fringes and shade-off phenomena are typical artifacts associated with phase contrast. Anisotropic object structures can be selectively contrasted if a polarizer, additional compensators and an analyzer are inserted at the usual points in the beam path. The polarizer is to be inserted below or in the area of the condenser, the analyzer above the lens or in the tube. Additional compensators (usually lambda or quarter lambda plates) should be placed above the polarizer. The polarizer and analyzer are to be arranged in such a way that they can be rotated in relation to one another and can therefore be brought into a cross position if necessary. In the usual technical arrangement, the polarizer is therefore provided as a rotary polarizer. Birefringent (anisotropic / optically active) object structures appear in maximum contrast when the polarizer and analyzer are placed in a crossed position relative to each other. On the other hand, an ideal cross position results in a very dark to black image background, so that accompanying structures that are not birefringent cannot be recognized or can only be recognized with little clarity when the polarization filters are crossed. Autofluorescent object structures or object structures marked with fluorochromes (fluorescent dyes) can be made visible selectively using fluorescence illumination, with additional non-fluorescent details remaining invisible, especially in reflected-light fluorescence, which has established itself as the standard method. It can therefore be advantageous to overlay a fluorescence image or a polarization image with a complementary, differently generated image that shows the non-fluorescent or non-birefringent components of an object in such a way that the clarity of the fluorescent or birefringent structures is not impaired. Only in this way can fluorescent or birefringent structures be observed and photographed in the context of their non-fluorescent accompanying structures with the highest possible contrast and best clarity in one image.

Since each of the types of illumination mentioned has specific advantages and disadvantages, characteristic optical limitations and artefacts, the overall visual information and imaging quality of a microscopic image can be radically improved if several of the methods mentioned are carried out simultaneously and the resulting methods are based on different illumination methods based individual sub-images are optically superimposed. It is advantageous here if the intensities of the individual images involved can be changed steplessly and independently of one another, so that the weighting of the respective partial images involved can be influenced to a large extent.

The respective method-specific information associated with bright and dark field, phase contrast, polarization, fluorescence and incident light illumination can thus be combined in an observable summation image, so that the overall visual information in the observed image is increased. In addition, typical artifacts of the individual processes involved (e.g. halo of the phase contrast, blooming in the dark field, diffraction lines in the bright field) can be weakened. The respective method-specific information differs in the types of illumination combined with one another according to the invention. Brightfield gives information about the structure, coloring and density of absorption objects; these have a relatively high layer thickness and optical density. Dark field provides linear contrast images of fine phase boundary lines and edge contours; In addition, small particles in the limit range of microscopic resolution can be visible due to scattering phenomena (Tyndal effect), even if they can no longer be distinguished in other methods due to their small size. Colored structures can also be observed in optically dense objects in their respective natural coloring, even if they only appear as dark silhouettes in other transmitted light methods due to their high density. Phase contrast displays very thin objects of low optical density in superior contrast, where halo artifacts in particular can limit the visual information. Optically active (anisotropic) structures can be selectively highlighted by means of polarization, and fluorescent details can be selectively displayed by means of fluorescence. When the described optical superimposition methods are used according to the invention, this wide range of method-specific information can be combined in an observable image without mutual interference occurring in the respective superimposed partial images.

In DE 10 2011 002 030 A1 , DE 10 2011 054 106 A1 . and DE 10 2012 005 911 A1 Methods have already been described which serve the aforementioned task of performing light and dark field, phase contrast and dark field, phase contrast and bright field or all three of these lighting techniques simultaneously and superimposing the resulting partial images in a variable manner. The methods presented in these documents are each based on specially designed condensers and/or lenses, while the tube in all methods corresponds to the usual technical standard. If phase contrast is involved in the respective method, special phase contrast lenses must be used in the methods presented in the aforementioned inventions, which are equipped with a phase plate and a phase ring in their rear focal plane. However, a phase ring in the lens can reduce the quality if the phase contrast lens in question is used for other lighting methods.

As further prior art, reference is made to the document DD 53 890 A1 pointed out, which describes an interphaco tube with two beam paths.

Incidentally, from the specialist article PIPER, Timm; PIPER, Jörg: "Universal Variable Brightfield-Darkfield Contrast: A Variant Technique for Improved Imaging of Problematic Specimens in Light Microscopy. In: Microsc. Microanal., 19, 2013, pp. 1092-1105, a variable bright field/dark field contrast is known, which is generated on a beam path axis.

From the above-mentioned inventions, the further task can be derived, bright and dark field, phase contrast and dark field, phase contrast and bright field or all three aforementioned types of illumination, if necessary, supplemented by polarization and / or fluorescence at the same time and optically to generate, among other things, a variable light-dark field - Contrast to superimpose a variable phase dark field contrast or a variable phase bright field contrast or in a corresponding manner also a variable polarization bright field contrast, a variable polarization dark field contrast or a variable polarization phase contrast, further all of these variable illumination techniques can be combined with fluorescence or incident light-bright-darkfield without the need for phase-contrast lenses.

Consequently, in accordance with this task, the possibility should be developed of creating modular microscopes which allow all of the above-mentioned illumination combinations to be implemented with full utilization of their respective application-related advantages on the basis of a uniform technical platform with one and the same set of lenses. The fact that phase rings in the lenses can be dispensed with in this way is a major technical advantage, since the phase ring in the lens represents an optical foreign body in any other imaging, i.e. also when generating brightfield, darkfield, phase contrast, polarization or fluorescence or other incident light methods which can have a detrimental effect on quality.

Consequently, the methods presented here offer the possibility of variably superimposing a “pure” phase contrast image with a “pure” phase ring-free, complementary illuminated image gladly. Even in the event that the multimodal illumination variants generated variably in the transmitted light are to be combined with reflected-light fluorescence, the omission of a phase ring in the lens has a particularly substantial quality-improving effect, since phase-contrast lenses which are provided with a phase ring lead to inferior results, especially in reflected light fluorescence; here the phase ring impedes the free passage of the illuminating excitation light as a result of reflection, partial absorption and scattering.

In order to solve the tasks outlined and to achieve the desired improvements, the tube is transformed into a special contrast-generating component, whereby two fundamentally different variants of special tubes can be considered: A double-barreled tube, in which two parallel beam paths are generated, which in two separate Light corridors run and a technically simpler designed single-section tube, which contains only one light corridor. In both embodiment variants, a phase-contrast image can be generated in a light corridor; without the need for phase contrast lenses, since phase contrast is generated in all versions in the special tube used.

In the respective special tube, partial images based on bright, dark field, polarization or phase contrast are generated, which interfere with one another and result in an observable summation image. If necessary, a fluorescence image can also be provided if a suitable incident light illuminator is provided below the special tube. The special tube is optically designed in such a way that the rear objective focal plane and the usually downstream intermediate image are each duplicated by projection lens systems, so that a second objective focal plane and a second intermediate image plane are created within the tube. In the type of the double-barreled special tube with two light corridors, the rays running to the tube are split inside the tube by a first splitter prism into two parallel beam paths, which enable different types of lighting to be generated simultaneously. The optically differently contrasted sub-images generated in the parallel beam paths are then recombined with one another by means of a second splitter prism that is connected downstream and positioned in the opposite direction, so that a summation image is obtained, which is produced by superimposing at least two different types of illumination (bright field and dark field or polarization, phase contrast and/or bright or dark field or polarization).

The phase-contrast-based partial image is generated by means of a light mask arranged in the area of the condenser, which contains a ring-shaped light passage, so that the object is irradiated by a light cone, which enters the entrance cross section of the objective, with the illuminating light entering the special tube after passing through the objective is phase-delayed in a known manner in relation to the imaging beams by a phase ring located there on a phase plate. If the phase ring is replaced by a suitably dimensioned ring-shaped light stopper, a central dark field image can be contributed instead of a phase contrast image by means of a phase plate modified in this way. If the phase ring and light stopper are removed from the beam path, a bright field image is generated. If a rotatable polarization filter is placed below or in the area of the condenser, if necessary additional compensators are introduced and an analyzer is also provided above the objective or in a light corridor of the special tube, a polarization image can be added as an additional component.

If the light mask in the area of the condenser is provided with an axially (centrically) arranged, small-area perforation to allow light to pass through, a narrow axial illumination beam is produced instead of the illumination light that shines in in a ring shape, which radiates through the object perpendicularly. If an optically congruent, disk-shaped light stopper is placed in a centric-axial position in the duplicated second rear objective focal plane inside the special tube, an axial dark-field image can be generated as a partial image.

If the light mask of the condenser is provided with a ring-shaped light passage, which is dimensioned so large that it is projected into the beam path outside the objective cross-section, a dark-field image is generated which is based on concentric-peripheral illuminating light. If the projection size of this ring of light is reduced slightly so that its inner zone is projected within the objective cross-section while its outer zone remains outside the objective cross-section, a light-dark field contrast based on concentric-peripheral illumination can be generated. If the projection size of this ring of light is further reduced, the entire ring of light is finally projected within the cross section of the lens, resulting in concentric-peripheral brightfield illumination.

Finally, the light mask can also be provided with two or more concentric light passages in the area of the condenser, either two or more annular passages of different diameters or one or more annular light passages and one centre-axial passage. Optically congruent to this, the corresponding phase plate can be arranged within the special tube, both a phase ring and one or more ring-shaped light stoppers or an axially, centrally arranged disc-shaped light stopper. With such an arrangement, phase contrast and central/axial dark field can be generated simultaneously on one beam path axis. The light passage, which is optically congruent with the phase ring, generates a phase contrast image, the additional light passage, which is optically congruent with the light stopper, generates a central/axial dark field image at the same time, which is optically overlaid with the phase contrast image to form a summation image. If the light mask is provided with a light passage that projects outside of the phase ring and light stopper, bright field can be added as an additional lighting component. If one of the ring-shaped light passages is dimensioned so large that it is projected outside the optically effective lens cross-section, dark field lighting based on concentric-peripheral light can be added.

As a technical modification, the light mask can be provided with two or more concentric light passages in the area of the condenser as described above, either two or more ring-shaped passages of different diameters or one or more ring-shaped light passages and a centric-axial passage, with the corresponding phase plate inside the special tube contains only one phase ring which is optically congruent with one of the annular light passages of the light mask. With such an arrangement, phase contrast and central/axial and/or concentric-peripheral bright field can be generated simultaneously on one beam path axis. If the aforementioned phase ring is replaced by a ring-shaped or centrally arranged disc-shaped light stopper, which behaves optically congruently with one of the two light passages, while the other light passage projects outside of the respective light stopper, both a central/axial dark field image and a light path axis are created axial or concentric-peripheral brightfield image. Of course, light masks with three separate light passages can also be created and combined with modified phase plates, so that brightfield, darkfield and phase contrast can be generated on one light axis or in a light corridor and superimposed on one another. In the latter case, the phase plate must of course contain a suitably dimensioned light stopper in addition to a phase ring.

In the latter arrangements, the light passages, which are associated with the phase contrast and the axial/central dark field or the axial (central) or concentric-peripheral bright field, can be provided with different color filters. In this case, for example, the phase contrast image appears in one color and the dark or bright field image generated at the same time in the other color. Furthermore, the light passages can be provided with adjustable multiple screens or polarization filter systems, in accordance with the relevant design proposals in DE 10 2011 054 106 A1 and DE 10 2012 005 911 A1 , so that the brightnesses or intensities of the respective light components let through can be regulated independently of one another and consequently the relative weightings of the phase contrast and dark field images or bright field or polarization images can be varied independently of one another. The light passages arranged on the light mask can also be provided with adjustable or exchangeable gray filters in order to vary the brightness of the associated light components and the resulting weighting of the associated partial images.

If a double-barreled tube with two parallel beam paths is used, the partial images assigned to the parallel beam paths can also be filtered at the tube level in the area of the two parallelized partial beam paths in different, visually easily distinguishable colors, for example red and blue, or two complementary colors. In the second intermediate image plane of the special tube, the partial images filtered in different colors are superimposed to form a summation image, as described above. This is then viewed in a known manner with the eyepiece at the desired magnification.

When using a double-barreled tube, polarization double filters are assigned to the two parallel beam paths in the tube for stepless and independent brightness regulation of the respective partial images polarization-optical means are infinitely variable in terms of their brightness. In a different design, the two imaging beam corridors running parallel to one another can be equipped with separate and independently adjustable (iris) diaphragms or adjustable or exchangeable gray filter sets be designed so that the light flow of the respective light passages can be changed, which also allows individual regulation of the brightness of the relevant beam components and their associated partial images.

To generate phase contrast or central darkfield, the annular light passage of the condenser corresponds to the phase ring or annular light stopper inside the tube, so that a phase contrast image or a central darkfield image can be generated simultaneously. A brightfield image can be contributed by removing the phase ring or light stopper from the light path. A polarization image can be contributed if a polarizer together with an analyzer and optional compensators are also provided in the beam path. The respective partial images involved in the summation image, which are assigned to the two partial beam paths guided parallel to one another in the double-barreled tube, can be infinitely adjusted in intensity, i.e. from zero to one hundred percent, by means of integrated, mutually adjustable polarizers or similarly acting (iris) diaphragms , so that without interrupting the observation you can switch between pure phase contrast, bright field or dark field and any possible overlays of any dominance of the partial images. This type of image formation makes it possible to make the details that are present in "problem objects" with changing optical density and layer thickness more clearly visible than is possible with the sole use of phase contrast, bright field, dark field or polarization or fluorescence.

In order to generate correct and maximally contrasted phase contrast, the condenser light annulus generating the phase contrast image must be dimensioned in such a way that it is optically congruent to the phase annulus in the special tube and at the same time all parts of the illumination light are covered by the phase annulus. If the phase ring is replaced by a ring-shaped light stopper, a maximally contrasted central dark field illumination results if all illuminating light components are covered by the light stopper. If a small proportion of this illumination light misses the light stopper or phase ring, bright field illumination is also added. This can be achieved in a technically advantageous manner if the condenser is designed as a zoom condenser, so that its aperture and consequently also the projection size of the light rings used can be continuously changed. Thus, leaving the phase ring or light stopper, a bright field image can be added as a third component if the projection size of the light ring is changed in small steps using the zoom condenser in such a way that a narrow zone of the light ring is projected outside of the light stopper or phase ring. If the condenser is not designed as a zoom system, the projection size of the light ring can also be varied by moderately adjusting the height of the condenser. Alternatively, a small illuminating light component can be guided past the respective light modulator (phase ring or light stopper) and onto it by minimal decentering of the light ring generate an additional brightfield image in this way.

If the ring-shaped light passage in the area of the condenser and the corresponding ring-shaped light stopper in the tube are replaced by a centric (axial) light passage and an optically congruent corresponding disc-shaped light stopper in an axial (centric) position, axial dark field can be contributed instead of central dark field. Axial and/or central dark field can be generated with phase contrast on the same beam path axis if the phase plate in the respective special tube is equipped with a phase ring and one or more light stoppers and the condenser light mask contains optically congruent light passages. Brightfield can be added if the light mask contains a light passage that does not touch either a phase ring or a light stopper at the phase plate level, or if, when using a zoom condenser, the projection size of the light passages is varied slightly as described above, so that a small part of the Illumination light runs past the light stopper or phase ring.

The two parallel beam paths assigned to different types of illumination can be filtered with different colors in the double-barreled special tube, with simple color filters or narrow-band monochromatic (monochrome) filters being able to be used for color filtering. If the light mask contains more than one light passage, these light passages can be lined with different color filters.

In order to achieve eccentric multimodal illumination from defined spatial directions, ring-shaped light passages in the condenser can be partially covered in the arrangements described above, so that more or less narrow light segments contribute to object illumination. With this optical arrangement, too, the weighting of the respective partial images and thus the character of the resulting superimposed image can be varied with the aid of the double polarizers or (iris) diaphragms on the tube side.

Instead of the above-described arc-shaped condenser light rings, which can be partially covered for oblique illumination, crescent-shaped delimited light columns for generating the illuminating light in the condenser can also be provided for oblique illumination, with width, length and position (solid angle) being steplessly variable. The light segment is to be precisely adapted to the geometry of the phase ring or light stopper. If this ring of light is arranged such that it can be rotated while maintaining congruence with the corresponding phase ring, the direction of light incidence in the resulting image can be further adapted to the positioning or orientation of the object in space.

Advantageously, the condenser has a rotatable turret disk with a plurality of light passages of different sizes, shapes and arrangements, which are arranged such that they can be rotated or displaced for selectable adjustment, with the aperture openings being selected in such a way that they are congruent with the phase ring or Light stoppers behave in the special tube. This provision takes account of the fact that the projection size of a light ring on the plane of a phase plate is influenced by the lens magnification.

For an advantageous further development, the condenser is designed to be adjustable in height to adapt the exact projection size of the respective light passages on the phase ring or light stopper. This makes it easier to coordinate the diameters of the lens cross section, phase ring and projection images of the condenser light passages, which are necessary during adjustment. An alternative solution for the adjustment is to design the condenser as a zoom system with a variable back focus and aperture of the lens system. With this procedure, the projection of the light passages in the beam path and the course of the illuminating rays determined thereby can be further adapted to the object conditions (position, size, layer thickness) by adjusting the height of the condenser and/or changing the condenser focal length or aperture . With the help of an adjustment eyepiece, the respective adjustment conditions can be checked visually in all the embodiment variants described above.

Finally, all of the above, on variable superimposition of bright, dark field. Processes based on phase contrast and polarization can be combined with reflected-light fluorescence if, preferably below the special tube, a reflected-light illuminator with a dichroic splitter mirror and fluorescence filter cubes according to technical standards is provided.

In addition to the double-barreled special tube described above, the types of illumination mentioned can also be generated in a partial beam path and superimposed on one another. For this purpose, phase ring and light stopper are to be placed on a modified phase plate within a one-way special tube with only one light corridor in the duplicated second rear objective focal plane. The associated light mask of the condenser has two light passages, which are optically congruent with the phase ring and light stopper in the tube, so that phase contrast and central/axial dark field arise in a beam path axis within the special tube. The weighting of the superimposed dark-field and phase-contrast sub-images can be changed in this arrangement if the light flow in the respective associated light passages is varied using the optical aids described above. Interchangeable ring-shaped gray filter sets, ring-shaped polarization filter systems and iris diaphragms can be considered as regulators. Of course, the light flow in the light passage placed too far can also be changed and, if necessary, weakened by means of the aperture diaphragm of the condenser. Bright field can be contributed if the condenser light mask contains a light aperture that is not projected into a phase ring or light stop, such that the illuminating light associated with that aperture bypasses the phase ring and light stop. In order to add concentric-peripheral dark field illumination, one of the ring-shaped light apertures must be designed with such a large diameter that it is projected outside the lens cross-section, so that the illuminating light bundles assigned to this light aperture pass the lens at an oblique angle.

If required, all of the design variants and special tubes described can also be combined with reflected-light illuminators, so that all the multimodal illumination techniques mentioned, which are based on the simultaneous execution and variable superimposition of various individual methods, can be combined with reflected-light methods (e.g. reflected-light fluorescence, reflected-light brightfield, reflected-light dark field) can be combined. The reflected-light illuminator is expediently placed above the objective, but below the respective special tube according to the invention. Under this condition, when performing reflected-light fluorescence, the excitation light can be guided to the object by bypassing the phase plate provided on the tube side by means of the dichroic splitter mirror in the reflected-light illuminator through the brightfield objective used in each case, without causing reflections, scattering and partial absorption of Excitation light comes through a phase ring. The fluorescence image is created as an additional tive partial image in the respective special tube, whereby it is generated in both light corridors in the case of a double-barreled special tube. The further sub-images generated with complementary methods can be superimposed on this fluorescence image with variable weighting. Since the brightness of the sub-images not based on fluorescence can be continuously adjusted, the intensities of all superimposed sub-images can be optimally adapted to the respective object conditions, so that impairments of the fluorescence image by the contributed, usually significantly brighter other sub-images can be largely avoided.

In an analogous manner, the multimodal techniques generated in transmitted light can in principle also be combined with incident-light-based bright or dark field. In this case, instead of a fluorescence reflected-light illuminator, a reflected-light illuminator is to be used, as is used according to the usual technical standard in material microscopy, for example for examining metal surfaces or semiconductor elements. If reflected light dark field is to be contributed, a special so-called HD lens must of course be used as the lens (HD = light/dark field). This consists of inner lenses, which are used for imaging and also guide the illumination light to the object that is assigned to the reflected light bright field illumination. Furthermore, such a lens contains a separate and circularly arranged outer light corridor, which directs an additional light component to the object, which generates concentric reflected light dark field. Especially semi-transparent and complex structured objects, which consist of transparent and non-transparent components, should be optimally examineable with the described techniques with regard to all existing structures.

If necessary, all of the design variants and special tubes described can be combined with reflected-light illuminators, so that all of the multimodal lighting techniques mentioned, which are based on the simultaneous execution and variable superimposition of various individual methods, can be combined with reflected-light methods (e.g. reflected-light fluorescence, reflected-light bright field, reflected-light dark field ) can be combined. The reflected-light illuminator is expediently placed above the objective, but below the respective special tube according to the invention. Under this condition, when performing reflected-light fluorescence, the excitation light can be guided to the object by bypassing the phase plate provided on the tube side by means of the dichroic splitter mirror in the reflected-light illuminator through the brightfield objective used in each case, without causing reflections, scattering and partial absorption of Excitation light comes through a phase ring. The fluorescence image is created as an additional partial image in the respective special tube, whereby it is generated in both light corridors in the case of a double-barreled special tube. The other sub-images generated with complementary methods can be superimposed on this fluorescence image with variable weighting. Since the brightness of the sub-images not based on fluorescence can be continuously adjusted, the intensities of all superimposed sub-images can be optimally adapted to the respective object conditions, so that impairments of the fluorescence image by the contributed, usually significantly brighter other sub-images can be largely avoided. In an analogous manner, the multimodal techniques generated in transmitted light can in principle also be combined with incident-light-based bright or dark field. In this case, instead of a fluorescence reflected-light illuminator, a reflected-light illuminator is to be used, as is used according to the usual technical standard in material microscopy, for example for examining metal surfaces or semiconductor elements. If reflected light dark field is to be contributed, a special so-called HD lens must of course be used as the lens (HD = light/dark field). This consists of inner lenses, which are used for imaging and also guide the illumination light to the object that is assigned to the reflected light bright field illumination. Furthermore, such a lens contains a separate and circularly arranged outer light corridor, which directs an additional light component to the object, which generates a concentric reflected light dark field. Especially semi-transparent and complex structured objects, which consist of transparent and non-transparent components, should be able to be optimally examined with the described techniques with regard to all existing structures.

All the methods presented here can also be carried out with any special lenses (e.g. mirror lenses, lenses with integrated iris diaphragm, with a long working distance, for heating and cooling tables), since the respective special tubes and condensers with corresponding light masks do not place any special technical requirements on the lenses.

• 1a*: Mikroskop mit doppelläufigem, zwei Parallelstrahlengänge (Lichtkorridore) beinhaltendem Spezialtubus und Kondensor zur gleichzeitigen Erzeugung von zentralem Dunkelfeld und Hellfeld;
• 1b*: Mikroskop mit doppelläufigem, zwei Parallelstrahlengänge (Lichtkorridore) beinhaltendem Spezialtubus und Kondensor zur gleichzeitigen Erzeugung von Phasenkontrast und Hellfeld;
• 1c*: Mikroskop mit doppelläufigem, zwei Parallelstrahlengänge (Lichtkorridore) beinhaltendem Spezialtubus und Kondensor zur gleichzeitigen Erzeugung von zentralem Dunkelfeld und Phasenkontrast;
• 1d*: Mikroskop mit doppelläufigem, zwei Parallelstrahlengänge (Lichtkorridore) beinhaltendem Spezialtubus und Kondensor zur gleichzeitigen Erzeugung von zentralem Dunkelfeld und Polarisation;
• 1e*: Mikroskop mit doppelläufigem, zwei Parallelstrahlengänge (Lichtkorridore) beinhaltendem Spezialtubus und Kondensor zur gleichzeitigen Erzeugung von Phasenkontrast und Polarisation;
• 2a*: Vereinfachte Ausführungsvariante mit speziellem, als Zoom-System ausgelegtem höhenverstellbaren Kondensor mit verschiebbaren ringförmigem Lichtdurchlass und technisch vereinfachtem einzügigem Tubus zur Erzeugung von zentralem Dunkelfeld und Beimischung von Hellfeld ohne stufenlose und unabhängige Helligkeitsregulierung mittels Strahlenaufspaltung;
• 2b*: Vereinfachte Ausführungsvariante mit speziell ausgestattetem, als Zoom-System ausgelegtem höhenverstellbaren Kondensor mit verschiebbaren ringförmigem Lichtdurchlass und technisch vereinfachtem einzügigem Tubus für eine Erzeugung von Phasenkontrast und Beimischung von Hellfeld ohne stufenlose und unabhängige Helligkeitsregulierung mittels Strahlenaufspaltung;
• 2c*: Vereinfachte Ausführungsvariante mit speziell ausgestattetem, als Zoom-System ausgelegtem höhenverstellbaren Kondensor mit ringförmigem Lichtdurchlass und technisch vereinfachtem einzügigem Tubus für eine Erzeugung von zentralem Dunkelfeld und Beimischung von Polarisation ohne stufenlose und unabhängige; Helligkeitsregulierung mittels Strahlenaufspaltung;
• 2d*: Vereinfachte Ausführungsvariante mit speziell ausgestattetem, als Zoom-System ausgelegtem höhenverstellbaren Kondensor mit ringförmigem Lichtdurchlass und technisch vereinfachtem einzügigem Tubus für eine Erzeugung von Phasenkontrast und Beimischung von Polarisation ohne stufenlose und unabhängige Helligkeitsregulierung auf der Basis einer Strahlenaufspaltung;
• 3a: Verschiedene Ausführungsbeispiele von Lichtmasken und Phasenplatten, vorgesehen zur variablen Überlagerung von Hell-Dunkelfeld mittels doppelläufigem Spezialtubus mit zwei parallel verlaufenden Lichtkorridoren K1 und K2;
• 3b: Verschiedene Ausführungsbeispiele von Lichtmasken und Phasenplatten, vorgesehen zur variablen Überlagerung von Phasenkontrast und Dunkelfeld mittels doppelläufigem Spezialtubus mit zwei parallel verlaufenden Lichtkorridoren K1 und K2;
• 3c: Verschiedene Ausführungsbeispiele von Lichtmasken und Phasenplatten, vorgesehen zur variablen Überlagerung von Hellfeld, Dunkelfeld und Phasenkontrast mittels technisch vereinfachtem einzügigem Spezialtubus mit nur einem Lichtkorridor.

*: Die 1 und 2 basieren auf einer Konstruktionsskizze eines Interphaco-Tubus Carl Zeiss Jena VEB aus Werksdruckschrift 30-305-1, DDR, 1965 (http://www.aesfga.com/interphako/PDF/30-305-1_lnterphako_Ampli.pdf)The essence of the invention is based on the 1a,b,c , 2a, b and 3a,b,c Schematically illustrated embodiments explained in more detail, showing:

• 1a* : Microscope with double-barreled special tube containing two parallel beam paths (light corridors) and condenser for simultaneous generation of central dark field and bright field;
• 1b* : Microscope with double-barreled special tube containing two parallel beam paths (light corridors) and condenser for simultaneous generation of phase contrast and bright field;
• 1c* : Microscope with double-barreled special tube containing two parallel beam paths (light corridors) and condenser for simultaneous generation of central dark field and phase contrast;
• 1d* : Microscope with double-barreled special tube containing two parallel beam paths (light corridors) and condenser for simultaneous generation of central dark field and polarization;
• 1e* : Microscope with double-barreled special tube containing two parallel beam paths (light corridors) and condenser for simultaneous generation of phase contrast and polarization;
• 2a* : Simplified version with a special, height-adjustable condenser designed as a zoom system with a movable ring-shaped light passage and a technically simplified single-section tube for generating a central dark field and adding bright field without stepless and independent brightness control by means of beam splitting;
• 2 B* : Simplified version with a specially equipped, height-adjustable condenser designed as a zoom system with a movable ring-shaped light passage and a technically simplified single-section tube for generating phase contrast and adding brightfield without stepless and independent brightness control by means of beam splitting;
• 2c* : Simplified version with a specially equipped, height-adjustable condenser designed as a zoom system with ring-shaped light passage and technically simplified single-section tube for generating a central dark field and adding polarization without stepless and independent; Brightness control using beam splitting;
• 2d* : Simplified version with specially equipped, height-adjustable condenser designed as a zoom system with ring-shaped light passage and technically simplified single-tube tube for generating phase contrast and adding polarization without stepless and independent brightness control based on beam splitting;
• 3a : Different embodiments of light masks and phase plates, provided for the variable superimposition of light and dark field by means of a double-barreled special tube with two parallel light corridors K1 and K2;
• 3b : Different embodiments of light masks and phase plates, provided for the variable superimposition of phase contrast and dark field by means of a double-barreled special tube with two parallel light corridors K1 and K2;
• 3c : Various embodiments of light masks and phase plates, intended for the variable superimposition of brightfield, darkfield and phase contrast using a technically simplified, single-section special tube with only one light corridor.

*: The 1 and 2 are based on a design sketch of an Interphaco tube Carl Zeiss Jena VEB from factory publication 30-305-1, GDR, 1965 (http://www.aesfga.com/interphako/PDF/30-305-1_lnterphako_Ampli.pdf)

The 1a-e show schematized different possible beam paths in a microscope, so show the beam path for variable bright/dark field illumination 1a , for phase contrast brightfield illumination 1b , for phase contrast darkfield illumination 1c ,for dark field polarization illumination , 1d and for phase contrast polarization illumination 1e , based on a division of the beam paths within the special tube and a stepless and independent controllability of the brightness of the partial images involved. A light beam is guided from the light source 1 via a collector lens 1a to the light mask 10 which is arranged in the area of the condenser 2 and which contains an annular light passage 10.1 which is necessary for generating phase contrast or a central dark field. The ring-shaped light passage is projected optically congruently into the plane of the modified phase plate 20 via a converging lens 11 .

In 1a the modified phase plate 20, which is located in the turret disc 19, carries an annular light stopper 20.2, so that a central dark-field partial image is generated by means of the light stopper 20.2. There is no light modulator in the second parallel beam path or second light corridor, ie neither a phase ring nor a light stopper. Consequently, a bright field image is generated simultaneously in this light corridor. The special tube is formed by all the components shown above the objective 4 or the tube lens 5 . Different projection lens groups 16 and 17 work together within the special tube, see above that the first rear lens focal plane OBE1 is duplicated inside the tube at the marked location and forms the second rear lens focal plane OBE2 there. The first intermediate image that usually occurs, which is projected into the first intermediate image plane ZBE1, is also duplicated inside the tube and projected as a second intermediate image into the second intermediate image plane ZBE2 at the location shown. The rear lens focal plane 2 OBE2 is preceded by a first splitter prism 18.1, which causes the beam path to be split into two parallelized partial beam paths. The light stopper 20.2 arranged in a rotatable turret disk 19 on a phase plate 20 is placed in the upper partial beam path shown in the position of the second rear objective focal plane OBE2. At the corresponding point there is no light modulator in the lower partial beam path, so that there is also bright field.

The intensities of the two partial images generated in this way (bright field and central dark field) can be continuously and independently regulated by the rotatable double polarizers 23, 24, with an adjustment range from 0 to 100 percent brightness being possible. Both partial beam paths are combined by the second splitter prism 18.2, which is arranged in the opposite direction to the prism 18.1, to form a summation image which lies as the second intermediate image in the second intermediate image plane ZBE2. The prism 6 arranged below releases the beam path to the observation eyepieces 8, or directs it to a photo eyepiece 9, which is optionally connected to a camera 9a. The respective eyepieces 8 enlarge the second intermediate image of the second intermediate image plane ZBE2 assigned to them in a conventional manner. The correct adjustment of the ring diaphragm 10.1 on the light mask 10 and the ring-shaped light stopper 20.2 on the modified phase plate 20 in the turret disc 19 can be checked by a Bertrand lens 7 that can be swiveled in and out, alternatively of course by a conventional focusing magnifier (not shown). , which is inserted into the eyepiece socket instead of one of the observation eyepieces. The (mirror) prism 25 is used for a technically and ergonomically expedient change in the course of the vertically incident beams into an almost horizontal alignment, so that the subsequent optical components can be arranged horizontally within the special tube. This limits the total height of the optical structures; this promotes the mechanical stability of the overall system.

If - as in 1b shown - instead of the ring-shaped light stopper, a phase ring of suitable size is introduced into the beam path, instead of a central dark field image, a phase contrast partial image results, which is overlaid with the bright field image additionally generated in the parallel beam path.

In addition, the double-barreled special tube can also be equipped with two rotatable turret discs, so that each of the two light corridors is assigned a separate turret disc, one of which contains the ring-shaped light stopper for central darkfield and the other the phase ring for phase contrast according to 1c . As shown in the figure, the ring-shaped light stopper used in each case and the corresponding phase ring should be dimensioned identically in both turret discs and be optically congruent with the associated ring-shaped light passage in the condenser. In this variant, too, the projection size of the light ring can be precisely adapted to the size of the two corresponding light modulators (light stopper and phase ring) if the condenser is designed as a zoom system. Brightfield can be added as a third lighting component and its intensity can be finely dosed if a small proportion of the lighting light is guided past the light modulators.

If in accordance with the regulations 1a and 1b an analyzer 22 is placed in the respective brightfield light corridor and a rotatable polarizer 12 and optional compensators, for example a quarter-lambda compensator 13 or a lambda compensator 14 are provided below or in the area of the condenser, a polarization image - such as 1d shows - with a central dark field image or - as in 1e shown - can be variably superimposed with a phase contrast image.

Different color filters (not shown) can be integrated into the beam paths in front of or behind the double polarizers 23, 24 for colored contrasting of the two light corridors assigned partial images. Both simple color filters and monochromatic band-pass or narrow-band filters or adjustable gradient filters or monochromators, which enable smooth color transitions, come into consideration.

Simplified variants of a microscope with a single-train special tube for contrast generation are in the 2a-d shown. The first beam splitting prism 18.1 is omitted, so that only one beam path remains in the tube, in which the turret disc 19 is inserted as described above. The two double polarizers 23, 24 are also omitted. The turret disk contains either an annular light stopper 20.2 according to 2a , which serves to generate a central dark field, or a phase ring according to 20.1 2 B to generate phase contrast. The remaining deflection prism 26 (in position from prism 18.2 1a-c ) is used only to direct the single beam path to the prism 6, which is used to forward it to the observation eyepieces 8 or photo eyepiece 9. This deflection prism causes a parallel displacement of the incoming rays by about the distance of its length downwards, so that observation and photo eyepieces can be arranged lower in an ergonomically advantageous manner and the height of the overall structure can be reduced by this distance. The rest of the arrangement corresponds to the 1a or b. In this simplified embodiment, a central dark field or phase contrast image is consequently realized by optically congruent adjustment of the condenser light ring 10.1 on the light mask 10 and the light stopper 20.2 or phase ring 20.1 in the turret disk 19. Brightfield is added when a small proportion of the illuminating light passes the respective light modulator (phase ring or light stopper); as described above, this can be achieved by means of a zoom condenser, by adjusting the height of the condenser or by moderately shifting (misadjusting) the light mask in the area of the condenser. Pure bright field can be generated if the light mask 10 and/or the phase ring 20.1 or light stopper 20.2 are removed. Polarization can be contributed if a rotatable polarizer 12 and optional compensators, e.g. a quarter lambda compensator 13 or a lambda compensator 14 are provided below or in the area of the condenser and an analyzer 22 is placed above the objective, for example inside the tube dem Tube turret 19 upstream or downstream, as shown. When equipping the tube turret with a light stopper 20.2 can - how 2c shows - polarization can be combined with dark field.

Will as in 2d shown, the light stopper replaced by a phase ring 20.1, a simultaneous execution of polarization and phase contrast is possible. The proportion of the polarization image in the respective summation image can be varied in the same way as described above for adding a bright field image (zoom condenser, height adjustment of the condenser, maladjustment of the light mask).

The in the 2a-d The simplified versions shown with a single-section special tube for contrast generation correspond in principle to the type of so-called "contrast tube" which was developed by Carl Zeiss Jena for the Microval and Jenaval series microscopes of the time and for the selective generation of pure phase contrast or pure central contrast Dark field was provided. In the case of this one-piece special tube, the developments specific to the invention therefore relate to devices which serve the simultaneous generation and variable superimposition of phase contrast and dark field or the simultaneous generation and variable superimposition of light field, dark field and phase contrast and the admixture of polarized light as required; they also relate to the development of specially designed light masks and phase plates, which contain several concentrically arranged light passages or light modulators, as well as to various technical devices for independently regulating the light flow in the multiple light passages used in each case.

With these 2a until 2d In the embodiment variants shown, a variable superimposition of central dark field or phase contrast with light field or polarization can be achieved by changing the degree of overlap of the light ring and light modulator (light stopper or phase ring). If the light ring 10.1 on the light mask 10 and the phase ring 20.1 or the ring-shaped light stopper 20.2 in the turret slide (19) behave completely congruently, so that all incident illumination light is covered by the respective light modulator, the result is a pure phase-contrast image or a pure central dark field without admixture by Hellfeld. As soon as small parts of the light ring run outside the light modulator, those parts of the illuminating light that are guided past the light modulator can superimpose a bright field image. The larger the transmission area outside the light modulator, the more brightfield dominates the superimposed image. As mentioned above, the light component required for brightfield can be generated in a simple manner if the light ring is slightly misaligned with respect to the light modulator. Alternatively, the inner and/or outer diameter of the light ring can deviate slightly from the corresponding diameters of the light modulator, so that a narrow marginal light fringe can pass through the light modulator in the area of its inner or outer zone. If the outer area of the light ring protrudes slightly beyond the light modulator, the resulting width of the brightfield or polarization-generating light fringe can also be regulated by means of an iris diaphragm in the condenser, which is placed as close as possible in front of or behind the light mask (not shown). If the light mask is in the immediate vicinity of the condenser's aperture diaphragm, the aperture diaphragm can of course be used for this regulation. Advantageously, the projection size of the centered light ring can also be used to generate an additional bright field or polarization image and to control its intensity be changed if the condenser is designed as a zoom condenser as described above.

As mentioned above, central/axial dark field can also be added to these technically simpler design variants if the light mask is provided with a ring-shaped or centric light passage, which turns into a corresponding ring-shaped or centric light stopper that is close to or in one of the rear objective focal planes OBE1 or OBE2 or in the center of the phase plate behaves optically congruently. If the light mask has several light passages for the simultaneous generation of axial/central dark field and phase contrast, these light passages can also be filtered in different colors as described above and their brightness and weighting can be regulated independently of one another using adjustable double diaphragms, polarization filters or adjustable gray filters. Of course, in such an embodiment with more than one light passage, the modified phase plate must contain optically congruent elements (phase ring and light stopper). In this case, one ring-shaped aperture produces a phase-contrast image, while the other aperture produces a central or axial dark-field image. If the luminous flux of these two light passages is varied separately and independently of one another by means of suitable devices, for example by means of adjustable polarization double filters, double diaphragm systems or interchangeable gray filter sets, the possibility opens up of the two phase and dark field images generated on a beam path axis being independent of one another with regard to their intensity and to change weighting without splitting into two parallel beam paths according to the 1 is required. Here, too, brightfield can be added as a third illumination component if small portions of the illumination light pass the light modulators.

Of course, instead of a light mask with two light passages, polarization double filter systems can also be provided, which consist of two components: two ring-shaped polarization filters arranged parallel to one another, which can be adjusted in opposite directions and are optically congruent with the phase ring, and two separately arranged polarization filters, which Optically cover the entire surface of the light stopper used. The two pairs of polarizers can each be adjusted independently of one another. Consequently, the two illuminating light components, which are assigned to the phase contrast and dark field image, can also be regulated steplessly and independently of one another in this way using optical polarization means. Instead of a light mask with two light passages, the entire cross section of the illumination light is functionally divided into two sectors by means of these polarization filters, which are assigned to the phase contrast and dark field image. Instead of the polarization filters described, exchangeable gray filter sets can also be used, which behave optically congruently with the respective light modulators. Finally, instead of filters, appropriately arranged double screen systems for the separate regulation of both light corridors could also be provided.

As a further design variant, the light mask in the condenser can also be equipped with a single ring-shaped light passage, the width and size of which is designed in such a way that its outer zone projects into the most peripherally located phase ring and its inner area into an adjoining, smaller-sized light stopper (or the opposite). Preferably, a significantly smaller proportion of the area should be congruent with the phase ring and a significantly larger proportion should be allotted to the light stopper. An iris diaphragm, e.g. the aperture diaphragm of the condenser, must be located in the immediate vicinity of this ring of light. Under these conditions, the iris diaphragm can be used to continuously regulate the proportions of phase contrast and dark field. If the iris diaphragm is fully open and the light transmission area for the phase contrast image by far exceeds that for the dark field partial image, the superimposed image is dominated by the phase contrast. The more the aperture diaphragm is narrowed, the more the area of the outer zone of the light ring is reduced, so that the portion of the phase-contrast illumination decreases continuously, provided that the phase ring is in the outer (peripheral) position on the phase plate. If the outer area, which is assigned to the phase ring, is completely covered by the iris diaphragm, pure central dark field illumination results.

Even with this last-described equipping of the light mask with a light ring, which is assigned to both the phase ring and the light stopper, the outer and inner zone of the light passage, which generate phase contrast and central dark field, can be equipped with concentric double polarizers of appropriate size or exchangeable ones with an appropriate shape Gray filters are provided so that the light flows in both zones can be regulated independently of one another by means of the respective filters, even if an iris diaphragm is not used.

Various embodiments of differently designed light masks and associated modified phase plates, their technical Design and application have been described, show the 3a-c . Different design variants of the light mask are shown in the lower row, and corresponding designs of the phase plates are shown in the upper row.

3a shows exemplary embodiments for different light masks and modified phase plates for generating various variants of a variable bright/dark field illumination, executed with a double-barreled special tube, which contains two light corridors K1 and K2. In concentric-peripheral dark field is generated by means of a large dimensioned ring of light, which is projected outside the cross-section of the lens. In addition, the light mask shown here includes a central round light passage. Light corridor K1 contains a centrally arranged, plate-shaped light stopper, which covers the illuminating light that comes from the central light passage. Consequently, peripheral and axial dark field arises in the corridor K1. Corridor K2 does not contain a light modulator, so that a bright field image based on axial illumination light is contributed in this beam path. In the light masks are shown with two light passages, all of which project within the lens cross-section. In the light corridor K1, there is a pure central/axial dark field. Pure brightfield is generated in corridor K2 ( ), axial brightfield plus central darkfield ( ) or concentric-peripheral brightfield plus axial darkfield ( ). shows a light mask with only one centrally arranged round light passage. In cooperation with a centrally arranged light stopper, this creates an axial dark field in corridor K1 and an axial bright field in corridor K2.

3b shows exemplary embodiments for different light masks and modified phase plates for generating various variants of a variable phase contrast dark field illumination, executed with a double-barreled special tube, which contains two light corridors K1 and K2. In a concentric-peripheral dark field is generated by means of a large dimensioned light ring, which is projected outside the lens cross-section. In addition, the light mask shown here includes a smaller ring-shaped light passage. Light corridor K1 contains a ring-shaped light stopper, which is optically congruent to the smaller ring-shaped light passage. As a result, peripheral and central dark field develops in corridor K1. Corridor K2 contains a phase ring, which corresponds to the smaller annular aperture and contributes a phase contrast image. In the light masks are shown with two light passages, all of which project within the lens cross-section. In the light corridor K1, there is a pure central or axial dark field ( ) or concentric-peripheral brightfield plus axial darkfield ( ). In the corridor K2, phase contrast is contributed in each case, and a central or axial dark field image is additionally generated by the necessary covering of the second light passage, which is not required for phase contrast. gives an example of the simultaneous generation of bright field, dark field and phase contrast. Here, a concentric-peripheral bright field and a central dark field are created in corridor K1 and a phase contrast image together with central dark field illumination in corridor K2.

3c finally shows several exemplary embodiments of light masks and light modulators for single-train special tubes with only one light corridor. The contain light masks, the outer light passage of which is projected outside the lens cross section and therefore produces peripheral dark field. The light passages on the light masks of are all projected within the lens cross-section.

In only concentric-peripheral dark field is generated, in additional axial brightfield is created, in concentric-peripheral brightfield. In the inner ring-shaped light passage is covered by an optically congruent ring-shaped light stopper, so that peripheral and central darkfield are performed simultaneously. In the inner ring of light behaves congruently to a phase ring, so that peripheral dark field and phase contrast are combined. If the condenser is designed as a zoom system, the projection size of the light passages can be changed slightly, so that concentric bright field lighting can be added as an additional lighting component.

The show combinations of phase contrast and axial or central darkfield, the (i and j) analogous examples for phase contrast and axial and concentric-peripheral brightfield illumination, respectively. In central darkfield is combined with axial brightfield, in central dark field with concentric peripheral bright field. Also in the examples shown here, which only produce phase contrast and dark field with exact and optically congruent centering, bright field can be added as a third type of illumination as described above if small portions of the illumination light pass the respective light modulators. If the condenser is equipped with an iris diaphragm (aperture diaphragm), which is located near the light mask, all of the shown off duct variants, the light passage located in the outer position can be reduced or completely covered by means of this iris diaphragm, as a result of which the brightness or weighting of the respectively assigned partial image can also be influenced. In all the exemplary embodiments shown, the intensities of the light flow in the respective light passages can of course also be varied at the condenser level by means of double diaphragm systems or double polarizers adjustable in opposite directions.

Reference List

01

light source

01a

collector lens

02

condenser

03

object

04

lens

05

tube lens

06

prism

07

Bertrand lens

08

observation eyepiece

08a

Eye

09

photo eyepiece

09a

camera

10

Light mask with light ring

10.1

ring-shaped light passage, ring diaphragm, condenser light ring

11

converging lens

12

polarizer

13

Quarter-wave compensator

14

Lambda compensator

16

first projection lens group

17

second projection lens group

18.1

first divider prism

18.2

second divider prism

19

rotating turret disc, turret slide

20

phase plate

20.1

phase ring, light modulator

20.2

Light stopper, light modulator

22

Analyzer A

23

Dual polarizers for the first light corridor

24

Dual polarizers for the second light corridor

25

(mirror) prism

26

deflection prism

ZBE1

first intermediate image level

ZBE2

second intermediate image level

OBE1

first rear lens focal plane

OBE2

second rear lens focal plane

K1

first light corridor

K2

second light corridor

Claims (9)

Method for generating a variable and simultaneous phase contrast image in combination with one of the images dark field image or bright field image or polarization image in the microscope, characterized by the following method steps: - illuminating an object with light from an illumination device in transmitted light and/or incident light; - splitting of the light after the passage of the object or the reflection from the object into two separate light beams associated with sub-images, each with method-specific information about the object; - Changing at least one of the light beams of the partial images according to phase position, polarization position, spectral composition and/or intensity; - Merging and superimposing the light beams assigned to the partial images to form a summation image by interference, the summation image being viewable or documentable in a manner known per se using the eyepiece. procedure after claim 1 , characterized by a stepless control of brightness and intensities of the light beams by attenuation, so that objects in pure phase contrast, pure central or axial dark field or pure bright field or pure polarization can be observed in the summation image, or that two or more of the aforementioned lighting methods with variable weighting of the participating partial images are executed simultaneously in combination. procedure after claim 1 , Characterized by color filtering of at least one of the light beams before it is combined with the other light beam, so that the light beams are individually filtered in different colors and thus changed in their coloring part of the summation image, with preferably the spectral range for coloring one partial image being complete is selected mentally to the spectral range for coloring the other partial image. procedure after claim 1 , characterized by adjusting the projection image projected approximately into the rear objective focal plane of at least one light passage located in the area of the condenser in relation to the position and size of the phase ring and/or other light modulators by adjusting the height of the condenser or the condenser lens, by changing the aperture stop of the condenser and/or or additionally existing, adjustable multiple diaphragm systems in the condenser to regulate the light transmission areas or by varying the focal length and aperture of the condenser lens system designed as a zoom. Procedure according to one of Claims 1 until 4 , characterized by generating a moderate oblique illumination by at least partially covering the light passages used for the two light beams. Microscope with illumination apparatus, object table with object, tube with eyepiece and lens for carrying out the method according to one of Claims 1 until 5 , whose illumination apparatus is equipped with a condenser, which is equipped with a condenser diaphragm having at least one light passage for generating phase contrast, characterized in that projection lenses (16, 17) are arranged inside the tube, through which a first objective focal plane (OBE1) and a downstream first intermediate image plane (ZBE1) are each duplicated to form a second rear lens focal plane (OBE2) and a second intermediate image plane (ZBE2), that for splitting the beam path into two parallelized partial beam paths, means for beam splitting are preferably provided as a splitter prism, with one of these two partial beam paths a phase ring (20.1) for phase contrast and in the other of the two partial beam paths a ring-shaped or centrally arranged, disc-shaped light stopper (20.2) for central or axial dark field is arranged or the light stopper (20.2) for generating bright field or polarization illumination from the respective light passage removed, and that in order to combine the partial images assigned to the two partial beam paths in the second intermediate image plane (ZBE2) to form a summation image that can be viewed enlarged in the usual way using eyepieces (8, 9), means for combining the partial beams of rays are provided, preferably designed as a combining prism. microscope with claim 6 , characterized in that in the tube for the partial beam paths generated by splitting there are rotatable polarizers or double polarizers and/or adjustable diaphragms or interchangeable light-attenuating gray filters and/or one color filter each with different spectral transmission ranges, which are preferably arranged so that the intensities and / or spectral composition of the two light beams are separately and independently adjustable. microscope after claim 6 or 7 , characterized in that in the tube for one of the two partial beam paths generated by splitting, a phase plate with a phase ring (20.1) for generating phase contrast, and for the other of the partial beam paths a modified phase plate (20) with a light stopper (20.2) or with an empty neutral aperture or are provided as a phase plate with a concentric phase ring and light stopper in such a way that phase contrast, central or axial dark field and/or bright field or polarization can be contributed at the same time. Microscope after one of Claims 6 until 8th , characterized in that the light passage (10.1) can be partially covered to change the angle of incidence of the illumination light between concentric and eccentric or obliquely.

Images