Method and microscope for generating variable light-dark field illumination

Abstract

Method for generating a high-contrast light-dark-field image in a microscope, comprising the following process steps: - Illuminating an object with light from a condenser in such a way that a beam of light penetrates the object in the manner of bright-field illumination, and a second beam of light, after passing through the object, is directed past the lens in the manner of dark-field illumination, - Merging the bright-field-dominated image created by bright-field illumination and the dark-field-dominated image created by scattered light in the manner of dark-field illumination in the plane of the intermediate image; - Generating a summation image by interfering with both superimposed light- and dark-field-dominated images in the intermediate image plane; - Viewing the superposition image thus created as a variable light-dark field image in a manner known per se using an eyepiece, wherein according to the method the illumination light is polarized by means of a polarizer and each light beam can be attenuated with respect to its intensity and thus its share in the summation image by means of an associated analyzer.

Description

[0001] The invention relates to a method for generating a microscopic image of an object using brightfield / darkfield illumination, wherein the illumination intensities of the brightfield image and the darkfield image are continuously variable independently of each other, it further relates to a microscope with illumination apparatus, stage with object, and tube with eyepiece and objective, in which the illumination apparatus comprises a condenser with a condenser diaphragm having at least one aperture located outside the optical axis and in which the objective is configured to generate both brightfield illumination and darkfield illumination, and in which the brightfield and darkfield images are superimposed.

[0002] The illumination of objects plays a crucial role in microscopy for resolution and image formation. Based on the well-known Köhler illumination apparatus, which is designed to produce a bright-field image, dark-field illumination was introduced, offering advantages over bright-field techniques in terms of resolution and image formation. From DE 10 2006 027 961 A1, a microscope is known in which improved structural representation is achieved to increase the contrast of images in phase contrast by having the light transmission aperture on an annular diaphragm of the condenser's aperture assembly be shaped like a circular sector, and by obliquely illuminating the object with a sector-shaped light beam lying on the surface of a hollow cone, where the central angle associated with the circular sector is at most 90°.Furthermore, DE 10 2007 029 814 A1 discloses a microscope in which, for improved contrast, enhanced brightness, increased depth of field, and increased resolving power, the illumination system has a diaphragm with a central aperture and a peripheral annular slit. The object is thus illuminated with a central light beam and a peripheral light beam. After the beams have passed through the object, at least a portion of either the central or the peripheral light beam is attenuated by a light-absorbing element in the objective or immediately behind it. In this way, different illumination variants can be seamlessly blended, achieving an image character and image information similar to conventional darkfield, phase contrast, interference contrast, and brightfield microscopy, resulting in improved detail rendering.Finally, a microscope is known from DE 10 2009 003 682 A1 in which, for phase contrast imaging, one or more beams of light concentric to the optical axis on the surface of a hollow cone are blocked from the beam path emanating from a light source by means of a condenser diaphragm with segment-like light transmission for "oblique" or multi-directional illumination of the object, wherein the direct and indirect image-forming light components emanating from the object are imaged in an intermediate image by means of an objective and can be viewed with an eyepiece.To enable the representation of objects with faint contours, the segments on the phase plate have areas of varying optical density within a central angle of no more than 180°. This allows the incident light, arriving obliquely to the optical axis from one or more discrete directions, to interfere with the background light, enabling the representation of details of the object that are unsatisfactorily contrasted under conventional phase contrast illumination. A disadvantage is the use of special light-attenuating agents, which necessitate special inserts or lenses.

[0003] According to US 2007 / 0014002 A1, a cardioid condenser for dark-field illumination is a prior art device which differs in principle from a conventional dark-field condenser in that a ring-shaped light aperture is provided on the underside of the condenser, so that the central area is covered opaquely by the opening surface of the underside of the condenser.

[0004] Regarding the objectives of the US teaching, the aim is to improve the resolution of high-resolution and high-magnification optical systems, while additionally reducing disruptive superposition caused by blurred object structures in spatially extended objects by means of ring-shaped illumination limited in the Z-axis. For this purpose, the illuminating ring light should be limited in the Z-axis in such a way that higher or lower object components are not illuminated at all and consequently cannot contribute to blurred superposition effects. Further prior art references in this context are DE 10 2007 029 814 A1 and DE 25 42 075 A1.

[0005] In order to avoid such special additives and to eliminate remaining deficiencies in resolution, the task arises to further develop the microscope so that, with the superposition of bright and dark field images under variable bright-dark field illumination, increased resolution as well as improved contrast and representation of structures with significantly increased depth of field are made possible for artifact-free observation of complexly structured three-dimensional objects.

[0006] This problem is solved by the characterizing features of the independent claims; advantageous further developments and preferred embodiments are described by the dependent subclaims.

[0007] To solve the problem and achieve the desired improvement, a bright-field-dominated and a dark-field-dominated sub-image are generated in the intermediate image plane. These superimpose and interfere with each other to produce an observable summed image. The bright-field-dominated sub-image is generated by illuminating the object with a first light component entering the objective's entrance cross-section. The dark-field-dominated sub-image is generated using the scattered light from a second light component that does not enter the objective's entrance cross-section. This second component is either blocked before reaching the object or prevented from entering after reaching it. In the intermediate image plane, the zero-order transmitted image is superimposed with a higher-order diffraction pattern from the dark-field component, resulting in a summed image.This is then viewed in the usual way with the eyepiece at the desired magnification. While the zeroth-order maximum is retained in the summed image, the dark-field image, lacking the zeroth-order diffraction maximum, also contributes to the overall image formation. In a further development, both the bright-field and dark-field dominated sub-images are given different polarization-optical properties using polarizing optics, allowing their brightness to be continuously adjusted by rotating the polarizing filters. If spectral filters are used, the two sub-images acquire different colorations, making certain features stand out more clearly against the background.

[0008] To achieve this, in one embodiment, a ring diaphragm downstream of the aperture diaphragm splits the object-illuminating light into two distinct light components, each optically resembling a brightfield or darkfield. The object is illuminated by the first light component, which produces a brightfield image, while the second light component, corresponding to the darkfield, illuminates it at such an angle that it continues beyond the lens after passing the object, thus preventing it from illuminating the background. This method of image formation allows for a clearer visualization of both outline and internal structures than is possible with simple brightfield or darkfield illumination alone. Furthermore, this method offers a greater depth of field than conventional brightfield, darkfield, phase contrast, or interference contrast illumination.Such a division into two light beams can also be achieved using a double condenser, whereby the two light beams are ultimately combined to illuminate the object and - as described above - produce the bright field image and the dark field image.

[0009] If a standard brightfield objective is used with a universal condenser constructed analogously to a phase contrast condenser, the size and width of the ring-shaped light passages in the condenser must be dimensioned so large that their inner zones are projected into the edge region of the optically effective objective cross-section, and the outer parts of the light rings lie outside the objective cross-section, in contrast to phase contrast microscopy.

[0010] The object is illuminated from all directions in a bright-field-like manner by a concentric and axially aligned cone of light incident at a relatively steep angle. Simultaneously, a dark-field analogous illumination, which does not penetrate the object, is provided by a cone of light from the outer portion of the illumination, incident at a less steep angle as concentric oblique illumination. The optically effective outer diameter of the light ring, and thus the remaining width of the outer zone corresponding to the dark-field component, can be reduced or narrowed as needed by adjusting the aperture diaphragm, thereby influencing the character of the resulting image. The ratio between the optically relevant objective diameter and the inner diameter of the condenser light ring determines the relative contribution of the bright-field component to image formation.The wider the translucent inner zone, i.e., the smaller the inner diameter of the light ring compared to the lens diameter, the brighter the image background will be; conversely, the narrower the light ring, the more the image will exhibit a dark-field aspect. Depending on the overlap between the edges of the lens cross-section and the condenser light ring, different contrast effects can be achieved. Stepless adjustment of the outer and inner diameters independently is advantageous. Such a light ring can also be used as a "multifunctional light ring" for lenses with different cross-sectional areas, thus eliminating the need for a set of individually sized rings.

[0011] To achieve eccentric bright-darkfield illumination from defined spatial directions, the light ring in the condenser is decentered in the aforementioned arrangement to such an extent that it only marginally overlaps with the optically relevant objective boundary, so that only a narrow segment of the light ring contributes to object illumination. The zone facing the object, which lies within the optically relevant objective cross-section, generates the brightfield-analogous image, while the illumination rays from the adjacent, more distant zone, which lies outside the objective cross-section, produce the darkfield-analogous image. In this optical arrangement, the proportions of the brightfield and darkfield components can also be varied using the aperture diaphragm and additionally by slight changes in the position or eccentricity of the light ring.

[0012] If, in the previously described arrangement, the illuminating light segment, along with a light cover located above the lens, is shifted from the equatorial plane towards the edge, oblique illumination results, the angle of incidence of which can be changed depending on the degree of eccentricity. Here, too, as previously described, bright-field or dark-field dominated images can be blended into one another depending on the degree of light attenuation. Maximum oblique illumination occurs when the edge zone of the lens cross-section is segmentally overlapped by a portion of the light transmission. In this situation, instead of a central bar, a circularly bounded area of ​​a double-aperture shutter, described in more detail later, can be used for light attenuation. Variable transitions between bright-field and dark-field are also continuously achievable with these settings.

[0013] Even with concentric illumination configurations where the condenser light ring is centrally aligned, moderate oblique illumination can be achieved using an illumination slider positioned above the objective lens, provided that part of the peripheral illumination beam path above the objective lens is covered. In this case, the brightfield component illuminating the object is generated under oblique illumination. Complete oblique illumination of both the brightfield and darkfield images is achieved by partially covering the condenser light ring within the condenser itself. Alternatively, instead of the previously described arc-shaped condenser light rings, a linear light slit can be provided within the condenser, where the width, length, and position are continuously adjustable. The corresponding light-blocking element, positioned above the objective lens, is adapted as precisely as possible to the geometry of the illumination slit.If the condenser and light cover are arranged to be rotatable while maintaining congruity, the direction of the light incidence can be further adapted to the positioning or orientation of the object in the room.

[0014] If the angles of incidence of both light-field and dark-field generating radiation components are to be changed independently of each other, this is possible with a special condenser, a "double condenser," which is equipped with two separate light apertures designed as light rings or adjustable slit apertures, or which may have other suitable geometric shapes. Both light apertures would have to be supplied with illumination light and be independently movable horizontally.

[0015] Advantageously, the condenser features an aperture with multiple openings, which are rotatable or slidable for selectable adjustment. The aperture openings are arranged linearly or annularly and are dimensioned in terms of size and width such that their inner zones project onto the edge of the optically effective lens cross-section, while the outer portions of the light rings lie outside the lens cross-section. Using apertures designed in this way, it is possible to provide a suitable light ring in the condenser for each lens used, even in cases with different lens cross-sectional areas.

[0016] For advantageous further development, the condenser is designed to be height-adjustable to precisely adapt the projection size of the respective light ring to the respective lens cross-section. This facilitates the necessary adjustments to the diameters of the lens cross-section and the projection image of the condenser light ring. An alternative solution for adjustment involves designing the condenser as a zoom system with a variable focal length of the lens system. With this approach, the projection of the light ring in the beam path and the resulting path of the illuminating rays can be further adapted to the object characteristics (position, size, layer thickness) by adjusting the height of the condenser and / or changing the condenser focal length. The proportions of the bright-field and dark-field components can also be adjusted using the aperture diaphragm and / or by slight changes to the position or...The eccentricity of the light ring can be varied. Using an adjustment eyepiece, the respective adjustment settings can be visually checked for all the aforementioned design variants. If the objective lens has an integrated iris diaphragm, and the light ring is appropriately designed, the optically effective objective cross-section can also be adapted to the dimensions of the light ring by moderately adjusting the objective iris diaphragm.

[0017] If the outer and / or inner zone of the condenser light ring is equipped with ring-shaped or partially ring-shaped spectral filters, different wavelengths and thus different colors can be assigned to the two light components. It is advantageous if the intensity and / or color of each of the light beams of the two light components can be varied independently of the other. The different colorations clearly highlight structures with different absorption characteristics. Ring-shaped or partially ring-shaped polarizing filters are also provided, which interact with a second rotatable polarizing filter integrated below in the condenser. The ring-shaped or partially ring-shaped polarizing filters are mounted independently of each other so that they can rotate freely.

[0018] This allows the brightness of the partial images to be changed independently of each other by rotating them.

[0019] An aperture slider inserted above the lens into the optical path can also be fitted with polarizing filters that act as analyzers. Advantageously, one or two separate analyzers can be incorporated, filling one or both aperture openings and rotatable from parallel to cross-position relative to each other. Alternatively, only half of the central strut between the two aperture openings is fitted with an analyzer, or the entire central strut consists of two adjacent analyzer plates in a cross-position. In these embodiments, a rotatable polarizer must also be provided at the condenser plane. This allows the various imaging possibilities using polarized light to be achieved by independently regulating the intensities of the light- and dark-field-generating light components using polarization optics.In this setup, the polarizing filter of the light-ring-bearing condenser acts as a polarizer, working in conjunction with another polarizing filter above the lens as an analyzer. Through all the configurations described above, the intensity, polarization plane, and / or color of each light beam from the two light components can be varied independently of the intensity, polarization plane, and / or color of the other.

[0020] The ability to change the incidence of the light beams from the two light components between concentric and eccentric or oblique angles is advantageous. With oblique illumination, the angle of incidence of the light beams from both components is aligned and / or continuously variable, independent of the other. For maximum eccentric bright-dark-field illumination from a defined spatial direction, the condenser light ring is decentered to such an extent that it marginally overlaps the optically relevant objective boundary. Under these conditions, only a narrow segment of the light ring contributes to object illumination; the object-facing zone of this segment generates the bright-field analog image, while the illumination rays from the adjacent, more distant zone produce the dark-field component.

[0021] Further illumination variations are achieved by inserting an aperture into the light path above the lens, in or as close as possible to the rear focal plane of the lens. This aperture preferably has two aperture openings located close together and separated by an opaque central bar. The incident illumination light is completely or partially blocked by the opaque areas of this aperture; any unblocked portion of the illumination light can pass through the aperture openings along with the imaging rays.

[0022] This aperture is advantageously designed as a shutter. This arrangement allows not only stepless transitions between brightfield and darkfield illumination, but also stepless adjustment of the eccentricity of the illumination light when the light is incident obliquely. To achieve this, the light ring in the condenser is shifted so that it runs approximately equatorially through the object field. In this position, the object is illuminated largely axially, similar to brightfield illumination. The length of the illuminating light segment, and thus the illumination aperture, can be regulated using the aperture diaphragm. If the double shutter is inserted into the beam path so that its central rib completely or partially covers the light transmission, complete coverage results in axial darkfield illumination, while partial coverage, where some of the transmitted light is added to the imaging beam path, moderately brightens the image background.The resulting brightfield image is superimposed on the additional axial darkfield image. Depending on the proportion of the transmitted light component, a wide variety of transitions between brightfield and darkfield can be generated. If necessary, the cross-section of the illuminating beam can be further restricted by the aperture diaphragm or another horizontally movable diaphragm within the condenser, which further influences the quality and character of the resulting summed image. This movement can also achieve a transition from a darkfield image to an image with brightfield components.

[0023] In an alternative embodiment, a movable aperture with at least one, preferably two, aperture openings is provided in the lens. In terms of its design and optical effect, this aperture slider corresponds to the aperture slider described above, which can be integrated into the beam path above the lens. To allow these aperture openings to be set to the selected position, the aperture slider is preferably designed as a linear slider with detents. The aperture openings themselves and the intervening opaque central bridge are dimensioned such that the illuminating rays are completely or partially blocked at the lens plane, while the bright-field-generating partial rays, together with the imaging rays, can pass through the lens.Under these conditions, different contrast effects can also be created with this variant, whereby stepless transitions between bright and dark field illumination are possible and the eccentricity of the illumination light can also be changed steplessly in the case of oblique light incidence.

[0024] To prevent the central illumination light from entering the objective lens cross-section in the second alternative, a microscope equipped with a catoptric or catadioptric mirror objective is suitable. In this type of microscope, the opaque back face of the central secondary mirror within the objective partially blocks this light component after it passes through the object. This results in a superimposition of a bright-field-dominated partial image and an axial and central dark-field-dominated partial image in the plane of the intermediate image. These superimposed images interfere with each other to form a summed image with the desired bright-dark-field illumination. Within the objective, the imaging rays are typically "folded" between the central and secondary mirrors and guided by the small, centrally located secondary mirror, via an additional lens system (present in catadioptric objectives), towards the eyepiece.An axial (central) dark field is created when the outer diameter of the condenser light ring is kept so small that all illuminating rays are intercepted by the back of the secondary mirror after passing through the object. Consequently, the zeroth-order maximum is also blocked, resulting in a reflection or diffraction image in the dark field in which the zeroth-order maximum is not involved. If the outer diameter of the illuminating light ring is slightly increased, or if the light ring is slightly decentered so that a narrow illuminating light segment can pass through the central mirror, a bright-dark field illumination results. In this case, too, the two bright-field and dark-field analogous partial images interfere to form the summed image as described above. If the light ring can be displaced from its central position relative to the optical axis, moderate oblique illumination can be achieved.

[0025] If the microscope is designed as an incident-light microscope, an incident-light illuminator and special objectives for incident-light darkfield imaging are used to generate an incident-light-based variable brightfield. For this purpose, an annular illumination beam is generated in the incident-light illuminator. The outer portion of this beam extends outside the imaging objective lens system and is guided through the outer zone of the objective in the usual manner to generate a concentrically illuminated incident-light darkfield image. Simultaneously, the inner portions of the illuminating light beam are designed to pass through the imaging objective lenses, thus creating a simultaneous incident-light brightfield image.

[0026] Under these conditions, bright-field and dark-field images superimpose in the reflected-light beam path and interfere to form the summed image. If the illuminating light beam is covered or blocked at a freely selectable point in the reflected-light illuminator, observation can also be carried out under oblique illumination. If the reflected-light illuminator is equipped with a polarizing filter in the area of ​​the light source and two polarizing filters acting as analyzers near the beam splitter, the brightness of the two illuminating light beams can be controlled independently of each other using polarizing optical means.

[0027] When additional color double contrasts are implemented in the described methods, the two illuminating beam components that generate the respective bright-field and dark-field analogue sub-images are filtered in different colors. Furthermore, as is possible with other illumination methods, the variants presented here can achieve further optimization of the image quality through monochromatic filtering of the illumination light, thereby eliminating any residual chromatic aberrations present in the entire optical system. In addition to a further increase in contrast, resolution and sharpness can be maximized specifically by using relatively short-wavelength light and narrowband filters with a low half-width.

[0028] Since the illumination aperture in the techniques presented here can be varied within wide limits by stopping down and / or blocking out illuminating beam components, the existing, computationally determinable relationships between aperture and resolution are highlighted. It can be deduced from this that when the aperture of the lens and / or condenser is reduced, the relative increase in axial resolution, and thus the gain in depth of field, is significantly greater than the loss in lateral resolution. Furthermore, it follows that the respective loss in lateral resolution can be reduced again by simultaneously using oblique illumination.These aspects underline that, especially with objects of high spatial depth, adequately stopping down the beam path can contribute to an overall superior image documentation despite reduced lateral resolution due to a more significant increase in depth of field, whereby the remaining loss of resolution can be reduced to a minimum if necessary by oblique lighting.

[0029] The essence of the invention will be explained by the information contained in the Fig. Examples 1 to 9, shown schematically, are explained in more detail; they show: Fig. 1: Microscope with light ring in the condenser with concentric beam path; Fig. 1a: Control image when adjusting a microscope according to Fig. 1 with focusing magnifier in concentric light-dark field with wide, light-field dominated inner zone, Fig. 1b: Control image when adjusting a microscope according to Fig. 1 with focusing magnifier in concentric healing dark field with narrow, dark field-dominated inner zone; Fig. 2: Microscope after Fig. 1, however with an eccentric beam path; Fig. 2a: Control images when adjusting a microscope according to Fig. 2 with focusing magnifier in concentric light-dark field with narrow, dark-field dominated outer zone; Fig. 3: Microscope after Fig. 1 with aperture slider above the lens; Fig. 4: Detail of the shutter; Fig. 4a: Shutter shutter with two rectangular aperture openings, Fig. 4b: Aperture slider with two circular aperture openings, Fig. 4c: Shutter shutter with two rectangular aperture openings, the ends of which are rounded; Fig. 5: Beam path and control images for partial masking of the illumination light with double aperture slider; Fig. 5a: Beam path for axially guided illumination light and central double aperture slider (axial light-dark field schematic), Fig. 5b: Beam path according to Fig. 5a, condenser light ring and aperture diaphragm shifted relative to the optical axis by “d” (eccentric bright-dark field), Fig. 5c: Control image with beam path according to Fig. 5a, light ring approximately equatorial to the object field, Fig. 5d: Control image with beam path according to Fig. 5c, light segment limited by aperture diaphragm, Fig. 5e: Control image with beam path according to Fig. 5d, the central bar of the double aperture slider covers the light transmission, Fig. 5f: Control image with beam path according to Fig. 5c with maximally eccentric incident illumination, light ring shifted to the peripheral edge area of ​​the lens cross-section, Fig. 5g: Control image with beam path according to Fig. 5f, Cover of the light ring segment with arc-shaped double aperture slider, Fig. 5h: Control image with beam path according to Fig. 1. Illumination light circularly covered by double shutter 10, partially peripherally; Fig. 6: Adjustment of a microscope for axial bright-dark field with modified aperture slider and condenser light rings for polarization-optically controlled bright-dark field illumination; Fig. 6a: Double aperture slider with a large-area analyzer, Fig. 6b: Adjustment with double aperture slider with two large-area analyzers, Fig. 6c: Adjustment with center bar with narrow analyzer plate, Fig. 6d: Adjustment with center bar with two narrow analyzer plates in cross position; Fig. 7: Polarization-optical developments of the condenser light ring for concentric bright-dark-field illumination; Fig. 7a: Design of the inner zone with a ring polarizer, Fig. 7b: Design of the inner and outer zones with two concentric ring polarizers in a cross position, Fig. 7c: Design of the inner zone with large-area polarization filters with an adjacent narrow outer zone; Fig. 8: Microscope with mirror objective and concentric beam path Fig. 1; Fig. 8a: Adjustment with focusing magnifier on a microscope with mirror objective at a setting for pure brightfield, Fig. 8b: Setting for axial dark field, Fig. 8c: Setting concentric variable light-dark field, Fig. 8d: Setting eccentric variable light-dark field; Fig. 9: Microscope with reflected light illuminator and concentric beam path, set up for bright-dark field illumination (schematic).

[0030] Fig. Figure 1 shows a highly schematic representation of the beam path in the microscope for concentric bright-dark-field imaging. A beam of light from the light source 1 leads to the aperture diaphragm 2, below which a rotatable polarizing filter 2.1 can optionally be inserted. The light transmitted through the filter passes via the light ring 4.1 in the light ring carrier 4 to the condenser optics 3. The light ring 4.1 in the light ring carrier 4 has such a width that only light transmitted from certain areas, acting as illumination 6, illuminates the object 5.1 placed on the slide 5 and contributes to the formation of a bright-field image. Illumination 6 transmitted from the other areas passes by the objective 7 and therefore does not contribute to background illumination. In this diagram, the optical axes of the condenser, the (not shown) tube, and the light ring 4.1 coincide to form a common optical axis (OAG).The light ring 4.1 provided in the light ring carrier 4 is designed and dimensioned such that the light transmitted from its outer region illuminates the object 5.1 placed on the slide 5 as illumination light 6 in the form of a concentric, hollow-cone-shaped light beam. After passing through the object 5.1, the light beam 8.3 bypasses the objective 7 and does not enter it; therefore, it cannot contribute to illuminating the image background. The light transmitted from the inner regions passes through the object 5.1 and reaches the objective 7, forming the basis for the bright-field image as light beam 8.1. However, a portion of the total light illuminating the object 5.1 is scattered. This scattered light 8.2 enters the objective 7 and forms the basis for the dark-field image. As a combined light beam 9, the light exits the objective 7 and enters the plane of the intermediate image ZWB (not shown in detail).There, the bright-field dominated image and the dark-field dominated image overlap and, through interference in variable concentric bright / dark-field illumination, form the structured summation image with higher resolution and improved depth representation.

[0031] A check using a focusing magnifier, similar to that used for adjusting a phase-contrast microscope, reveals – as in the Fig. 1a and Fig. 1b shows the object-transmitting component as a narrow bright edge on the outer surface of the lens cross-section; the portion of the light transmission from the dark-field illumination that extends outwards remains invisible in the image of the focusing magnifier. Fig. Figure 1a shows an image with bright-field dominance. Here, corresponding to the relative bright-field dominance, a relatively wide, translucent inner zone is visible. If the bright-field component is reduced, the dark-field component comes to the fore and the image becomes dark-field dominated ( Fig. 1b) and the dark-field dominated image is created by using a larger light ring with a narrower translucent inner zone. The two light-field or dark-field dominated images can be combined into one another, as described above, depending on the degree of light attenuation.

[0032] Will - how Fig. 2 shows - in the arrangement according Fig. 1. By shifting the light ring support 4.1 from the equatorial plane towards the edge, the optical axis OAL of the light ring 4.1 and the optical axis OAG of the tube and lenses of the condenser optics 3 are laterally separated, resulting in eccentric object illumination and oblique illumination of the object 5.1. The angle of incidence of this oblique illumination can be changed depending on the degree of eccentricity. Here too, as described above, the light components transmitted from the inner areas after passing the object 5.1 form the basis for the bright-field-dominated image as a light beam 8.1. Together with the light scattered by the object 5.1, as a scattered light beam 8.1,As indicated in Figure 2, which forms the basis for the dark-field dominated image, the light leaves the lens 7 in the light beam 9 to reach the plane of the intermediate image, where it is superimposed and forms the summed image through interference. Depending on the degree of light coverage or blockage, the bright-field or dark-field dominated images can be superimposed. An adjustable iris diaphragm in the condenser, used to limit the cross-sectional area of ​​the illuminating beam, allows the illumination aperture to be further reduced, the depth of field to be increased, and any overexposure in the dark-field sub-image to be attenuated.

[0033] Because of this lateral displacement, only a portion of the light ring 4.1, originating from one part facing the optical axis OAG, carries the illumination light 6. The light beam 8.1 transmitted from the object 5.1 contributes to the brightfield image, while the complementary component, light beam 8.3, passes by the objective 7. The scattered light 8.2 again forms the basis of the darkfield-dominated image, which, when superimposed on the brightfield-dominated image through interference, then yields the image structured in variable bright-darkfield illumination with a higher resolution and improved depth representation. For adjustment, the light ring 4.1 is shifted in the condenser so that it peripherally touches the object field and illuminates the object obliquely in a brightfield manner. The representative control image for such an arrangement with eccentric oblique illumination and a reduced brightfield component shows Fig. 2a, in which the illumination is dark-field dominated. The length of the illuminating light segment, and thus the illumination aperture, can be regulated using the aperture diaphragm 2, and the relative intensities of the bright- and dark-field partial images by moderately shifting the illuminating light segment.

[0034] If an additional aperture is provided downstream of the lens 7, additional effects can be achieved. Such an aperture is advantageously designed as a double aperture slider 10. Some embodiments are described in the Fig. 4 shown. Fig. Figure 4a shows such a double aperture slider 10 with aperture openings 11 that are rectangular in shape and separated by a central bar 12. The double aperture slider 10 of the Fig. 4b has circular apertures of 11'. Finally, it shows Fig. 4c a double aperture slide 10 with aperture openings 11'' in rectangular shape, the outer boundaries of which are rounded.

[0035] This design is particularly advantageous when the microscope has a filter holder above the objective 7, which accommodates a double diaphragm slider 10 with at least one, preferably two, closely spaced apertures 11 separated by a central bar 12. By designing the double diaphragm slider 10 as a slidable element, its position can be precisely adjusted to the size and location of the respective light ring. The double diaphragm slider 10 is positioned above the objective, in or near the rear focal plane of the objective 7, and is inserted into the beam path such that the central bar 12 between the two apertures 11 completely or partially covers the through beam 8.1. The imaging rays of the scattered light beam 8.2 pass through the apertures 11 of the double diaphragm slider 10.If its aperture openings 11 are designed as circular openings 11', different contrast effects can be advantageously generated by moving them, and stepless transitions between bright-field and dark-field illumination are also possible. In the plane of the intermediate image, the bright-field-dominated image and the dark-field-dominated image are superimposed to form the summed image. The eccentricity of the illumination light can also be continuously varied when the light is incident obliquely.

[0036] The double aperture slider 10 above the objective lens can be inserted into the beam path such that the central bar 12 between the two aperture openings 11 completely or partially covers the through beam 8.1, while the image-forming rays of the scattered light beam 8.2 pass through it. Under these conditions, different contrast effects can be produced, with stepless transitions between brightfield and darkfield illumination possible, especially since the eccentricity of the illumination light can also be continuously adjusted when the light is incident obliquely. Here, too, the respective adjustments can be checked using the focusing eyepiece. Schematic beam path diagrams are shown. Fig. 5a for an axially guided illumination light 6 and a central double aperture shutter 10. In the embodiment shown here, its continuous beam 8.1 is limited in such a way that it is completely covered by the central web 12 of the centrally arranged double aperture shutter 10, resulting in axial dark-field illumination. If the double aperture shutter 10 is moved a small distance in the arrangement shown here, so that only a small proportion of the rays of the light beam 8.1 passing through the object 5.1 can pass through the aperture 11, an intensity-controllable background brightening and a simultaneous representation of the object 5.1 in the light-dark-field beam path result. Fig. In 5b, the condenser light ring 4.1 is displaced perpendicular to the optical axis OAG by the distance "d". This creates oblique illumination. In the embodiment shown here, part of the now obliquely incident radiating light beam 8.1 passes through one of the aperture openings 11, so that by superimposing two light-field and dark-field-based partial images, a representation of the object 5.1 in light-dark-field contrast is created.

[0037] If the light ring 4.1 in the condenser is shifted until it runs approximately equatorially through the object field, the object 5.1 is illuminated largely axially in a bright-field manner. The corresponding control image shows Fig. 5c. The length of the illuminated light segment, and thus the illumination aperture, can be regulated with the aperture diaphragm 2; the control image shows Fig. 5d. If the double aperture slider 10 is now inserted into the beam path so that its central web 12 covers the light transmission, the following is obtained as a control image: Fig. 5e. In this complete overlap (see Fig. 5a) Only the scattered light 8.2 associated with the dark-field illumination passes through the aperture openings 11' to the intermediate image plane, where the resulting image can be magnified and viewed using the eyepiece. The entire beam of light from the illumination 6 passes through the object 5.1. The light component 8.1 associated with the bright-field illumination is only intercepted after the objective 7, thus allowing fine internal structures of the object 5.1 to become more clearly visible and reducing edge flare on angular structures.

[0038] When the illumination light is partially obscured by the central rib 12, a portion of the transmitted light is released in the imaging beam path, the image background is moderately brightened, and the resulting bright-field-dominated absorption image is superimposed on the additional axial dark-field-dominated image. Depending on the proportion of the released transmitted light component, a wide variety of stepless transitions between bright and dark fields can ultimately be generated. To further increase the depth of field and reduce any edge flare at boundary contours, the illumination aperture is reduced. This is achieved by limiting the cross-section of the illuminating beam with the aperture diaphragm or an iris diaphragm that is horizontally and perpendicularly to the optical axis and can be moved within the condenser.

[0039] If the condenser iris diaphragm and the segment of the light ring 4.2 that contributes to illumination are now displaced relative to the optical axis – approximately as in Fig. 5b shown, to the distance “d” - results in oblique illumination of the object 5.1 and, depending on the positioning of the double aperture slider, a partial overlap, in which part of the light beam 8.1 assigned to the brightfield illumination is released, as in Fig. Figure 5b illustrates this. The angle of incidence of the illumination light can be changed depending on the degree of eccentricity determined by repositioning the light ring 4.1 in the condenser. Depending on the size of the distance "d" and the position of the double aperture slider, the released proportion of the transmitted light associated with the brightfield illumination is changed, thus ultimately allowing for the creation of a wide variety of stepless transitions between brightfield and darkfield.

[0040] Maximum oblique illumination can be achieved when the edge zone of the lens cross-section 7 is segmentally overlapped by a portion of the aperture opening 11 of the double aperture slider 10. The control image shows Fig. 5f. Advantageously, a double shutter 10 is used for light control, whose central bar 12 is bounded by a circular arc, roughly analogous to the circular aperture openings 11'. The corresponding control image shows Fig. 5g. Variable transitions between brightfield and darkfield illumination with stepless, seamless transitions are also possible with this setting. Furthermore, the cross-sectional area of ​​the light illuminating object 5.1 can be further limited by an additional (not shown) aperture, in particular an iris diaphragm in the condenser, which can be moved perpendicular to the optical axis OAG.

[0041] The effect of oblique illumination can also be achieved, with the light ring 4.1 positioned concentrically, by partially covering part of the peripheral illumination beam path with the double shutter 10. The control image for this is shown in Fig. 5h is reproduced. This results in an increased relief effect, a reduction in the illumination aperture, an increase in depth of field, and the suppression of any overexposure in the dark-field sub-image. Maximum oblique illumination is achieved when the edge zone of the cross-section of the lens 7 is segmentally overlapped by a portion of the light transmission. Instead of the central rib 12 between the aperture openings 11, a circular arc-shaped area 11' of the double aperture slider 10 can also be used for light blocking.

[0042] Variable transitions between brightfield and darkfield are also seamlessly achievable with these settings. Furthermore, with concentric adjustment of the light ring 4.1 in the condenser, moderate oblique illumination of the brightfield image can be achieved if part of the peripheral illumination beam path above the objective 7 is covered by the double diaphragm 10. To generate oblique illumination affecting both the brightfield and darkfield images, the light ring 4.1 is partially covered in the condenser itself in a similar manner.

[0043] To achieve further control over the beam paths, the condenser is fitted in the usual manner with a rotatable polarizing filter 2.1 as a polarizer, and the double aperture shutter DBS is fitted with one or two analyzers. The fitting with one analyzer 13' can be achieved with only one aperture 11 ( Fig. 6a). Alternatively, - as Fig. Figure 6b shows both aperture openings 11 provided with separate analyzers 13' and 13'', whose polarization planes are crossed with respect to each other. In a further embodiment, the central bridge 12 is wholly or partially ( Fig. 6c) designed with an analyzer, or the entire central web consists of two analyzer plates arranged side by side with polarization planes crossed towards each other ( Fig. 6d). The condenser light ring (LR) is to be adjusted so that it overlaps with the central bar of the double aperture slider (DBS). This allows the different imaging possibilities using polarized light to be achieved, and it is advantageous if, in addition to the polarizing filter in the condenser, at least one of the analyzers is also rotatably mounted. In this way, the intensities, and thus the proportions of the bright-field and dark-field images in the summed image, can be changed in very small increments.

[0044] Polarizing optics can also be used to adjust the weighting of the light components for bright-field and dark-field dominated images, even with concentric illumination of the object. For this purpose, the inner zone of the light ring, which illuminates the object and generates the bright-field dominated image, is fitted with a narrow, ring-shaped polarizing filter ( Fig. 7a) Another polarizing filter of standard design is to be placed below the light ring in the illuminating beam path. With the brightness of the outer zone, and thus of the dark-field dominated image, remaining constant, the brightness of the bright-field sub-image can now be continuously varied by adjusting the lower polarizing filter.

[0045] In an alternative configuration, the outer zone of the light ring is also equipped with a separate ring-shaped polarizing filter, which is inserted concentrically to that of the inner zone in such a way that the two polarization planes are crossed with each other ( Fig. 7b), by rotating the polarizing filter located below it, the brightness of both the bright-field and dark-field dominated sub-images can be continuously adjusted in opposite directions. Even more independent control of the brightness of both sub-images is achieved by mounting at least one of the two ring-shaped polarizing filters rotatably within the condenser's light ring. From a technical design perspective, it is advantageous to equip the outer ring-shaped polarizing filter with a continuously adjustable ring mount for easier adjustment. This allows the intensity of the light component for the bright-field dominated sub-image to be adjusted using the polarizing filter located below the condenser's light ring, and subsequently, the intensity of the dark-field image to be continuously adjusted by rotating the outer ring polarizer.This allows for stepless adjustment of the light component's intensity for the dark-field dominated sub-image simply by rotating the outer ring-shaped polarizing filter. If the axially directed illumination light is also used to generate a bright-field dominated image, an alternative development involves inserting a large-area polarizing filter with a diameter extending to the outer edge of the inner zone of the former light ring into the opaque zone of the condenser light ring. Fig. 7c) is used, to which a narrow outer zone is attached, which generates the dark-field dominated partial image. If a second large-area polarizing filter is rotatably arranged below this construction, the brightness of the bright-field dominated partial image can be controlled independently of that of the dark-field dominated partial image.

[0046] Is the microscope - as in Fig. 8 with reference to the beam path of the Fig. As shown in Figure 1, a variable light-dark-field illumination can be achieved in both concentric and eccentric beam paths using a catoptric or catadioptric mirror objective 20 to generate a variable light-dark-field contrast, as described above. In a mirror objective 20, the imaging rays are typically "folded" between the two mirrors 27.1 and 27.2. The light passing through the object is received by the concentrically arranged, concave central mirror 27.1 and reflected onto the concentrically arranged convex secondary mirror 27.2, which directs it via an optional lens system 27 of the mirror objective 20, which also participates in the imaging process, to the plane of the intermediate image ZWB.

[0047] To obtain the light-darkfield illumination, the object 25.1 lying on the slide 25 is illuminated by the illumination light 26 coming from the condenser. The central light passes as a light beam 28.3 onto the opaque back side of the secondary mirror 27.2 of the mirror objective 20 and is absorbed. Thus, this portion of the light cannot contribute to illuminating the image background. Light incident at a less steep angle enters the mirror objective 20 as a light beam 28.1, is reflected by the concave central mirror 27.1 onto the reflective convex side of the secondary mirror 27.2, and is directed from there into the lens system 27. The light scattered by the object 25.1 also enters the central mirror 27.1 as a scattered light beam 28.2, is reflected onto the secondary mirror 27.2, and then passes on to the lens system 27.The bright-field dominated image and the dark-field dominated image are guided via the lens system 27 in the common beam 29 to the intermediate image ZWB, where both images are combined and interfere to form the desired summation image in variable bright-dark-field illumination, which exhibits increased resolution, improved contrast, and enhanced structure rendering. If the outer diameter of the illumination light 26 is kept so small, for example by reducing the opening angle of the light beam coming from the light source using the aperture diaphragm in the condenser, that all illuminating rays are intercepted by the rear face of the small secondary mirror 27.2 after passing through the object, an axial (central) dark-field image can be achieved.Both light beams leave the mirror lens 20 as a common light beam 29 and reach the plane of the intermediate image ZWB, where they superimpose and interfere to produce the summation image, which can then be viewed magnified through the eyepiece in the known manner.

[0048] The in Fig. The control image shown in Figure 8a depicts pure brightfield illumination. The light ring in the condenser is dimensioned so that all components of the beam contribute directly to the illumination of the object. The image in Fig. The control image shown in Figure 8b refers to an axial dark field, which is achieved by keeping the outer diameter of the light ring in the condenser so small that all illuminating rays are intercepted by the rear face of the small secondary mirror after passing through the object. Here, too, the zeroth-order maximum is consequently suppressed, so that a reflection or diffraction image is formed in the dark field in which the zeroth-order maximum is not involved. A concentric variable light-dark field, whose control image the Fig. Figure 8c shows that this is caused by a slightly enlarged outer diameter of the illuminating light ring. If the light ring is slightly off-center, this will be shown in Fig. The control image shown in Figure 8d is obtained. Due to this decentering, only a narrow illuminating light segment can pass through the central mirror, resulting in a bright-dark-field illumination. In this case as well, the two bright-field and dark-field analogous partial images interfere to form a summed image as described above.

[0049] A reflected light illuminator 30, designed for technology with a variable light-dark field, is in Fig.Figure 9 is shown schematically. Analogous to microscopes with transmitted light, in the reflected light illuminator 30 the light coming from the light source 31 is parallelized in the lens system 33, which acts as a collector and condenser. An aperture diaphragm 32 allows the illumination aperture to be adjusted. By means of an illuminator diaphragm 34 with light rings 34.1, the two ring-shaped illuminating light components of the illumination light 36 are blocked and deflected onto the object 35.1 via a semi-transparent mirror 36.1. In this process, the outer ring-shaped beam of the illumination light 36 is directed outside the imaging lens system of the objective lenses 37.1 to generate a concentrically illuminated reflected light dark-field image and is deflected by means of the ring mirror 36.2 in order to strike the object 35.1 arranged on the slide 35 at such an oblique angle that direct light cannot enter the imaging lenses 37.1 of the objective.The portion of light scattered or reflected at object 35.1 creates the dark-field dominated image. The inner ring-shaped beam of the illumination light 36 passes peripherally through the imaging objective lenses 37.1, thus creating a simultaneous bright-field dominated image in the incident light. Both images are directed as a beam 39 through the semi-transparent mirror 36.1 and the tube lens system 37.2 to the intermediate image ZWB, where they interfere to form the summed image. If the illuminating rays are covered or blocked in the incident-light illuminator 30, at its aperture diaphragm 32, or at another freely selectable location, the observation could also be carried out under oblique illumination.

[0050] To separately regulate the illumination intensities of the light components generating a bright field and a dark field, rotatable polarizing filters are integrated at suitable locations in the reflected-light illuminator 30 as polarizers and analyzers. A larger, ring-shaped analyzer is suitable for regulating the outer zone of the illumination light, which generates the dark field, and a smaller analyzer for regulating the inner zone, which generates the bright field. Observations under oblique illumination can also be carried out if the light rings 34.1 are arranged centrally on the outside and only a narrow illuminating light segment reaches the semi-transparent mirror 36.1, resulting in oblique illumination of the object 35.1. Reference symbol list 01 Light source 02 Aperture stop 02.1 Rotatable polarizing filter 03 Condenser optics 04 Light ring carrier 04.1 Light ring 05 Slides 05.1 Object 06 Lighting light 07 Lens 08.1 Transmitting light beam (bright-field dominated image) 08.2 Scattered light beam (dark-field dominated image) 08.3 Light beam 09 common light beam (for the intermediate image) 10 double shutters 11 aperture settings 11 aperture openings (in a circular shape) 11'' apertures (rectangular, outer edges rounded) 12 Middle walkway 13' Analyzer 13'' Analyzers 13' and 13" 20 mirror lens 25 slides 25.1 Object 26 Lighting light 27 lens system 27.1 concave central mirror 27.2 convex secondary mirror 28.1 Light beam 28.2 Scattered light beam 28.3 Light beam 29 Common beam 30 incident light illuminator 31 Light source 32 Aperture stop 33 lens system 34 Illuminator aperture 34.1 Light rings 35 slides 35.1 Object 36 Lighting light 36.1 semi-transparent mirror 36.2 Ring mirror 37.1 Objective lenses, imaging lenses 37.2 Tube lens system 38.1 Lighting component 38.2 Lighting component 39 beams “d” distance “d” DBS double shutter slider LR condenser light ring OAL optical axis OAL (of the light ring 4.1) OAG optical axis OAG (tube and lenses of condenser optics 3) ZWB Intermediate image ZWB

Claims

[1] Method for producing a high-contrast light-dark-field image in a microscope, comprising the following process steps: - Illuminating an object with light from a condenser in such a way that a beam of light penetrates the object in the manner of bright-field illumination, and a second beam of light, after passing through the object, is directed past the lens in the manner of dark-field illumination, - Merging the bright-field-dominated image created by bright-field illumination and the dark-field-dominated image created by scattered light in the manner of dark-field illumination in the plane of the intermediate image; - Generating a summation image by interfering with both superimposed light- and dark-field-dominated images in the intermediate image plane; - Viewing the superposition image thus created as a variable light-dark field image in a manner known per se using an eyepiece, wherein according to the method the illumination light is polarized by means of a polarizer and each light beam can be attenuated with respect to its intensity and thus its share in the summation image by means of an associated analyzer. [2] Method according to claim 1, characterized by , that the light beams are separated before the illumination light passes through the object. [3] Method according to claim 2, characterized by , that the light beams separated before the passage of the object are separated immediately after the aperture diaphragm of the condenser and in front of its lens system by means of a light ring. [4] Method according to claim 3, characterized by, that the projection image of the light ring is adjusted in relation to the optically effective entrance cross-section of the lens, whereby the optically effective diameter of the lens cross-section is further adapted to the geometry of the illuminating beams. [5] Method according to claim 4, characterized by , that the projected image of the light ring is adjusted in relation to the optically effective entrance cross-section of the lens by changing the distance of a condenser lens from the object by raising or lowering it, or by changing the focal length of the condenser lens system designed as a zoom. [6] Method according to claim 4, characterized by , that the projection image of the light ring is adjusted with respect to the optically effective entrance cross-section of the objective by adjusting an aperture diaphragm of the condenser or a diaphragm in the condenser that is preferably arranged to be displaceable at right angles to the optical axis of the microscope. [7] Method according to any one of claims 3 to 6, characterized by , that the ring of light, as is usual with phase contrast, is projected approximately into the rear focal plane of the lens. [8] Method according to any one of claims 1 to 7, characterized by that the light beams are separated after the illumination light has passed through the object. [9] Method according to claim 8, characterized by , that a mirror lens is provided as the lens, whose central secondary mirror with an opaque back prevents the central part of the illumination light from entering the lens. [10] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 9, the illumination apparatus of which is provided with a condenser having a condenser diaphragm having at least one aperture and which is equipped with the objective for generating bright-darkfield illumination, wherein in the condenser an aperture diaphragm (2) is provided an annular diaphragm with a light ring (4.1) arranged in a light ring carrier (4), wherein the light (8.1) passing through the interior of the light ring illuminates the object (5.1) in the manner of brightfield illumination and is received by the objective (7) to the plane of the intermediate image (II) to generate a brightfield-dominated image in the plane of the intermediate image, and wherein the light (8.3) passing through the exterior of the light ring (4.1) is directed at the object (5.1) at such an acute angle as to generate a darkfield illumination.1) is such that this light beam (8.3) after passing through the object outside the lens (7) does not contribute to brightening the image background, while the light scattered and / or reflected at the object (5.1) as a scattered light beam (8.2) reaches the plane of the intermediate image (II) via the lens (7) to generate a dark-field image-dominated image, so that in the plane of the intermediate image the bright-field image-dominated image based on the zeroth diffraction order and the dark-field-dominated image formed without participation of the zeroth-order diffraction maximum interfere superimposed to form the summation image, wherein the opening of the light ring (4.1) is dimensioned in size and width such that one part of the illumination light is projected into the edge region of the optically effective lens cross-section, while the other part lies outside the lens cross-section, . characterized by, that the condenser is equipped with a polarizing filter (2.1) as a polarizer and the outer and / or inner zone of the light ring (4.1) is designed with an annular or a partial annular polarizing filter, with which the polarization plane of each of the light beams of the two light components can be varied independently of the intensity or polarization plane of the other. [11] Microscope according to claim 10, characterized by , that the light ring carrier (4) has several light rings (4.1) and is arranged to be rotatable or displaceable for selectable adjustment, wherein the light rings (4.1) are dimensioned with respect to the size and width of their openings such that their inner zones are projected into the edge region of the optically effective cross-section of the respective lens, while the outer parts of the light rings lie outside this cross-section. [12] Microscope according to claim 11, characterized by, that for moderate oblique illumination the light ring (4.1) can be displaced from its central position perpendicular to the optical axis (OAG) and thus moved to an eccentric position. [13] Microscope according to claim 11, characterized by , that each of the light beams can be individually modified with respect to its color and thus its share of the summation image can be changed in color, and / or that the outer and / or inner zone of the light ring (4.1) is equipped with a ring-shaped or, in each case, a partially ring-shaped spectral filter, with which the color of each of the light beams of the two light components can be varied independently of the intensity or color of the other. [14] Microscope according to any one of claims 10 to 13, characterized by , that a second polarization filter is integrated in the condenser below the polarization filter (2.1) provided in the condenser, wherein both polarization filters are rotatably mounted independently of each other. [15] Microscope according to any one of claims 10 to 14, characterized by , that the incidence of the illumination light can be changed between concentric and eccentric or oblique by relocating the light ring carrier (4) with light ring (4.1) perpendicular to the optical axis (OAG) or by introducing a second light beam based on a double capacitor. [16] Microscope according to claim 15, characterized by , that in the case of oblique illumination, the angle of incidence of the two light beams of the illumination light is aligned and / or can be continuously changed independently of that of the other. [17] Microscope according to any one of claims 10 to 16, characterized by, that the condenser is designed to be height-adjustable for adjusting the exact projection size of the respective light ring to the respective cross-section of the lens (7), wherein preferably the condenser for adjusting the exact projection size of the respective light ring (4.1) to the respective optically effective cross-section of the lens (7) is provided with a lens system designed as a zoom system with variable focal length. [18] Microscope according to any one of claims 10 to 17, characterized by , that in the lens (7) an aperture is provided in or immediately near the rear focal plane, which has at least one, preferably two aperture openings (11) separated by a central bridge (12). [19] Microscope according to claim 18, characterized by , that the aperture is designed as an aperture slide, preferably as a double aperture slide (10), which is displaceable perpendicular to the optical axis (OAG). [20] Microscope according to claim 19, characterized by , that the width of the central bridge (12) between the aperture openings (11) of the aperture slide or double aperture slide (10) is at least equal to the width of the image of the light ring (4.1), covering it. [21] Microscope according to any one of claims 18 to 20, characterized by , that one or more of the aperture openings (11, 11', 11'') of the aperture above the lens (7) each have a polarizing filter as an analyzer, the plane of oscillation of which is continuously variable between crossed and parallel positions relative to that of the polarizing filter (2.1) in the condenser. [22] Microscope according to claim 21, characterized by , that the analyzers in the aperture openings (11) of the double aperture slider (10) are positioned opposite each other and line the inner areas of both aperture openings (11) in such a way that the edge areas remain free, preferably with the polarization planes of both analyzers being independently adjustable. [23] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 9, the illumination apparatus of which is provided with a condenser having a condenser diaphragm and which is equipped with the objective for generating light-dark-field illumination, wherein a catoptric or catadioptric mirror objective (20) is provided for generating variable light-dark-field illumination, wherein the opaque rear face of the central secondary mirror (27.2) in the mirror objective (20) partially blocks the central illumination light (26) after it has passed through the object (25.1) in such a way that this light does not enter the entrance cross-section of the objective (20) and thus does not contribute to brightening the image background, wherein the portion of the illumination light (26) incident at the secondary mirror (27.2) at a not too steep angle is reflected by the central mirror (27.1) reflected to the secondary mirror (27.2) as a bright-field dominated light beam (28.1) together with the light scattered and / or reflected at the object (25.1) as a dark-field dominated scattered light beam (28.2) onto the objective lens system (27) and via this to generate the bright-field dominated image to the plane of the intermediate image (ZWB), . characterized by , that in the plane of the intermediate image the bright-field image-dominated image based on the zeroth diffraction order and the dark-field-dominated image formed without participation of the zeroth-order diffraction maximum interfere superimposed to form the summation image, wherein the light ring is dimensioned according to the size and width of its aperture such that one part of the illumination light (26) is guided past the edge region of the secondary mirror (27.2), while the other part is blocked at the rear front of the secondary mirror (27.2). [24] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 8, the illumination apparatus of which is provided with a condenser having a condenser diaphragm and which is equipped with the objective for generating a bright-dark-field illumination, characterized by, that the microscope, designed as a reflected-light microscope, has an illumination apparatus designed as a reflected-light illuminator, as well as special objectives with imaging lenses (37.1) and illuminating elements for reflected-light darkfield, and that a ring diaphragm (34) with at least two light rings (34.1) is connected downstream of the reflected-light illuminator (30) emitting parallel light to generate ring-shaped limited illumination, wherein the light passing through the inner of the light rings (34.1) as the inner illumination light component (38.1) illuminates the object (35.1) via input lenses (37.1) in the manner of brightfield illumination after deflection by a semi-transparent mirror (36.1), and the light passing through the outer of the light rings (34.1) as the outer illumination light component (38.2) illuminates the object (35.1) past the input lenses (37.1) via a near-object (35.1) provided ring mirror (36.2) illuminate in the manner of dark-field illumination, wherein the light reflected from the object (35.1) to generate a bright-field dominated image and the light scattered from the object (35.1) to generate a dark-field dominated image are directed via an output lens system (37.2) to the plane of the intermediate image (II), and that in the plane of the intermediate image the bright-field dominated image based on the zeroth diffraction order and the dark-field dominated image formed without participation of the zeroth-order diffraction maximum interfere superimposed to form the summation image, wherein the openings of the light rings (34.1) are dimensioned according to their size and width such that one part of the illumination light (26) is projected into the centrally located imaging objective lens system (37.1), while the other part is projected outside this lens system (37.1) passes through the outer zone of the lens, and that the illumination light emanating from the light source (21) is polarized by means of a polarizer, and furthermore two polarization filters acting as analyzers are provided near the semi-transparent mirror (36.1), which are preferably arranged concentrically and rotatably so that the brightness of both the inner and outer illumination light components (38.1, 38.2) can be controlled independently of each other.

Images