Method and apparatus for generating variable phase contrast / dark field illumination

Abstract

Method for generating a high-contrast phase contrast / dark field image in a microscope, characterized by the following process steps: - Illuminate the object with light from the condenser in such a way that a beam of light penetrates the object in the manner of phase contrast illumination and a second beam of light, after passing through the object, is guided past the phase contrast objective in the manner of dark field illumination. - Merging the phase-contrast-dominated image created by phase-contrast illumination and the dark-field-dominated images created by scattered light in the manner of peripheral and axial dark-field illumination in the plane of the intermediate image; - Generating a summation image by interfering with the overlapping partial images (phase contrast, dark field) in the intermediate image plane, the superposition image thus created as a variable phase contrast dark field image can be viewed in a known manner using the eyepiece.

Description

[0001] The invention relates to a method for generating a microscopic image of an object using phase contrast / dark field illumination, wherein the illumination intensities of the phase contrast image and the dark field 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 at least two aperture openings located outside the optical axis and in which the objective is configured to generate both phase contrast illumination and dark field illumination, and in which the phase contrast and dark field images are superimposed.

[0002] The illumination of objects plays a crucial role in microscopy for resolution and image formation. Starting with the well-known Köhler illumination apparatus, 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. This was followed by the development of phase-contrast illumination, in which existing, weak path differences in objects of low optical density are contrasted and visualized using a wave-optical method.From DE 10 2006 027 961 A1, a microscope is known in which improved structural representation of images in phase contrast is achieved by having the light transmission aperture on an annular diaphragm of the diaphragm assembly in the condenser be shaped like a circular sector and by illuminating the object obliquely with a sector-shaped light beam lying on the surface of a hollow cone, wherein the central angle associated with the circular sector is at most 90°. Furthermore, from DE 10 2007 029 814 A1, a microscope is known in which, for better contrast with improved brightness, increased depth of field and increased resolving power, the illumination device has a diaphragm with a central aperture and a peripheral annular slit opening. The object is thus illuminated with a central light beam and a peripheral light beam.After the light beams pass through the object, at least part of either the central or peripheral light beam is attenuated by a light-absorbing element in the lens or immediately behind it. This allows different illumination variants to be seamlessly blended, resulting in image characteristics and information similar to conventional darkfield, phase contrast, interference contrast, and brightfield imaging, and leading to 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 lying 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] To avoid such special additives and eliminate remaining resolution deficiencies, DE 10 2011 002 030 A1 solved the problem of further developing the microscope in such a way that, by superimposing bright-field and dark-field images with variable bright-dark-field illumination, increased resolution, improved contrast, and enhanced visualization of structures with significantly increased depth of field are achieved for artifact-free observation of complex three-dimensional objects. Since the techniques described in DE 10 2011 002 030 A1 involve the interference of bright-field and dark-field images, despite several technical advantages, potentially subtle path differences may be displayed with less contrast than when using conventional phase contrast.

[0004] Further documents are known from the prior art, such as DE 199 49 029 C2, which document a method and a device for characterizing a culture fluid, DE 1 123 134 B, which relates to an interference microscope, and DE 30 28 418 A1, which in particular protects an illumination arrangement for microscopes.

[0005] Therefore, a further task arises to improve a phase contrast image in such a way that existing path differences within a phase object remain fully recognizable, yet typical phase contrast-associated halo artifacts are suppressed as effectively as possible, the lateral resolution of the phase contrast image is increased so that even very small structures at the limit of the respective optical resolution can be recognized as far as possible, the visible depth of field is increased compared to conventional phase contrast, and in the case of additional optically denser areas, possibly also with their own color, which cannot usually be adequately represented in conventional phase contrast, a detailed image reproduction is achieved.

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

[0007] To solve the problem and achieve the desired improvement, a phase-contrast-dominated and a dark-field-dominated sub-image are generated in the intermediate image plane. These images are superimposed and interfere with each other to produce an observable summed image. The phase-contrast-dominated sub-image is generated by illuminating the object with a first light component entering the lens's entrance cross-section. As the illumination light passes through the lens via a phase ring located there, it is phase-delayed relative to the imaging rays in a known manner. The dark-field-dominated sub-image is generated using the scattered light from a second light component that does not enter the lens's entrance cross-section. This second component is either blocked before reaching the object or prevented from entering after reaching it.In the plane of the intermediate image, the zero-order transmitted image of the phase-contrast component is superimposed with a higher-order diffraction pattern of the dark-field component, which interfere to form a summed image. This is then viewed in the usual way with the eyepiece at the desired magnification. While the zero-order maximum is retained in the summed image, the dark-field image, without the zero-order diffraction maximum, also contributes to the overall image formation. In a further development, both the phase-contrast-dominated and the dark-field-dominated sub-images are given different polarization-optical properties using polarization-optical means, so that their brightness can be continuously adjusted by rotating the polarizing filters.When spectral filters are used, the two partial images acquire different colorations, causing certain features to stand out more clearly from the background. In a further development, the respective light apertures in the condenser are equipped with separate and independently adjustable (iris) diaphragms, allowing the area of ​​each aperture to be changed, thus enabling individual regulation of the brightness of the respective beam components.

[0008] To achieve this, a ring diaphragm downstream of the aperture diaphragm, consisting of two concentric elements of different diameters, splits the object-illuminating light into two distinct light components that function optically similarly to phase contrast and dark-field imaging. The object is illuminated by the first light component, which produces a phase-contrast image, in a phase-contrast analogous manner. The second light component, corresponding to the dark-field effect, illuminates the object 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 phase contrast alone or dark-field imaging alone.Additionally, the depth of field is greater with this method than with conventional brightfield, darkfield, phase or interference contrast illumination, because the illumination aperture is reduced and the aperture diaphragm of the condenser can be used to modulate the resulting image.

[0009] When a standard phase-contrast lens is used with a universal condenser constructed analogously to a phase-contrast condenser, the inner condenser light ring that generates the phase-contrast image must be dimensioned so that it behaves optically congruently to the phase ring of the lens. Additionally, a second light ring, significantly larger in diameter, must be dimensioned in terms of size and width such that its inner diameter is just projected outside the optically effective lens cross-section.

[0010] The object is illuminated from all directions in a phase-contrast-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 component generating the dark-field image, can be reduced or narrowed as needed using the aperture diaphragm, thereby influencing the character of the resulting image. The size or area ratio of the inner and outer condenser light rings determines the relative contributions of the phase-contrast and dark-field images to the image formation. The wider the penetrating inner light ring, i.e.,The larger the area of ​​this light ring, the brighter the image background will be; conversely, the narrower the light ring, the more the image can exhibit a dark-field aspect. Depending on the area ratio of the two light rings, different contrast effects can be achieved. Stepless and independent adjustment of the outer and inner diameters of both light rings is advantageous. Such variable-sized and variable-area light rings can also be used as "multifunctional light rings" for lenses with differently sized phase rings and cross-sectional areas, thus eliminating the need for a set of several individually sized ring combinations.

[0011] To achieve eccentric phase-contrast dark-field illumination from defined spatial directions, one or both light rings in the condenser are partially covered in the aforementioned arrangement, so that only narrow segments of the respective light rings contribute to object illumination. The inner condenser light ring, which lies within the optically relevant objective cross-section and behaves congruently with the objective's phase ring, generates the phase-contrast analog image, while the illumination rays of the outer light ring, which extend outside the objective cross-section, produce the dark-field analog image. With this optical arrangement, the proportion of the dark-field component can also be varied using the aperture diaphragm.

[0012] Instead of the previously described arc-shaped condenser light rings, which can be partially covered for oblique illumination, crescent-shaped light slits can also be provided in the condenser for generating phase contrast and dark-field images. The width, length, and position (solid angle) of these slits can be continuously varied. The inner phase-contrast-generating light segment must be precisely aligned with the geometry of the phase ring. If this light ring is rotatable while maintaining congruence with the corresponding phase ring, the direction of light incidence in the phase-contrast image can be further adjusted to the positioning or orientation of the object in space. Similarly, by rotating the outer dark-field-generating light segment, the illuminating dark-field rays can also be adjusted to the object's orientation in space.

[0013] Advantageously, the condenser features an aperture with multiple pairs of aperture openings, which are rotatable or slidable for selectable adjustment. The aperture openings are arranged linearly or annularly and are dimensioned in terms of their size and width such that the inner light ring is projected onto the phase ring of the lens, while the outer light ring lies outside the lens cross-section. Using these apertures, it is possible to provide a suitable concentrically arranged pair of light rings in the condenser for each lens used, regardless of the size of the phase rings or lens cross-sectional areas.

[0014] For advantageous further development, the condenser is designed to be height-adjustable to precisely match the projection size of the respective light rings to the lens cross-section and phase ring. This facilitates the necessary adjustments to the diameters of the lens cross-section, phase ring, and the projection images of the condenser light rings. 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 rings 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 proportion of the dark-field component can also be adjusted using the aperture diaphragm and / or by slight changes to the position or...The eccentricity of the outer light ring can be varied. Using an adjustment eyepiece, the respective adjustment settings can be visually checked for all the aforementioned variants. If the objective lens also has an integrated iris diaphragm in addition to a phase ring, the quality of the dark-field sub-image can be further optimized (further reduction of existing edge flare, sharper contour boundaries, further increase in depth of field).

[0015] If the outer and / or inner light ring of the condenser 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 generating phase contrast and dark field. 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 make structures with different absorption or reflection more clearly visible. Ring-shaped or partially ring-shaped polarizing filters are also provided, which interact with a second rotatable polarizing filter integrated below or directly below the condenser. The ring-shaped or partially ring-shaped polarizing filters are mounted to rotate independently of each other.This allows the brightness of the partial images to be changed independently of each other by rotating them.

[0016] If the microscope is designed as a reflected-light microscope, a reflected-light illuminator and special reflected-light darkfield objectives are used to perform the procedure. These objectives, unlike their usual design, are equipped with an additional phase ring, enabling the generation of a reflected-light-based variable phase-contrast darkfield. For this purpose, the reflected-light illuminator generates two ring-shaped illuminating light components. The outer component of this component 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 reflected-light darkfield image. Simultaneously, the inner component of the illuminating light beam is designed and aligned so that it passes through the imaging objective lenses, is optically superimposed with the phase ring integrated within the objective, and thus produces a simultaneous reflected-light phase-contrast image.

[0017] Under these conditions, phase contrast 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, the 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 concentric 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 optics (analogous to the method already described for transmitted light).

[0018] When additional color double contrasts are implemented in the described methods, the two illuminating beam components that generate the respective phase-contrast 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.

[0019] 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"—even in the case of a light cone with obliquely incident illumination from all sides.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.

[0020] In addition to the previously described designs, all of which are based on two conical illuminating light components or sectors incident obliquely at different angles, one generating the phase contrast image and the other the dark-field image, an axial dark-field image can also be superimposed on a phase contrast image, either additionally or instead of peripheral dark-field illumination. To achieve this, the condenser's light mask is provided with an additional small perforation, preferably located precisely in the center of the optical axis, which allows a narrow illuminating light beam to pass through, preferably axially and perpendicular to the object, and which is congruent with the optical axis.Furthermore, in addition to the phase ring, a small-area light stopper is to be integrated into the rear focal plane of the respective phase contrast objective, preferably centered exactly along the optical axis. The axially directed central illumination light aimed at this light stopper is to be limited in area so that it is blocked as completely as possible by the light stopper located in the rear focal plane of the objective.

[0021] Depending on the aperture diaphragm's opening width, this arrangement yields three different illumination settings, which can be continuously adjusted between them. With the aperture diaphragm fully open, three partial images are superimposed: a dark-field image based on obliquely incident rays from the periphery, a phase-contrast image, and an axial dark-field image. When the aperture diaphragm is closed enough to cover the outer ring of light generating the dark field, a phase-contrast image and an axial dark-field image remain, which interfere with each other. Further narrowing of the aperture diaphragm beyond the respective phase-contrast light ring results in object illumination solely in the axial dark field.

[0022] In analogy to the previously described embodiments with two concentric condenser light rings, the three condenser-side light apertures can also be designed with separate, preferably independently rotatable, polarizing filters when axial darkfield is additionally integrated. Again, an additional rotatable polarizer must be provided below the condenser. In this way, the intensities of all three partial beams can be controlled separately using polarizing optics, regardless of the aperture width. Naturally, in this extended embodiment, all three partial beams can also be filtered in different colors to create additional color contrasts.

[0023] Without polarization filters, separate regulation of the respective illuminating beams can also be achieved using iris diaphragms. This is accomplished by integrating one or two sufficiently small iris diaphragms, centrally located on the optical axis, into the condenser light mask in such a way that they allow stepless adjustment of the cross-section of the central beam associated with the axial dark field and / or the width of the phase-contrast-generating light ring. The iris diaphragms also allow for changes to the outer diameter of existing condenser light rings, as well as the aperture of an optional central light passage within the condenser. Adjustable diameter opaque elements can further enable an increase in the inner diameter of existing light rings, if required.This allows the optically effective objective diameter to be adapted to the geometry of the illuminating beams and the geometry of the light passages to be adapted to the size and position of phase rings and objective-side or objective-near light stoppers.

[0024] Provided that the phase contrast lenses used are equipped as special lenses with an additional iris diaphragm located in their rear focal plane, in analogy to darkfield illumination alone, an influence on the character and quality of the darkfield components can also be exerted in variable phase contrast darkfield by moderate narrowing of this lens iris diaphragm (reduction of darkfield-associated edge flares at boundary structures, moderate increase in contrast and depth of field).

[0025] Oblique illumination from freely definable spatial directions can be generated for both the phase contrast image and the peripheral and axial dark field image if the respective light apertures in the condenser are partially covered.

[0026] Mirror objectives are ideally suited for generating axial darkfield images because they incorporate a centrally located secondary mirror along the optical axis, whose opaque back surface, facing the object, acts as a light barrier. To additionally generate a phase contrast image, a phase ring, for example of the Cassegrain-Schwarzschild type, is preferably positioned directly adjacent to the edge of the secondary mirror within the mirror objective. With such a modified mirror objective, a variable phase contrast darkfield based solely on axial illumination rays can be generated using a simple brightfield condenser, provided the condenser can be sufficiently stopped down.If the illumination light is restricted by an aperture diaphragm such that its peripheral components pass exclusively through the phase ring in the mirror objective, while its central components are blocked by the rear face of the central secondary mirror, a largely axially illuminated phase contrast image interferes with an axial dark-field image. The more the aperture diaphragm is closed, the more the axial dark-field illumination dominates. If the aperture diaphragm is narrowed so that all illumination light strikes only the rear face of the secondary mirror and the phase ring extending outwards in the objective is blocked, the result is illumination solely in axial dark-field.

[0027] Instead of a brightfield condenser, a condenser with a modified light mask can also be used when employing a mirror objective equipped with a phase ring as described above. This condenser contains a small central aperture through whose center the optical axis runs, and which serves to generate the axial darkfield image. Immediately adjacent to this is an annular light aperture, which is optically congruent with the phase ring of the mirror objective and generates the phase contrast image. A third, larger light ring is projected in the beam path outside the optically effective cross-sectional area of ​​the mirror objective and serves to generate peripheral darkfield illumination. Consequently, with this design variant, up to three partial images can be optically superimposed even when using a mirror objective (axial and peripheral darkfield as well as phase contrast). By blocking out or...By covering one of these three light apertures, summation images can also be generated if required, which consist of two partial images (axial dark field plus phase contrast, peripheral dark field plus phase contrast, axial and peripheral dark field).

[0028] If the size of the phase-contrast-generating light ring in the condenser is adjusted, a larger phase ring can also be provided in the corresponding mirror objective in the plane of the central collecting mirror. This allows the paraaxial zone, which is directly adjacent to the outer edge of the central collecting mirror, to be available for imaging rays without a phase ring obstructing them. Even when using mirror objectives equipped with a phase ring, the respective alignment conditions can be checked with a focusing magnifier.

[0029] Naturally, even when using a mirror objective equipped with a phase ring, the intensities of the various illuminating beam components can be regulated with polarizing filters arranged concentrically in the condenser. In this case, a plate polarizer must be placed in the condenser in accordance with the optical axis. This polarizer is optically congruent with the rear face of the collecting mirror in the mirror objective and serves to regulate the brightness of the axial dark-field illumination. Furthermore, a sufficiently wide ring polarizer must be provided adjacent to this polarizer in the condenser. This ring polarizer is optically congruent with the phase ring in the mirror objective and allows for brightness regulation of the phase-contrast illumination. At least the outer ring polarizer should be rotatably mounted. Additionally, as described above, a further polarizer must be placed below the aforementioned polarizers.Below the condenser, another rotatable polarizer can be placed so that the intensities of the axial darkfield and phase contrast illumination can be continuously adjusted independently of each other. It goes without saying that even when using such polarizing filters, in the case of a brightfield condenser, the aperture diaphragm must remain closed at least to the extent that no illumination light outside the phase ring and the adjacent collecting mirror passes through the mirror objective. It also goes without saying that when using a light mask in the condenser, the outer light ring, which generates the peripheral darkfield, can be reduced in width or completely covered as needed by means of the aperture diaphragm, or it can also be fitted with a rotatable ring polarizer so that its transparency can be adjusted by polarization optics as described above.Naturally, in this axial design variant with a mirror lens, the different components of the illumination light, which are assigned to the phase contrast image and the axial or peripheral dark-field image, can also be filtered in different colors. Complex iris diaphragm systems, as described in the exemplary embodiments, can also be used in conjunction with the aforementioned modified mirror lenses.

[0030] If, when using a modified lens objective, the axial rays within the objective are to be preserved for imaging purposes, and at the same time paraaxial 360° dark-field illumination of the object from all spatial directions is to be achieved, an annular light stopper can be provided instead of a disc-shaped light stopper preferably positioned along the optical axis as described above. This annular light stopper is integrated concentrically into the phase plate of the objective and has a diameter smaller than that of the phase ring. In optical consistency with such an annular light stopper, this embodiment requires an additional inner annular light passage instead of a centrally located round aperture in the condenser.Consequently, in this variant, the condenser-side light mask consists of three concentrically arranged ring-shaped light apertures: an outer light ring for generating peripheral dark field, a middle light ring for phase contrast, and an inner light ring for paraaxial concentric dark field.

[0031] The previously described variants of variable phase contrast darkfield illumination, realized in transmitted or incident light using modified lens or mirror objectives, are based, with regard to axial darkfield, on a fixed light-blocking element (rear front of a central collecting mirror or centrally adjusted light stopper in a lens objective), which is preferably located in the center of the respective optical system along the optical axis.

[0032] Further illumination variations with regard to the axial dark-field component can be achieved by mounting the small-area light stopper located in the rear focal plane of a suitably modified lens objective in a way that allows it to be moved horizontally. Such a movement can be implemented in various ways. For example, a transparent slider could be used, which carries the opaque stopper at a suitable location and is inserted into the objective close to, preferably directly below, the phase ring plate. Alternatively, a suitably designed, larger transparent slider can also be inserted into the beam path above the objective, provided the distance to the rear focal plane of the objective remains sufficiently small.An aperture, preferably comprising two closely spaced aperture openings separated by an opaque central bar, can be designed as a slider and inserted into a suitably prepared lens or directly above the lens, in or as close as possible to the rear focal plane of the lens. The incident illumination is completely or partially blocked by the opaque areas of such an aperture; unblocked portions of the illumination can pass through the aperture openings along with the imaging rays, thus illuminating the background or generating an additional bright-field analog image.

[0033] Advantageously, this aperture is designed as a shutter. This arrangement allows the eccentricity of the illumination light to be continuously adjusted even at oblique angles. To achieve this, the light aperture in the condenser that generates the axial or paraaxial dark field must be moved in the same direction as the corresponding light stopper so that it is congruent with the light stopper in the respective target position. Naturally, horizontal adjustability of this condenser light aperture requires technically suitable light masks within the condenser. For example, the central light aperture for axial or paraaxial dark field can be positioned on an eccentric disk, allowing it to be adjusted centrally or eccentrically, independent of the position of the light rings that generate the phase contrast and peripheral dark field.Alternatively, a double condenser can be considered, which contains two separately light-fed components, one of which includes the two light apertures for phase contrast and peripheral dark field, and the other the central diaphragm that can be variably moved horizontally for axial or para-axial oblique dark field.

[0034] If elongated, rectangular or biconcave central struts act as light stops, a length- and width-adjustable and horizontally movable slit aperture can also be provided in the condenser as a central light passage for axial or paraaxial illumination, which must be adapted to the shape and position of the light stop with regard to its size and positioning.

[0035] If the axial rays are to be preserved within the objective lens for imaging purposes, and if paraaxial 360° dark-field illumination of the object from all spatial directions is to be achieved simultaneously, a ring-shaped light stopper can be provided instead of a disc-shaped light stopper positioned along the optical axis. This ring-shaped light stopper is concentric to the phase ring and has a diameter smaller than that of the phase ring. Such a light stopper can also be mounted on a transparent slide, which, depending on its design and size, can be inserted within the objective lens, preferably directly below the phase plate or at least very close to it, or directly above the objective thread into a filter holder present on the microscope.Optically congruent to the respective ring-shaped light stopper, this design variant requires an additional inner ring-shaped light passage instead of a centrally located round passage opening in the condenser.

[0036] It is advisable to visually check the adjustment conditions of the axial or paraaxial condenser light transmission and the corresponding movable light stopper, as well as their complete or partial overlap, using a focusing magnifier.

[0037] Under the outlined conditions, the object is illuminated either in an axial or oblique-paraaxial dark field, with the respective dark field image thus generated being superimposed on a simultaneous phase contrast image and an additionally generated peripheral dark field image. If the respective light stopper, which is movably inserted in or near the objective lens, is only partially aligned with the corresponding illuminating beam, a moderate brightening of the image background results instead of axial or paraaxial-oblique dark field illumination due to partial overlap, along with a simultaneously superimposed bright field-like transmission image, since part of this transmitted light is added to the imaging beam path without passing through the phase ring.The resulting brightfield image is superimposed on the additionally present axial or paraaxial darkfield image, the phase contrast image, and any peripherally illuminated darkfield that may also be generated. Depending on the proportion of transmitted light, the brightness of the aforementioned additional brightfield-like image can be continuously adjusted. If necessary, the cross-section of the illuminating beams can be further restricted by the aperture diaphragm or another horizontally movable diaphragm within the condenser, or parts of the light-transmitting elements in the condenser can be covered, which further influences the quality and character of the resulting summed image.

[0038] In an alternative embodiment, as mentioned, a sufficiently small, movable aperture with at least one, preferably two, aperture openings can also be provided within the lens itself. This aperture, in terms of its design and optical effect, corresponds to the (larger) aperture sliders that, as described above, 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 bar are dimensioned such that the illuminating axial or paraaxial rays are completely or partially blocked at the lens plane, and, if necessary, bright-field-generating partial rays that bypass the light blocking element can pass through the lens together with the other imaging rays.Under these conditions, different contrast effects can also be created with this variant, whereby stepless weighting of the individual lighting components is possible and the eccentricity of the lighting light can also be changed steplessly in the case of oblique light incidence.

[0039] The essence of the invention will be explained by the information contained in the Fig. Examples 1 to 11, shown schematically, are explained in more detail; they show: Fig. 1: Microscope with two differently sized light rings in the condenser with concentric beam path for generating variable phase contrast darkfield illumination; Fig. 2: Exemplary embodiments of suitable double light rings for variable phase contrast darkfield illumination, arranged on slides for insertion into a condenser; Fig. 3: Control image when adjusting a microscope after Fig. 1 with focusing magnifier for concentric phase contrast darkfield (perforated version of the inner light ring for phase contrast illumination), Fig. 4: Polarization-optical further developments of the condenser light rings for concentric phase-contrast dark-field illumination; Fig. 4a: Design of the inner light ring with a polarizer, Fig. 4b: Design of the inner and outer light rings with two concentric polarizers (inner plate and outer ring polarizer) in a cross position, Fig. 5: Microscope with reflected light illuminator and concentric beam path, set up for phase contrast darkfield illumination (schematic). Fig. 6: Microscope with three differently designed light apertures in the condenser with concentric beam path for generating variable phase contrast darkfield illumination, based on axial darkfield, phase contrast and peripheral darkfield, additionally equipped with a special objective which, in addition to a phase ring, contains a small-area light stopper arranged centrally in the rear focal plane. Fig. 7: Exemplary embodiments of suitable double light rings for variable phase contrast darkfield illumination, additionally equipped with a central small-area light aperture for generating axial darkfield, arranged on slides for insertion into a condenser. Fig. 8: Phase ring plate of the special lens from Fig. 6, equipped with phase ring and additional centrally arranged small-area light stopper for generating axial dark field. Fig. 9: Construction sketches for light transmissions for the simultaneous generation of axial and peripheral dark field as well as phase contrast according to Fig. 7, in which the central light aperture for axial dark field and / or the adjacent light ring for generating the phase contrast image are adjustable in width by means of iris diaphragms. Fig. 10: Mirror lens (catoptric system according to Cassegrain-Schwarzschild) with concentric beam path according to Fig. 7, equipped with a phase ring in the immediate vicinity of the inner central mirror. Fig. 10a: Generation of variable axial phase contrast darkfield using an adequately stopped-down brightfield condenser Fig. 10b: Generation of variable phase contrast dark field, based on peripheral and axial dark field as well as phase contrast by means of a condenser light mask, consisting of a centrally arranged small light aperture for axial dark field and two adjoining concentric light rings for phase contrast and peripheral dark field. Fig. 11: Control images of the beam path in a mirror lens equipped with a phase ring, observable through a focusing magnifier Fig. 11a: Adjustment with focusing magnifier of a microscope with a phase-ring equipped mirror objective at a setting for pure brightfield Fig. 11b: Setting for axial dark field Fig. 11c: Setting for concentric phase contrast darkfield illumination. Fig. 12: Slider with light stop or double aperture and center bar for insertion into a lens or filter holder above the lens. Fig. 12a: Transparent slider with flat light stopper Fig. 12b: Double aperture slider with square aperture openings and rectangular center bar Fig. 12c: Double aperture slider with circular aperture openings and biconcave center bar Fig. 12d: Double aperture slider with square aperture openings, rounded outer edges and rectangular center bar Fig. 12e: Transparent slider with ring-shaped light stopper. Fig. 13: Modified phase contrast objective with integrated phase ring on phase plate and insertable slider with light stopper according to Fig. 12a.

[0040] Fig. Figure 1 schematically shows the beam path in the microscope for concentric phase-contrast dark-field illumination, based on peripheral dark-field. From the light source 1, a beam of light passes to the two concentric condenser light rings 2.1 and 2.2 (2.1 for dark-field, 2.2 for phase contrast) located on the condenser-side light mask 2, thus generating two conical illumination beams: an outer dark-field-generating beam 3.1 and an inner phase-contrast-generating beam 3.2. Both light beams reach the condenser optics 4 and strike the object 5. After passing through the objective lenses 6, the illuminating phase-contrast-generating light cone strikes the phase ring 7, which is embedded on a phase ring plate 8 in the rear objective focal plane. The illumination light 3 transmitted through the outer condenser light diaphragm...Light 1 passes completely past the lens 6, so it cannot enter it and therefore does not contribute to background illumination. The light 3.2 transmitted by the inner condenser diaphragm passes through the object 5 and reaches the lens 6, forming the basis for the phase contrast image as light beam 3.2. However, the object 5 is illuminated by all the light originating from the outer condenser light ring, and some of this light is scattered by the object. This scattered light 9.1 enters the lens 6 and forms the basis for the dark-field image. As a combined imaging light beam 9, consisting of a scattered light component 9.1 associated with the dark field and an additional transmitted light component 9.2 associated with the phase contrast after directly passing through the object, the light leaves the lens 6 and, after passing through the phase ring plate 8, enters the plane of the intermediate image 10.There, the phase contrast-dominated image and the dark field-dominated image overlap and, through interference in variable concentric phase contrast / dark field illumination, form the structured summation image with higher resolution and improved depth representation, which can be viewed via the eyepiece 11 with the eye 12.

[0041] How Fig. As shown in Figure 2 using the example of aperture sliders, the condenser apertures for generating variable phase-contrast dark-field illumination are to be designed as a concentrically arranged pair of apertures. The inner light ring 2.2 must be dimensioned so that it optically overlaps completely with the phase ring of the respective objective lens. The diameters of the phase rings used usually differ depending on the objective magnification; therefore, several aperture inserts with differently dimensioned inner light rings are required to adapt to the respective phase rings of the different objectives. The outer light ring 2.1 must be dimensioned so that all light emanating from it passes by the objective lens, thus enabling the simultaneous generation of a dark-field image.Since only light scattered by the object contributes to image formation in darkfield spectroscopy, whereas in phase-contrast illumination the light passing directly through the object is image-forming, the brightness of a darkfield image is significantly lower than that of a phase-contrast image at a constant light source intensity. Therefore, when optically designing suitable light ring pairs for generating balanced phase-contrast and darkfield images, it is essential to ensure that the necessary brightness adjustment between phase-contrast and darkfield illumination is achieved. This can be accomplished either by keeping the inner light ring 2.2 sufficiently narrow, thus limiting its total area, or by constructing this light transmission ring from several small perforations arranged on a circular arc. Narrow circular light transmission rings 2.2 are described in... Fig. 2 a and c shown ( Fig. 2 a for 10x and 16x, Fig. 2 c for 25x and 40x magnifying phase contrast objectives). Analogous designs, in which the inner light ring 2.2 consists of small-area, circularly arc-shaped perforations, show the Fig. 2 b and d.

[0042] Fig. Figure 3 shows the correct adjustment of the condenser light rings shown when observed through a phase-contrast focusing magnifier (focusing eyepiece for adjustment check). The inner phase-contrast-generating light ring 2.2 must be in alignment with the phase ring 7 of the objective lens, whether it is in a continuous circular shape ( Fig. 3a) or in perforations ( Fig. 3b) arranged. The outer light ring 2.1 must in any case be projected outside the objective cross-section so that it remains invisible when looking through the focusing eyepiece.

[0043] Polarizing optics can also be used to adjust the weighting of the light components for phase-contrast-dominated and dark-field-dominated images ( Fig. 4) For this purpose, the inner light ring 2.2, which illuminates the object and generates the phase-contrast-dominated image, is equipped with a relatively small, plate-shaped polarizing filter 13 ( Fig. 4a). Alternatively, a ring-shaped polarizer can be used, provided it completely covers the inner light ring. Another polarizing filter 14 of conventional design is rotatably positioned further down in the illuminating beam path. With the brightness of the outer light ring, and thus of the dark-field dominated image, remaining constant, the brightness of the phase-contrast sub-image can now be continuously varied by adjusting the lower polarizing filter 14. No analyzer is located above the condenser-side double-ring aperture 2, which generates the illumination light for the two phase-contrast and dark-field analog sub-images. This ensures that birefringent structures can be contrasted in the same way as non-birefringent objects.

[0044] In an alternative embodiment, the outer light ring 2.1 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. 4b), by rotating the polarizing filter located below it, the brightness of both the phase-contrast and the dark-field-dominated sub-images can be continuously and oppositely adjusted. Even more independent control of the brightness of both sub-images is achieved by mounting at least one of the two ring- or plate-shaped polarizing filters rotatably within the double light ring of the condenser. From a technical and design perspective, it is advantageous to equip the outer ring-shaped polarizing filter of light ring 2.1 with a continuously adjustable ring mount for easier adjustment. This allows the intensity of the light component for the phase-contrast-dominated sub-image to be adjusted using the rotatable polarizing filter located below the condenser, and subsequently, the intensity of the dark-field image to be continuously adjusted by rotating the outer ring polarizer.This opens up the possibility of stepless adjustment of the intensity of the light component for the dark-field dominated sub-image by simply rotating the outer ring-shaped polarizing filter, as well as stepless brightness adjustment of both sub-images.

[0045] A reflected light illuminator designed for the technique with a variable phase contrast dark field is in Fig. Figure 5 is shown schematically. Analogous to microscopes with transmitted light, in the reflected-light illuminator 15 the light coming from the light source 16 is parallelized in the downstream lens system 17, which acts as a collector and condenser. An optional aperture diaphragm 18 allows the illumination aperture to be adjusted. By means of an insertable double diaphragm slider 19 with inner and outer light rings, the two ring-shaped illuminating light components 20 and 21 of the illumination light are blocked and deflected onto the object 25 via a semi-transparent mirror 22. The outer ring-shaped beam of the illumination light 20 is directed outside the imaging lens system 23.1 of the objective lenses 23 to generate a concentrically illuminated reflected-light dark-field image and, after passing through further optional ring-shaped objective-side condenser lenses 23, which serve to further focus the illumination light.The light is deflected by means of the objective-side ring mirror 24 to strike object 25 at such an oblique angle that direct light cannot enter the imaging lenses 23.1 of the objective 26. The portion of light scattered or reflected by object 25 creates the dark-field dominated image. The inner ring-shaped beam 21 of the illumination light passes peripherally through the imaging objective lenses 23.1, is congruent with the phase ring 27 of the objective 26, and thus creates a simultaneous phase-contrast dominated image in the incident light. Both images are guided by the semi-transparent mirror 22 and the tube lens system 28 as a beam 29 to the intermediate image (not shown here), where they interfere to form the summation image. If the illuminating rays in the reflected light illuminator are covered at its optional aperture diaphragm 18 or at another freely selectable location, orIf the light is hidden, the observation can also be carried out under oblique lighting.

[0046] To separately regulate the illumination intensities of the light components generating phase contrast and dark field, rotatable polarizing filters are integrated into the reflected light illuminator at suitable locations, acting 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 phase contrast. A rotatable polarizer is placed upstream of both analyzers. Observations under oblique illumination are also possible if the light rings are designed in a circular sector or crescent shape, allowing only narrow illuminating light segments to reach the semi-transparent mirror 22, thus resulting in oblique illumination of the object.

[0047] Fig. Figure 6 schematically shows the beam path in the microscope for concentric phase-contrast dark-field illumination, based on axial and peripheral dark-field as well as phase contrast. From the light source 1, a beam of light passes to the three concentric condenser light apertures 2.1, 2.2 and 2.3 located on the condenser-side light mask 2 (2.1 for peripheral dark-field, 2.2 for phase contrast, 2.3 for axial dark-field), so that three separate illumination beams are generated: an outer beam 3.1 generating peripheral dark-field illumination, an inner beam 3.2 generating phase-contrast illumination, and an axial beam 3.3 generating axial dark-field illumination. All light beams reach the condenser optics 4 and strike the object 5. After passing through the objective lenses 6, the illuminating phase contrast-generating light cone strikes the phase ring 7.1, which is embedded on a phase ring plate 8 in the rear focal plane of the objective lens.The illumination light 3.1 transmitted through the outer condenser aperture completely bypasses the lens 6, so it cannot enter it and therefore does not contribute to background brightening. The illumination light 3.3 from the axial light aperture strikes the light stop 7.2 located in the rear focal plane of the lens at the center of the phase plate 8 and is completely blocked by it, so this light also does not contribute to background brightening. The light transmitted through the inner condenser light ring passes through the object 5 and reaches the lens 6, forming the light beam 3.2, which constitutes the basis for the phase contrast image. However, the total amount of light from the outer condenser light ring and the axial light aperture illuminates the object 5 in such a way that some of this light is scattered by the object.This scattered light 9.1, resulting from the peripheral and axial darkfield illumination, enters the objective 6 and forms the basis for the darkfield image. As a combined imaging light beam 9, consisting of a scattered light component 9.1 associated with the darkfield images and an additional light component 9.2 associated with the phase contrast after passing directly through the object, the light exits the objective 6 and, after passing the phase ring plate 8, enters the plane of the intermediate image 10. There, the phase contrast-dominated image and the two images dominated by axial and peripheral darkfield, respectively, are superimposed and, through interference in variable concentric phase contrast / darkfield illumination, form the structured summation image with higher resolution and improved depth representation, which can be viewed with the eye 12 via the eyepiece 11.

[0048] Fig. Figure 7 shows modified aperture sliders according to Fig. 2, in which, in addition to the two concentric light rings 2.1 and 2.2 required for phase contrast and peripheral dark field, a centrally arranged light aperture 2.3 is provided in the course of the optical axis, which according to the beam path diagram of Fig. 6 serves to generate axial darkfield. Consequently, peripheral darkfield illumination is generated by light from the annular aperture 2.1, phase contrast by illumination light from the annular aperture 2.2, and axial darkfield by light from the central aperture 2.3.

[0049] Fig. Figure 8 illustrates the path of the illuminating beams in the area of ​​the phase plate of a special lens according to Fig. 6, which is designed for phase contrast and simultaneous axial darkfield illumination. The phase contrast-generating conical illumination light 3.2 penetrates the phase ring 7.1; the axially illuminating small-area central beam 3.3, which originates from the central light aperture of the condenser, strikes the light stopper 7.2, is completely prevented from passing through further by it, and generates the axial darkfield illumination.

[0050] Various embodiments of double aperture systems, which serve to separately regulate the illumination intensities of phase contrast and axial dark field, are described in Fig. Figure 9 shows that the centrally located light aperture 2.3, situated along the optical axis, for generating the axial dark-field image, can be adjusted in diameter by means of a small iris diaphragm. The outer light ring 2.2, for generating the phase-contrast image, can be of constant width if a fixed light mask is attached to the inner iris diaphragm, the inner diameter of which corresponds to the outer diameter of the central iris diaphragm. Fig. 9a). Conversely, the central light aperture 2.3 for generating axial dark-field illumination can be of constant size, while the outer ring-shaped light aperture 2.2 for phase-contrast illumination can be adjusted in width by means of an iris diaphragm ( Fig. 9b). By combining both in the Fig. The iris diaphragms shown in 9 a and b result in a double diaphragm system in which both the central light transmission for axial dark field 2.3 and the light ring 2.2 required for phase contrast are continuously adjustable with respect to the respective aperture areas by means of iris diaphragms ( Fig. 9c).

[0051] In principle, all the design variants shown can be configured according to the Fig. 9 ac the respective outer light rings 2.1, which serve to generate a peripheral dark-field image, are narrowed by means of the aperture diaphragm if necessary. Naturally, instead of the aperture diaphragm, a similarly functioning third iris diaphragm can be added to the outer edge of the respective light mask, so that instead of the one described in the Fig. 9 ac, shown double aperture systems triple aperture systems with a third outer iris aperture are created.

[0052] Is the microscope - as in Fig. As shown in Figure 10, a variable phase-darkfield illumination can be achieved by equipping the object with a catoptric or catadioptric mirror objective 30 to generate a variable phase-darkfield contrast. In a mirror objective 30, the imaging rays 9 and the background-illuminating rays 3.2 associated with the phase contrast are typically "folded" between the two mirrors 31.1 and 31.2. The light 3.2 passing through the object and generating a phase-contrast image is received by the concentrically arranged, concave central mirror 31.1 and reflected onto the concentrically arranged convex secondary mirror 31.2, which directs it via a light aperture 33 located in the center of the mirror 31.1 to the plane of the intermediate image or to the eyepiece.The imaging rays 9, which are assigned to the phase contrast and dark field-based partial images, take the same path.

[0053] To obtain phase-contrast dark-field illumination, the illumination light 3 from the condenser shines through the object 5, which lies on the slide 35 under the coverslip 36 at the intersection of the illumination rays. The central light passes as a light beam 3.3 onto the opaque back of the secondary mirror 31.2 of the mirror objective 30 and is captured. Thus, this portion of the light cannot contribute to illuminating the image background. Light rays 3.2, which are incident at a less steep angle, enter the mirror objective 30 as a light beam 3.2, pass through the phase ring 32 located there, are reflected by the concave central mirror 31.1 onto the reflective convex side of the secondary mirror 31.2, and are directed by it into the light aperture 33. The light scattered by the object also enters the central mirror 31.1 as a scattered light beam 9, is reflected onto the secondary mirror 31.2 and then passes to the light aperture 33.The phase-contrast-based image and the axial dark-field-based image are guided to the intermediate image via the light aperture 33. There, both images are combined and interfere to form the desired summation image under variable phase-contrast dark-field illumination, which offers increased resolution, improved contrast, and enhanced structure rendering. If the outer diameter of the illumination light 3.3 is kept so small, for example by reducing the opening angle of the light beam coming from the light source 1 using the aperture diaphragm 34 in the condenser, that all illuminating rays are intercepted by the rear face of the small central mirror 31.2 after passing through the object, an axial (central) dark-field image can be achieved. The light beams 9 and 3.2 leave the mirror lens and reach the plane of the intermediate image, where they superimpose and interfere to produce the summation image, which can then be viewed magnified through the eyepiece in the usual way.

[0054] If a three-component light mask 2 is used in the condenser, which, in addition to the apertures for axial dark field 2.3 and phase contrast 2.2, also contains a further concentric light ring 2.1, whose light 3.1 is guided past the aperture of the mirror objective, a peripherally illuminated dark field image can also be superimposed.

[0055] As in Fig. As shown in Figure 10a, a largely axially illuminated variable phase contrast darkfield image can consequently be produced when using a mirror objective 30 equipped with a phase ring, if the illumination light is guided through a commercially available brightfield condenser and limited by means of the aperture diaphragm 34 to such an extent that the outer parts 3.2 of the illuminating light cone pass exclusively through the phase ring 32, while the inner parts 3.3 of the light cone are intercepted by the rear face of the central mirror 31.2.

[0056] According to the in Fig. In the arrangement shown in 10b, a modified condenser with a light mask 2 according to the construction examples of can alternatively be used instead of a bright-field condenser. Fig. 6, Fig. 7 or Fig. 9. In this case, the three differently illuminating partial beams 3.1, 3.2, and 3.3 are separated from each other by the existing light apertures 2.1, 2.2, and 2.3. The width of the outer light ring 2.1, which generates a peripherally illuminated dark-field image, can be reduced by means of the aperture diaphragm 34 if necessary. Provided the light apertures for phase contrast (2.2) and axial dark-field illumination (2.3) are equipped with adjustable iris diaphragms as shown in the examples from Fig. Since 9 are equipped with this, the width of these openings can also be varied by the respective apertures. In analogy to the ones in Fig. In the embodiments shown in 4, the three apertures on the light mask 2 can also be designed with rotatable polarizers, so that, with the integration of an additional rotatable polarization filter below the condenser, the intensities of all three partial beams can be continuously adjusted independently of each other using polarization-optical means.

[0057] Starting from Fig. 10b, the inner diameter of the phase ring 32 can also be enlarged to such an extent that a free space remains between the outer edge of the centrally arranged collecting mirror 31.2 and the inner edge of the phase ring 32, which is not covered by the phase ring. Naturally, the size of the associated phase-contrast-generating condenser light ring 2.2 must be adapted to the size of the corresponding phase ring 32. In this embodiment, imaging rays can pass through the mirror objective directly next to the collecting mirror 31.2, i.e., as close as possible to the optical axis, without touching the phase ring.

[0058] Fig. Figure 11 shows typical control views of the beam path in a mirror lens designed for phase contrast according to Fig. 10a, as they can be observed in a phase-contrast focusing magnifier. This in Fig. The control image shown in Figure 11a depicts pure brightfield illumination. In this case, the light transmission in the condenser is dimensioned so large, or the aperture diaphragm 34 is opened so wide, that all components of the radiation contribute directly to the illumination of the object. The image in Fig. The control image shown in Figure 11b refers to an axial dark field, which is achieved by keeping the cross-section of the illumination light 3.2 and 3.3 so small by adequately closing the aperture diaphragm 34 in the condenser that all illuminating rays are intercepted by the rear face of the small secondary mirror 31.2 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 phase-contrast dark-field illumination, whose control image the Fig. Figure 11c shows that this effect arises from a slightly enlarged outer diameter of the illuminating light ring or light beam. In this case, the two phase-contrast and dark-field analogue partial images interfere to form a summed image as described above.

[0059] Fig. Figure 12 shows exemplary embodiments of aperture sliders (37) suitable for generating an axially or paraaxially variable dark-field image with lens objectives and which, depending on their dimensions and lens design, can be inserted either into the lens itself or directly above the lens into the beam path. Such aperture sliders can, for example, be designed as transparent sliders with a small-area light stopper or as double aperture sliders. Fig. Figure 12a shows a transparent slider (37) with light stopper (38), Fig. 12b a double aperture slider (37) with aperture openings (39) that are rectangular in shape and separated by a central bar (40). The double aperture slider of the Fig. 12c has circular aperture openings (39') separated by a central bridge (40'). Finally, it shows Fig. 12d a double aperture slider (37) with aperture openings (39'') in a rectangular shape, the outer edges of which are rounded. These openings are also separated by a central bar (40). Fig. 12e presents a transparent slide (37) with an annular light stopper 38', which is projected concentrically to the phase ring in the beam path and is optically congruent with a suitably dimensioned separate annular light aperture in the condenser.

[0060] These designs are particularly advantageous when the microscope has a filter holder above the objective lens that accommodates a double diaphragm slider (37) with at least one, preferably two, closely spaced apertures (39, 39', 39'') separated by a central bar (40 or 40'). By designing the double diaphragm slider as a slidably arranged element, its position can be precisely aligned with the position of the corresponding light aperture in the condenser. The double diaphragm slider is positioned above the objective lens, in or near the rear focal plane of the objective lens, and is inserted into the beam path such that the central bar between the two apertures completely or partially covers the passing beam of light. The imaging rays of the scattered light beam pass through the apertures of the double diaphragm slider.If its apertures are circular, different contrast effects can be advantageously generated by moving them, whereby an additional bright-field-like image can be added in the case of partial overlap of the light stopper and the associated illumination light. In the plane of the intermediate image, the resulting partial images (phase contrast, peripheral and axial / paraaxial dark field, optional bright field) are superimposed to form the respective summed image. The eccentricity of the illumination light can also be continuously varied when the axial or paraaxial light beam is incident at an oblique angle.

[0061] Fig. Figure 13 schematically shows a specially modified lens objective (41) which, in addition to a phase plate (8) with phase ring (7), has an insertable slider (37) which, according to embodiments of Fig. 12 is designed either as a transparent slide (37) with a suitably applied small-area light transmission (38) (this design type is used in Fig. 13 shown), or as a double aperture slide (37) with an opaque central bar (40 or 40'). The degree of eccentricity of the respective light stopper results from the insertion depth of the slider. Complete or partial congruence of the light stopper with the corresponding condenser-side light transmission must be ensured and can be checked using a focusing magnifier.

[0062] In the arrangement shown here, illuminating rays 3.1 pass through the outer light ring 2.1, bypass the objective 41, and illuminate the object 5 in the manner of peripheral dark-field illumination. Through the inner light ring 2.2, the illuminating beams 3.2 pass through the object 5, pass the phase ring 7 on the phase ring plate 8 in the objective 41, and, if the light ring 2.2 and phase ring 7 are congruent, generate a phase contrast image. A narrowly defined illuminating beam 3.3 passes through the central light aperture 2.3 at right angles in the optical axis through the object 5 and the objective lenses 6 to the light stop 38 located in the objective on an insertable slide 37. Provided this axial illumination light 3.If the light stopper 38 completely covers the object 5, an axial darkfield illumination results; if the light stopper 38 is slightly displaced from its position, a small portion of the illuminating beam 3.3 can bypass the light stopper 38, moderately brightening the image background and generating an additional brightfield-analogous partial image. The imaging rays generated by scattering at the object 5, indicated as light beam 9, reach the intermediate image plane (IIP) together with the background rays 3.2. In this way, a phase-contrast image interferes with an axial and a peripheral darkfield image, and an optional brightfield image is generated in addition—in the case of slight decentering of the light stopper 38. Depending on the aperture diaphragm 34, the peripherally propagating light beam 2 can...1 can be successively narrowed or completely blocked, and with further narrowing of the aperture diaphragm 34, the phase contrast-generating light beam 2.2 can also be continuously narrowed or completely blocked in the same way, so that in the latter case, with complete blocking of the light beams 2.1 and 2.2, only axial dark field or light-dark field illumination remains. Reference symbol list 1 light source 2 Light mask with multiple light apertures in the condenser 2.1 Light ring for peripheral darkfield illumination 2.2 Light ring for phase contrast illumination 2.3 Central light transmission for axial dark field 3 Illuminating beams 3.1 Illumination beams for peripheral dark field 3.2 Illumination beams for phase contrast 3.3 Centrally oriented illumination beam for axial dark field 4 Condenser lens 5 Object on slide 6 lens 7 Phase ring 7.1 Phase ring 7.2 Central light stopper 8-phase plate 9 Common light beam (for the intermediate image) 9.1 Stray light component (associated with dark field) 9.2 Transillumination component (associated with phase contrast) 10 Intermediate image 11 Eyepiece 12th eye 13 polarizing filters (to cover the inner light passages) 14 Rotatable polarizer (mount below 13) 15 incident light illuminator 16 light sources 17 lens system 18 Illuminator aperture 19 light rings for reflected light phase contrast and darkfield on a slider 20 Illumination light component for reflected light dark field 21 Illumination light component for incident light phase contrast 22 semi-transparent mirrors 23 objective lenses 23.1 Imaging (image-forming) objective lenses 23.2 Ring-shaped objective lenses that focus (illuminate) the dark-field illumination light 24 ring mirrors 25 objects 26 lens 27 Phase plate with phase ring 28 Tube lens system 29 Imaging rays (summation image, consisting of incident light phase contrast and dark field) 30 Mirror lens (example shown: Cassegrain-Schwarzschild catoptric system) 31.1 Concave central mirror (main mirror) 31.2 Convex secondary mirror 32-phase ring 33 Light transmission to intermediate image plane and eyepiece 34 Aperture stop 35 slides 36 Cover glass 37 aperture sliders for insertion into lenses or into a filter holder located above the lens 38 flat light stoppers on sliders 38' ring-shaped light stopper on slider 39 rectangular or square apertures 39' circular apertures 39'' rectangular apertures with rounded outer edge 40 Central walkway, rectangularly bordered 40' central walkway, biconcave limited 41 modified phase contrast lens with slider ZBE Intermediate image plane

Claims

[1] Method for generating a high-contrast phase contrast / dark-field image in a microscope, characterized by the following procedural steps: - Illuminate the object with light from the condenser in such a way that a beam of light penetrates the object in the manner of phase contrast illumination and a second beam of light, after passing through the object, is guided past the phase contrast objective in the manner of dark field illumination. - Merging the phase-contrast-dominated image created by phase-contrast illumination and the dark-field-dominated images created by scattered light in the manner of peripheral and axial dark-field illumination in the plane of the intermediate image; - Generating a summation image by interfering with the overlapping partial images (phase contrast, dark field) in the intermediate image plane, the superposition image thus created as a variable phase contrast dark field image can be viewed in a known manner using the eyepiece. [2] Method according to claim 1, characterized by , that the object is illuminated with light from the condenser in such a way that a third illuminating planar or conical central beam of light running in or near the optical axis penetrates the object at right angles or almost at right angles and, after passing through the object in the objective, is completely or partially covered by a light stop located there, so that additional axial darkfield illumination and, optionally in the case of partial coverage, moderate background brightening results from simultaneous brightfield-like illumination. [3] Method according to any one of the preceding claims, characterized by, that the light beams are separated before the illumination light passes through the object. [4] The method of claim 3, insofar as it relates to claim 1, characterized by , that the projection images of the light transmissions, as is usual with phase contrast, are projected approximately into the rear focal plane of the lens and are adjusted with respect to the optically effective entrance cross-section of the lens as well as the size and position of the phase ring. [5] The method of claim 3, insofar as it relates to claim 2, characterized by , that the projection images of the light transmissions, as is usual with phase contrast, are projected approximately into the rear focal plane of the lens and are adjusted with respect to the optically effective entrance cross-section of the lens as well as the size and position of the phase ring and the centrally mounted light stopper. [6] Method according to claim 4 or 5, characterized by, that to adjust the projection images of the light transmissions, the distance of the condenser lens from the object is changed by raising or lowering it, or the focal length of the condenser lens system designed as a zoom. [7] Method according to claim 4 or 5, characterized by , that to adjust the projection images of the light transmissions, the aperture diaphragm of the condenser and, if applicable, additional adjustable multiple aperture systems present in the condenser are adjusted. [8] Method according to any one of the preceding claims, characterized by that the light beams are separated after the illumination light has passed through the object. [9] Method according to any one of the preceding claims, characterized by , that 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 of the summation image by means of an associated analyzer. [10] Method according to any one of the preceding claims, characterized by , that each of the light beams can be individually changed in terms of its color and thus its share of the summation image can be changed in color. [11] Method according to any one of the preceding claims, characterized by , that for moderate oblique lighting the light passages (2.1, 2.2 and possibly 2.3) are partially covered. [12] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 11, the illumination apparatus of which is provided with a condenser with a condenser diaphragm having an aperture and which is provided with the objective for generating phase contrast darkfield illumination, characterized by, that in the condenser of the aperture diaphragm (34) an annular diaphragm with at least two light rings (2.1 and 2.2) arranged in a light ring carrier (2) is provided, wherein the light (3.2) passing through the inner light ring 2.2 illuminates the object (5) in the manner of phase contrast illumination and is received by the lens (6) to the plane of the intermediate image (10) to generate a phase contrast-dominated image in the plane of the intermediate image, and wherein the light (3.1) passing through the outer light ring (2.1) is directed to the object (5) at such an acute angle in the manner of dark-field illumination that this light beam (3.1) does not contribute to the brightening of the image background after passing through the object outside the lens (6), while the light scattered or reflected at the object (5) is formed as a scattered light beam (9.1) to generate a dark-field image-dominated image via the objective (6) to the plane of the intermediate image (10), so that in the plane of the intermediate image the phase-contrast-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 (2.1 and 2.2) are dimensioned in size and width such that one part of the illumination light is projected outside the optically effective objective cross-section, while the other part is congruent with the phase ring of the objective. [13] Microscope according to claim 12, characterized by, that a light ring carrier has several light masks (2) with each matched light transmissions (light rings 2.1 and 2.2, optionally central aperture 2.3) and is arranged rotatably or slidably in the condenser for selectable adjustment, wherein the light rings (2.1 and 2.2) are dimensioned with respect to the size and width of their apertures such that the inner light ring is projected into the phase ring of the lens, while the outer light ring lies outside the lens cross-section. [14] Microscope according to claim 12 or 13, characterized by, that in the center of the respective light mask an additional planar or ring-shaped light aperture 2.3 is arranged to generate an axial or paraaxial dark field, and that the illuminating beam of light passed through it, which runs axially or paraaxially, is completely or partially covered by an optically congruent light stopper (7.2 or 38, 38', 40, 40') arranged in the objective or directly above the objective. [15] Microscope according to claim 12, 13 or 14, characterized by , that to separate the light beam before the passage of the object, light rings or differently shaped separate light passages are arranged immediately after or before the aperture diaphragm of the condenser and in front of its lens system. [16] Microscope according to any one of claims 12 to 15, characterized by, that the outer and / or inner light ring (2.1 and / or 2.2) as well as the optional axial light transmission 2.3 are designed with an annular or with a partial annular or planar spectral filter, with which the color of each of the light beams of the illuminating light components can be varied independently of the intensity or color of the others. [17] Microscope according to any one of claims 12 to 16, characterized by , that the condenser is equipped with a polarization filter (13) as a polarizer. [18] Microscope according to claim 17, characterized by , that a second polarization filter (14) is integrated in the condenser below the polarization filter (13) provided in the condenser, wherein both polarization filters are rotatably mounted independently of each other. [19] Microscope according to claim 17 or 18, characterized by, that the outer light ring (2.1) is additionally equipped with an annular or a partial annular polarizing filter, so that 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. [20] Microscope according to any one of claims 12 to 19, characterized by , that in order to change the angle of incidence of the illumination light, the light passages can be partially covered, alternating between concentric and eccentric or oblique positions. [21] Microscope according to any one of claims 12 to 20, characterized by, that in order to adjust the exact projection size of the respective light ring to the respective cross-section of the lens (6) as well as the size and position of the phase ring (7 or 7.1) and to adjust the projection area of ​​the optional central light aperture (2.3) to the size of the optional light stops (7.2, 38, 38', 40, 40') the condenser is designed to be height-adjustable, wherein preferably the condenser is provided with a lens system designed as a zoom system with variable focal length for adjusting the exact projection sizes of the respective light apertures (2.1, 2.2 and 2.3) to the respective optically effective cross-section of the lens (6) as well as to the size and position of the phase ring (7 or 7.1) and light stops (7.2, 38, 38', 40, 40'). [22] Microscope according to any one of claims 12 to 21, characterized by, that to adjust the exact projection size of the respective light ring to the respective cross-section of the lens (6) as well as the size and position of phase ring (7 or 7.1) the condenser is provided with a lens system designed as a zoom system with variable focal length. [23] Microscope according to any one of claims 12 to 22, characterized by , that in the lens (6) an iris diaphragm is provided in or immediately near its rear focal plane, with which the lens aperture can be varied. [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 11, the illumination apparatus of which is provided with a condenser with a condenser diaphragm having an aperture and which is provided with the objective for generating phase contrast darkfield illumination, characterized by, that as objective (6) a catoptric or catadioptric mirror objective (30) with a central secondary mirror (31.2) is provided, the opaque rear face of which partially blocks the central illumination light after passing through the object (5) embedded between the slide (34) and the coverslip (35) in such a way that this light does not enter the entrance cross-section of the objective (30) and thus does not contribute to the brightening of the image background, wherein at the secondary mirror (31.2) the portion of the illumination light incident at a not too steep angle penetrates the phase ring (32), is reflected from the central mirror (31.1) to the secondary mirror (31.2) as a phase-contrast-dominated light beam (3.2) together with the light scattered at the object (5) orreflected light as a dark-field dominated light beam (9) is reflected onto the objective-side light transmission (33) and passes through it to the plane of the intermediate image to generate the phase-contrast dominated image, and that in the plane of the intermediate image the phase-contrast 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 condenser-side light transmission and / or the aperture diaphragm are dimensioned in size and width such that one part of the illumination light is directed past the edge region of the secondary mirror (31.2), while the other part is blocked at the rear front of the secondary mirror (31.2). [25] Microscope according to claim 24, characterized by, that the central secondary mirror of the mirror lens is provided with a phase ring attached to it, wherein the back of the secondary mirror is opaque to trap the central part of the illumination light. [26] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 11, the illumination apparatus of which is provided with a condenser having a condenser diaphragm and which is provided with the objective for generating phase contrast darkfield illumination, characterized by, that a reflected-light lens (26) with an illumination apparatus designed as a reflected-light illuminator (15) is provided as the lens, wherein the reflected-light lens (26) has imaging lenses and illuminating elements for reflected-light darkfield, and that an insertable ring diaphragm slider (19) with at least two diaphragm rings is connected downstream of the reflected-light illuminator (15) emitting parallel light to generate ring-shaped limited illumination light, wherein the light passing through the inner of the light rings as the inner illumination light component (21) penetrates the phase ring (27) after deflection by a semi-transparent mirror (22),The object (25) is illuminated via the input lenses in a phase-contrast manner, and the light passing through the outer of the light rings as the outer illumination light component (20) is deflected by the semi-transparent mirror (22) and illuminates the object (25) past the input lenses via a ring mirror (24) located near the object (25) in a dark-field manner, and the light reflected from the object (25) to produce a phase-contrast-dominated image, as well as the light scattered from the object (25) to produce a dark-field-dominated image, as imaging light (29) via the output lens system (28) to the plane of the intermediate image and to the eyepiece, and in the plane of the intermediate image, the phase-contrast-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 two light rings are dimensioned in terms of their size and width such that one part of the illumination light is projected congruently with the phase ring into the centrally located imaging objective lens system, while the other part passes outside this lens system through the outer zone of the lens. [27] Microscope according to claim 26, characterized by , that the reflected light illuminator (15) is provided with a polarizing filter in the area of ​​the light source (16) and two polarizing filters acting as analyzers near the semi-transparent mirror (22), which are preferably rotatably arranged so that the brightness of both the inner and outer illumination light components (20 and 21) can be controlled independently of each other. [28] Microscope with illumination apparatus, stage with object, tube with eyepiece and objective for carrying out the method according to one of claims 1 to 11, the illumination apparatus of which is provided with a condenser having a condenser diaphragm and which is provided with the objective for generating phase contrast darkfield illumination, characterized by , that a lens objective (6) is provided as the objective, which includes a phase ring plate (7) with a phase ring (7.1) and additionally an insertable transparent slide (37) with a disc- or ring-shaped light stopper (38, 38') that can be inserted into the lens objective (6) near the phase plate (7), wherein a separate light passage (2.3) is provided in the light mask (2) of the condenser (4), which is optically congruent with respect to the respective light stopper (38, 38'). [29] Microscope according to claim 28, characterized by, that an opaque aperture slide (37) with preferably two adjacent light diaphragms (39, 39', 39") separated from each other by a central bar (40 or 40') can be inserted into the beam path as close as possible to the rear focal plane of the lens, and that a separate light passage (2.3) is to be provided in the condenser (4) which is optically congruent with the respective central bar (40 or 40') of the aperture slide (37) which acts as a light stop. [30] Microscope according to any one of claims 12 to 29, characterized by, that complex adjustable aperture systems are used to adapt the optically effective diameter of the objective cross-section to the geometry of the illuminating beams and to adapt the geometry of the light passages to the size and position of phase rings and objective-side or objective-near light stops, consisting of an iris diaphragm in the phase contrast objective, optionally modified with an additional light stop, for reducing the optically effective objective cross-section as required, and consisting of several concentrically arranged light diaphragms in the condenser, adjustable in size and position, with which the size and position of the light passages (2.1, 2.2 and 2.3) can be continuously adjusted. [31] Microscope according to claim 30, characterized by, that the respective outer diameters of these light passages can be reduced as required with the iris diaphragms and the respective inner diameters of the ring-shaped light passages can be enlarged with opaque elements adjustable in the outer diameter.

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