WO2012028292A1 - Light microscope and optical module - Google Patents

Abstract

The present invention relates to a light microscope, more particularly a differential interference contrast microscope and/or polarization microscope and/or fluorescence microscope, comprising a microscopic beam path for transmitted light and/or reflected light microscopy, in which illumination light can be applied to a sample, more particularly via a condenser, comprising a polarizer arranged in the beam path and serving for polarizing the illumination light, comprising an analyzer arranged in the beam path and serving for analyzing light coming from the sample, and comprising an objective arranged in the beam path and serving for imaging the sample to be arranged in a sample region. The light microscope is characterized in that, for at least partial compensation of depolarization effects brought about by optical components arranged between the polarizer and the analyzer, more particularly by the objective, a compensation device is present in the beam path, said compensation device being arranged between the sample region and the objective in the case of reflected light microscopy and between the condenser and the objective in the case of transmitted light microscopy. The invention additionally relates to an optical module for arrangement in a microscopic beam path of a light microscope.

Description

 Light microscope and optical module

The present invention relates in a first aspect to a light microscope according to the preamble of claim 1.

In a second aspect, the invention relates to an optical module according to the preamble of claim 19.

Generic light microscopes, in particular differential interference contrast microscopes and / or polarizing microscopes and / or fluorescence microscopes, have a microscopic beam path for transmitted light and / or Auflichtmikroskopie in which a sample, in particular via a condenser, can be acted upon with illumination light, arranged in the beam path polarizer for polarizing the illuminating light, an analyzer arranged in the beam path for analyzing light coming from the specimen and an objective arranged in the beam path for imaging the specimen to be arranged in a specimen region.

Known optical modules for arranging in a microscopic beam path of a light microscope between a polarizer and an analyzer have an objective, wherein a sample side of the objective faces a sample area in a state installed in the beam path.

In light microscopes that measure with polarized light, such as differential interference contrast microscopes or polarization contrast microscopes, it is important that a predetermined by the polarizer of the light microscope polarization of the illumination light is not unintentionally changed by optical components of the microscope. In general, optical components, such as the lenses of an objective, affect the polarization of the illumination light or of the light coming from the sample. By crossed polarizers, that is, polarizers whose polarization directions are rotated by 90 ° to each other, then there is no complete extinction of the intensity of the light coming from the sample. For a high image quality of an image generated in the microscope, however, as complete as possible extinction of the intensity of the light is required by crossed polarizers.

An interference microscope, in particular a differential interference microscope, has within this beam path means for splitting and merging the light, which are needed for contrasting samples. These means can be brought into a centered position to the optical axis of the microscope so that they have substantially no effect on the extinction of the light. In the differential interference microscope, these means are so-called Wollaston or Nomarski prisms. The effects described below apply regardless of the use of these prisms.

In particular, when using lenses with high numerical aperture, there may be changes to the polarization towards the pupil edge, so that these areas appear partly bright. As a result, a so-called Maltese cross is often visible. This reduces the image contrast. In the following, the term depolarization is used for this situation. The depolarization refers to influencing both the phase and the amplitude of the light or of components of the light when the light is split into sub-beams polarized perpendicular to one another.

No. 6,924,893 B2 describes a polarizing microscope in which delay elements are provided in the beam path of the microscope in front of the condenser lens in order to compensate for a depolarization caused by optical components between a polarizer and an analyzer. In this case, the delay elements each have a plurality of sectors, which are individually controlled and their properties can be set independently. This makes it possible that the polarization of the light is changed only in those areas in which a depolarization is compensated by the optical components. The preparation of these delay elements is expensive and requires a lot of effort. In addition, when changing from optical Components, such as the lens, a re-adjustment of the sectors of the delay elements necessary.

US 3,904,267 describes a compensation plate for use in differential interference contrast microscopes. In these microscopes, linearly polarized illumination light is split with a Wollaston prism into two mutually perpendicular polarized partial beams. With oblique incidence on the Wollaston prism, it can lead to an undesirable change in polarization, a depolarization. To compensate for this, the compensation plate comprises a birefringent crystal, by which the two mutually perpendicularly polarized partial beams are influenced differently. In this case, the compensation plate is positioned in a section with convergent beam path, namely behind the tube lens downstream of the objective. The disadvantage here is that the analyzer must also be arranged behind the tube lens, which can likewise cause depolarization effects, for example by stress birefringence. In addition, the area behind the tube lens is often difficult to access, so that the compensation plate is only consuming interchangeable. Finally, a compensation plate would have to be arranged in each tube or intermediate tube.

Further optical arrangements with birefringent components are described in DE 103 55 725 A1, DE 10 2006 017 350 A1, DE 10 2009 023 166 A1.

As an aspect of the invention, it can be considered to provide a light microscope and to provide an optical module for arranging in a microscopic optical path of a light microscope in which a depolarization of polarized light is easily compensated.

This object is achieved by the light microscope with the features of claim 1 and by the optical module having the features of claim 19.

Preferred embodiments of the light microscope according to the invention and of the optical module according to the invention are the subject matter of the dependent claims and are also described in the following description, in particular in conjunction with the figures.

In the light microscope of the abovementioned type, according to the invention, at least partial compensation of depolarization effects, which is caused by and the analyzer arranged optical components, in particular by the lens, are provided in the beam path, a compensation device which is arranged in incident light microscopy between the sample area and the lens and in transmitted light microscopy between the condenser and the lens.

In the case of the optical module of the abovementioned type, according to the invention a compensation device arranged on the sample side of the objective is present for the at least partial compensation of at least depolarization effects which are brought about by the objective. A basic idea of the invention can be seen in arranging the compensation device at the point in the beam path at which the steepest angles for the illumination light or the light coming from the sample are present. Since the compensation achievable by the compensation device is dependent on the angle, a particularly good compensation of depolarization effects can be achieved at this point, ie in front of the objective or between the condenser and the sample.

The details of the course of the light coming from the sample relate to the imaging beam path, that is, to a beam path emanating from a point of the sample. Thus, the light coming from the sample diverges at an angle that depends on the numerical aperture of the objective. The term illumination light encompasses the light that flows onto the sample, ie at

Incident light microscopy, the light guided to the sample with the objective and, in transmitted-light microscopy, the light directed onto the sample with the condenser. The term "light coming from the sample area", that is to say "light coming from the sample", comprises the light influenced by the sample, ie the light to be measured which reflects light transmitted through the sample by transmitted light microscopy or reflected by reflected light microscopy on the sample scattered light, in fluorescence microscopy also from the sample of fluorescence light and in other contrast methods, the light generated in each case by the sample. The general term light includes the illumination light as well as the light coming from the sample. Particularly advantageous is the arrangement of the compensation device directly adjacent to the lens. As a rule, the objective makes up a high proportion of the caused velvet depolarization effects, which vary in different lenses. By positioning the compensation device on the lens, the compensating device can be exchanged in a simple manner when changing the objective, so that there is always a compensating device tuned for the respective objective in the beam path.

As a significant advantage of the invention may also be considered that the compensation device is arranged comparatively far forward in the beam path. As a result, the analyzer can also be arranged relatively far in the front of the beam path. Advantageously, thus the number of optical components, which are arranged between the polarizer and the analyzer and can cause a Depolari- sationsseffekt be minimized. For example, a tube lens located behind the objective in the beam path can be arranged behind the analyzer, so that depolarization effects caused by the tube lens need not be compensated. This is particularly important since tube lenses are generally not tension-free and cause a depolarization of light over its entire cross-sectional area. By arranging the compensation device in front of the objective, finally, the depolarization effects can be compensated independently of the choice of the tube lens.

The compensation by the compensation device is effected by a polarization change of the light, in particular by a polarization change, which is different for different regions of a cross-sectional area of the light.

In a preferred embodiment of the light microscope according to the invention, the compensation device is arranged in a convergent or divergent part of the beam path. As a result, light that is parallel to the beam path and thus perpendicular to the compensation device is advantageously influenced differently by the compensation device than light which strikes the compensation device obliquely, ie at an angle to the microscopic beam path. Thus, it is possible to change the polarization in certain areas of a cross-sectional area of the light, so that a substantially uniformly directed over the cross-sectional area of the light polarization is achieved. The optical module according to the invention can also in front of the lens in a divergent or convergent part of the beam path, whereby the above explanations also apply to the optical module.

The arrangement of the compensation device between the condenser and the objective allows an arrangement between the condenser and the sample area and / or an arrangement between the sample area and the objective. In principle, an arrangement in the condenser or in the lens is possible. However, the compensation device is preferably arranged between a front optics of the condenser facing the sample area and a front optic of the objective. In the case of transmitted-light microscopy, the compensation device, in particular exclusively, is also particularly preferably arranged between the sample region and the objective.

In an advantageous alternative of the light microscope according to the invention or the optical module according to the invention, the compensation device has at least one birefringent plate, so it is a material of the plate birefringent. As a result, a polarization change of the light caused by the plate is dependent on the polarization of the light. Thus, a proper component of light having a direction of polarization perpendicular to a major axis of the birefringent plate is differently affected than an extraordinary component of light having a polarization direction component parallel to a major axis of the plate. Polarization direction is understood to be the direction of oscillation of the electric field of a light beam.

Since the change of the polarization depends on the material, in particular on the dielectric property of the material, in particular the birefringence, and on its thickness, advantageously material, position of the major axis and thickness of the plate can be chosen so that the depolarization effects are at least partially compensated. For this purpose fitting algorithms can be used. The thickness of the platelet should be understood to mean the expansion of the platelet in the direction of the beam path and / or an optical axis of the objective.

An advantageous alternative of the light microscope according to the invention or the optical module according to the invention provides that at least one birefringent plate is a parallel plate which has a main axis which is aligned parallel to an optical axis of the objective. Under parallel plates are supposed here any birefringent platelets having a major axis aligned parallel to the optical axis of the objective are understood. The platelets can have two opposite sides, ie surfaces, which are perpendicular to the optical axis of the objective and / or to the microscopic beam path. For vertical incidence of light on a parallel plate this has no dependent on the direction of polarization of the incident light effect. But if polarized light falls obliquely on the parallel plate, the light is split into two partial beams, a proper beam of light and an extraordinary beam of light. Depending on the refractive index of the birefringent material of the platelet and its thickness, a light beam incident obliquely on the parallel platelet experiences a path difference between its ordinary and extraordinary components. Thus, the polarization is influenced depending on the angle of incidence of the light on the parallel plate. Thus, a phase shift based depolarization effect can be at least partially compensated by a parallel plate. A phase shift between the perpendicularly polarized incident beam components results in elliptically polarized light.

In an advantageous alternative of the light microscope according to the invention or the optical module according to the invention, the material and thickness of the at least one birefringent parallel plate are chosen such that a path difference between polarization components of light at an angle to a normal of a surface of the platelets, in particular at an aperture angle of the objective incident on the parallel plate is just large enough to cause a polarization change of the light which substantially compensates for a depolarization effect caused by phase shift.

Here, the half-aperture angle of the objective should be understood as the aperture angle. By arranging the birefringent platelets between the sample area and the objective, the maximum angle at which incident light from the sample is incident on the platelets is precisely the aperture angle of the objective. By selecting the material and thickness of the parallel plate as a function of the aperture angle, compensation can be made specifically for the outer regions of a cross-sectional area of the light coming from the sample for which a depolarization effect is greatest. Alternatively, material and thickness of the mini- at least one parallel plate also be chosen so that averaged over the entire cross-sectional area of the light polarization is maximum. For example, the average polarization can be understood to mean the integral of the electric field strength of the light over the cross-sectional area of the light at a particular point in time.

The necessary for the desired phase shifts path differences between ordinary and extraordinary light beam are relatively low. That is, with a phase shift of a few degrees, the path difference is only a few nanometers, depending on the wavelength of the light used. Therefore, the required thickness of a parallel plate is very small. However, the production of such a thin plate is difficult and the plate is very fragile. In an advantageous embodiment of the light microscope according to the invention or the optical module according to the invention therefore two parallel plates are present, of which one is optically birefringent positive and the other is optically negatively birefringent. This causes the two parallel plates opposite effects on incident light. If the refractive indices of the positively birefringent and of the negative birefringent parallel platelet are not equal in magnitude and / or the thicknesses of the platelets differ, an effect can be achieved which corresponds to that of a single, very thin parallel platelet.

As synonyms for optically positive birefringent and optically negatively birefringent, the terms "optically positive" and "optically negative" as well as "positively birefringent" and "negatively birefringent" are also used.

In a preferred variant of the light microscope according to the invention or of the optical module according to the invention, to compensate for a depolarization effect based on differential transmission, a birefringent plate is present which is a vertical plate having a major axis oriented perpendicular to an optical axis of the objective. By vertical platelets is meant each platelet with a birefringent material having a major axis oriented perpendicular to an optical axis of the objective. Differential transmission occurs when optical components have different transmissions for light perpendicular to the input. case level is polarized at the optical component, and for light which is polarized parallel to the plane of incidence on the optical component. Diffusive transmission rotates the plane of oscillation of a linear polarization. An elliptical polarization undergoes a rotation of its major axis.

For the perpendicular wafer, the refractive index, thickness and azimuth angle of the main axis about the optical axis of the lens can be chosen such that in those regions of a cross-sectional area of the light in which the plane of oscillation or principal axis of the polarized light is rotated by differential transmission of optical components, the perpendicular wafer causes a change in polarization which causes the light coming from the sample at the analyzer over its entire cross-sectional area to be substantially collinear with the direction of passage of the analyzer, with respect to the plane of oscillation or major axis plane. In this case, a depolarization caused by the sample remains essentially unchanged.

A polarization change for the entire cross-sectional area of the light, such as through a vertical platelet, does not represent depolarization. In this case, complete complete extinction of the light between polarizer and analyzer can be achieved without a sample. For this purpose, the transmission directions of the polarizer and the analyzer can be aligned with each other by an angle differing from 90 ° and / or an additional vertical plate can be arranged in the compensation position in front of the analyzer.

For a perpendicular wafer to compensate for a differential transmission-based depolarization effect, the angle of the major axis of the perpendicular wafer to the polarization direction, that is, the forward direction, of the polarizer is significant. For this purpose, it is provided in an advantageous embodiment of the light microscope according to the invention or of the optical module according to the invention that the perpendicular plate acts as a λ / 4 plate or as a λ / 2 plate. In this case, the main axis of the vertical plate is aligned so that it affects a rotation angle, in particular canceled, by which a direction of polarization generated on the polarizer would be rotated due to differential transmission through the optical components or through at least the lens. A λ / 2 plate causes a rotation of the polarization direction by twice the angle between the polarization direction of the incident light and the direction of the major axis of the λ / 2 plate in the case of linearly polarized incident light. In other words, the polarization direction is mirrored on the major axis of the λ / 2 plate.

A λ / 4 plate is commonly used to convert linearly polarized light into circularly or elliptically polarized light, or vice versa.

Thus, a λ / 4 plate generally converts linearly polarized incident light into elliptically polarized light. In this case, either the small or the large half-axis of the ellipse is parallel to the main axis of the λ / 4 plate.

In the case of a vertical plate which acts as a λ / 4 or λ / 2 plate, the main axis is preferably aligned such that a rotation angle is compensated in order to compensate for the polarization direction produced at the polarizer due to differential transmission through the optical components or at least would be turned through the lens. The direction of polarization of the light rotated due to differential transmission should be understood as the polarization direction in the region of the cross-sectional area of the light where the differential transmission is strongest or, in other words, where the change of the polarization direction is strongest.

In the case of a vertical plate, which acts as a λ / 2 plate, the direction of the main axis lies exactly between the polarization direction predetermined by the polarizer and a polarization direction which is present in the region of a cross-sectional area of the light which has the greatest influence through differential transmission becomes. In other words, here the main axis of the perpendicular plate is aligned so that it halves an azimuth angle. In this case, the azimuth angle is an angle between a polarization direction of the light immediately after passing through the polarizer and a changed polarization direction, which would take the light coming from the sample without the compensation device directly in front of the analyzer, if the changed polarization direction by differential transmission through the optical Components or the lens is conditional. Advantageously, vertical platelets and parallel platelets can be combined in such a way that their summed effects, namely the rotation of the polarization plane or the major axis plane of an elliptical polarization and the phase change between two mutually perpendicular polarization components, for an optimized compensation of said depolarization by the Lens guide.

The birefringent platelets may advantageously be designed for a wavelength range which is in the visible range, in the near infrared range and / or in the near ultraviolet range. The determination of the material and the thickness of the platelets can be designed in this case such that, at a wavelength between 500 and 600 nm, the compensation effected by the compensation device is best.

Appropriately, it can be provided that materials and thicknesses of the platelets are selected so that a dispersion of light coming from the sample area is kept as low as possible, in particular an achromatic or apochromatic behavior of the objective or several or all arranged in the optical beam path components through the compensation device remain unchanged. Alternatively or additionally, at least one further platelet may be present for this purpose. By the compensation device, the lens class of the lens and thus among other things, the flattening and the color correction of the lens over the field of view, in particular an achromatic or an apochromatic color correction, not changed.

Depending on the lens class, the working distance, which is the distance between the sample area and the side of the objective facing the sample area, is between about 0.1 mm and a few millimeters. Typically, the working distance for a 40x magnification objective and an oil immersion having a refractive index of n = 1.52, whereby the numerical aperture may be 1.3, is 0.2mm to 0.3mm. Especially with lenses with high magnification and high numerical aperture, the working distance can be very small. Advantageously, the thicknesses of the platelets can be selected so that the total thickness of the compensation device is smaller than the working distance of a lens. The thicknesses of the platelets are therefore preferably between 5 μm and 1 mm, in particular dere between 10μηη and ΙΟΌμιτι. On the other hand, in dry lenses without imersion oil larger platelet thicknesses can be provided.

In order to ensure simple production, in a further embodiment of the light microscope according to the invention or the optical module according to the invention, the birefringent platelets have plane-parallel surfaces. Thus, the platelets have no imaging effect, which would have to be considered in the design of the lens. To ensure that the plates are stress-free and can be used in common sockets, they can also have a circular shape.

Conveniently, the plane-parallel surfaces of the birefringent plates may be aligned substantially perpendicular to the optical axis of the objective. The platelets can have the same optical properties over the entire plane-parallel surfaces. A segmentation of the platelets is therefore not necessary, but in principle also possible.

In order to reduce light reflections, it is provided in a preferred embodiment of the light microscope according to the invention or the optical module according to the invention that the platelets are tempered, in particular have a multiple coating.

In an advantageous alternative of the light microscope according to the invention or of the optical module according to the invention, the compensation device, in particular one of the platelets, is fixedly connected to the objective on the sample side of the objective, in particular cemented. Alternatively, the compensation device can also be detachably connected to the lens.

It can also be several or all platelets firmly connected. Advantageously, therefore, the platelets must not be used separately in the microscopic beam path, but can be introduced together with the lens in the beam path. In addition, the risk of unintentional breakage of the thin platelets can be reduced.

A large proportion of the depolarization effects is being caused by the lens. If several lenses are to be usable in the light microscope, for example via a lens revolver, a separate compensation device can be used for each lens because of the different depolarization effects of the different lenses. be available. Thus, a compensating device which is set up to compensate for depolarization effects of the respective objective can be attached to each objective in an objective revolver of the light microscope.

Thus, the platelets show a birefringent effect, in a preferred variant of the optical microscope according to the invention or of the optical module according to the invention each plate on a crystal, in particular a uniaxial crystal, in particular from the group magnesium fluoride MgF2, calcite, CaCO _3, sapphire Al2O3 or crystal quartz SiO _2. Alternatively, however, the platelets may also comprise any other birefringent material, in particular a polymer.

Additionally or alternatively, a device may be provided for generating a mechanical stress, an electric field or a magnetic field in at least one of the platelets in order to effect a birefringent effect in the at least one platelet. Thus, it can be provided that the platelets are birefringent only through the device.

In order for the azimuth angle of the vertical platelet to be adjustable relative to a transmission direction of the polarizer, it is advantageous if the vertical platelet and / or the compensation device are rotatable. Conveniently, therefore, it may be provided that a mechanical means, in particular a mechanical adjusting ring, is provided to allow rotation of the compensating means about the optical axis of the objective for adjusting the polarization change caused by the compensating means. In this case, the compensation device, in particular together with the objective, is preferably rotatable about the optical axis of the objective, even if the objective is permanently installed in the light microscope. Thus, the lens can be rotated during operation. An angle scale may also be provided on the objective. In addition, there may be a device for automatically adjusting the compensation device, for example by rotating the compensation device and determining for which rotational position the best possible compensation is achieved.

In a preferred embodiment of the light microscope according to the invention, a tube lens is present and the analyzer is arranged between the objective and the tube lens. Depolarisation effects through the tube lens are therefore insignificant and are not taken into account by the compensation device. moreover a compensation device can be used independently of the tube lens used.

In particularly simple embodiments of the light microscope according to the invention or of the optical module according to the invention, the compensation device consists exactly of a parallel plate or exactly two parallel plates or of exactly one or two perpendicular plates and one or two parallel plates.

The light microscope and / or the objective can be designed for measurements with transmitted light or incident light.

In order to reduce the polarization effects as far as possible, the optical components, in particular the lens, and also the platelets of the compensation device are manufactured stress-free.

In a preferred variant of the optical module according to the invention, the compensation device is adapted, in addition to depolarizing the lens and depolarization effects of other optical components, which are arranged in the beam path between the polarizer and the analyzer, in particular of a condenser to compensate.

The compensation device according to the invention should not be confused with a compensator known in polarization microscopy. This is a λ / 4 plate at 45 ° to the polarizer and the analyzer, which is used, for example, to judge whether measured interference colors are actually caused by a sample in the sample area, or depolarization effects of optical components are attributed. By a compensator thus the depolarization, so a reduction over the cross-sectional area of the light averaged polarization, not compensated. In contrast, the polarization of certain areas of the cross-sectional area of the light are selectively changed by the compensation device according to the invention. This increases a polarization averaged over the cross-sectional area of the light.

Further advantages and features of the invention will be described below with reference to the accompanying schematic figures. Herein show: Fig. 1 is a schematic representation of light in the pupil of a

 Microscope between a polarizer and an analyzer;

Fig. 2 is a graph of depolarization in dependence of the sine of

 Aperture angle of a lens;

3 shows a schematic representation of essential components of a light microscope according to the invention and of an optical module according to the invention;

4 shows a further illustration of a light microscope according to the invention;

Fig. 5 is a schematic view of an optical according to the invention

 module,

Figures 6a to 6e embodiments of compensation means on the sample side of the lens according to the invention and

FIGS. 7a to 7d show a refractive index ellipsoid.

Equivalent components are identified by the same reference numerals in all figures.

FIG. 1 shows a schematic representation of a cross-sectional area 1 of light in a pupil of a light microscope 100. In the microscopic beam path 5, a polarizer 50, 51 and an analyzer 52 are present, whereby the passage direction of the analyzer 52 is perpendicular to the passage direction of the polarizer 50, 51 stands.

The cross-sectional area 1 of the light should be understood to mean a surface perpendicular to the microscopic beam path 5 and to the optical axis 15 of the objective 10.

In order to achieve the best possible image contrast, in this crossed arrangement of polarizer 50, 51 and analyzer 52, the light in the pupil should be completely extinguished. Without compensation device 20, however, only an area 2 from the cross-sectional area 1 of the light is dark. At cross-sectional edges 3, however, light is visible. The resulting form is called Maltese Cross. The brightening at the cross-sectional edge 3 is due to a depolarization of the light, that is, a change in the polarization in certain areas of the cross-sectional area of the light, namely at the cross-sectional edges 3.

The depolarization is caused by various Depolarisationseffekte on optical components in the beam path 5 of the microscope 100. The optical components, the lens 10, the condenser 55 and the Wollaston prisms 60, 61 count.

The strengths of the depolarization effects are generally dependent on the numerical aperture of the objective 10 used. According to Fresnel's rules, light components whose polarization direction is perpendicular and parallel to an incidence plane of the light on an optical component are refracted differently. In addition, the optical components 10, 60, 61, 55 may be stress birefringent. Here, the voltage may already be present in the material of the optical components or be generated by the pressure of the version of the optical components. Also, optical multilayers, which are used for example for anti-reflection on optical components 10, 60, 61, 55, can cause a phase rotation of the incident light, ie a path difference between perpendicular and parallel polarized light. Due to these differences in the phase and transmission of the parallel and perpendicular polarized portions of the light, a depolarization of the light occurs. As a result, in the pupil of the microscope 100, the brightened cross-sectional areas 3 of the cross-sectional area 1 of the light are visible, whereby the image contrast is reduced.

FIG. 2 shows a simulation of the dependence of the depolarization of the light on the sine of the aperture angle of the objective 10.

The aim is to compensate for the depolarization shown here with the help of the compensation device as far as possible, so that a dark pupil is visible through crossed polarizers. In the depolarization illustrated in FIG. 2, light passes through crossed polarizers, between which inter alia a lens is arranged. Since there is no sample in the sample area, ideally no light coming from the sample area should be able to pass through the analyzer. However, the objective leads to a depolarization, as a result of which the proportion of the light coming from the sample area, which portion is changed in its polarization, can pass through the analyzer. FIG. 2 shows the dependency of the depolarization occurring on the sine of the aperture angle α.

In this case, an imaging beam path is considered for which light emanating from a point of a sample area diverges and strikes the lens at the aperture angle α.

Graph 70 shows the depolarization by phase shifting between the portion of the light polarized perpendicular to and parallel to an incidence plane on the objective. By phase shifting, linearly polarized light becomes elliptically polarized light. Here, the degree specification of the depolarization for the graph 70 indicates the phase difference between the two polarized components of the light perpendicular to each other.

Graph 71 indicates the depolarization by differential transmission as a function of the sine of the aperture angle α. Differential transmission causes in linearly polarized light rotation of the polarization direction, the light is still linearly polarized. However, since light coming from the specimen diverges on the objective, that is, different areas of the cross-sectional area of the light coming from the specimen hit the objective at different angles, differential transmission causes depolarization across the cross-sectional area of the light. Here, the degree of depolarization indicates the rotation of the linearly polarized light.

In Fig. 2 it is illustrated that in approximately perpendicular to optical components incident light is hardly depolarized. On the other hand, light incident at an angle to a normal of a surface of an optical component is changed by several degrees in its polarization. As a result, the light across its cross-sectional area is no longer polarized in the same direction; it is depolarized. This depolarization is to be compensated by the compensation device 20. 3 shows a schematic representation of a light microscope 100 according to the invention. The light microscope 100 includes the compensation device 20 as well as components required for polarization measurements or differential interference contrast measurements, namely the polarizer 50, the Wollaston prisms 60, 61, the objective 10 and the analyzer 52 ,

In addition, a condenser 55 is provided which directs illumination light onto the sample area 30. The structure shown here corresponds to that of a transmitted-light microscope. All statements made for this purpose, except for the condenser 55, but are also valid for a reflected-light microscope. The microscope 100 according to the invention may also be suitable for fluorescence contrast methods in which the polarization properties of the sample are used.

Indications such as "before", "behind" and "after" with respect to the components 10, 50, 51, 52, 55, 60, 61 or the microscopic beam path 5 are defined by the running direction of the light 42, 43, 44.

Illumination light is irradiated along the microscopic beam path 5. The illumination light may initially be unpolarized. In addition, it can be either monochromatic or spectral broadband in the visible, in the near UV and / or in the near IR region.

The illumination light passes through the polarizer 50 and is linearly polarized along the transmission direction of the polarizer 50. In differential interference contrast microscopy, behind the polarizer 50, a Wollaston prism 60 or a comparable optical component is arranged to divide the polarized illumination light into two spatially spaced or split perpendicularly polarized sub-beams. In addition, in the case of circular differential interference contrast microscopy, a λ / 4 plate may be present to produce circularly polarized light.

Via the condenser 55, the polarized illumination light is then directed to a sample area 30, in which a sample to be examined can be located.

The light coming from the sample passes through the illumination aperture, the diffraction, the refraction or scattering on the structures of the sample or on the ground the fluorescence of the sample diverges and hits the lens 10. First, the light coming from the sample passes through the compensation device 20. This is arranged on the sample side 16 of the lens 10. The exact effect of the compensation device 20 on the polarization of the light will be explained in detail with reference to FIG.

Alternatively, it is also possible to arrange the compensation device 20 between the condenser 55 and the sample area 30. Since at this point the illumination light is generally convergent, a compensation of depolarization effects can also be achieved here.

After passing through the compensation device 20, the light coming from the sample strikes the objective 10 in the illustrated embodiment, whose optical axis 15 is arranged parallel to the microscopic beam path 5.

The objective 10 and the compensation device 20 represent the optical module 90 according to the invention.

In the case of differential interference contrast microscopy, another Wollaston prism 61 or a comparable optical component is arranged in the beam path behind the objective 10 in order to combine and to bring the two partial beams of the light coming from the sample into interference.

Finally, an analyzer 52 is arranged in the microscopic beam path 5. It is a polarizer whose direction of passage is rotated to that of the polarizer 50, usually by 90 °. The analyzer 52 is intended to transmit only light from the sample whose polarization has been changed by the sample.

In the case of a polarization contrast microscope, the Wollaston prisms 60, 61 are omitted. The components otherwise shown in Fig. 3 are identical for polarization contrast microscopy and differential interference contrast microscopy.

A schematic overall representation of a light microscope 100 according to the invention is shown in FIG. 4.

In this case, a light source 40, 41 emits illumination light 42, 43 onto a sample region 30. From the sample region 30, the light 44 passes through the light 44, which is not shown here. Finally, the light coming from the sample 44 is coupled out of the light microscope 100.

The beam cross-section shown here indicates the maximum occurring beam cross section, ie the total envelope of all occurring light, which in transmitted light microscopes roughly corresponds to the pupil beam path.

The light microscope 100 may include a light source 40 for incident light and / or a light source 41 for transmitted light. The light sources 40, 41 emit unpolarized illumination light 42, 43 along a microscopic beam path 5. The illumination light 42, 43 is polarized by the polarizers 50, 51. In this case, linearly polarized light can be generated or also circularly polarized light for circular differential interference contrast microscopy.

The polarized illumination light 42, 43 is then focused on a sample area 30, to which a condenser 55 can be used.

From the sample area 30, the light 44 passes through a compensation device, not shown in FIG. 4. When viewing the imaging beam path also not shown here, the light coming from the sample diverges below the aperture angle of the objective 10.

An objective 10 is arranged in the microscopic beam path 5 behind the compensation device. Various objective lenses 10 of an objective revolver are shown in FIG. 4, with only the optical elements 11, 12, 13 of the objective 10 being shown for the objective 10 located in the beam path 5 ,

The beam path can have further elements, such as a DIC slider for differential interference contrast microscopy.

The light coming from the sample strikes the optical elements 11, 12, 13 of the objective 10 on an analyzer 52.

Behind the analyzer 52, a tube with a tube lens is arranged. In this case, the light 44 coming from the sample can leave the microscope 100 via an eyepiece 46 or a light extraction 47. A schematic view of an optical module 90 according to the invention is shown in FIG.

The optical module 90 has the compensation device 20 and the objective 10. The descriptions of the compensation device 20 and the objective 10 also apply with respect to the light microscope 100 according to the invention.

In the illustrated imaging beam path, the light 44 coming from the sample area 30 extends divergently at an aperture angle of the objective 10.

First, the light 44 coming from the sample passes through the compensation device 20. This can have one or more birefringent platelets. The compensation device 20 is arranged on a sample area 30 facing sample side 16 of the lens 10.

Subsequently, the light 44 coming from the sample encounters optical elements, namely the lenses L1 to L14, of the objective 10. The compensation device 20 can be firmly connected to the objective 10, in particular to the lens L1 of the objective 10, for example by cementing or be glued. The lens L1 is here a daughter ball, which engages in a parent ball L2. In this case, the compensation device 20 does not cover the entire surface of the parent ball L2, but only the daughter ball L1.

The compensation device 20 and the platelets of the compensation device 20 not shown here are arranged such that they have surfaces which are perpendicular to the optical axis 15 of the objective 10 and the microscopic beam path 5.

Depending on the angle at which light 44 coming from the sample passes out of the sample area 30, its polarization is influenced differently by the compensation device 20.

Embodiments of the compensation device 20 according to the invention are shown schematically in FIGS. 6a to 6e.

In each of the exemplary embodiments, the compensation device 20 has one or more plates 21, 22, 23 which are located on a sample side 16 of the objective 10 are arranged. In each case, the front optics of the objective 10, namely the optical element 11, are shown.

The platelets 21, 22, 23 may each comprise a birefringent, preferably uniaxial, crystal.

The embodiments differ in material, thickness and number of platelets 21, 22, 23 and in the crystal direction of the platelets 21, 22, 23. The crystal direction of the platelets, that is, the direction of the optical axis of the crystal, parallel 25 and / or perpendicular 26 with respect to the optical axis 15 of the lens 10 extend. The terms "main axis" and "main axis of the plate" are synonymous with crystal direction.

In principle, however, it is also possible for one or more platelets to be present whose major axes, that is to say crystal axes, are aligned obliquely, ie, neither parallel nor perpendicular, to the optical axis 15 of the objective 10. In principle, the platelets can also have a non-constant thickness in the cross-sectional direction. The decisive factor is that the desired compensation of the depolarization is achieved by the platelets.

FIG. 6 a shows an exemplary embodiment of the invention, in which the compensation device 20 comprises a plate 22 made of magnesium fluoride, which has a main axis 25 parallel to an optical axis 15 of the objective 10 and the optical element 11. Light, which passes through the plate 22 parallel to the optical axis 15, undergoes no polarization change by the plate 22. In an oblique incidence of light, a partial beam of light may have a polarization perpendicular to the major axis of the plate. Another partial beam may have a polarization, which in turn is perpendicular thereto. These sub-rays are called neat and extraordinary ray. Through the plate 22, a path difference between the ordinary and extraordinary beam is generated. As a result, a phase rotation is achieved, that is, the polarization of that portion of the light which obliquely meets the compensation device 20 is changed.

The magnitude of the phase rotation depends on the refractive index n ₀ for the ordinary ray and on the refractive index n _a for the extraordinary ray. To illustrate the ordinary and the extraordinary beam, an indicatrix, ie a refractive index ellipsoid, of an optically uniaxial crystal is shown in FIGS. 7a and 7b. The refractive indices ni, n ₂ and Π3 describe the refractive indices for light which has a polarization, ie an electric field, in the direction of the vector ni, Π2 or n ₃ . The refractive index n ₃ is greater than Γ ^^ and n ₂ for positively birefringent crystals, while it is smaller for negatively birefringent crystals. Fig. 7c shows the ordinary ray and Fig. 7d the extraordinary ray. The propagation direction k of the beams assumes an angle of incidence Θ to the optical axis 15. As shown in Fig. 7c, the electric field of the ordinary ray is perpendicular to the optical axis 15. The electric field of the extraordinary ray shown in Fig. 7d is perpendicular to that of the ordinary ray. Thus, the extraordinary ray has a polarization component parallel to the propagation direction k, that is, its polarization lies in a plane which is spanned by the optical axis 15 and the propagation direction k.

The extraordinary refractive index n _a is dependent on the angle of incidence Θ, which is measured relative to the optical axis 15, ie to a perpendicular of a surface of the compensation device.

The dependence of the extraordinary refractive index n _a (9) on the angle of incidence Θ is given in the following formula, wherein the refractive index n _a without dependence on Θ is intended to indicate the refractive index in the event that the polarization of the extraordinary beam is completely parallel to the main axis of the birefringent material stands:

1 / n _a ² (0) = cos ² (0) / n ₀ ² + sin ² (0) / n _a ² .

By way of example, the following table lists, for various materials, the ordinary refractive index n ₀ , the extraordinary refractive index n _a and the extraordinary refractive index n _a (58 °) for an incident angle of 58 °. This angle of incidence corresponds to a typical aperture angle of a light microscope 100. Material n ₀ n _a n _a (58 °)

MgF ₂ 1, 38 1, 385 1, 3836

CaC0 ₃ 1, 658 1, 486 1, 5288

Al ₂ 0 ₃ 1, 768 1, 76 1, 7622

Si0 ₂ 1, 544 1, 553 1, 5506

Magnesium fluoride MgF ₂ and quartz Si0 ₂ have an ordinary refractive index smaller than their extraordinary refractive index, and are thus positively birefringent. Calcite and sapphire, calcium carbonate CaCO 3 and aluminum oxide Al ₂ O ₃ , are, however, negatively birefringent.

FIGS. 6b and 6c each show a compensating device 20 which has two plates 22, 23 whose main axes are aligned parallel to the optical axis 15 of the objective 10. Here, in each case a positive birefringent plate 23 and a negatively birefringent plate 22 are present.

In FIG. 6b these are MgF ₂ and CaCO ₃ , whereas in FIG. 6c they are SiO ₂ and Al ₂ O ₃ .

The positive and negative birefringent platelets 22, 23 cause opposite effects. For a balance of depolarization effects to be achieved, the platelets 22, 23 can therefore have greater thicknesses than a single platelet with a parallel main axis. Due to the greater thickness, the plates 22, 23 are easier to manufacture and less fragile. In addition, an achievable path difference between ordinary and extraordinary light beam through the use of two plates 22, 23 can be achieved very precisely. Finally, with two platelets 22, 23 with a suitable choice of material with regard to the refractive indices n ₀ and n _a, and with a suitable thickness of the platelets, the dispersion can also be kept low.

In the following table are exemplified the phase rotations of polarized light at an incident angle of 58 ° for different material combinations and Thickness of the platelets indicated. The angle of incidence may just be the aperture angle.

 The table illustrates that any phase rotation can be achieved with comparatively large platelet thickness. By contrast, a single plate of magnesium fluoride for a phase rotation of 4 ° would have an extremely small thickness of only 2.9μιτι.

FIG. 6d shows a compensation device 20 arranged on a sample side 16 of an optical element 11 of the objective 10. It has two plates 22, 23 with a parallel main axis, and another plate 21 with a main axis 26 perpendicular to the optical axis 15 of the objective 10 ,

The platelets 22, 23 with a vertical main axis may have the materials MgF ₂ and Al ₂ O ₃ . The main vertical axis plate 21 may comprise MgF ₂ .

The plate 21 with a vertical main axis can compensate for a differential transmission-based depolarization effect. By differential transmission, linearly polarized light is rotated in its oscillation direction, and in the case of elliptically polarized light, the position of the ellipse main axis is rotated. The platelet 21 with a vertical crystal axis can again produce linearly polarized light when suitably positioned from elliptically polarized light. In addition, the direction of oscillation is influenced, so that both ellipticity and oscillation direction of the resulting linearly polarized light can be adjusted together with the effect of one or more parallel plates. For this purpose, advantageously material and thickness of the plate 21 can initially be selected be that it acts for the light as a λ / 4 plate or λ / 2 plate. In addition, the direction of the major axis within a plane perpendicular to the optical axis 15 of the objective 10 is significant. This direction is indicated by the azimuth angle, which describes the angle between the forward direction of the polarizer 50, 51 and the direction of the major axis of the plate.

The following table shows two embodiments with a vertical plate and two parallel plates. For the sake of simplicity, an incident-angle-independent effect is initially assumed in the case of the vertical plate.

Although causes the plate 21 with a vertical main axis in an orientation with azimuth angle not equal to 0 °, even for perpendicularly incident light a polarization change. In this case, however, the analyzer 52 can be aligned accordingly and / or an additional vertical plate between the objective 10 and the analyzer 52, in particular in a parallel part of the beam path 5, be present. This polarization change thus causes no disadvantage.

In case of oblique incidence on a vertical plate 21, in turn, a phase angle dependent on the angle of incidence is generated. This adds to the phase change through the parallel plate 22, 23 added. For example, a parallel plate 22, 23 followed by a vertical plate 21 at the same thickness, namely the thickness as it would be for a vertical plate 21 with λ / 4 property, in case of oblique incidence again give a phase difference of 90 °, ie a phase shift as a λ / 4 plate under normal incidence alone. A thickness of the parallel plate 22, 23, which exceeds this thickness, consequently causes an additional phase change, which can be used to achieve the overall phase change to be achieved.

FIG. 6 e shows an exemplary embodiment in which the compensation device 20 shows exactly one plate 21 with the main axis direction 26 perpendicular to an optical axis 15 of an objective 10. Already by a single plate 21 is an at least partial compensation of Depolarisationseffekten possible.

However, the compensation device 20 preferably has at least one small plate 22, 23 with a parallel main axis and at least one plate 21 with a vertical main axis. As a result, depolarization effects, which are based on differential transmission and / or phase shift, can be largely compensated. Advantageously, it can thus be achieved that, without a sample, the light can not pass the analyzer 52 over its entire cross-sectional area 1.

The achievable image contrast in a polarizing microscope and / or a differential interference contrast microscope is thus significantly improved.

Claims

PATENT CLAIMS 1. Light microscope, in particular differential interference contrast microscope and/or polarization microscope and/or fluorescence microscope, with a microscopic beam path (5) for transmitted light and/or reflected light microscopy, in which a sample can be exposed to illuminating light (42, 43), in particular via a condenser (55), with a polarizer (50, 51) arranged in the beam path (5) for polarizing the illuminating light (42, 43), with an analyzer (52) arranged in the beam path (5) for analyzing light (44) coming from the sample, and with a lens (10) arranged in the beam path (5) for imaging the sample to be arranged in a sample area (30), characterized, that for at least partial compensation of depolarization effects caused by optical components (10, 60, 61, 55) arranged between the polarizer (50, 51) and the analyzer (52), in particular by the lens (10), in which Beam path (5) there is a compensation device (20), which is arranged between the sample area (30) and the objective (10) in reflected light microscopy and between the condenser (55) and the objective (10) in transmitted light microscopy. 2. Light microscope according to claim 1, characterized, that the compensation device (20) is arranged in a convergent or divergent part of the beam path (5). Light microscope according to claim 1 or 2, characterized, that the compensation device (20) is arranged between the sample area (30) and the objective (10) in transmitted light microscopy. Light microscope according to claim 1 or 3, characterized, that the compensation device (20) has at least one birefringent plate (21, 22, 23). Light microscope according to claim 4, characterized, that the material, position of a main axis (25, 26) and thickness of the birefringent plate (21, 22, 23) are selected so that the depolarization effects are at least partially compensated. Light microscope according to claim 4 or 5, characterized, that at least one of the birefringent plates (22, 23) is a parallel plate which has a main axis (25) which is aligned parallel to an optical axis (15) of the objective (10). Light microscope according to claim 6, characterized, that the material and thickness of the at least one parallel plate (22, 23) are selected such that that a path difference between polarization components of light (42, 43, 44), which is incident on the plates at an angle to a normal of a surface of the plates (21, 22, 23), in particular at an aperture angle (a) of the lens (10). (21, 22, 23) is just large enough to cause a polarization change of the light (42, 43, 44), which essentially compensates for a depola sation effect caused by a phase shift. 8. Light microscope according to one of claims 6 or 7, characterized, that two parallel plates (22, 23) are present, one of which in particular is optically positively birefringent and the other is optically negatively birefringent. 9. Light microscope according to one of claims 4 to 8, characterized, that to compensate for a depolarization effect based on differential transmission, a birefringent plate (21, 22, 23) is present, which is a vertical plate (21) which has a main axis (26) which is perpendicular to an optical axis ( 15) of the lens (10) is aligned. 10. Light microscope according to claim 9, characterized, that the vertical plate (21) acts as a λ/4 plate or λ/2 plate, and i5 that the main axis (26) of the vertical plate (21) is aligned in such a way that it influences, in particular cancels, a rotation angle, by which a polarization direction generated on the polarizer (50, 51) would be rotated due to differential transmission through the optical components (10, 60, 61, 55) or through at least the lens (10). 20 11. Light microscope according to one of claims 4 to 10, characterized, that the birefringent plates (21, 22, 23) have plane-parallel surfaces. 12. Light microscope according to claim 11, 25 characterized in that the plane-parallel surfaces of the birefringent plates (21, 22, 23) are aligned perpendicular to the optical axis (15) of the objective (10). 13. Light microscope according to one of claims 1 to 12, characterized, that the compensation device (20), in particular one of the birefringent plates (21, 22, 23), is firmly connected to the objective (10), in particular cemented, on the sample side (16) of the objective (10). 1 . Light microscope according to one of claims 4 to 13, characterized, that the birefringent plates (21, 22, 23) are firmly connected to one another. 15. Light microscope according to one of claims 4 to 14, characterized, that each of the birefringent plates (21, 22, 23) has a crystal, in particular a uniaxial crystal, in particular from the group of magnesium fluoride, calcite, sapphire or crystal quartz _. 16. Light microscope according to one of claims 4 to 15, characterized, that materials and thicknesses of the birefringent plates (21, 22, 23) are selected so that dispersion of light (42, 43, 44) is kept as low as possible, in particular achromatic or apochromatic behavior of the objective (10) remains unchanged. 17. Light microscope according to one of claims 1 to 16, characterized, that a mechanical means, in particular a mechanical adjustment ring, is present to enable rotation of the compensation device (20) about the optical axis (15) of the objective (10) to adjust the polarization change caused by the compensation device (20). 18. Light microscope according to one of claims 1 to 17, characterized, that a tube lens is present and that the analyzer (52) is arranged between the objective (10) and the tube lens. 19. Optical module for arranging in a microscopic beam path (5) of a light microscope (100), in particular a light microscope according to one of claims 1 to 18, between a polarizer (50, 51) and an analyzer (52) with an objective (10), a sample side (16) of the objective (10) facing a sample area (30) when installed in the beam path (5), characterized, that a compensation device (20) arranged on the sample side (16) is present for at least partial compensation of at least depolarization effects caused by the objective (10).