WO2023186558A1 - Illumination for a microscope, microscope having dark-field illumination, use for examining blood, and method for illuminating a sample - Google Patents

Abstract

The invention relates to: an illumination device for microscopic illumination, in particular for dark-field illumination; a microscope having this kind of illumination device; a use for examining blood; and a method for illuminating a sample. For this purpose, light emitting diodes arranged in a cone shape are provided which irradiate onto a condenser body.

Description

Illumination for a microscope, microscope with dark field illumination, use for blood testing and method of illuminating a sample

Technical area

The invention relates to transmitted light illumination for a microscope with particular suitability for dark field microscopy, fluorescence microscopy and for evanescent sample illumination.

State of the art

An LED-based dark field illumination is known from CN 105739075 A, but is only intended episcopically.

Cardioid condensers, for example from US20070127117 A1, are known for dark field illumination, but they are expensive and not very energy efficient.

From US1999240 A a dark field condenser with an ellipsoid reflector is known. The disadvantage is the low energy efficiency of the lighting.

From US20160363753 A1 a microscope illumination with an LED ring light for fluorescence and darkfield transmitted light illumination is known. The light emitting diodes for dark field illumination are positioned and collimated separately using a non-reflective collar. This light is guided past the condenser via the collar and a free beam path and fed to the sample. The disadvantage is the small numerical aperture that can be achieved.

This means that this dark field illumination is not suitable for high magnifications.

From WO2014/041820 A1, among other things, dark field illumination with light emitting diodes is known, the light of which is guided past the bright field condenser and fed to the sample via a free beam path. The disadvantage is the small numerical aperture that can be achieved.

Methods for dark field microscopy are known from a publication by the company Helmut Hund GmbH and various condenser types are described (Haus, Jörg; Technical Basics of Dark Field Microscopy, Wetzlar 2008 published at “https://www.hund.de/images/pdf/Microscopy/Dunkelfeldmicroscopie_Naturheilpraktiker_mit_Hund -Logo.pdf”). This publication shows that Abbe condensers with a central aperture for dark field microscopy at high magnification due to poor condensation Contrasts are only suitable to a limited extent. Stopping down the microscope objective improves the contrast, but reduces the resolution. From this publication, native blood tests using dark field microscopy with conventional condensers are known.

From the internet publication “https://correctiv.org/faktencheck/2021/12/20/dieses-video-be-weise-nicht-dass-in-covid-19-impfstoffen-wuermer-sind-oder-das-blut Blood tests using dark field microscopy are also known. However, there is currently no standardized method for heparin-free capillary blood preparation and for examining such preparations in the dark field. Optical blood tests, in particular recording in several focal planes and evaluating the images, are known from US 2017 / 0 292 905 A1.

From CN 1 02 004 307 A an illumination device for fluorescence microscopy intended for epiillumination is known. A ring beam is first generated from the light from a fiber-coupled light source using a conical reflector, which is then projected onto the sample through the microscope objective using an imaging system. The disadvantages are the complexity of the system and the low light output.

From US 2019/0 064496 A1 a microscope for examining biological samples is known, which has an eyepiece for observing the samples.

Task of the invention

The object of the invention is to provide an inexpensive, efficient illumination device suitable for dark field microscopy and high numerical aperture (NA), to provide such a microscope, and to specify a method for such illumination of a sample.

Solution to the task

The object is achieved by an illumination device according to claim 1, a microscope according to claim 14 and a method for illumination according to claim 16. In addition, a use of the illumination device according to claim 17 is specified.

Advantages of the invention

The illumination device is inexpensive, achieves a high NA and is suitable for immersion microscopy at high magnification. In contrast to unshielded ring light arrangements, an NA of over 1 can be achieved with the lighting device presented here. In addition, it can be designed with a low overall height and can be mounted on the microscope table if necessary. In addition, inexpensive aperture-less lenses, for example immersion lenses with high magnification, for example 100x with NA 1.25, can be used with the microscope presented here in order to be able to observe samples in the dark field. The illumination device can also be used for fluorescence microscopy and, with a correspondingly high NA, for evanescent sample illumination.

Description

An illumination device according to the invention which is particularly suitable for dark field microscopy is described below.

The invention includes a transmitted light illumination device for a microscope, comprising

• a rotationally symmetrical condenser body with an axis of symmetry, a flat light exit surface and several convex light entrance surfaces, the arrangement of a sample and/or a sample carrier with a sample on the light exit surface being provided, several light emitting diodes (LED), from each of which a light bundle with a central beam can be emitted is, the central rays coming from the several light emitting diodes emerging light bundles are arranged in a first beam section between the light emitting diode and the condenser body on a first cone shell of a first truncated cone, and each of the light bundles is assigned one of the light entry surfaces.

The light entry surfaces can be the surface sections of a curved lateral surface of the condenser body, through which the light beam of a light emitting diode enters the condenser body. The light entry surfaces can be separated by surface sections which are not intended for light entry. However, the light entry surfaces can also be connected to one another if the light bundles from adjacent light emitter diodes overlap when entering the condenser body. If the condenser body is shaped as a spherical segment (spherical frustum), this lateral surface can be referred to as a spherical zone. If the condenser body is shaped as a spherical segment (spherical mandrel), which can be viewed as a special case of a spherical disk, or specifically as a hemisphere, this lateral surface can be referred to as a spherical cap, also as a dome or hood. It should be noted that in English usage the term “spherical segment” can be understood as a spherical disk, not the spherical segment. A spherical segment or spherical section is a part of a spherical body that is separated by cutting with a plane. A spherical segment has the shape of a dome and has a circular disk as its base. A hemisphere is a special case of a spherical segment in which the cutting plane contains the center of the sphere.

The axis of symmetry can be arranged in a z-direction. The z direction can be the direction of an observation beam path. The optical axis of the observation beam path can coincide with the axis of symmetry. Perpendicular to the z-direction you can define an x-direction and a y-direction, so that the xyz directions form a Cartesian coordinate system. The light exit surface can be designed as a flat surface, which is arranged perpendicular to its axis of symmetry due to the rotational symmetry of the condenser body.

Beams of rays can be emitted from the light emitting diodes. If the respective light emitting diode is supplied with an electrical operating current, a beam of light can actually be emitted. The central ray can be understood as the ray that represents the center of gravity of the angular distribution of the light output of the beam. This beam can also be referred to as the main emission direction. According to the invention, the central rays are Light bundles are arranged in a second beam section in the condenser body, which extends in a straight line from the respective light entry surface to the light exit surface, on a second cone shell of a second truncated cone. This can mean that the central rays converge on one point, namely the second tip of the cone. The first and second truncated cones are arranged concentrically to the axis of symmetry. An obtuse cone can be understood as one with a tip angle of more than 90°, measurable as a full angle. Advantageously, the angle of incidence of each central ray onto the light entry surface, ie the angle of the central ray to the surface normal of the light entry surface at the point of impact, can be smaller than 15°, which can mean that the angle of incidence of the central ray deviates from the vertical incidence by a maximum of 15°. Then polarization, astigmatic and chromatic artifacts can be minimized. The second cone shell can be identical to the first cone shell or different from it. The first and second cone shells can be identical if the angle of incidence of the central rays is 0°, which corresponds to a vertical incidence on the condenser body. The second cone can advantageously be provided in such a way that at the light exit surface a numerical aperture (NA) of the central rays is between 0.8 and 1.4, advantageously between 1.1 and 1.4, particularly advantageously between 1.25 and 1.4 occurs. The NA can be viewed here as a medium ring aperture; this NA can also be effective on the sample. The middle ring aperture can represent an average value between an inner and an outer aperture of the oblique illumination that can be generated by the lighting device. The sine of the cone half angle a ₂ /2, ie half of the second cone angle a ₂ can be calculated from the NA divided by the refractive index n of the condenser body. The cone half angle a ₂ /2 can be the angle of the central rays to the z direction. You can calculate the suitable second cone angle a ₂ (full angle) as a ₂ = 2 * arcsin (NA/n)

For example, if you use a condenser body with a refractive index of 1.518 and provide an NA of 1.3, you can calculate the appropriate second cone angle as 117.82°. Advantageously, the sample can be illuminated by means of the transmitted light illumination device with a numerical aperture, to be determined as a mean ring aperture of more than 1, in particular of more than 1.25. It can be advantageous to provide the NA of less than 1.33. The latter can be useful if the sample includes objects to be observed in an aqueous environment. Namely, if the NA exceeds the refractive index of the water, total internal reflection could occur, reducing the light output on the sample and/or leading to more unwanted scattered light. For other sample media, for example glycerol, resin or oil, higher numerical apertures can also be used. The second cone angle can advantageously be less than 140°, particularly advantageously between 110° and 125°. The first cone angle can advantageously be less than 140°, particularly advantageously between 100° and 125°.

kann man aus dem zweiten Kegelwinkel a₂ und dem Winkel zwischen dem zweiten Strahlabschnitt und der Normale der Lichteintrittsfläche des Kondensorkörpers nach dem Brechungsgesetz berechnen. Auch kann man dazu ein Raytracing Programm verwenden. Ebenso kann man den Ort berechnen, an dem die Lichtemitterdioden vorteilhaft angeordnet werden können, so dass die Zentralstrahlen der Lichtemitterdioden nach dem Passieren des Kondensorkörpers und gegebenenfalls des Probenträgers am Probenort zusammenlaufen. Zum Ermitteln der optimalen Position der Lichtemitterdioden kann man den zweiten Kegel so legen, dass die Spitze in der Probe liegt. Die Schnittlinie des zweiten Kegelmantels mit der Außenfläche des Kondensorkörpers, auf der die Lichteintrittsflächen liegen, kann somit festgelegt werden. In einer xz Schnittebene kann das der Schnittpunkt eines Zentralstrahls mit der zugehörigen Lichteintrittsfläche sein. Dann kann der erste Kegel so gelegt werden, dass der erste Kegelmantel ebendiese Schnittlinie mit dem Kondensorkörper hat. In der xz Schnittebene kann dadurch jeweils der Punkt auf der Lichteintrittsfläche festgelegt werden, in welchem der erste Strahlabschnitt und der zweite Strahlabschnitt geometrisch aufeinandertreffen. The first cone angle

can be calculated from the second cone angle a ₂ and the angle between the second beam section and the normal of the light entrance surface of the condenser body according to the law of refraction. You can also use a ray tracing program for this. You can also calculate the location at which the light emitting diodes can be advantageously arranged so that the central rays of the light emitting diodes converge at the sample location after passing through the condenser body and, if applicable, the sample carrier. To determine the optimal position of the light emitting diodes, you can place the second cone so that the tip lies in the sample. The intersection line of the second cone shell with the outer surface of the condenser body, on which the light entry surfaces lie, can thus be determined. In an xz cutting plane, this can be the intersection of a central ray with the associated light entry surface. Then the first cone can be placed so that the first cone shell has this same line of intersection with the condenser body. In the xz cutting plane, the point on the light entry surface can be determined in which the first beam section and the second beam section meet each other geometrically.

The light emitting diodes can be arranged at a predetermined distance from the condenser body with their main emission direction on the line of the first beam section determined in this way. It can be advantageous to choose the distance between the light emitting diodes and the capacitor body as small as possible, for example between 0 and 3mm, particularly advantageously less than 2mm. The distance can be determined as the length of the free beam path of the respective central beam. By keeping the distance short, the overall height of the lighting device can be minimized. In addition, the homogeneity of the lighting can be better than at greater distances.

Advantageously, 3 to 128 light emitting diodes can be provided, particularly advantageously 8 to 128. In an advantageous exemplary embodiment, a number of 12 light emitting diodes with a housing diameter of 3 mm has proven successful. These can be grouped around a spherical segment-shaped condenser body with a radius of 7.5mm. Light emitting diodes with an even smaller diameter can also be advantageous, in which case even more copies would be placed on the intended first cone shell at the intended distance from the condenser body fit. This means that a higher light intensity can be achieved. Light emitting diodes with a larger diameter can also be advantageous if, for example, a larger object field in the sample plane, ie a larger sample area, is to be illuminated.

The plurality of light emitting diodes can be present as light emitting diodes in a light emitting diode housing and can advantageously each have a housing lens (front lens) with which the respective light exit beam can be collimated to a divergence (FWHM) of 30° or less, in particular between 10° and 25°. In principle, the light emitting diodes can also be collimated with even smaller divergence or almost ideally. However, light emitting diodes that are collimated to, for example, 15° to 25° can be considerably cheaper than better collimated light emitting diodes. In addition, the light is bundled again as it enters the condenser body because of the convex light entry surface. If the light radiation from the light emitting diodes at the light entrance surface of the condenser body were ideally collimated or even convergent, this could lead to uneven illumination of the sample area to be observed in the sample plane. In the worst case scenario, the light emission surfaces of the light emitting diodes could be imaged onto the sample, so that the image of the light emitting diodes could cause superimposed artifacts in the image of the sample. Therefore, a divergence of the beams of the light emitting diodes in the above-mentioned framework can be advantageous compared to ideal collimation.

The housing lens can simultaneously represent the light exit surface of the light emitting diode. The beam path between the light exit surface of the light emitting diodes and the sample to be illuminated can advantageously be provided free of reflections over the entire course. This can have the advantage that, in contrast to known cardioid capacitors, no reflection surfaces have to be produced.

The sample can be arranged directly on the light exit surface. For this purpose, objects to be observed can, for example, be applied directly to the light exit surface in a liquid medium. Alternatively, a sample carrier can be provided. The sample carrier can, for example, be a slide that is common in microscopy. The sample carrier can also be a transparent plate-shaped body with at least one fluid channel through which a sample flows. In the latter case, the sample flowing through can be observed continuously. The sample carrier can be optically coupled to the light exit surface using an immersion medium. The sample carrier can also be optically coupled to the light exit surface by blowing it on. The sample carrier can also be optically coupled to the light exit surface by means of a gap. In the latter case, the thickness of the gap can advantageously be smaller than a quarter of a mean wavelength (design wavelength) of the illuminating light in order to avoid light losses. For example, an immersion oil can be suitable as an immersion agent or, especially in the hobby sector, glycerin (glycerol) from the hardware store.

The sample can be covered with a coverslip. For this purpose, a commercially available cover glass for microscopy, for example with a thickness between 0.08mm and 0.25mm, can be used. The thickness of the cover glass can advantageously be 0.17 mm, for which common standard lenses for microscopes can be corrected. To observe the sample, a space can be provided between the cover glass and the microscope objective. In the case of a dry objective, the gap can be designed as a free-jet path, but in the case of an immersion objective, it can advantageously be filled with an immersion medium.

Apertures can advantageously be arranged between the light emitting diodes and the condenser body. These can be provided to dim the edge rays of the light bundles. This can improve the contrast in the darkfield image. The aperture openings can be circular, for example advantageously with a diameter of 0.5mm to 2mm. The aperture openings can also advantageously be designed oval or rectangular, with the smaller dimension, i.e. the short axis in an elliptical design or the short side in a rectangular design, being able to be arranged perpendicular to the first cone shell. The sample can then be illuminated over a larger area with a narrow ring aperture.

It can be advantageous that the plurality of light emitting diodes are each arranged at a distance from the light entry surfaces at a distance of less than the average radius of curvature of the light entry surfaces, in particular less than half the radius of curvature. The first jet section can advantageously be designed as a free jet path. The first jet section can run in air.

The condenser body can have the basic shape of an ellipsoid segment disk of revolution or an elliptical paraboloid segment disk. The basic shape of a spherical disk, in particular a spherical segment, is particularly advantageous. Under a spherical disc in the sense of The invention presented here can be understood as a rotationally symmetrical body that emerges from a spherical segment by separating a smaller spherical segment from the spherical segment. The surface of the spherical disk created by cutting off the smaller spherical segment can be referred to as the top surface. The intersection lines of the segment base surface and the lateral surface (cap) can lie in parallel planes due to the rotational symmetry of the spherical disk. The top surface can have the function of a bright field light entry surface. The spherical segment or the spherical segment on which the spherical disk is based can advantageously be smaller than or equal to a hemisphere. Particularly advantageously, the condenser body can have the basic shape of a hemisphere or a spherical segment that has a segment height (cap height) of the spherical radius minus a predetermined target thickness d of a sample carrier. The condenser body can deviate from the basic shape at places that are not optically effective, but it can also be completely designed as this basic shape. The condenser body can advantageously have a spherical radius between 3mm and 15mm, particularly advantageously between 5mm and 10mm. The light exit surface can be circular and advantageously have a radius between 3mm and 15mm, particularly advantageously between 5mm and 10mm. The light exit surface can be the base area, also referred to as the segment base area, of the spherical segment. The convex light entry surfaces can advantageously be arranged on the lateral surface (spherical zone or spherical cap). A spherical cap can be understood as the curved part of the surface of the spherical segment, which in this case can also represent the spherical zone. In the case of the spherical disk, the term spherical zone can be understood as the part of the cap of the spherical segment on which the disk is based, which belongs to the disk. The convex light entry surfaces can advantageously have a radius of curvature between 3mm and 15mm, particularly advantageously between 5mm and 10mm

The second cone shell can advantageously have the same cone angle as the first cone shell. This can be particularly advantageous if the condenser body has the basic shape of a spherical segment that has a segment height of the spherical radius minus a predetermined target thickness d of a sample carrier. In this case, the basic shape of a spherical disk can also be advantageous, in which case its segment height can be related to the spherical segment on which the spherical disk is based.

The second cone shell can also advantageously have a larger cone angle than the first cone shell. This can be particularly advantageous if the condenser body has the basic shape of a hemisphere or a hemispherical disk, ie a spherical disk which the center of the sphere can lie in a sectional surface, namely the light exit surface.

A bright field light entry surface for a further light beam for paraxial illumination of the sample can advantageously be provided on the condenser body opposite the light exit surface. Paraxial illumination can be Köhler illumination. The bright field light entry surface can be designed, for example, as a surface of a spherical disk-shaped condenser body opposite the light exit surface. The bright field light entry surface can advantageously be flat or also advantageously concave. The bright field light entry surface can also be designed to be convex. Such a condenser body with a convex or concave, i.e. non-flat, top surface can be referred to as a generalized spherical disk. It should be noted that a spherical disk in the sense of the present invention can also be understood as a generalized spherical disk, i.e. with a flat segment base surface and a top surface that can be flat, convex or concave. It should also be noted that a spherical segment can be viewed as a special case of a spherical disk. In this special case, the spherical zone can be designed as a spherical cap.

A microscope according to the invention can advantageously comprise:

• a transmitted light illumination device according to the invention described above,

• a lens and

• an eyepiece and/or an image sensor.

The microscope can advantageously be used for dark field microscopy if the objective (26) has a smaller NA than the ring aperture of the transmitted light illumination device. The sample can also be illuminated evanescently, which is also advantageous for special tasks. The latter can be achieved in particular by providing the NA of the transmitted light illumination device to be greater than the refractive index of the sample medium. In this case, total reflection of the light bundles can occur on the sample medium and the sample is only illuminated by evanescent (transversely attenuated) light waves.

The transmitted light illumination device can advantageously also comprise a housing with a receiving trough for the condenser body. It can be particularly advantageous if the condenser body can be removed for a second operating mode and the transmitted light illumination device is available in this second operating mode without immersion medium without the condenser body Transmitted light illumination of the sample can be operated. Depending on the microscope objective used, the second operating mode can be oblique bright field illumination or further dark field illumination of the sample. Without the condenser body, the NA of the illumination can be NA = sinfai/2), where a _r is the first cone angle (determinable as a full angle). The trough can advantageously be manufactured with positive tolerance, dd slightly larger than the condenser body. The condenser body can then be attached to the sample carrier in a detachable and, if necessary, displaceable manner using a thin layer (approx. 0.5pm -100pm) of immersion medium. The sample carrier can be placed on the housing so that the condenser body protrudes into the positively tolerated trough without mechanical overdetermination occurring.

It can be advantageous if the light emitting diodes can be controlled individually and/or divided into several groups. You can then take several images with different lighting and use image processing to enhance contrasts.

It can be advantageous if the condenser body is equipped to scatter and/or absorb light on surfaces outside of optically functional surfaces, in particular matt and/or blackened. For this purpose, the condensation body can advantageously be coupled to the housing material in the trough of the housing by means of an immersion medium or a transparent adhesive. The housing can, for example, be made of black plastic or black anodized aluminum. The housing can advantageously have a low overall height, for example from 5mm to 12mm. The transmitted light illumination device according to the invention can then be arranged on the microscope stage of a commercially available microscope, with the overall height being so low that the microscope can be focused on the sample within the existing focusable area of the microscope.

The microscope can advantageously include an immersion objective, which can be operated with an immersion medium.

It can be advantageous to use a transmitted light illumination device according to the invention or a microscope according to the invention for examining a preparation or a sample which contains biological examination objects in, for example, an aqueous sample medium. Biological examination objects can, for example, be cells or tissue sections that are so thin that they are transparent. It can be particularly advantageous to use Application of a transmitted light illumination device according to the invention or a microscope according to the invention for dark field examination of a blood sample, in particular a native blood sample.

According to the invention, a method for illuminating a sample is also provided, comprising:

• Generating several light bundles by means of a light emitting diode, each light bundle having a central beam, and the central rays emerging from the light emitting diodes are arranged in a first beam section on a first cone shell of a first truncated cone,

• Coupling the light bundles into a convex light entry surface of a rotationally symmetrical condenser body with an axis of symmetry, which has a flat light exit surface.

• Straight transmission of the light beams to a light exit surface of the condenser body,

• Coupling the light bundles from the light exit surface,

• Coupling the light bundles emerging from the light exit surface into a sample arranged directly or by means of a sample carrier on the light exit surface.

The sample carrier can be placed on the light exit surface of the condenser body or can be optically coupled to the light exit surface of the condenser body using a thin layer (thickness e.g. 0.5pm -100pm) of an immersion medium.

The figures show the following:

Fig. 1 shows a first exemplary embodiment.

Fig. 2 shows a second exemplary embodiment.

Fig. 3 shows a third exemplary embodiment.

Fig. 4 shows the second operating mode of the third exemplary embodiment.

Fig. 5 shows a fourth exemplary embodiment.

Fig. 6 shows a fifth exemplary embodiment.

Fig. 7 shows a sixth exemplary embodiment.

Fig. 8 shows a seventh embodiment.

Fig. 9 shows the seventh exemplary embodiment without a condenser body.

Fig. 10 shows a condenser body designed as a hemispherical disk.

Fig. 11 shows a condenser body designed as a generalized hemispherical disk. Fig. 12 shows a condenser body designed as a generalized spherical disk.

Examples of embodiments

The invention is explained below using exemplary embodiments.

Fig. 1 shows a first exemplary embodiment. A transmitted light illumination device 1 for a microscope is shown. This comprises a rotationally symmetrical condenser body 2 with an axis of symmetry 3 which is arranged in the z direction. The condenser body 2 has a flat light exit surface 4, which is arranged in an xy plane.

The condenser body 2 is shaped as a spherical segment, here specifically as a hemisphere, and has several convex light entry surfaces 5 (5a, 5b, ...) on the lateral surface, also as a cap, here specifically as a spherical cap.

A sample 6, which is arranged on a sample carrier 7, is also shown. The sample 6 is covered with a cover glass 8. The sample carrier 7 is arranged on the light exit surface 4. A possibly existing gap 22 can be bridged with an immersion medium 23,

The arrangement comprises a plurality of light emitting diodes (LED) 9, from each of which a light bundle 13 with a central beam 14 can be emitted, the central rays 14 of the light bundles 13 emerging from the plurality of light emitting diodes 9 in a respective first beam section 15 between the light emitting diode and the condenser body 2 a first cone shell 16 of a first truncated cone are arranged. The first cone shell 16 has a first cone angle 18. Each of the light bundles, represented by its central beam 14, is assigned one of the light entry surfaces 5 (5a, 5b, ...). Also indicated are the electrical connections 12 of the light emitting diode and the housing lens 10 designed as a front lens, which at the same time represents the light exit surface of the light emitting diode 9. In this figure, as well as in the following one, for the sake of clarity, not the complete beams of rays are shown, but only their central rays 14.

A first beam section 16 is designed as a free beam path. According to the invention, the central rays 14 of the light bundles are arranged in a second beam section 19, which extends in a straight line in the condenser body 2 from the respective light entry surface 5a, 5b to the light exit surface 4, on a second cone shell 20 of a second truncated cone. The first 16 and the second 20 truncated cones are arranged concentrically to the axis of symmetry 3. The second cone shell 20 is different here from the first cone shell 16. This is due to the refraction of the light because the central ray does not fall perpendicular to the surface normal. The second cone shell 20 has a second cone angle 21.

In the further exemplary embodiments, the reference numbers introduced here continue to apply accordingly.

Fig. 2 shows a second exemplary embodiment. The second example is similar to the first, with the difference that the light emitting diodes 9 are arranged closer to the capacitor body 2.

Fig. 3 shows a third exemplary embodiment. The condenser body 2 is shaped as a spherical segment, which is smaller than a hemisphere. The segment height is chosen so that it represents the sphere radius minus the thickness of the sample carrier. The first 16 and second cone shell 20 are identical and have the same cone angle 21. The first and second cone shells can be identical, since in this example the central rays 14 of the light bundles 13 are incident in the direction of the normal vector of the respective light entry surface of the condenser body 2.

In this example, the beams 13 are shown in more detail. The beams 13 can be restricted by optional apertures 11. It is also visible that the divergence of the beams 13 is reduced by the refraction of light as it enters the condenser body 2.

Fig. 4 shows the second operating mode of the third exemplary embodiment. Here the condenser body is removed and the arrangement 1 is operated in a second operating mode without the condenser body. If the NA of an observation lens (not shown in the figure) is smaller than the NA of the illumination device, the second operating mode can be a dark field illumination device 1. The NA of the illumination on the sample is reduced compared to the first mode. This can be seen from the fact that the central rays 14 follow the refraction the underside of the sample carrier 7 falls more steeply onto the sample 6. If the NA of an observation lens is greater than the NA of the illumination device, the second operating mode can be oblique bright field illumination.

Fig. 5 shows a fourth exemplary embodiment. A further group of second light emitting diodes 37 is also provided here. Their central rays lie on a free beam path on a third cone jacket 38. Within the condenser body they lie on a fourth cone jacket 39. This allows the illuminable ring aperture area to be expanded.

Fig. 6 shows a fifth exemplary embodiment. A microscope 24 is shown with a transmitted light illumination device 1 for dark field illumination of the sample 6, which is arranged on a microscope table 25. A paraxial bright field illumination device 32 can optionally be arranged under the microscope table 25.

The sample carrier 7 is optically coupled to the condenser body 2 by means of a first immersion medium 23a.

The condenser body 2 has, opposite the light exit surface 4, a bright field light entry surface 36 for a further light bundle for paraxial bright field illumination of the sample 6. In a modification of the exemplary embodiment, which is not shown in the figure, the bright field light entry surface can be designed as a flat top surface of a spherical disk-shaped condenser body 2. However, the bright field light entry surface 36 is shown to be concave in this example. The bright field light entrance surface is the top surface of the hemispherical disk. The condenser body 2 shown here is a generalized hemispherical disk with a concave top surface. The transmitted light illumination device 1, which is preferably used for dark field illumination, and the optional bright field illumination 32 can be switched on independently of one another. This means that pure bright field observation and pure dark field observation are optionally possible. For bright field illumination, a first bright field lens 33 and a second bright field lens 34 are provided, which are illuminated by means of a bright field light source 35. A bright field illumination 32 arranged under the microscope table 25 is usually part of the basic equipment of a microscope. Advantageously, this bright field lighting can be used unchanged in this exemplary embodiment. The lighting device comprises a housing 30 with a receiving trough for the condenser body 2. A cavity 40 is provided in the housing, which can be used for wiring the light emitting diodes 9 (not shown in the figure). Apertures 11 are integrated into the housing in order to limit the beams of light emitting diodes 9.

The microscope 24 includes a microscope objective 26. If this is an immersion objective, an immersion medium 23b can be used between the cover glass 8 and the objective 26. The microscope includes a further optical component 27, which can include an eyepiece or an image sensor or a microscope camera. Also indicated is a microscope tube 28 with an observation beam path 29 running therein.

The other reference numbers have already been explained in the previous exemplary embodiments.

Fig. 7 shows a sixth exemplary embodiment. Here the condenser body 2 is designed as a hemisphere. The optional bright field illumination 32, comprising a bright field light source 35 and a first lens 33, is also bundled here by means of the convex bright field light entry surface 36. To reduce the effective height of the transmitted light illumination device 1, the bright field light entry surface 36 of the condenser body can protrude into a recess in the microscope table 25.

A bright field illumination 32 arranged under the microscope table 25 is usually part of the basic equipment of a microscope. Advantageously, this bright field lighting can be used in this exemplary embodiment either unchanged or, as shown, with the second bright field lens removed compared to the previous example.

In a modification of the fifth and sixth exemplary embodiments, the sample 6 can be illuminated evanescently. The latter can be achieved in particular by providing the NA of the illumination to be greater than the refractive index of the sample medium.

Fig. 8 shows a seventh embodiment. A transmitted light illumination device 1 for a microscope is shown in a broken-out view. This comprises a rotationally symmetrical condenser body 2. The lighting device comprises a housing 30 with a receiving trough 31 for the condenser body 2. The condenser body 2 can be removed for a second operating mode and the transmitted light illumination device can be removed in this second operating mode Can be operated without immersion medium without the condenser body 2 for transmitted light illumination of the sample 6. Depending on the microscope objective used, the second operating mode can be oblique bright field illumination or dark field illumination of the sample 6. Fig. 9 shows the seventh exemplary embodiment without a condenser body. Without the condenser body, the NA of the illumination can be NA = sin(α1 _/ 2), where α ₁ is the first cone angle (full angle). The light emitting diodes 9 are arranged on the first cone shell so that they shine obliquely upwards into the plenum with respect to the specified z direction. The central rays meet in the first cone tip 17.

Fig. 10 shows a condenser body designed as a hemispherical disk. The top surface of the condenser body 2, which is just formed here and is shown below, can, but does not have to, be provided as a bright field light entry surface 36. The light entry surfaces 5 lie on the curved lateral surface (spherical zone). The center of the sphere lies in the flat light exit surface 4, which represents the segment base area. The hemispherical disc allows for a reduced overall height compared to a hemisphere.

Fig. 11 shows a condenser body designed as a generalized hemispherical disk. In contrast to the previous example, which was designed as a hemispherical disk, the top surface is designed as a concave bright-field light entry surface 36.

Fig. 12 shows a condenser body designed as a generalized spherical disk. Compared to the previous example, the height is reduced. The light exit surface 4 shown above is the segment base area, with the center of the sphere lying above this surface. This means that the underlying spherical segment is smaller than a hemisphere.

The reference symbols used uniformly in all figures are as follows:

1 . Transmitted light illumination device

2. Condenser body

3. Axis of symmetry

4. Light exit area, segment base area

5. Light entry surface

6. Sample

7. Sample carrier

8. Cover slip 9. Light emitting diode

10. Housing lens

11. Aperture

12. Electrical connections

13. Light beam

14. Central ray

15. first beam section

16. first cone shell

17. first cone tip

18. first cone angle (full angle)

19. second beam section

20. second cone shell

21. second cone angle (full angle)

22. Gap

23. Immersion media

24. Microscope

25. Microscope table

26. Lens

27. Eyepiece / image sensor or microscope camera

28. Microscope tube

29. Observation beam path

30. Housing

31. Hollow

32. Coaxial lighting device

33. First bright field lens

34. Second bright field lens

35. Bright field light source

36. Bright field light entrance surface, truncated surface

37. Second light emitting diodes

38. Third cone shell

39. Fourth cone shell

40. Cavity

Claims

Patent claims 1 . Transmitted light illumination device (1) for a microscope, comprising a. a rotationally symmetrical condenser body (2) with an axis of symmetry (3), a flat light exit surface (4) and several convex light entry surfaces (5, 5a, 5b), the arrangement of a sample (6) and / or a sample carrier (7) with a sample (6) is provided on the light exit surface (4), b. a plurality of light emitting diodes (9), in particular 8 to 128 pieces, from each of which a light bundle (13) with a central beam (14) can be emitted, the central rays (14) of the light bundles (13) emerging from the plurality of light emitting diodes (9) in one each first beam section (15) is arranged between the light emitting diode and the condenser body (2) on a first cone shell (16) of a first truncated cone, and each of the light bundles (13) is assigned one of the light entry surfaces (5), the central rays ( 14) the light bundles (13) are arranged in a second beam section (19) which extends in a straight line in the condenser body (2) from the respective light entry surface (5) to the light exit surface (4) on a second cone jacket (20) of a second truncated cone and the The first and second truncated cones are arranged concentrically to the axis of symmetry (3). 2. Transmitted light illumination device (1) according to claim 1, characterized in that the sample (6) is intended to be arranged directly on the light exit surface (4) or that the sample carrier (7) is optically attached to the light exit surface (23, 23a) by means of an immersion medium (23, 23a). 4) can be coupled or that the sample carrier (7) can be optically coupled to the light exit surface (4) by blasting or that the sample carrier (7) can be optically coupled to the light exit surface (4) by means of a gap (22) with a thickness of less than a quarter of a design wavelength. can be coupled. 3. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the plurality of light emitting diodes (9) each have a housing lens with which the respective light exit bundle has a divergence (FWHM) of 30° or less, in particular between 10° and 25 °, is collimable. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that it comprises apertures (11) arranged between the light emitting diodes (9) and the condenser body (2), which are provided for dimming edge rays of the light bundles. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the plurality of light emitting diodes (9) are each arranged at a distance of less than half the average radius of curvature of the light entry surfaces (5) from the light entry surfaces (5) and / or the first Jet section (16) is designed as a free jet path. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the sample (6) can be illuminated by means of the transmitted light illumination device (1) with a numerical aperture of more than 1, in particular more than 1.25. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the condenser body (2) has the basic shape of a spherical disk, in particular a spherical segment, in particular a hemisphere or such a spherical disk, which has a segment height of the spherical radius minus a predetermined target thickness d of a sample carrier ( 7). Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the second cone shell (20) has the same cone angle as the first cone shell (16) or that the second cone shell (20) has a larger cone angle than the first cone shell (16) . Transmitted light illumination device (1) according to one of the preceding claims, characterized in that a bright field light entry surface (36) is provided on the condenser body (2) opposite the light exit surface (4) for a further light beam for paraxial illumination of the sample (6). Transmitted light illumination device (1) according to one of the preceding claims, also comprising a housing (30) with a receiving trough (31) for the condenser body. per (2), characterized in that the condenser body (2) can be removed for a second operating mode and the transmitted light illumination device can be operated in this second operating mode without immersion medium without the condenser body (2) for transmitted light illumination of the sample (6). 11. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the light emitting diodes (9) can be controlled individually and/or divided into several groups. 12. Transmitted light illumination device (1) according to one of the preceding claims, characterized in that the condenser body (2) is equipped to scatter and/or absorb light on surfaces outside of optically functional surfaces, in particular matt and/or blackened. 13. Microscope with a transmitted light illumination device (1) according to one of the preceding claims, characterized in that this is provided for dark field illumination of the sample (6) and / or for evanescent illumination of the sample (6). 14. Microscope (24) for dark field microscopy, comprising a. a transmitted light illumination device (1) according to one of the aforementioned claims 1 to 12, b. a lens (26) and c. an eyepiece and/or an image sensor (27), characterized in that the objective (26) has a smaller NA than a ring aperture of the transmitted light illumination device (1). 15. Microscope (24) with a transmitted light illumination device (1) according to claim 10, characterized in that it can be operated in the second operating mode without the condenser body (2) for further dark field illumination or for oblique bright field illumination of the sample (6). 16. Use of a transmitted light illumination device or a microscope (24) according to one of the preceding claims for dark field examination of a blood sample, in particular a native blood sample. ahs for illuminating a sample (6) comprising a. Generating a plurality of light bundles (13) by means of a light emitting diode (9), each light bundle (13) having a central beam (14), and the central rays (14) emerging from the light emitting diodes (9) in a first beam section (15). a first cone shell (16) of a first truncated cone are arranged b. Coupling the light bundles (13) into a convex light entry surface (5) of a rotationally symmetrical condenser body (2) with an axis of symmetry (3), which has a flat light exit surface (4), c. Conducting the light bundles (13) in a straight line up to a light exit surface (4) of the condenser body (2) d. Coupling the light bundles (13) from the light exit surface (4) e. Coupling the light bundles (13) emerging from the light exit surface (4) into a sample (6) arranged directly or by means of a sample carrier (7) on the light exit surface (4).