EP3762707A1

EP3762707A1 - Optical device, optical module and microscope for scanning large samples

Info

Publication number: EP3762707A1
Application number: EP19707738.1A
Authority: EP
Inventors: Florian Fahrbach; Werner Knebel; Peter Pott; Wernher FOUQUET
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2018-03-05
Filing date: 2019-02-20
Publication date: 2021-01-13
Also published as: CN111819434A; JP2021515910A; US20210063714A1; DE102018203247A1; WO2019170413A1; US12019227B2

Abstract

The invention relates to an optical device (1) for illuminating a sample (25) located in a sample volume (23) with illumination light (15) and for detecting scattered and/or fluorescent light (27) from the sample (25). The device (1) comprises an illumination assembly (3) for transmitting the illumination light (15) along an illumination path (21) into the sample volume (23) and comprises a detection assembly (5) for collecting and relaying the scattered and/or fluorescent light (27) along a detection path (29). The invention also relates to an optical module (95) and a microscope (37), in particular a confocal microscope (39), comprising a mechanical receiving device (97) for optical modules (95). Solutions from the prior art are geometrically limited in terms of the arrangement of the illumination assembly (3) and the detection assembly (5), offer a low depth of immersion and have the disadvantage of more frequent collisions with the sample (25). The optical device (1) according to the invention improves the solutions from the prior art in that an attachment element (7) is provided, in which at least some portions of the illumination path (21) and/or the detection path (29) run.

Description

Optical device, optical module and microscope for scanning large samples

The invention relates to an optical device for illuminating a sample arranged in a sample volume with illumination light and for detecting scattered and / or fluorescent light from the sample, the device comprising an optical illumination arrangement for the transmission of the illumination light along an illumination path into the sample volume and an optical detection arrangement for collecting and forwarding the scattered and / or fluorescence light along a detection path from the sample volume.

Furthermore, the invention relates to an optical module and a microscope, in particular a confocal microscope, comprising a mechanical recording device for optical modules. Prior art optical devices, such as those used for light-leaflet microscopes, are preferred because of the preferred geometrical configuration of a light-beam microscope, i. the orientation of the illumination arrangement and detection arrangement perpendicular to each other, not suitable for scanning large samples. In the solutions of the prior art, a sample is illuminated, for example, from the side, particularly preferably with a light sheet, and in particular perpendicular thereto, the scattered and / or fluorescent light emitted by the sample is detected. The illumination arrangement may limit a possible mechanical experience of the sample.

The aim of the present invention is therefore to provide a compact optical device, a compact optical module and a microscope, which allow to scan large samples.

The optical device mentioned in the introduction solves this problem by having at least one attachment element in which the illumination path and / or the detection path extend at least in sections. The optical module according to the invention solves this task by virtue of the fact that it has an optical device according to the invention and the microscope mentioned above achieves the above object by accommodating therein an optical module according to the invention in the receiving device. The attachment element makes it possible to modify the orientation or the distance of the illumination and / or detection optics from the sample volume, which allows a more flexible and compact construction of the optical device. In that regard, the attachment element can have a beam-deflecting property. Furthermore, the solutions according to the invention allow investigations of sample sections, since the solid angle, which is occupied by the optical illumination arrangement and the optical detection arrangement, with attachment element is <180 °. The optical device, the optical module and the microscope can be further improved by the respective advantageous embodiments described below. Technical features of the embodiments can be arbitrarily combined with one another or omitted, as long as the technical effect achieved with the omitted feature does not matter.

The optical illumination arrangement or optical detection arrangement can comprise a plurality of individual lenses or a lens system. Such an embodiment allows easy access and replacement of the optical components and offers a high degree of flexibility. In particular, the illumination arrangement and / or the detection arrangement can each be designed as an objective. The illumination path can be defined by the optical elements of the illumination arrangement and in particular end in the sample volume or in the sample. Likewise, the detection path can be defined by the optical elements of the detection arrangement and also the detection path can end in the sample volume.

In particular, the terms illumination path or detection path denote those transmission paths which are arranged on a sample side of the illumination arrangement or on the sample side of the detection arrangement. The optical paths which run on the side of the illumination arrangement or detection arrangement facing away from the sample are not encompassed by these terms.

Furthermore, illumination light which passes through a sample or through a sample volume without a sample can also run beyond the sample volume along an optical path. Again, this is not understood as part of the lighting path.

The attachment element is therefore preferably located on the sample side of the illumination arrangement and the detection arrangement, wherein the attachment element may be a three-dimensional, transparent to the light used body in which the light (illumination light and / or scattered and / or fluorescent light) is transmitted ,

Preferably, light in the ultraviolet, visible and near-infrared spectral range is used. For example, the light of the illumination can have an ultraviolet excitation wavelength of a fluorescent substance and the fluorescent light can comprise wavelengths in the boring visible spectral range or in the near-infrared spectral range. Consequently, the illumination arrangement for the ultraviolet and the detection arrangement for the near-infrared spectral range is optimized. The deflection element may preferably be located only in the illumination path and be optimized for the ultraviolet spectral range. The attachment element can be configured such that both the illumination path and the detection path run in it, or else only the illumination path or the detection path.

In one possible embodiment, the attachment element can have an input-side optical cross-section and an output-side optical cross-section, wherein the input-side optical cross section can be larger than the output-side optical cross section. Such a cross-section is to be understood as usable for the transmission of light surface.

The attachment element may for example be frustoconical or truncated pyramidal.

The optical device can be further improved by attaching the attachment element to the optical illumination arrangement and / or to the optical detection arrangement. Consequently, the attachment element can be moved with one of the optical arrangements or with both arrangements.

Such an embodiment has, for example, the advantage of increased stability when illuminating / viewing the sample in a liquid medium without having to immerse the optical arrangements themselves. Contact of the usually expensive optical arrangements with unknown or undesired liquids is thus avoided.

In a further advantageous embodiment, at least the detector arrangement can be an imitation optics. Likewise, the illumination arrangement can be an immersion optics.

The illumination arrangement can preferably have a greater working distance than the detection arrangement, so that the illumination arrangement can be positioned at a greater distance from the sample volume than the detection arrangement. In addition, illumination arrangements can be used which have a smaller numerical aperture (NA) than the detection arrangement. The lower NA may be a slimmer, that is, slimmer, with respect to an optical axis. allow smaller size and thus further reduce mechanically limited angle when positioning the lighting assembly and the detection device.

If an immersion optics is used as detection arrangement, then it dips into a immersion medium with its proximal end, in particular with a front surface of an optical element. In an advantageous embodiment, the attachment element is located in the illumination path and immersed in the immersion medium, the attachment element protruding from the immersion medium. It should be noted at this point that an immersion medium is to be understood as meaning that medium which is located between the attachment element and the sample and in which the sample is embedded. The position of the immersion medium shown in this disclosure is not explicitly limited to the area between the optical arrangement in a cover glass, but also includes the medium surrounding the sample.

The illumination path preferably occurs outside of the immersion medium in the attachment element and preferably in the immersion medium from the attachment element. Thus, no disturbances occur in the illumination path, which originate from a moving surface of the immersion medium. When scanning the sample, the illumination arrangement can thus illuminate the sample without interference and a calming down of the surface movement of the immersion medium does not have to be awaited.

Also, airborne vibrations that can interfere with the surface of the immersion medium do not affect lighting or detection. The attachment element can be configured cup-shaped, whereby it opens to the illumination arrangement. Compared to a prefabricated element made of solid material, a cup-shaped attachment element is lighter and can thereby contribute to a faster possible scanning movement. In particular, the advantage lies in the fact that the optical path (product of refractive index and distance in the material of the attachment element) is not significantly changed, whereby no adaptation of the optical arrangement is necessary.

In a further advantageous embodiment, the attachment element can have a transparent element towards the sample volume. This transparent element can be a window and can be transparent in particular to the illumination light and / or to the scattered and / or fluorescent light. In particular, it is advantageous if the transparent element is oriented perpendicular to the respective beam path of the illumination or the detection. The perpendicular orientation to a beam path has the advantage of reducing aberrations caused by slanting the beam onto an illumination index interface.

The cup-shaped attachment element can preferably be introduced into the illumination path and be immersed in the immersion medium with a cup bottom or bottom surface. At least a part of the attachment element or the entire attachment element may have an optimal have refractive index adapted to the optical refractive index of the sample or of the medium surrounding the sample.

The intent element of the optical device may have at least two interfaces, which are oriented substantially perpendicular to the illumination path and / or to the detection path. This is particularly advantageous if the light to be transmitted comprises several wavelengths or is broadband.

By a vertical passage of the light through the interfaces, a wavelength-dependent angular separation (angular dispersion) is avoided by the dispersion of material. When monochromatic light is used for illumination, the incidence of the light into the material of the attachment element can also deviate from the vertical incidence. However, it should be noted here that the passage of a focused light beam through an oblique boundary surface leads to aberrations (coma, etc.) if the refractive index of the material differs on both sides of the interface. An important criterion at this point is therefore whether the refractive index of the attachment element is adapted to the sample / immersion medium. Such an adaptation can be achieved with a further advantageous embodiment of the optical device according to the invention, in which the cup-shaped attachment element can be filled with a further optical medium. For example, the attachment element can be filled with a further optical medium whose refractive index corresponds to that of the material of the attachment element. Furthermore, the further optical medium can be the medium of the attachment element, in other words a solid attachment element made entirely of a material is obtained.

The cup-shaped attachment element can also be designed as a chamber. The object-side entry surface into the chamber is preferably transparent and may in particular be perpendicular to the optical axis of the optical arrangement. The volume of the chamber can now be filled, for example, with the immersion medium. Likewise, of course, any other medium in question, for example, a mixture in which the refractive index on the concentration of a component can be significantly changed. The advantage then lies in the fact that the passage of the light from the optical arrangement takes place in the sample medium at the entrance surface in the attachment element, on which the interface is already perpendicular to the optical axis. Thus, aberrations are minimized. The exit can now basically take place at any angle, provided there is no jump in the refractive index. The front interface need not necessarily be oriented perpendicular to the optical axis, if, for example, thereby the space for the sample is increased. In terms of however, for possible optical errors, it may be advantageous to orient the sample side window of the chamber perpendicular to the optical axis of the optical assembly.

If the attachment element, which consists entirely of a material, is fastened in front of the optical arrangement, then it is advantageous if the entry and exit surfaces are perpendicular to the optical axis of the optical arrangement. If an attachment produced from a single material comprises a material with a refractive index which is greater than that of air (refractive index> 1 or more preferably refractive index> 1.33), then the refraction at the entry surface becomes such an element obtained an extension of the working distance of the optical arrangement. This can be advantageously used to the effect that the optical arrangement can be reset even further in relation to the sample in order to avoid a collision with a further optical arrangement. It is likewise possible that the angle between the optical illumination arrangement and the optical detection arrangement can be chosen to be smaller, since these can be spatially separated from one another. The two boundary surfaces of the cup bottom, one facing the illumination arrangement, the other to the sample volume, can therefore also be oriented perpendicular to the illumination path, so that no wavelength-dependent angular dispersion occurs when the illumination light passes through the cup bottom.

In a further embodiment, the attachment element of the optical device can be a deflecting element deflecting the illumination light and / or scattering and / or fluorescent light. This has the advantage that optical axes of the illumination arrangement and the detection arrangement need not be oriented perpendicular to one another, but an angle smaller than 90 ° can be selected, with a deflection of the light by the deflection element in the vicinity of an arrangement (illumination or detection) can. The second arrangement (detection or illumination) may be reset away from the sample volume, i. be further away from the sample volume.

Since an optical arrangement preferably extends rotationally symmetrically about the corresponding transmission path, for example in the form of holders and housing parts of the optical elements, the smallest possible angle between the illumination arrangement and the detection arrangement can be limited by these same housing parts.

Resetting one arrangement in relation to the other arrangement can mitigate this geometric limitation, so that both arrangements can be joined at a smaller angle. that can be oriented. Only the corresponding transmission path of the recessed arrangement passes the non-recessed arrangement and can be deflected in the deflection element such that the illumination path is preferably oriented perpendicular to the detection path. In a further advantageous refinement, a transmission path for the illumination light and / or the scattered and / or fluorescent light can be oriented in the deflecting element at an acute gloss angle to a side surface of the deflecting element. For the side surface and the transmitted light, the conditions for total reflection can be satisfied.

Thus, in a simple way, only by the choice of the geometry of the deflecting a deflection.

The gloss angle is defined as the angle between the incident light (along the corresponding transmission path) and the surface of the side surface. The gloss angle is the complementary angle to the angle of incidence to 90 °.

The conditions for the occurrence of total reflection are known from the prior art and include, for example, the transition from an optically dense to an optically less dense medium.

In particular, when using an immersion medium, it may be possible that the refractive index difference between the material of the deflecting element and the immersion medium is too low to allow total reflection below the desired gloss angle. In such a case, the side surface preferably has a reflective layer for the incident light. Such a coating may be a vapor deposited metal or a dielectric coating. In particular, a dielectric coating may be optimized for a predetermined glancing angle and wavelength of light used.

The attachment element designed as a deflection element also preferably has the two boundary surfaces oriented essentially perpendicular to the illumination path and / or to the detection path. This ensures that the deflection element does not introduce any angular dispersion and that the deflection is based solely on a reflection.

In particular, such a deflection element can also protrude from the immersion medium, wherein the entry of the illumination light into the deflection element occurs outside of the immersion medium and the exit from the deflection element within the immersion medium. killed. In order to avoid Fresnel reflections at the interfaces, antireflection coatings may be provided thereon.

Since a sample can be located completely in the immersion medium, it can be observed by the detection arrangement likewise immersed, at least in sections, in the immersion medium.

The sample can be moved by means of the translation stage, i. Although this scanning can induce disturbances in the surface of the immersion medium, it does not lead to disturbances in the illumination or in the detection.

Furthermore, due to the boundary surfaces oriented perpendicular to the corresponding transmission path, no distortions are introduced into the illumination light or the scattered and / or fluorescent light.

The scanning can in particular be done over a distance of several centimeters, which makes it possible, for example, to microscopy large brain sections. It is also possible to record three-dimensional cell clusters (organoids) in locally delimited volumes (so-called wells), which extend into the spatial directions over several 100 pm.

Since, in contrast to confocal microscopy, the area of a light sheet, that is to say an area of 500 μm by 500 μm, for example, can be detected in one step and not just a diffraction-limited point, one area is scanned in one scanning step and not just one point. which makes it possible to quickly microscopy the large-area object. A skewed light sheet may allow a penetration depth of up to about 300 pm when examining an unclarified sample. In the case of a clarified, i. chemically treated (brightened) sample, the penetration depth can be several millimeters.

The illumination of the sample can take place by means of a so-called static light sheet, which is preferably generated by a cylindrical lens. In transparent or semitransparent samples, a light sheet illuminates a cross-section; in the case of non-transparent objects, only the part of the circumference of the object reached by the illumination light is illuminated.

It is likewise possible to produce a so-called virtual light sheet in which focused illumination light is moved periodically (preferably) by means of a scanning device, for example a scanning mirror, along a direction perpendicular to the propagation direction. The frequency of scanning is preferably greater than an image acquisition frequency. Scanning a sample and detecting the scattered and / or fluorescent light emitted by it results in a stack of inclined cuts through the sample, which enable a 3D reconstruction.

The attachment element can preferably be attached to the illumination arrangement and / or to the detection system, so that after a single adjustment no subsequent adjustment of the deflection element is necessary.

The optical module according to the invention comprises an optical device according to the invention, it being possible for the illumination light and the scattered and / or fluorescent light to have a jointly used access or exit opening in the optical module. Such a shared opening can make it possible to accommodate the module according to the invention in a receiving device of a microscope.

Confocal microscopes, in particular, can have a collinear transmission path for the illumination light and the scattered and / or fluorescent light, so that illumination light emitted by the microscope on the optical module is transmitted in the module through the optical illumination arrangement via the illumination path to the sample volume.

Likewise, the scattered and / or fluorescent light emitted by the sample volume, especially from a sample arranged in it, is fed along the detection path through the optical detection arrangement through the common opening into the microscope, where it can be detected.

The optical module can thus be understood as a retrofit kit, by means of which light sheet microscopy with a confocal microscope is possible. By simply and quickly removing the optical module, the confocal microscope can again be used for confocal microscopy. Both the illumination arrangement and the detection arrangement can be mechanically fixed to the module via an adapter block and, in particular, be exchangeable. Likewise, it is conceivable that the individual optical elements of the illumination arrangement or the detection arrangement are each arranged and fastened separately in the optical module and can thus be variably replaced. The optical module can furthermore have an actuator, which makes it possible to vary the distance of the module to the sample and thus the position of the sheet of light to be formed, ie to set the sample level. Furthermore, the module can alternatively fall back on already provided in a micro-scope actuators.

To combine the transmission paths of the illumination and detection, in a further embodiment of the optical module according to the invention at least one wavelength-selective optical element can be provided, which has transmission and / or reflection properties for the illumination light, which differs from the transmission and / or Distinguish reflection properties of the fluorescent light.

The wavelength-selective optical element may be a dichroic mirror or a partial reflector. It is also conceivable to design the module according to the invention as a confocal illumination and / or detection module by removing a divider element (dichroic divider 101, 102, 35).

In the microscope according to the invention, an optical module according to the invention can now be accommodated by means of the mechanical recording device. This is preferably done by means of a standardized mechanical recording device, which is designed for one, the optical module mechanically fixed and secure in the microscope and on the other hand via a pick-up mechanism to precisely align the optical module with respect to the transmission paths of the microscope.

In the following, the present invention will be explained in more detail with reference to exemplary embodiments. The embodiments are advantageous in each case, wherein the technical features of the embodiments shown can be arbitrarily combined with each other and / or omitted. The same technical features and technical features of the same function or technical effect are provided with the same reference numerals.

Show it:

Fig. 1 shows a first embodiment of the optical arrangement according to the invention;

2 shows a second embodiment of the optical arrangement according to the invention;

3 shows an optical module according to the invention; and

4 shows the optical device according to the invention in two different configurations.

FIG. 1 shows an optical device 1 which comprises an optical illumination arrangement 3, an optical detection arrangement 5 and an attachment element 7. The optical illumination 3 comprises a schematically shown lens system 9 with an image-side main plane 10, whereas the optical detection arrangement 5 comprises a single lens 1. Both the optical illumination arrangement 3 and the optical detection arrangement 5 are shown with a housing 13, in other embodiments only lens systems 9 or individual lenses 1 1 can be arranged in the optical device 1.

The optical illumination arrangement 3 transmits illumination light 15, which is guided from a device side 19 to a sample side 17 and is transmitted there along a illumination path 21 through a sample volume 23. In the sample volume 23, a sample 25 is at least partially recorded. Starting from the sample 25, scattered and / or fluorescent light 27 is transmitted along a detection path 29 to the optical detection arrangement 5 through the sample volume 23 and collected by the optical detection arrangement 5 and forwarded to the device side 19.

The attachment element 7 is configured cup-shaped and has two boundary surfaces 31 oriented perpendicular to the illumination path 21. In the sample 25, the illumination path 21 and the detection path 29 are oriented substantially at an angle of 90 ° to each other. The two boundary surfaces 31 have an output-side optical cross-section 8b, which limits the size of the light passing through. In the illustrated embodiment, the output side optical cross section 8b is smaller than an input side optical cross section 8a facing the optical illumination device. If the attachment element 7 is assigned to an optical detection arrangement 5, then these cross sections 8a, 8b are defined analogously.

On the device side 19 a plurality of deflection mirrors 33 and dichroic mirrors 35 are shown. The dichroic mirrors 35 are special embodiments of wavelength-selective optical elements 101, which have different transmission and / or reflection properties 102 for the illumination light 15 and the scattered and / or fluorescent light 27.

Furthermore, FIG. 1 schematically shows the detail of a microscope 37, which in the embodiment shown is a confocal microscope 39. This comprises, for example, an optic 41 which collimates the illumination light 15 and conducts it to a cylindrical lens 43 via the two dichroic mirrors 35. In the cylindrical lens 43, the illumination light 15 is focused in one direction only, so that a light sheet 45 is formed in the sample 25. The light sheet is clearly shown in FIG. 4 in magnifications 47.

Perpendicular to the light sheet 45, the scattered and / or fluorescent light 27 is thus emitted and guided along the detection path 29 to the optical detection arrangement 5. The latter transmits the scattered and / or fluorescent light 27 via two deflection mirrors 33 and through the two dichroic mirrors 35 to a detector system 49 of the microscope 37.

The forming light sheet 45 extends along a propagation direction 51 which, in the embodiment shown, is oriented parallel to the illumination path 21 and along a width direction 53 which extends into the plane of the drawing. The light sheet 45 further has a thickness 55 measured along a thickness direction 57, wherein the thickness direction 57 and the detection path 29 are parallel to each other.

Both the illumination path 21 and the detection path 29 may be referred to as transmission paths 59. To distinguish the paths between the sample side 17 and the device side 19, the paths on the device side are referred to as input path 61 and as output path 63, respectively.

In a flange region 65, both the input path 61 and the output path 63 are collinear.

The operation of the attachment element 7 is shown in Fig. 2. The optical device 1 of FIG. 2 comprises an optical illumination arrangement 3 or an optical detection arrangement 5 designed in each case as an objective 67. The optical detection arrangement 5 is configured in particular as immersion optics 68 and in the embodiment shown in FIG. 2 as immersion objective 69, which is provided with a Front 71 is completely submerged in an immersion medium 73. By contrast, the optical illumination arrangement 3 is located only partially below a surface 75 of the immersion medium 73.

So that vibrations 77 which can form on the surface 75 do not disturb the illumination path 21 and the illumination fluctuates by means of the illumination light 15, the attachment element 7 is provided. Its first end 79 is located above the surface 75 of the immersion medium 73, whereas its second end 81 is below the surface 75 of the immersion medium 73. The cup-shaped attachment element 7 is not filled with the immersion medium 73 and allows interference-free illumination of the sample 25 through the interfaces 31.

In other embodiments (not shown), the attachment element 7 can be filled with a further optical medium or made solid in order to ensure adaptation to the refractive index of the sample and / or the immersion medium.

In particular, the vibrations 77 can occur on the surface 75 of the immersion medium 73 when a sample holder 83, on which the sample 25 is located, is moved along one of two possible scanning directions 85.

In contrast to the microscope 37 of FIG. 1, the microscope 37 of FIG. 2 has a scan mirror 87, which is held movably about an axis 87 and forms a so-called virtual light sheet 91 by a scanning movement 93 in the sample 25.

FIG. 3 shows a further embodiment of the optical device 1 according to the invention, which is accommodated in an optical module 95.

Such an optical module 95 can be accommodated in the microscope 37 by means of a suitable receiving device 97 and optionally comprise a confocal scanner. In the receiving device 97, as well as in the module 95 itself, a shared access opening 99 is formed, through which both the input path 61 and the output path 63 are co-linear.

This is realized by previously described dichroic mirrors 35, which constitute a special embodiment of the wavelength-selective optical elements 101.

In the embodiment of the optical device 1 shown in FIG. 3, the attachment element 7 is designed as a deflection element 103.

The deflecting element 103 comprises the boundary surfaces 31 and side surfaces 31a.

An interface 31 (the one which is located above the surface 75 of the immersion medium 73) is oriented perpendicular to the illumination path 21, whereas the interface 31 located in the immersion medium 73 is oriented at an angle not equal to 90 ° to the illumination path 21. The transmitted by the deflecting element 103 illumination light 15, that is, transmitted light 105 is reflected on a side surface 31 a and forms the light sheet 45, which is oriented perpendicular to the detection path 29.

However, the illumination arrangement 3 and the detection arrangement 5 are arranged relative to one another in an adjustment angle 107, which is significantly smaller than 90 °, compared to the embodiments shown above.

FIG. 4 shows the optical device 1 according to the invention in different configurations 109.

The configurations 109 relate, in particular, to differently running transmission paths 59, which lead to differing illumination paths 21.

In Fig. 4, a paraxial transmission path 59a and a peripheral transmission path 59b are shown, resulting in a paraxial illumination path 21a and in a peripheral illumination path 21b.

A configuration of paraxial illumination with deflection element 109a and with attachment element without deflection function 109b, as well as a configuration of peripheral illumination 109b are shown in the two enlargements 47.

In the configuration of paraxial illumination 109 a, the paraxial illumination path 21 a is collinear with an optical axis 11 1 of the optical illumination arrangement 3 and enters via the interface 31 into the attachment element 7 configured as a deflection element 103. The deflecting element 103 is fastened to the optical detection arrangement 5 by means of suitable fastening elements 121 in the shown configuration 109a, but in other embodiments may also be fastened to the optical lighting arrangement 3.

The transmitted light 105 strikes a side surface 31a and encloses a gloss angle 13 with it. Total reflection 115 can occur on the side surface 31a or a reflective layer 17 can be provided, which reflects the transmitted light 105.

In Fig. 4, the reflective layer 1 17 is shown schematically below the position of the total reflection 115. In real applications, either total reflection 115 or a reflective layer 117 is generally used to redirect the transmitted light 105 and the paraxial illumination path 21a. For these considerations, both the gloss angle 1 13, and the refractive index 103 a of the deflecting element 103 and the refractive index 73 a of the immersion medium 73, since, given similarly large refractive indices 73a, 103a, no total reflection 1 15 under the desired gloss angle 113 may be possible.

As a result of the deflection in the deflection element 103, the further paraxial illumination path 21a extends substantially perpendicular to the optical axis 11 of the optical detection arrangement 5. A dotted line shows an unchanged illumination path 21u which does not have the optical axis 11 of the optical detection arrangement 5 in the right Angle cuts.

In the peripheral illumination configuration 109b, the illumination light 15 is not collinearly irradiated to the illumination optical assembly 3, so that the peripheral illumination illumination path 21a does not coincide with the optical axis, unlike the paraxial illumination configuration 109a

1 11 of the optical illumination arrangement 3 coincides or matches.

The illumination light 15 is transmitted along the peripheral illumination path 21b in the cup-shaped attachment element 7 and passes perpendicularly through the two boundary surfaces 31 of a bottom surface 1 19 of the attachment element 7. As a result of this vertical orientation, no angular dispersion occurs during the transition from the attachment element 7 into the immersion medium 73.

Due to the non-collinear irradiation into the optical illumination arrangement 3, the peripheral illumination path 21 b is already oriented substantially perpendicular to the optical axis 11 of the optical detection arrangement 5 without any deflection. In the configuration 109 c, the attachment element 7 is solid, ie designed as a volume body 123. This configuration has the advantage that a distance (not shown) between the front of the optical arrangement and the sample can be increased.

reference numeral

1 optical device

3 optical illumination arrangement

5 optical detection arrangement

7 attachment element

8a input optical cross section

8b output optical cross section

9 lens arrangement

10 pictorial main level

1 1 lens

13 housing

15 illumination light

17 sample side

19 Device side

21 illumination path

21a paraxial illumination path

21 b peripheral illumination path

21 u unchanged illumination path

23 sample volumes

25 sample

27 scattered and / or fluorescent light

29 detection path

31 interface

31a side surface

33 deflecting mirror

35 dichroic mirror

37 microscope

39 confocal microscope

41 Optik43 cylindrical lens

45 light sheet

47 Magnification

49 detector system

51 propagation direction

53 width direction

55 thickness

57 thickness direction

59 T ransmissionspfad

59a paraxial transmission path

59b peripheral transmission path

61 input path

63 output path

65 flange area 67 lens

68 immersion optics

69 immersion objective

71 front

73 immersion medium

73a refractive index immersion medium

75 surface

77 vibrations

79 first end

81 second end

83 sample holders

85 Scan direction

87 scanning mirror

89 axis

91 virtual light sheet

93 Scanning motion

95 optical module

97 receiving device

99 access or exit opening

101 wavelength-selective optical element

102 transmission and / or reflection properties

103 deflecting element

103a refractive index deflecting element

105 transmitted light

107 adjustment angle

109 configuration

109a Configuration of paraxial illumination

109b Configuration of peripheral lighting

1 1 1 optical axis

113 gloss angle

115 total reflection

117 reflective layer

119 floor area

121 fastener

123 solids

Claims

claims

1. Optical device (1) for illuminating a sample (25) arranged in a sample volume (23) with illumination light (15) and for detecting scattered and / or fluorescent light (27) from the sample (25), the device (1) comprises an optical illumination arrangement (3) for transmitting the illumination light (15) along a illumination path (21) into the sample volume (23) and an optical detection arrangement (5) for collecting and relaying the scattered and / or or fluorescence light (27) along a detection path (29) from the sample volume (23), characterized by at least one attachment element (7) in which the illumination path (21) and / or the detection path (29) extends at least in sections.

2. Optical device (1) according to claim 1, characterized in that the ancillary element (7) has an input-side optical cross-section (8a) and an output-side optical cross-section (8b), the input-side optical cross-section (8a) being larger as the output side optical cross section (8b). 3. Optical device (1) according to claim 1 or 2, characterized in that the

Attachment element (7) is attached to the optical illumination arrangement (3) and / or to the optical detection arrangement (5).

4. Optical device (1) according to one of claims 1 to 3, characterized in that the at least the detector arrangement (5) is an immersion optics (68). 5. Optical device (1) according to one of claims 1 to 4, characterized in that the attachment element (7) has at least two boundary surfaces (31) substantially perpendicular to the illumination path (21) and / or to the detection path (29 ) are oriented.

6. Optical device (1) according to one of claims 1 to 5, characterized in that the attachment element (7) is an illumination light (15) and / or scatter and / or

Fluorescent light (27) deflecting deflecting element (103).

7. Optical device (1) according to claim 6, characterized in that in the deflection element (103) a transmission path (59) for the illumination light (15) and / or the scattered and / or fluorescent light (27) at an acute glancing angle (1 15) to a page surface (31 a) of the deflecting element (103) is oriented, wherein for the side surface (31 a) and the transmitted light (105) the conditions for total reflection (115) are met.

8. An optical device (1) according to claim 7, characterized in that the side surface (31a) for the light (15, 27) reflective layer (117). 9. Optical device (1) according to one of claims 1 to 5, characterized in that the attachment element (7) is designed cup-shaped and opens to the optical illumination arrangement (3) out.

10. Optical device (1) according to claim 9, characterized in that the ancillary element (7) has a transparent element towards the sample volume (23). 1 1. An optical device (1) according to claim 9 or 10, characterized in that the

Attachment (7) is filled.

12. An optical module (95) comprising an optical device (1), characterized in that the optical device (1) is an optical device (1) according to any one of claims 1 to 11. 13. Optical module (95) according to claim 12, characterized in that for the illuminating light (15) and the scattered and / or fluorescent light (27) a jointly used access or exit opening (99) in the optical module (95).

14. An optical module (95) according to claim 12 or 13, characterized in that at least one wavelength-selective optical element (101) is provided, which has different transmission and / or reflection properties (102) for the illumination light (15) which differ from the transmission and / or reflection properties (102) for the scattered and / or fluorescent light (27).

15. A microscope (37), in particular a confocal microscope (39), comprising a mechanical recording device (97) for optical modules (95), characterized in that an optical module (95) according to any one of claims 12 to 14 in the receiving device

(97) is recorded.