WO1999003008A1

WO1999003008A1 - Device for the optical analysis of samples

Info

Publication number: WO1999003008A1
Application number: PCT/EP1998/004227
Authority: WO
Inventors: Stefan Hummel; Rolf Günther; Norbert Garbow
Original assignee: Evotec Biosystems Ag
Priority date: 1997-07-09
Filing date: 1998-07-08
Publication date: 1999-01-21
Also published as: DE19729245C1

Abstract

A device for the optical analysis of samples, comprising at least one light source (80) for generating electromagnetic radiation (12) and a mirror lens (10) arranged on a first optical axis (11), for receiving the electromagnetic radiation (12) generated by said light source, defining a focus (14) in a sample (16) of interest and receiving the electromagnetic radiation generated in said focus (14). Said mirror lens (10) is provided with at least one optical element (18) consisting of a transparent optical material having a first outer side (20) and a second outer side (22) opposite said first side and facing said focus (14). Said first outer side (20) has a first, concave reflecting surface (24) including an opening (28) for letting light through, while said second outer side (22) has a second, convex reflecting surface (32) located on said optical axis (11), in face to face relationship with said opening (28) for letting light through, provided in said first outer side (20), and a surface (34) for letting light through, provided around said second reflecting surface (32).

Description

Device for the optical detection of samples

The present invention relates to a device for the optical detection of samples, which has a mirror lens, and to the use of this device for different application areas.

In addition to the pure analysis of individual molecules, statements about the state parameters of the molecules, such as their conformation and interaction with other molecules or molecular structures, are important for many areas - such as in the screening for pharmacologically active substances. Modern methods of evolutionary biotechnology deal with highly complex collectives of molecules. It is important to identify molecules with specific interaction properties against target structures, i.e. to measure a certain fitness with regard to a desired function. Such fitness can be traced back to thermodynamic parameters such as binding constants or speed constants.

To solve certain problems, a very large number of samples often has to be dealt with, which should be analyzed more or less simultaneously. The molecules to be analyzed are often only present in small concentrations. The methods of confocal fluorescence spectroscopy have proven to be advantageous analysis methods.

WO 94/16313 describes a method and a device for identifying one or a few molecules by using laser-excited fluorescence correlation spectroscopy (FCS). The FCS measuring principle is based that fluorophoric molecules are measured in extremely dilute solutions by exposing a relatively small measuring volume of preferably ≤ 10 ^"14 1 of the solution to the excitation light of a laser. Molecules with a corresponding excitation spectrum that are in this volume are excited emitted fluorescence from this measurement volume can then be imaged on a detector of high sensitivity, the change in the fluorescence intensity resulting from the changing number of molecules in the measurement volume due to the diffusion movement is analyzed, the device described in WO 94/16313, equipped with confocal optics, is drawn is characterized in particular by a pinhole arranged in the emission beam path in the image plane of the objective or by a detector replacing the pinhole, in particular the optics used have a high numerical aperture.

WO 96/13744 describes a method and a device for determining substance-specific parameters of one or fewer molecules by means of correlation spectroscopy, the principles of near-field optics being used.

The coupling of optical fiber arrays to lens systems has been described (Kufner et al., Micro-optics and lithography, VUBPress, Brussels, 1997; Sazaki et al., Put-in micro-connectors for alignment-free coupling of optical fiber arrays, IEEE Photon Technol. Lett. Vol. 4, p. 908-911, 1992).

Catadioptric systems (Mangin mirror, Cassegrain systems) are described in the literature (Warren J. Smith, Modern Optical Engineering, The Design of Optical Systems; Mc-Graw-Hill Inc., 1990). Due to the construction of these systems from several individual optical elements, miniaturization is structurally limited. GB 21 19 112 A relates to an optical system for reflective focusing of the electromagnetic radiation entering the optical system through an aperture onto a detector shielded from undesired background radiation by means of reflective surfaces.

The object of the present invention is to enable the use of a single objective for both the illumination and the detection beam path, it being intended to ensure that the “fields of view” of the excitation radiation source and the detector overlap in the sample to be examined or are essentially identical.

The problem underlying the invention is solved by a device according to claim 1. Advantageous refinements and applications of the invention are specified in the remaining claims.

The invention provides a device for optical

Detection of samples provided with at least one light source for generating electromagnetic radiation, a mirror objective arranged on a first optical axis, which receives electromagnetic radiation generated by the light source and generates a focus in a sample to be examined and receives the electromagnetic radiation generated therein, wherein the mirror objective is provided with at least one optical element made of an optically transparent material, the optical element has a first outer side and a second outer side facing away from it and facing the focus, the first outer surface has a concave first mirror surface with a light transmission opening, and the second outer surface has a convex second mirror surface which is arranged on the optical axis of the light transmission opening opposite the first outer surface and has a light transmission surface arranged around the second mirror surface, and on the optical axis At least one aperture and / or at least one detector element and / or at least one optical fiber is arranged in the area of the common beam path of the electromagnetic radiation generated by the light source and the radiation generated in the sample between the light source and the mirror lens or in the area of the light passage opening of the mirror lens .

The device according to the invention shows its advantage particularly when "small" measurement volumes, in particular less than 10 ^"12 1, are to be generated. The measurement volume is determined by the intersection of the" field of view "of the transmitter and detector The dye of the sample is excited to glow in the area of the entire transmitted beam, ie the field of view of the excitation radiation source. The excitation density at every point of the sample is proportional to the brightness of the beam, ie in the area of the focus, much larger than in the environment However, the proportion of the focus area in the total fluorescence light generated is small, since the focus has only a small extent. The "field of view" of the detector can be viewed in a completely analogous manner. Only if it is ensured that the detector fluorescence light mainly comes from the focus of the transmitted beam receives, one receives well-defined "small" measuring volume. In this case, the bright, high-weight areas of the focus enhance their contribution to the measurement volume; the areas with less weight outside the focal point are mutually reduced.

Mathematically, the measurement volume is defined as an integral over the product of the so-called point spread functions (PSF) of the transmitter and detector:

V e _ß \ A - = j JljJijJ PSF _transmitter (x, y, z) PSF _detector (x, y, z) dx dy dz V

The point spread functions have their maximum in focus and have only small values outside of it, so that their product is very small and negligible in the integral. The construction according to the invention using an aperture, a detector or an optical fiber in the region of the light transmission opening or in the above-mentioned common beam path ensures that the two functions are essentially identical. The arrangement according to the invention advantageously enables measurements without the need for complex readjustments of individual optical elements.

In a preferred embodiment, in particular a diaphragm is arranged in the area of the light transmission opening. With a suitable choice of the size of the light transmission opening, this can itself take on the function of an aperture. It can also be preferred to replace the diaphragm by a detector, in particular by miniaturized detector elements. This fills only a part of the light transmission opening, so that it is ensured that electromagnetic radiation generated by the light source can enter the mirror objective 10. The mirror objective has only very low chromatic and transverse aberration, so that it is particularly advantageous to use an optical fiber in the area of the light transmission opening, in particular especially a single-mode fiber to connect. The emission radiation emanating from the sample can be collected with this fiber and, for example, fed to a suitable detection device via a dichroic mirror. It has proven to be advantageous to couple the glass fiber to the mirror lens, which has a suitable coupling material, by fusing it with the fiber or using a so-called plug-in microconnector (Kufner et al., Micro-optics and lithography, VUBPress, Brussels, 1997 , Pp. 103 and 104; Sazaki et al., Put-in microconnectors for alignment-free coupling of optical fiber arrays, IEEE Photon. Technol. Lett. Vol. 4, p. 908-911, 1992). In general, means for variable coupling of the optical fiber can be provided in the region of the light transmission opening 28 and / or along the optical axis.

The structure according to the invention is particularly suitable for parallelization or for the design of an array. For this purpose, a large number of light sources for generating electromagnetic radiation can be provided; Alternatively, however, the electromagnetic radiation generated by one or a few light sources can be split into light paths that are essentially parallel to one another by means of suitable optical elements. This results in a large number of optical axes on which the mirror lenses already described can be arranged. These each focus the radiation into samples to be examined, which are preferably arranged in an array. The samples to be examined can be located, for example, in known micro- or nanotiter plates. Radiation emanating from the samples to be examined - for example fluorescence radiation - is received by the respective mirror objective and fed to an associated detector. According to the invention, each of the plurality of optical axes is also located in the area of the common one Beam path of the electromagnetic radiation generated by the respective light source and the radiation generated in the respective focus between the respective light source and the respective mirror lens or in each case in the region of the light passage opening of the relevant mirror lens at least one diaphragm and / or at least one detector element and / or at least one optical fiber.

It has also proven to be particularly advantageous that when an optically transparent film is used, the distance between it and the focus is less than 1000 μm and in particular between 50 μm and 300 μm. As a result, adsorption effects of the substances to be examined on the optically transparent film and interference effects, such as Minimize scattering effects when the excitation and / or emission radiation passes through the sample. For applications that require the use of an optically transparent film, the shape of the light transmission surface 34 and the choice of materials (mirror lens body / immersion liquid between lens and film / film) can be adapted to the desired optical set-up in such a way that chromatic errors are almost impossible become. For this purpose, the person skilled in the art can, for example, use optical software available on the market (e.g. Zemax, Focus Software Inc., USA; Oslo, Sinclair Optics Inc., USA) to adapt the design of the mirror lens to the optical set-up. Spherical aberrations, which are caused in particular by sample carriers, can be completely compensated for by a preferably aspherical configuration of the mirror surface.

In a preferred embodiment, the light transmission surface is curved, in particular hyperbolic or spherical. However, it can also be advantageous to emboss the light transmission surface in a planar manner. It can also be provided with diffractive optical elements. The light transmission surface can in particular have a coating of dielectric and / or polarization-selective and / or colored materials.

The optically transparent material of the optical element can have a homogeneous refractive index. In a further embodiment, however, the optical element consists of optically transparent materials with different refractive indices. The refractive index of the optical element preferably varies in a radially symmetrical manner with respect to the optical axis and / or along the optical axis.

It is further preferred to design the first mirror surface to be elliptical or spherical, while the second mirror surface is in particular spherical.

The imaging properties of the mirror objective are preferably to be adapted to the thickness of the film and / or the refractive indices of the immersion liquid, the film, which can in particular be a cover glass, and / or the sample. For this purpose, the radii of curvature of the first mirror surface and / or the second mirror surface and / or the light transmission surface can be designed in a suitable manner. To this extent, it is advantageous if the imaging properties of the mirror objective with regard in particular to the radii of curvature of the first and second mirror surfaces and / or the light transmission surface and / or the refractive index of the optical element to the thickness of the film and / or the refractive index of the immersion liquid, the film and / or are adapted to the sample.

In a further embodiment, the mirror objective can also be used without a film and / or immersion liquid, the sample in this case in particular as so-called hanging drop is in contact with the outside. Furthermore, an optically transparent material with a suitable refractive index can be selected for the optical element. These can be glass materials known in the prior art or also suitable plastic materials, such as polycarbonates, of optical quality.

It can also be preferred to arrange further optical elements, in particular lenses and / or mirrors and / or optical filters along the optical axis of the mirror objective.

In order to compensate for possible variations in the thickness of the optically permeable film and / or to vary the surface shape of the mirror objective, it may be preferred to arrange piezo actuators and / or electrostrictive actuators in particular on the first outside. A change in the radius of curvature of the mirror surfaces and / or the light transmission surface can also be achieved by a suitable arrangement of an expandable material, for example a metal frame that can be tempered, in the region of the optical element.

In a further embodiment, the second outside is completely or at least partially coated with an optically transparent material. This has in particular the same refractive index as the immersion liquid used. A planar surface of the mirror lens on the side facing the immersion liquid can be achieved by suitable coating.

In a preferred embodiment, the position of the mirror objective according to the invention in relation to a vessel containing the sample to be examined can be controlled three-dimensionally, for example by means of piezo technology. For this purpose, means for positioning the mirror objective relative to the sample can be provided. be seen. It is also possible to rasterize the sample.

The mirror lens according to the invention is particularly useful in optical scanning microscopy, e.g. laser-excited fluorescence scanning microscopy. It can also preferably be used in spectroscopy. Luminescence spectroscopy such as fluorescence correlation spectroscopy (FCS) or molecular-independent fluorescence techniques, Raman spectroscopy, light scattering and absorption spectroscopy should be mentioned here in particular. The molecules in sample 16 can be excited by single or multi-photon excitation. A method based on a molecular brightness analysis that is independent of the molecular weight is disclosed in detail in WO 98/16814, the disclosure content of which is hereby expressly incorporated by reference. Applications of the FCS are discussed in particular in European Patent EP 0679 251 B1, the entire disclosure content of which is also hereby incorporated by reference.

Another field of use of the device according to the invention can be seen in medical technology and here in particular in endoscopy.

Exemplary embodiments of the invention are explained in more detail below with reference to the drawings. Show individually n:

1 is a side view of a miniaturized mirror lens,

2 shows a schematic representation of a large number of mirror lenses arranged in arrays for quasi-parallel examination of a large number of samples, 3 shows an optical construction with an aperture arranged in the region of the light passage opening of the mirror objective,

4 is a side view of a mirror lens with fiber coupling,

5 is a side view of a further embodiment of a mirror lens with fiber coupling,

Fig. 6 is a side view of another embodiment of a mirror lens with fiber coupling and

Fig. 7 is a side view of another embodiment of a mirror lens.

In Fig. 1, a mirror lens is shown schematically in side view. In the case shown in FIG. 1, the mirror objective 10 is used to focus incident excitation light or radiation 12 in a focus 14 which is arranged within the sample 16 to be examined, with the result of the excitation light 12 in the sample 16 being emission light or Emission radiation is generated, which leaves the mirror lens 10 in the opposite direction to the direction of incidence of the excitation light. The mirror objective 10 has at least one optical element 18 made of an optically transparent material and has a first outer side 20 facing the incident excitation light 12 and a second outer side 22 facing the sample 16 and thus the focus 14 and facing away from the first outer side 20. The first outer side has a concave first outer surface 24, which carries an essentially light-reflecting coating 26. The first outer surface 24 has a region 28 enclosed on all sides by the coating 26, in which it is free of the coating 26. The area 28 represents thus a through opening for the excitation and emission light. The material of the coating 26 is selected such that the first outer surface 24 acts in its area provided with the coating material 26 for radiation incident from the inside like a mirror surface (first mirror surface).

The second outer side 22 has a second outer surface 30, which comprises differently curved sections. In its central region and opposite the opening 28, the second outer surface 30 is provided with a relatively strongly convexly curved section 32, to which a less strongly curved surface section 34 adjoins on the outside in a ring shape. The section 32 of the second outer surface 30 is coated from the outside with a coating material which is essentially light-reflecting. In its section 34, the second outer surface 30 is translucent, so that light can enter or leave the element 18 via this section 34, in particular with refraction of light. To this extent, section 34 of second outer surface 30 is a light transmission surface, in particular a refraction surface, while section 32 acts like a mirror surface (second mirror surface) for light that strikes from the inside.

As can be seen from Fig. 1, is? the mirror lens 10 is in the immediate vicinity of a glass or plastic plate or film 38, the area between the film 38 and the second outside 22 of the element 18 being filled with an immersion liquid 40, which is in particular water or always - Sion oil is trading. The sample 16 to be examined is located on the side of the film 38 facing away from the mirror objective 10. The thickness of the film is preferably between 100 and 200 μm. The operation of the mirror lens 10 and in particular the propagation of light within the mirror lens are as follows. Excitation light 12 enters the optical element 18 via the opening 28. This excitation light 12 is reflected divergently on the second mirror surface 32 until it strikes the first mirror surface 24. From there, the excitation light 12 is reflected in the direction of the light transmission surface 34. The excitation light 12 emerges through this light transmission surface 34, in particular with refraction of light, and through the immersion liquid 40. Furthermore, the excitation light 12 penetrates the film 38 and is focused in the focus 14. The focus 14 located in the sample 16 to be examined has in particular a volume in the range of 10 10 ^"12 1, preferably <10 ^{~ 14} 1. In the sample 16, emission radiation excited by the excitation light 12 reaches the direction of propagation of the excitation light 12 opposite direction back into the optical element 18 and out of the opening 28.

The advantage of the mirror objective 10 described here and shown in FIG. 1 is that the reflection surfaces and the light transmission surface 34, which is designed in particular as a refraction surface, are formed on a common element. This allows the mirror - objectively in one piece, i.e. realize monolithic. This in turn has advantages in terms of miniaturization of the mirror lens. In this way, for example, mirror lenses 10 can be produced, the extensions of which lie transversely to the direction of light incidence and in the direction of light incidence in the millimeter or sub-millimeter range.

FIG. 2 schematically shows a large number of mirror lenses arranged in arrays for quasi-parallel examination of a large number of samples. The mirror lenses are arranged here in the form of a multi-array, in order to be able to examine a large number of samples 16, which are present in suitable so-called multi-well plates - which may have a 96 format, for example - at the same time. This results in particular uses in the area of high-throughput screening for pharmacologically active substances or for diagnostic examinations. The optical elements 18 arranged in columns and rows are integrally formed on a common carrier body 44 and have light guides 46, which lead to an optical lens array 48 with a plurality of lenses 50, are coupled into the light guides 46 via the excitation radiation and emit emission radiation of the samples the optical fibers 46 is coupled out.

Figure 3 shows a further embodiment of the device according to the invention. Electromagnetic radiation generated in a light source 80 (for example a He-Ne laser with a wavelength of 543 nm, Uniphase Ine,, USA) strikes a dichroic mirror or beam splitter 70 and, after passing through a lens arrangement 62 and diaphragm 66, reaches the light transmission opening 28 of the mirror lens 10, which is an optical element 18 from, for example Has quartz glass. The mirror lens has in particular a numerical aperture ≥ 0.9. The laser light that enters the sample through a cover glass BK7 excites the molecules in the observation volume, for example, to fluoresce. The resulting fluorescent light passes through the mirror lens 10, the aperture 66 and the lens arrangement 62 onto the dichroic mirror or beam splitter 70. From there it is fed to a further lens arrangement 64 and by means of a detector 90 (eg avalanche photo diode SPCM-AQ-131 , EG & G Inc., Canada).

In FIG. 4, the mirror objective 10 is embedded in a body made of quartz, for example consists of a first part 104 and a second part 102 serving as a "front panel". This configuration offers advantages in terms of production technology in particular if the optical element 18 is filled with an immersion liquid. In this embodiment, the fiber tip 47 of the coupled single-mode fiber 46 serves as a common field of view diaphragm for excitation and detection light. The refractive surfaces of the front plate 102 are chosen to be flat. With a suitable choice of the immersion liquid, the remaining axial focus shift, which is caused by the dispersion of the front plate and sample holder, can be reduced to below 0.1 μm for the entire visual range. The micro-optics map two diffraction-limited points, the core mode on the fiber front surface and the focal point in the sample. The focus diameter is inversely proportional to the wavelength and proportional to the numerical aperture of the focused beam. In a good approximation, the divergence angle of the light emerging from the fiber is proportional to the wavelength. The numerical aperture (on the focal point side) therefore increases with the wavelength, so that the focus diameter is almost independent of the wavelength.

FIG. 5 shows a further preferred embodiment of the mirror objective 10. This is embedded in a monolithically designed body 104. The optical element 18 consists of two different “optically transparent materials. This can be, for example, immersion liquid in the part of the optical element 18 facing the body 104, while quartz glass can be used as a further material. This embodiment also has mounting plates 120 that connect the monolithically shaped body 104 to the mirror lens 10. In the embodiment shown in FIG. 6, the mirror objective 10 is in turn embedded in a body which consists of a first part 104 and a second part 102 serving as a “front plate”. In contrast to FIG. 4, a curved light transmission surface 34 is arranged around the mirror surface 32.

FIG. 7 shows, compared to FIG. 6, an optical setup without a sample carrier. The sample 16 to be examined - such as an assay solution - is in direct contact with the front plate 102.

Claims

EXPECTATIONS

1. Device for the optical detection of samples with at least one light source (80) for generating electromagnetic radiation (12), a mirror lens (10) arranged on a first optical axis (11), which receives electromagnetic radiation (12) generated by the light source and generates a focus (14) in a sample (16) to be examined and receives the electromagnetic radiation generated therein, the mirror objective (10) being provided with at least one optical element (18) made of an optically transparent material, the optical element ( 18) has a first outer side (20) and a second outer side (22) facing away from it and facing the focus (14), the first outer side (20) has a concave first mirror surface (24) with a light transmission opening (28), and the second Outside (22) has a convex second mirror surface (32) which is on the optical axis (11) of the light passage opening (28) of the first outside (20) is arranged above and has a light transmission surface (34) arranged around the second mirror surface 032), and on the optical axis (11) in the region of the common beam path of the electromagnetic radiation (12) generated by the light source (80) and that in the sample (16) generated radiation between the light source (80) and the mirror lens (10) or in the region of the light passage opening (28) of the mirror lens (10) at least one diaphragm (66) and / or at least one detector element and / or at least one optical fiber (110) is arranged.

2. Device according to claim 1, characterized in that the light transmission surface (34) is curved, in particular hyperbolic or spherical, or flat and / or is provided with diffractive optical elements.

3. Apparatus according to claim 1 and / or 2, characterized in that the light transmission surface (34) is coated in particular with dielectric and / or polarization-selective and / or colored materials.

. Device according to one of claims 1 to 3, characterized in that the optically transparent material of the optical element (18) has an inhomogeneous refractive index which varies in particular radially symmetrically to the optical axis (11) and / or along the optical axis (11).

5. Device according to one of claims 1 to 4, characterized in that the optically transparent material of the optical element (18) consists at least partially of an immersion liquid.

6. Device according to one of claims 1 to 5, characterized in that the second mirror surface (32) is spherical.

7. Device according to one of claims 1 to 6, characterized in that the first mirror surface (24) is elliptical, spherical or aspherical.

8. Device according to one of claims 1 to 7, characterized in that between the sample (16) and the second outside (22) an optically transparent film (38) and / or immersion liquid (40), in particular water or immersion oil, is arranged.

9. Device according to one of claims 1 to 8, characterized in that a single-mode fiber is located on the optical axis (11) in the region of the common beam path of the entering into the light passage opening (28) and emerging from this electromagnetic radiation, one end of the fiber is arranged in particular in the region of the light passage opening (28).

10. Device according to one of claims 1 to 9, characterized in that means for variable coupling of an optical fiber are arranged in the region of the light passage opening (28) and / or along the optical axis (11).

11. The device according to one of claims 1 to 10, characterized in that along the optical axis

(11) further optical elements, in particular lenses and / or mirrors and / or optical filters, are arranged.

12. Device according to one of claims 1 to 11, characterized in that in particular on the first outer side (20) of the mirror objective (10) means, in particular piezo actuators and / or electrostrictive actuators, arranged to vary the surface shape of the mirror objective (10) are.

13. Device according to one of claims 1 to 12, characterized in that the second outside (22) is completely or partially coated with an optically transparent material, which in particular has the refractive index of the immersion liquid (40) used, the surface of the material towards the immersion liquid (40) being designed to be particularly flat.

14. Device according to one of claims 1 to 13, characterized in that means for positioning the mirror objective (10) relative to the sample (16) are provided.

15. The device according to one of claims 1 to 14, characterized in that a plurality of mirror lenses

(10) with their optical elements (18), in particular as an array (42) in preferably orthogonal columns and rows, in particular monolithically arranged on a common carrier body (44).

16. The device according to one of claims 1 to 16, characterized in that the mirror objective (10) is embedded in a body (102, 104).

17. The apparatus according to claim 16, characterized in that the body has a first part (104) which receives the mirror lens (10), and a second part

(102), which covers the second outside (22), and that the two parts are fixedly or releasably connected to one another.

18. Use of a device according to one of claims 1 to 17 for optical scanning microscopy, in particular for laser-excited fluorescence scanning microscopy.

19. Use of a device according to one of claims 1 to 18 for spectroscopy, in particular luminescence spectroscopy such as fluorescence correlation spectroscopy, molar mass-independent fluorescence techniques, Raman spectroscopy, light scattering, absorption spectroscopy, in particular with single or multi-photon excitation.

20. Use of a device according to one of claims 1 to 19 in medical technology, in particular in endoscopy, in diagnostics or in screening processes for finding pharmacological active ingredients.