EP1384104A2

EP1384104A2 - Device and method for the optical measurement of chemical and/or biological samples

Info

Publication number: EP1384104A2
Application number: EP02738002A
Authority: EP
Inventors: Achim Kirsch; Roland Stange; Jürgen Müller
Original assignee: Evotec OAI AG
Current assignee: Evotec OAI AG
Priority date: 2001-04-28
Filing date: 2002-04-26
Publication date: 2004-01-28
Also published as: DE10121064A1; WO2002088819A3; US20040257576A1; WO2002088819A2; AU2002312858A1; US7474777B2

Abstract

An optical measuring device for the measurement of chemical and/or biological samples comprises an illumination device (16), for the illumination of the sample (20) for measurement. A lens arrangement (22) is further provided, for imaging a region of the sample in an image field (10). A fist partial region (12) of the image field is recorded by a first detector (28) and a second partial region (14) of the image field (10) is recorded by a second detector (34). It is thus possible to change between two measuring techniques without switching or to carry out two measurements simultaneously on one sample.

Description

Device and method for the optical measurement of chemical and / or biological samples

The invention relates to a device and a method for the optical measurement of chemical and / or biological samples. The device according to the invention and the method according to the invention are particularly suitable for use in high and medium throughput screening systems.

There are different measurement methods for the microscopic optical examination of samples, which provide different information. Either image or point measurements can be carried out. In the case of image measurements, the value of a physical quantity is generally recorded as a function of the measurement location. This physical quantity is measured either at many different points or measuring locations in parallel or only at one point and the location of this point, i.e. the measurement location is varied. The latter is often referred to as "scanning" the sample area. Each individual measurement must be carried out very quickly in order to be able to complete the image measurement within a reasonable time.

Examples of image measurement methods that can be used are: bright field, dark field, total internal reflection, fluorescence, 2-photon fluorescence, fluorescence lifetime, fluorescence emission spectroscopy, polarization and fluorescence polarization microscopy. In addition, these methods can be carried out with different detectors, such as line or area cameras and there is also the possibility to use them in both conventional and confocal arrangements.

In contrast to image measurement, in the case of point measurements, the measurement is only carried out at a single location in the sample, the location information is not evaluated or it is averaged over a large number of measurement locations at the same time (conventional measurement with a large-area detector). Accordingly, the single measurement can be both complex and time consuming. The complexity can relate to both the measuring apparatus and the resulting data (e.g. entire spectra). For example, The point measurement method fluorescence correlation spectroscopy (FCS) records the fluctuation of a fluorescence signal coming from a small volume over a longer period of time and derives information about the photophysical, chemical and physical properties of fluorescent particles and molecules in this volume. These measurements are often based on the assumption that the volume under consideration is representative of the sample. This is certainly true for homogeneous samples, but only to a limited extent if there are also structured components in the sample. Possible point measuring methods include but not exclusively FCS, FIDA, fluorescence emission, fluorescence excitation and absorption spectroscopy, fluorescence lifetime spectroscopy and fluorescence anisotropy measurements as described in many scientific and technical publications. The measurements can often be carried out in a conventional as well as in a confocal arrangement.

For the characterization of substances for possible pharmaceutical or medical applications, an investigation using both image and point measurement methods is often useful and necessary. Then information about all components can be extracted from samples that contain both homogeneous and structured components. For example, the reaction of a sample of cells thought to be specific molecules in cells dispense the surrounding solution, be examined for an added test substance. Via FCS, a possibly occurring biochemical reaction can be detected in the solution, while fluorescence microscopy allows an examination of the cell layer and, for example, gives indications of a possible toxicity of the test substance.

With the help of multichannel microscopy, several measurements of the same sample area can be carried out under different measurement conditions or with different detectors for a sample with image measurement methods. This is e.g. the distribution of different fluorophores in the sample, which differ based on their photophysical properties, such as the excitation, emission spectra and / or fluorescence lifetime, are determined.

The FCS instrument for intracellular FCS from Brock (see e.g. Brock, "Fluorescence Correlation Microscopy and Quantitative Microsphere Recruitment Assay", Dissertation, 1999) can be mentioned as devices that combine image measurement methods with point measurement methods. This device consists of a fluorescence microscope, which has been supplemented with the parts necessary for FCS. The choice between the measurement methods of microscopy or FCS is made by a folding mirror arranged in the beam path.

Such a mechanical switching of the beam path with the aid of a folding mirror leads on the one hand to the fact that the positions of the different measurement volumes relative to one another have to be determined. In addition, in high-throughput applications, this switching cycle must be carried out frequently, so that mechanical wear of the components occurs. This wear process in particular also influences a previously determined relative position of the measurement volumes, so that a new determination would have to be repeated at regular intervals. Possibly. the entire switching unit must even be replaced due to wear. Especially for high-throughput applications, there is also the disadvantage that the switching process Measurement time is lost, and thereby the achievable sample throughput is reduced.

So-called laser scanning microscopes (LSM) generate a two-dimensional image of a sample by sequentially scanning the object plane with a single small measurement volume. From the knowledge of the position of the measurement volume at any point in time and the measured value recorded for this purpose, an image of this level can be generated. Possible signals are reflection and fluorescence intensity, but also anisotropy and fluorescence anisotropy. These microscopes can be combined with point measurement methods, e.g. fluorescence emission spectroscopy, relatively simple, since only the movement of the measurement volume has to be switched off in order to obtain a sufficient measurement time.

The use of laser scanning microscopes has the disadvantage that the measuring times of the individual images are relatively long. The use of such microscopes is therefore not possible for high-throughput screening, since the required short measuring times cannot be achieved with sufficient signal quality. With short measurement times, the measurement results have only a low signal-to-noise ratio, so that it is necessary to center over several images. This averaging is carried out to improve the signal-to-noise ratio and is therefore necessary for short measurement times for a single image. With long measuring times, a sufficient signal-to-noise ratio can be obtained even without averaging. This in turn increases the measuring time. Furthermore, the laser scanning microscope is limited to the use of relatively complex imaging methods.

In order to distinguish several fluorophores in a microscope based on their excitation and emission spectra, special optical filters, color cameras and imaging spectrographs are used in multi-channel microscopes. If optical filters are used, at least three filters are usually used. The first filter defines the excitation wavelength range, the second filter is a dichromatic mirror that reflects the excitation light and transmits the emission light (or vice versa). Another filter is inserted in front of the detector, which only allows the emission area to pass. For the detection of different fluorophores, there is the possibility to replace all three filters, as is done as standard in the commercially available microscopes. Alternatively, a single filter set can be built up for several dyes if the excitation and emission wavelengths are far enough apart, as is the case, for example, for the dyes DAPI, FITC and TRITC. If these dyes are to be recorded separately, a color film or a color CCD camera can be used. A black and white camera can also be used in combination with a selectable excitation spectrum, as is also done, for example, in the so-called Pinkel filter sets. With filter sets, it is also possible to differentiate dyes based on their Stokes shift. Different dyes can be excited with the same wavelength and differ in the different shift of the emission spectrum.

In multi-channel microscopes, one or more filters often have to be switched between the individual measurements. This has considerable disadvantages for use in high-throughput systems, such as wear of the components or loss of measuring time, which result from the switching times. Systems that can detect several dyes at the same time are limited to only a few dye combinations, since the emission spectra must differ sufficiently and must not overlap with the excitation spectra of the other dyes. In addition, the production of such optical filters, which are suitable for several dyes, is very complex, so that the change to other dyes due to the considerable development and Manufacturing costs is hindered. This considerably limits the choice of possible dyes. This disadvantage also applies to systems based on spectrographs, since the excitation light also has to be attenuated in spectrographs. In addition, imaging spectrographs are not particularly suitable for taking an image of the sample, since the acquisition - and possibly also the processing times - often takes several seconds.

The object of the invention is to provide a device and a method for measuring chemical and / or biological samples, with which or with which different measurements of a sample, in particular an image measuring method and a point measuring method, can be carried out in a simple manner.

According to the invention, the object is achieved by the features of claims 1, 11 and 19.

The invention is based on the finding that when imaging a sample area in an image field, part of the image field is always not used for the examination. In conventional optics, a round image field 10 (FIG. 1) is generated. The field of a CCD camera is arranged within the image field, for example in a first partial area 12 of the image field 10. The part of the image area 10 surrounding the first partial area 12 is thus not used for the detection of reactions occurring in the sample. The essence of the invention is to detect a second partial area of the image field in the unused area of the image field by means of a second detector.

The optical measuring device according to the invention, which is particularly suitable for use in high-throughput screening systems, can have an illumination unit for illuminating the sample to be measured. Furthermore, an optical device is provided for imaging a sample area in the image field. Furthermore, a first detector is provided which covers a first partial area of the Image field captured. According to the invention, a second detector is provided which detects a second partial area of the image field.

By providing at least two detectors, each of which detects a partial area of the image field, it is possible to carry out different measurements simultaneously on a sample. In particular, it is possible, for example, to provide a CCD camera as the first detector with which one of the image measurement methods described above is carried out. With the aid of the second detector, a point measurement method can then preferably be used, by means of which e.g. the time course of a signal can be recorded.

The provision of a folding mirror to switch between the detectors used for different measuring methods is not necessary in the device according to the invention. The consequence of this is that no mechanical wear can occur which can impair the measurement results, in particular the relative position between the two measurement areas in the sample. Furthermore, the switching process, i.e. folding the mirror so that the required measurements can be carried out in a short period of time. This is particularly advantageous in the case of extremely cost-intensive methods, such as high-throughput screening. The possibility of carrying out two different measurements on a sample at the same time also has the advantage that effects in a sample that only occur over a short period of time can also be perceived by two measurement methods. When using known devices, it is necessary to carry out the two different measurements in succession with the aid of two samples.

According to the invention, a spatially limited decoupling of at least a partial area of the image field thus takes place. This decoupling is preferably carried out by a totally reflecting mirror, the decoupled one Partial area is detected by a point detector, such as a fiber end. Since the device according to the invention is particularly suitable for use in medium and high-throughput screening methods, individual wells of sample carriers, such as titer plates or the like, are preferably observed with the aid of the device. A part of the liquid in a well is thus preferably imaged by the device in an image, a partial region of this well image being coupled out. It is thus possible, in particular when a totally reflecting mirror is provided to couple out the partial area, to detect different areas of the image with different detectors, such as area and point detectors. The provision of a folding mirror or a dichroic mirror is not necessary here. On the one hand, this has the advantage that no mechanical wear and the like. occurs, and on the other hand no expensive dichroic mirrors have to be used.

Multi-channel microscopy can also be carried out in a simple manner with the device according to the invention. Since the different partial areas of the image field can be deflected, for example by providing mirrors, it is possible to arrange different filters in the individual beam paths. There is no need to change the filters. Likewise, filters that are specifically tailored to the requirements can then be used in the two beam paths. The provision of expensive combination filters and the like. not necessary. Likewise, the number of dyes that can be used is considerably larger, since these are no longer the same on a special filter system. must be coordinated.

In particular, in the device according to the invention, the separation of the two partial areas does not take place via spectral differences due to dichromatic mirrors or sequentially in time by pivoting a mirror in and out.

All of the above-mentioned methods can be considered as measurement methods for image and / or point measurement. In a preferred embodiment of the invention, the first detector is located on the optical axis of a lens of the optical unit. Furthermore, there is a mirror in the edge region of the beam path generating the image field, via which the second partial region is coupled out and directed onto the second detector. This has the advantage that the second detector does not have to be arranged directly next to the first detector. As a result, different detectors can be used, in which it may not be possible to arrange two detectors directly next to one another, since control elements of the detector or the like are arranged next to the detector surface.

Of course, it is also possible not to arrange the first detector directly on the axis of the objective, but also to deflect the beam path by means of a mirror. In any case, in this preferred embodiment, a mirror coupling out the second partial region is arranged in the beam path generating the image field. The mirror is preferably arranged such that it lies outside the beam path that is detected by the first detector. An overlap of the individual subareas is preferably avoided. Since the position of the mirror coupling out the second partial area ensures that it does not protrude into the beam path that strikes the first detector, a totally reflecting mirror can preferably be used. This has the advantage that the mirror does not influence the radiation perceived by the second detector.

Since generally only a relatively small part of the image field is used as the first partial area, the remaining image field can be used by several detectors. The image field is thus divided into a number of two, three, four or more partial areas. Each of these sub-areas can in turn have one Mirror can be coupled out. This avoids space problems in the area of the image field.

The further detectors or the further mirrors, which couple out corresponding partial areas for these detectors, are preferably arranged such that an overlap of the partial areas is excluded.

Since an image measurement and a point measurement are preferably carried out with the aid of the device according to the invention, at least one of the detectors, preferably the second detector, is designed as a point detector. This can be, for example, the open fiber end of an optical fiber. With such a point detector, for example, temporal courses of the fluorescence of a substance can be measured.

In the case of measuring systems with high axial resolution, such as, for example, in confocal microscopes, it is also necessary to be able to adjust the axial position of the measuring point in a reproducible manner. In the case of high-throughput screening methods in particular, the axial adjustment, i.e. focusing is done automatically and quickly. Intervention by operating personnel is not possible because this takes too much time and high-throughput screening methods would be uneconomical. Since the quality of the received signal is strongly dependent on the axial position, it is generally necessary to repeat the focusing in each sample to be examined.

For focusing, it is possible, for example, to use the contrast changes in an image recorded by a detector or to evaluate them statistically. It is therefore possible in the device according to the invention to design one of the detectors as a focusing device for the axial detection of the sample area depicted in the image field. One of the detectors on which a partial area of the image field is imaged can thus be used for focusing. This has the advantage that none additional focusing devices are required, which generally require a separate light source, by which the measurement results are influenced, since this light source also introduces light into the sample.

Furthermore, active focusing devices can also be used, which then have, for example, their own light source in order to introduce light into the sample for focusing. For example, a light beam incident obliquely into the sample can be used, which generates a fluorescence line in the fluorescent sample. The reflection of this obliquely incident light beam can be used for focusing. A corresponding device is described for example in US 6 025 601. Such focusing by means of a line appearing in the sample is particularly unsuitable for brightly fluorescent samples. The lowest possible excitation power should be used in these samples in order to avoid bleaching of the sample and possibly damage to any living cells present in the sample. In this autofocus system, however, a reduction in the excitation power also results in the height signal being weakened. This will make the focus setting worse. Furthermore, such a focusing device can only be used with relatively small numerical apertures, since the light is irradiated at an angle to the optical axis. This increases the excitation focus, so that the axial resolution is lower.

In a preferred embodiment of the invention, a focusing device with an aperture diaphragm is used. The smallest possible point in the sample is illuminated with the aid of the aperture diaphragm. The light reflected from this point reaches a suitable detector through the same aperture diaphragm. Due to the confocality of the arrangement, focusing has only taken place if there is a reflecting surface in the position corresponding to the aperture in the sample. For example, the reflection occurs on a glass bottom of the sample holder in which the samples are are arranged, simple focusing on the glass bottom is possible with the device according to the invention. Since the thickness of the glass bottom and the optimal distance between measuring points in the sample and the sample bottom are known, after focusing on the glass bottom, a corresponding relative displacement between the sample carrier and the objective can ensure that the measurement within the sample is at a predetermined distance from the sample bottom he follows. Furthermore, it is possible to select the plane in which the aperture diaphragm is located so that in the event of a reflection signal from the glass bottom through the aperture, the other detectors are arranged in such a way that their focus lies in the sample area of interest.

The invention thus further relates to an optical measuring device with an illumination unit and an optical unit as described above. As described above, a first partial area of the image field is detected by a first detector. Instead of or in addition to a second detector, a focusing device is provided for axially fixing the sample area depicted in the image field. The focusing device has a light source generating a focusing beam, the focusing beam running at least partially within the beam path generating the image field. A focusing mirror is preferably provided, which is arranged in the image field beam path in accordance with the above mirror. The arrangement of the mirror is in turn preferably such that the mirror does not overlap the portion of the image field perceived by the first detector, i.e. does not cast a shadow on the first detector.

Preferably, the focusing device is arranged, if necessary using a correspondingly arranged focusing mirror, in such a way that the optical device directs the focusing beam in the direction of the sample. Preferably, the focusing beam reflected by the sample or an interface of a sample carrier is likewise directed in the direction of the optical device Focusing device or directed in the direction of the focusing mirror. The reflection of the focusing beam can take place at different interfaces. For example, the reflection of the outer surface of the sample holder, on the bottom surface of the sample holder, ie the inside of the sample holder bottom on which the sample liquid is applied, or also on the surface of the sample itself, ie on the interface between the sample and the surrounding medium, can generally be air , be reflected. Depending on the interface that is used to reflect the focusing beam, the lens device must be appropriately adjusted so that it is ensured that the measurement within the sample takes place at a corresponding distance from the interfaces. This ensures an optimal measurement result.

Of course, a combination of the two devices described above is possible, so that several detectors are provided which detect different sub-areas of the image field, and a focusing device is additionally provided which preferably does not cover any of the sub-areas of the image field, but uses the optical device for guiding the focusing beam.

The invention further relates to a method for the optical measurement of chemical and / or biological samples, in particular by means of high-throughput screening systems. In the method according to the invention, the sample to be measured can be illuminated. A sample area is then imaged in an image field with the aid of an optical device. Both the sample area and the image field are each a spatially limited area. In a first partial area of the image field, the image depicted in the partial area is detected by means of a first detector. Furthermore, a second partial area of the image field is detected by means of a second detector. This method has the same advantages over known methods that are explained using the device according to the invention described above.

The method according to the invention preferably not only captures two, but three, four, five or more sub-areas of the image field. The individual partial areas preferably do not overlap.

The invention is explained in more detail below on the basis of preferred embodiments with reference to the accompanying drawings.

Show it:

1 is a schematic representation of an image field,

Fig. 2 is a schematic representation of a preferred

Embodiment of the invention with three detectors,

FIGS. 3-5 schematic representations of possible arrangements of the second detector,

FIGS. 6-8 schematic representations of design options of the first detector together with an illumination device and

Fig. 9 is a schematic representation of another preferred

Embodiment of the invention in combination with a focusing device.

In Fig. 1, an image field 10 is shown, which is usually from a microscope and the like. used optical device is generated. The image field 10 is usually a circular two-dimensional one Area. A first partial area 12 is provided within the image field 10. In the example shown, the first partial area 12 is a rectangular partial area, for example a camera field of a CCD camera. A second partial area 14 is also arranged within the image field 10. In the example shown, the second partial area 14 is shown as a circle. For example, it is a sub-area in which a point measurement is carried out. For example, this is a detector designed as an optical fiber, so that a fiber end of an optical fiber is arranged in the partial region 14.

The schematic structure of an optical measuring device according to the invention (FIG. 2) has an illumination device 16 which illuminates a sample 20 arranged, for example, in a titer plate 18. The light emitted by the sample 20 or the radiation emitted by the sample 20 is directed in the direction of a first detector 28 with the aid of an optical device 22 which has an objective 24 and at least one tube lens 26. The image field 10 (FIG. 1) is generated in an image plane located at the level of the detector 28. The image field is generated by a beam path 30 shown in dashed lines. The first detector 28 detects the first partial area 12 (FIG. 1). This is delimited by a first beam path 32, which is part of the image field beam path 30.

In the exemplary embodiment shown, a second detector 34, which serves to detect the second partial area 14 of the image field 10 (FIG. 1), is arranged rotated through 90 ° to an optical axis 36. In order to steer the relevant part of the image field beam path 30 in the direction of the second detector 34, a totally reflecting mirror 38 is provided. The mirror 38 couples out a part 40 of the image field beam path 30 and directs it onto the second detector 34.

A further, third detector 42 can be arranged opposite the second detector 34. This is perpendicular to the second detector 34 the optical axis 36 is arranged. The third partial area of the image field 10 imaged on the third detector 42 is coupled out of the image field beam path 30 by a mirror 44. Of course, the third detector 42 can also be arranged in a different position together with the mirror 44 and can detect a different partial area of the image field 10.

It is essential in the invention that the partial region 12 imaged on the first detector 28, which is generated by an optical path 46, is not influenced by the second and third detectors 34, 42. 2, the beam path 46 runs between the two mirrors 38, 44, so that neither of the two mirrors 38, 44 protrude into the beam path 46 and would thereby impair the part of the sample 20 imaged on the first detector 28.

The lighting device 16 has a light source 50 and a lens arrangement 52. The lens arrangement 52 bundles the light emitted by the light source 50 onto the sample so that the light is concentrated in the sample 20. In the exemplary embodiment shown, the illuminating device 16 is arranged opposite the objective device 22, so that the sample is illuminated from the side opposite the objective device 22. This is the so-called transmission lighting. It is also possible to illuminate the sample 20 by incident light illumination. For this purpose, a light emitted by a light source is coupled into the lens 24 via a corresponding lens and mirror arrangement and is guided by the latter into the sample.

The first detector 28 is, for example, a CCD or CMOS camera. In combination with a suitable line illumination, the first detector 28 can also be a line camera. A spectrograph can also be used as a detector. A detector corresponding to the first detector or a combination of the detectors mentioned above can be provided as the second detector. There is also the possibility of providing one or more of the detectors 28, 34, 42, for example as a fluorescence emission spectroscope, possibly in combination with suitable filters. The detectors can also be designed as FCS or FIDA detectors.

In Figs. 3-5 different arrangement options of the second detector 34 are shown. The lighting device 16 is not shown in each of these figures.

3, the mirror 38 is arranged in front of an image plane 54 in which the image field 10 is located. A beam 40 is deflected as described above with reference to FIG. 2. The second detector 34 is arranged in an image plane 56, which is part of the image plane 54, which has been deflected by the mirror 38.

It is also possible to arrange the mirror 38 in an intermediate image plane 58 (FIG. 4). In order to arrange the detector 34 again in the image plane 56, a lens 60 is provided between the mirror 38 and the detector 34. Correspondingly, a further lens 62 is provided between the intermediate image plane 58 and the image plane 54, through which the first partial region 10 is imaged on the detector 28. If the detectors 28, 34 do not require imaging of the sample (e.g. point intensity measurements or direct coupling into a spectrograph), the lenses 60, 62 can also be dispensed with.

As shown in FIG. 5, it is also possible to arrange the mirror 38 behind the intermediate image plane 58 in relation to the beam path. 4 is again between the mirror 38 and the image plane 56 of the detector 34, the lens 60 is arranged. Likewise, the lens 62 is provided between the intermediate image plane 58 and the image plane 54 of the detector 28.

In Figs. 6-8, the lighting device 16 is arranged between the intermediate image plane 58 and the image plane 54. The lighting device has a lens or lens arrangement 52. The light emitted by the light source 50, which can also be a line illumination 64 (FIGS. 7 and 8), is coupled into the beam path running between the objective 24 and the first detector unit 28 via a partially transparent or dichromatic mirror 66. The second detector 34 is together with the mirror 38 in the in FIGS. 6-8 illustrated embodiments each arranged in front of the intermediate image plane 58.

In the conventional light source 50 shown in FIG. 6, this is combined with a CCD or CMOS camera as the first detector 28. 7, line lighting 64 is provided, which is combined with a line detector as the first detector 28. To obtain a complete image of a sample plane, the line must be moved relative to sample 20. This is done either by moving the sample or by moving the light beam incident on the sample, e.g. over an oscillating mirror. Instead of a line scan camera, an imaging spectrograph can also be used as the first detector 28 (FIG. 8). In addition to an image recording device 68, the latter has a mirror arrangement comprising two planar mirrors 69 and a concave mirror 71. The planar mirrors 69 can be tilted against one another.

In the exemplary embodiment shown in FIG. 9, a focusing device 70 is arranged instead of the second detector 34. Of course, one or more detectors 34, 42 can be arranged in addition to the focusing device 70. The mirror 38 is arranged as described above and in particular as can be seen in FIG. 2. A light source 72 directs light through a lens 74 onto a partially transparent mirror 76. Of the light is deflected towards an aperture diaphragm 78. The aperture diaphragm is located in or near an image plane. This illuminates a point in the sample. The light reflected from this point in the sample likewise arrives in the direction of the aperture diaphragm 78 via the mirror 38. After passing through the aperture diaphragm 78, the light returning from the sample 20 is transmitted through the partially transparent mirror 76 and via a lens 80 to a detector 82 steered. With the aid of the focusing device shown, focusing on a reflecting surface of the sample, for example an interface of the titer plate or a surface of the sample, is possible. With appropriate focusing, a maximum of light reflected from the sample reaches the detector 82 through the aperture diaphragm. If it is known that, for example, focusing has been carried out on the glass bottom of a sample carrier 18, the optical unit can subsequently be shifted by a certain amount, so that in the Image area 10, an area lying within the sample is imaged. The aperture diaphragm 78 can also be shifted so far out of the image plane 56 that a signal at the detector 82 is accompanied by a focusing of the sample region of interest on the detector 28.

Other focusing devices are also possible in which the focusing takes place, for example, via the contrast or via a fluorescence line generated in the sample.

For example, 2x2 phase couplers can be used as focusing sensors. The fiber itself is then used as the aperture determining the autofocal measuring location. It is also possible to use an additional aperture diaphragm. For example, in scanning systems such as line detectors, the measuring point for the autofocus can also lie in front of the line in the scanning direction. In this case, it is possible to carry out the height control in the control loop without a phase offset, since the measuring point records the measured value in a position which the actual detector will only reach later. There If the autofocus measuring point is located next to the area captured by the image detector, it makes sense to use a correction of the height according to the skew of the sample. Such a correction is always useful if there is a lateral offset of measuring points.

An essential feature of the device according to the invention, as can be seen in particular from the figures, is that the decoupling mirror or the detectors, i.e. the fiber or other point detectors are arranged in front of, in or behind the image plane. In particular, the detectors are arranged near the image plane.

Claims

Optical measuring device for measuring chemical and / or biological samples, in particular for high-throughput screening systems, with

an optical device (22) for imaging a sample area in an image field (10),

a first detector (28) and a first partial area (12) of the image field (10)

a second detector (34) which detects a second partial area (14) of the image field (10).

2. Optical measuring device according to claim 1, characterized by an illumination device (16) for illuminating the sample to be measured (20).

3. Optical measuring device according to claim 1 or 2, characterized in that the first detector (28) on the optical axis (36) of an objective (24) of the optical unit (22) is arranged.

4. Optical measuring device according to one of claims 1-3, characterized in that a mirror (38) coupling out the second partial region (14) is arranged in the beam path (30) generating the image field (10).

5. Optical measuring device according to claim 4, characterized in that the mirror (38) is totally reflecting.

6. Optical measuring device according to one of claims 1-5, characterized by further detectors (42) for detecting further partial areas of the image field (10).

7. Optical measuring device according to one of claims 1-6, characterized in that the partial areas (12, 14) do not overlap.

8. Optical measuring device according to one of claims 1-7, characterized in that at least one detector (34) is a point detector.

9. Optical measuring device according to claim 8, characterized in that the point detector is an open fiber end of an optical fiber.

10. Optical measuring device according to one of claims 1-9, characterized in that the optical device (22) has only one lens (24).

11. Optical measuring device according to one of claims 1-10, characterized in that one of the detectors (28, 34, 42) is designed as a focusing device (70) for axially fixing the sample area depicted in the image field (10).

12. Optical measuring device for measuring chemical and / or biological samples (20), in particular for high-throughput screening systems, with

an optical device (22) for imaging a spatially limited sample area in a spatially limited image field (10),

a first detector (28) and a first partial area (12) of the image field (10) a focusing device (70) for axially fixing the sample area imaged in the image field (10), with a light source (72) generating a focusing beam, the focusing beam running at least partially within the beam path (30) generating the image field (10).

13. Optical measuring device according to claim 12, characterized by an illumination device (16) for illuminating the sample to be measured (20).

14. Optical measuring device according to claim 12 or 13, characterized by a focus mirror (38) arranged in the image field beam path (30) for coupling and / or decoupling the focus beam.

15. Optical measuring device according to claim 14, characterized in that the focus mirror (38) is arranged outside a first beam path incident on the first detector (28).

16. Optical measuring device according to one of claims 12-15, characterized in that the optical device (22) directs the focusing beam in the direction of the sample (20).

17. Optical measuring device according to one of claims 12-16, characterized in that the optical device (22) the focusing beam reflected from the sample (20) or an interface of a sample carrier (18) in the direction of the focusing device (70) or in the direction of Focusing mirror (38) steers.

18. Optical measuring device according to one of claims 12-17, characterized in that the focusing device has an aperture diaphragm (78) through which both the focusing beam directed onto the sample (20) as well as the light reflected from the sample (20) or an interface.

19. Optical measuring device according to one of claims 12-18, characterized in that the illumination device (16) generates the focusing beam.

20. Optical measuring device according to one of claims 12-19, characterized in that the focusing device has a glass fiber through which both the focusing beam directed onto the sample (20) and the light reflected from the sample (20) or an interface extends.

21. Optical measuring device according to one of claims 1-11, characterized by a focusing device (70) according to one of claims 12-20.

22. A method for the optical measurement of chemical and / or biological samples (20), in particular by means of high-throughput screening systems, with the steps:

Imaging a sample area in an image field (10),

Detecting a first partial area (12) of the image field (10) by means of a first detector (28) and

Detecting a second partial area (14) of the image field (10) by means of a second detector (34).

23. The method of claim 22, wherein the sample (20) is illuminated.

24. The method according to claim 22 or 23, in which further partial areas (12, 14) of the image field (10) are detected.

25. The method according to any one of claims 22-24, in which the sample area to be imaged is focused in the image field (10).

26. The method according to claim 25, in which a focusing beam is generated which extends at least partially within a beam path (30) generating the image field (10).