WO2015044035A1

WO2015044035A1 - Confocal light microscope and method for examining a sample using a confocal light microscope

Info

Publication number: WO2015044035A1
Application number: PCT/EP2014/069953
Authority: WO
Inventors: Peter Westphal; Alexander Gaiduk; Helmut Lippert
Original assignee: Carl Zeiss Microscopy Gmbh
Priority date: 2013-09-30
Filing date: 2014-09-19
Publication date: 2015-04-02
Also published as: DE102013016367A1; WO2015044035A8

Abstract

The invention relates to a light microscope comprising a light source device for emitting illumination light in the direction of a sample, a detector device for recording sample images of the sample and optical imaging means for focusing illumination light as light spot pattern onto a height plane at the sample and for guiding sample light from the sample to the detector device. The light source device and the optical imaging means are designed to illuminate a plurality of mutually spaced apart lateral regions of the sample with the light spot pattern during a camera integration time of the detector device. According to the invention, the light microscope is characterized in that the light source device, the optical imaging means and the detector device are designed to enable recordings of at least two sample images in which the light spot patterns are focused onto different height planes and/or in which different height planes are imaged onto the detector device, wherein the light source device, the optical imaging means and the detector device are arranged spatially identically for each of the recordings of the different sample images. In this case, identical lateral regions of the sample are illuminated in each of the at least two sample images. In addition, electronic evaluation means are present and designed to calculate height information of the sample with the aid of the at least two sample images. In addition, the invention relates to a corresponding method for examining a sample using a light microscope.

Description

CONFOCUS LIGHT MICROSCOPE AND METHOD FOR EXAMINING A SAMPLE WITH A CONFOCAL

LIGHT MICROSCOPE

The present invention relates in a first aspect to a light microscope according to the preamble of claim 1. In a second aspect, the invention relates to a method for examining a sample with a light microscope according to the preamble of claim 12.

A generic light microscope includes light source means for emitting illumination light toward a sample, detector means for recording sample images of the sample, and optical imaging means for focusing illumination light as a spot pattern onto a height plane on the sample and for directing sample light from the sample to the detector means. The light source device and the optical imaging means are configured to illuminate a plurality of spaced-apart lateral regions of the sample during a camera integration time of the detector device with the light spot pattern. In a generic method for examining a sample with a light microscope, it is provided that illumination light is emitted in the direction of a sample with a light source device, that sample images of the sample are recorded with a detector device and that illuminating light is focused as a light spot pattern onto a height plane on the sample with optical imaging means and sample light is passed from the sample to the detector device. In this case, the light source device and the optical imaging means are designed to illuminate a plurality of spaced-apart lateral regions of the sample simultaneously or successively during a camera integration time of the detector device with the light spot pattern. Autofocusing is carried out in particular with such light microscopes and methods. In this case, a sample table on which the sample is held, adjusted in a height direction. As a result, a sharp image can be generated on a camera of the light microscope. Autofocusing is particularly advantageous in the case of reflective or light-scattering samples, which cause an uneven surface. have surface. Thus, due to the non-planar surface, a focus adjustment to the height of a sample area of interest is imperative.

From DE 102 42 373 A1 a sensor for fast optical Abstnadsmessung according to the confocal imaging principle is known. DE 197 13 362 A1 describes a confocal scanning microscope with a confocal measurement of the dimensions.

From DE 10 2004 014 048 A1 a measuring device for objects according to the principle of confocal microscopy is known.

In known autofocus methods, usually only a single light spot is generated on the sample and the sample light emitted by this sample point is measured. Such a procedure is disadvantageous in inhomogeneous samples with large differences in reflectivity or light scattering. For these samples, the amount of sample light returned depends strongly on which area of the sample the spot is blasted. For a given intensity of the illumination light, it may therefore happen that the intensity of the reflected sample light brings the detector device to saturation or is too low.

This problem can be alleviated by illuminating a plurality of spaced-apart lateral regions of the sample with one light spot each. As a result, it is probable that at least some of the illuminated lateral regions reflect a quantity of sample light which neither saturates nor is too low for the detector device.

Light microscopes and methods of the aforementioned type are known for example from US 201 1/0017902 A1 and US 7,570,795 B2 and are excited in the scanning microscope from US 7,858,91 1 B2. Another light microscope with an autofocus function is described in US 2010/003381 1 A1.

However, in particular with samples having a pronounced height profile, faults or inaccuracies can frequently occur with known light microscopes and methods, whereby the time required for height measurements of the sample is also high. This is disadvantageous for the determination of a height profile and / or the performance of an autofocusing.

As an aspect of the invention, it can be considered to provide a light microscope and a method for examining a sample with a light microscope, which easily and quickly enable precise height measurement of the sample.

This object is achieved by the light microscope with the features of claim 1 and by the method having the features of claim 12.

Advantageous variants of the method according to the invention and of the light microscope according to the invention are the subject of the dependent claims and are also explained in the following description.

In the light microscope of the above-mentioned kind, the light source device, the optical imaging means and the detector device are designed to enable images of at least two sample images in which the light spot patterns are focused on different height planes and / or in which different height planes are imaged onto the detector device wherein the light source means, the optical imaging means and the detector means are spatially equally spaced for each of the images of the different sample images. In each case, the same lateral areas of the sample are illuminated for the recordings of the at least two sample images. In addition, electronic evaluation means are provided and designed to calculate height information of the sample with the aid of the at least two sample images.

In the method of the abovementioned type, it is provided according to the invention that the light source device, the optical imaging means and the detector device are arranged spatially the same during recording of at least two sample images, the light spot patterns being focused on different height planes in the at least two sample images and / or Different height levels are imaged on the detector device, and wherein for the at least two sample images each same lateral regions of the sample are illuminated. Furthermore, height information of the sample is calculated with the aid of the at least two sample images. A basic idea of the invention can be seen in that a spatially identical arrangement is used for recording the at least two sample images. It is therefore carried out between the images of these images no scanning movement in the height direction. Such a scanning movement is carried out in conventional light microscopes, in particular by moving a sample stage relative to the beam path or by changing an illumination or detection plane relative to the sample. By not making such a height scan motion, a speed advantage is achieved. In addition, improved precision is possible since inaccuracies of mechanical movements are eliminated and vibrations of optical components have less effect.

As a spatially identical arrangement for receiving the at least two sample images on the one hand a spatially fixed arrangement can be used. In this case, no movement of the light source device, the optical imaging means or the detector device takes place during or between the recordings of the two sample images.

On the other hand, it can also be provided to direct a light beam successively to different lateral areas in order to generate the light spot pattern. In this case, although there is a movement of, for example, a scanning mirror, but the exact same arrangement with the exact same movement is also used to record the second sample image. For this, therefore, the same scan movement is performed over the lateral areas. However, no height scanning motion is performed that would distinguish the arrangements for the images of the two sample images.

For the calculation of the height information from the sample images, it is important that the same lateral areas are illuminated for both sample images and that they are illuminated

Detector device are measured. A lateral area should be understood to mean a sample area with an extension transverse, in particular vertical, to a height direction. The height direction may correspond to the optical axis from one objective to the next. The lateral areas illuminated by the light spot pattern are preferably separated from one another, that is, there are non-illuminated sample areas between them. Generally, however, it is sufficient that the light spot pattern causes the sample to have a spatially inhomogeneous light intensity distribution is irradiated. Thus, a grid or stripe image can be generated as a light spot pattern. Preferably, however, a light spot pattern consists of a plurality of mutually separate light spots, which are each round, for example.

By illuminating the same or the same lateral regions for the images of the at least two sample images, it is to be understood that the lateral region which is illuminated by a light spot during the recording of the first sample image illuminates with the lateral region which illuminates from a light spot during the recording of the second sample image will, at least overlapped. Preferably, the centers of these two lateral regions are identical. The extent of the two lateral areas, however, may be different. At a common center, therefore, one lateral area is completely contained in the other. For example, the two light spots for the two images of the two sample images can be focused in different height levels. As a result, the light spots generated on the sample surface, that is, the illuminated lateral areas, are of different sizes, but have a common center.

The invention is particularly suitable for the investigation of samples with a height profile, ie a non-uniform height. In principle, structures within a particular liquid sample can also be investigated. In both cases, the exact heights of regions of interest of the sample are unknown. One goal is the determination of these heights, ie either the surface profile or a determination of the course and position of sample parts, for example cell structures of biological samples. More generally, therefore, the profile of sample parts within a sample can also be understood by a height profile, and the sample surface can be understood as the surface of a sample part. In order to determine information about the height profile, the light spots are focused on at least one height level on the sample. A height level refers to a plane which is transverse, in particular perpendicular, to an optical axis from an objective to the sample. Since the position, geometry or structure of the sample are initially unknown, such a plane may be either spaced from the sample, that is, between the objective and the sample, the plane may be within the sample, or the plane may intersect, ie partially within, the sample surface and partially outside the sample. The focusing of the light spots on a height plane indicates that the height plane is an optically conjugate plane to a plane in which the light spot pattern is generated. This may be the light source itself or, for example, a pinhole disk, which is irradiated by the illumination light. In examining a surface profile of the specimen, the size of an illuminated lateral region depends on the height of the elevation plane at which the light spot is focused relative to that lateral region. Depending on the surface profile, differently sized spots of light on the sample surface are therefore produced with one height level.

This can be used to determine altitude information. In a sample image, sample light is received by the illuminated lateral areas. With a relatively small illuminated lateral area, a relatively small area with high sample light intensity is illuminated on the detector device. On the other hand, a larger illuminated lateral area leads to a comparatively large area with lower sample light intensity being illuminated on the detector device.

From this, it can be determined which illuminated lateral regions lie within or outside the height plane onto which the light spot pattern is focused. However, it is not yet possible to determine from a single image whether the lateral regions lying outside the height plane are above or below this height level. This is possible with the help of the second sample image.

In one of the alternatives of the inventive concept, the light spot patterns are focused on different height levels for the two images of the sample images. In this case, the detector elements which record the two sample images can be located in the same image plane for the recording of the two images. Whether a lateral area illuminated for the second sample image is larger or smaller than the corresponding lateral area illuminated for the first sample image depends on whether the height of this lateral area is above or below the first height plane on which the light spot pattern focuses on the first sample image becomes. Therefore, from a comparison of the measurement information, the same Lateral area are obtained in both sample images, it can be determined whether the height of this lateral area is above or below the first height level.

On the other hand, if the measurement information of different lateral areas had to be compared with one another, varying degrees of reflectivity and / or light scattering over the sample would falsify the result.

In the other alternative of the inventive concept, an identical light spot pattern is generated on the sample surface for the acquisition of both sample images. In order to gain extensive altitude information, different altitude levels are mapped to the detector device. The detector device accordingly receives at least two sample images in which a detector plane in which the detector elements of the detector device are located is optically conjugate to a first height level and a second height level different therefrom. The height level to which the light spot pattern is focused may be identical to the first or second height level or to both. The above alternative can also be described as follows: A specific height level on the sample is imaged onto two spatially offset image planes. The two image planes are thus each optically conjugate to the height level. The image planes are different with respect to the detector plane of the detection direction and are measured separately, with which the two sample images are recorded. The height level is thus imaged differently sharply on the detector plane. As described for the first inventive alternative, it can then be determined from a comparison of the measurement information obtained at the same lateral area in both sample images, whether the height of this lateral area is above or below the first height level. Advantageously, therefore, height information can be obtained precisely by the invention, without mechanical movements would be required.

The sample light to be detected may be illumination light reflected by the sample, in particular reflected or scattered illumination light. However, the sample light can also be luminescent light, ie fluorescent or phosphorescent light, which is produced by excitation by means of the illumination light. The light source device used can in principle be designed as desired. It may comprise one or more light source units, for example lasers or LEDs. For generating the light spot pattern, a plurality of light sources may be arranged next to each other in a corresponding pattern. Alternatively, a light source may illuminate a mask that generates the light spot pattern. The mask can be formed for example by pinhole or mirror. As a mirror, an electronically adjustable micromirror array (DMD, English: Digital Mirror Device) can be used. Other electronically controllable light modulators can be used which, for example, can be based on switchable liquid crystal regions, such as an LCoS (English: Liquid Crystal on Silicon). As a mask, a grid can also be used. This can be adjustable transversely and / or in the propagation direction of the light. This increases the number of different lateral areas that can be illuminated. In addition, the height level can be changed, in which the light spot pattern is sharply imaged. The grating may have a periodic structure with one or two lattice constants. Alternatively, the grid may also have an irregular structure, whereby light spots of different dimensions are generated simultaneously.

Furthermore, the light source device or the optical imaging means can also have a scanner, by means of which an illumination light beam illuminates different lateral areas one after the other and thus generates the light spot pattern.

In a preferred embodiment, the light spots of a light spot pattern have different sizes and / or shapes. As a result, with even greater certainty, at least one lateral area of the sample is illuminated with a light intensity which is suitable for this sample area, in order to avoid the detector device

To bring saturation.

In order to determine height information, a light intensity or an image sharpness are preferably determined in the at least two sample images for each of the lateral regions. The light intensities or image sharpnesses which were determined in the at least two sample images at the same lateral area then become the

Obtain a height information of the lateral area offset against each other. For example, in the two sample images, light spot patterns are focused on different height levels. If different sharpenings are now determined for the measurement signals of the two sample images for a specific lateral region, then it can be concluded that the height of this lateral region lies closer to the height plane of the sample image with the higher sharpness.

Quantitative statements are possible with the help of pre-stored reference data. These may have been determined in a previous reference measurement on an object with a known height profile. By comparing the image sharpening, the reference data can be used to make a statement as to how far the height of the examined lateral area is from the two height levels.

In an analogous manner, instead of the sharpness, the light intensity for the different lateral areas can also be evaluated.

Preferably, the light source device for focusing the light spot pattern on different height levels comprises a first and at least one second light source unit. In this case, the first light source unit is arranged in a plane that is optically conjugate to a first height level on the sample, and the second light source unit is arranged in a plane that is optically conjugate to a second height level on the sample. Thus, a spatially rigid arrangement can be used to generate light spot patterns in the different elevation planes.

As light source units here also illuminated masks can be viewed with particular punctiform holes.

For a simple design, the at least two light source units can be switched on one after the other. As a result, one and the same detector region of the detector device, that is, for example, the same camera sensor elements, can be used to receive the at least two sample images.

If the illumination light of the two light source units is distinguishable from each other, the at least two sample images can also be recorded simultaneously. In particular for this purpose, in a preferred embodiment with beam splitting means, which are arranged between the sample and the detector device, sample light is split into at least two spatially different detection beam paths. The detector device in each case comprises a detector area for each of the different detection beam paths.

With the detector areas, the at least two sample images can be recorded simultaneously. As a result, a speed gain can be achieved.

The beam splitting means may in principle be of any type and comprise, for example, a semitransparent mirror. In a preferred embodiment, the beam splitting means are formed by a light diffractive element, wherein the two detection beam paths are generated by different diffraction orders of the light diffractive element. As a result, a construction with a low number of components and thus low cost is possible at very low light losses. In order for the illumination light of the two light source units to be distinguishable from each other, the illumination light emitted by the at least two light source units may differ in wavelength. For combining the illumination light of the at least two light source units onto a common illumination beam path to the sample, a dichroic element may be present which reflects or transmits light as a function of wavelength. Losses of light are thereby reduced compared to merging the illumination light of the light source units through a semitransparent mirror.

Alternatively or additionally, polarization means may be present, by means of which the illumination light emitted by the at least two light source units is differently polarized. For merging the illumination light of the at least two light source units onto a common illumination beam path to the sample, a polarization beam splitter may be present, which reflects or transmits incident illumination light as a function of its polarization. In order to focus light spot patterns on different height levels, the light source device may also be configured to illuminate light from at least send out two different spectral ranges. The optical imaging means in this case have a longitudinal chromatic aberration by which illumination light is wavelength-dependent focused at different height levels on the sample. For emitting illumination light of different spectral ranges, the light source device can have different light source units. Alternatively, a single spectrally broadband light source unit can also be provided.

According to the invention, two sample images can also be taken, in which a light spot pattern is focused on one and the same height level, but different height levels are imaged on the detector device. For this purpose, beam splitting means can be present between the sample and the detector device, with which sample light is split up to at least two spatially different detection beam paths. The detector device in each case has a detector area for each of the different detection beam paths. Different altitude levels are mapped to the different detector areas.

In other words, in each of the detection beam paths with the sample light, an image of the illuminated height plane is generated in each case one image plane, wherein the image planes of the different detection beam paths are generated in different planes relative to the respective detector area.

The two detector areas can be formed, for example, by different cameras. However, it is particularly preferred that the different detector areas are different sections of a common camera, that is, a common camera chip. If the light source device comprises at least two light source units whose illumination light differs in wavelength, then the beam splitting means, with which sample light is directed to at least two spatially different detection beam paths, preferably comprises a dichroic element which reflects or transmits light in a wavelength-dependent manner. As a result, light losses when dividing the sample light onto the different detection beam paths are low, which results in improved measurement accuracy. If there are polarization means by which the illumination light emitted by the at least two light source units is differently polarized, then the beam splitting means with which sample light is directed to at least two spatially different detection beam paths preferably comprises a polarization beam splitter which reflects incident sample light depending on its polarization direction or transmitted. As a result, light losses can also be kept low and thus the measurement accuracy can be improved.

In particular, a three-dimensional image of the sample can be obtained with the obtained height information. After the at least two sample images have been recorded, a height-scanning movement can also be carried out, for example a height adjustment of a sample table. Subsequently, at least two sample images are taken again by the method described. This allows height information to be collected over a larger altitude range.

Instead of or in addition to measuring a three-dimensional image but can also be designed electronic Auswertemittel for performing an autofocus method. In this method, it is calculated from the at least two sample images whether the lateral areas of the sample illuminated by the light spot pattern are above or below a plane which is sharply imaged onto a main camera of the light microscope, and an adjustment direction is determined in which the sample is moved relative to To move an imaging beam path of the sample light is to achieve a sharp image on the main camera.

Then, a relative movement in the adjustment direction between the sample table, on which the sample is durable, and an imaging beam path of the sample light from the sample to the main camera can be performed. The main camera may be different from the detector device used for the autofocus method.

For an iterative autofocus method, a scanning device may be present. This can be used to shift a focus of illumination light in its propagation direction or to shift an imaging plane from the sample light. be guided. The electronic evaluation means are designed to control the scanning device in the autofocus method so that the displacement takes place in accordance with the determined adjustment direction to achieve a sharp image. This means that the sample is moved in the adjustment direction or the focus of the illumination light is shifted in the opposite direction to the adjustment direction.

A dynamic range of the detector device is preferably larger than a dynamic range of the main camera. The dynamic range indicates the respective range of measurable light intensities without saturation occurring. A particularly large dynamic range is especially important for the autofocus method. This is done before the actual sample measurement with the main camera, so that sample properties are still largely unknown. Therefore, a predetermined light intensity of the light source device is used, which may be relatively high or low for the still unknown sample. As a result, the light intensity received by the detector means may also have very low or high values. Therefore, a higher dynamic range is advantageous for the detector device used for the autofocus method.

For a high dynamic range, the detector device preferably has at least one HDR camera (HDR: High Dynamic Range). This can take, for example, several raw images with exposure times of different lengths in succession and charge them to a sample image. The HDR camera can also be designed to record the raw images simultaneously with different camera sensors or different camera pixels of the same camera sensor. In this case, the exposure times may in turn be different for the raw images. Alternatively, the sensitivities of the camera sensors may differ. Furthermore, an HDR camera can also be formed with a camera chip whose photosensitivity is not linear but, for example, logarithmic.

A light microscope can also be understood as any optically operating topology measuring device. In this respect, it suffices if observation means, such as a camera or an eyepiece, and optical imaging means, such as a lens, are present. Further advantages and features of the invention will be described below with reference to the accompanying schematic figures.

Fig. 1 shows a first embodiment of a light microscope according to the invention, in which two light source units are arranged in different planes.

FIG. 2 shows a second exemplary embodiment of a light microscope according to the invention, in which two different height levels are imaged side by side on a detector device. 3 shows a third exemplary embodiment of a light microscope according to the invention, in which two different height levels are imaged onto two detector units.

4 shows a fourth exemplary embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes.

5 shows a fifth exemplary embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes and in which two different height planes are imaged side by side on a detector device.

6 shows a sixth exemplary embodiment of a light microscope according to the invention, in which two light source units are arranged in different planes.

Fig. 7 shows a seventh embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes and in which two different height levels be imaged side by side on a detector device.

8 shows an eighth exemplary embodiment of a light microscope according to the invention, in which two light source units focus illuminating light of different wavelengths on different height levels and in which two different height levels are imaged side by side on a detector device.

9 shows a ninth exemplary embodiment of a light microscope according to the invention, in which a light spot pattern is generated by a scanning illumination light beam and in which two different height planes are imaged side by side on a detector device.

10 shows a tenth exemplary embodiment of a light microscope according to the invention, in which a light spot pattern is generated by a scanning illumination light beam and in which two different height levels are imaged onto two detector units.

Identical and identically acting components are generally provided with the same reference numerals in the figures.

1 shows a first exemplary embodiment of a light microscope 100 according to the invention.

This first comprises observation means 28, which may comprise an eyepiece and / or a main camera, a lens 22 and further imaging means 27. With these, a focal plane 24 is imaged onto the observation means 28. in the

In the area of the focal plane 24, a sample 26 can be arranged on, for example, a sample table or other sample holding means.

A basic object of the invention is to obtain height information about the sample 26. In contrast to conventional measuring methods, no scanning movement in the height direction is required for this purpose. As altitude direction, the direction of the sample 26 to the lens 22 are understood, that is, the direction of the optical axis.

For recording altitude information, a light source device 10 and a detector device 30 are used. The light source device 10 in this embodiment includes two light source units 1 1 and 12. Each of them emits illuminating light, which is passed to the sample 26 as a light spot pattern. The light source units 1 1 and 12 are arranged in different optical planes, which are not optically conjugate to each other. In FIG. 1, the light source unit 1 1 is located behind a confocal plane 14 and the light source unit 12 in front of the confocal plane 14, which is a plane conjugate to the focal plane 24. As a result, the light spot patterns of the two light source units 1 1 and 12 are imaged or focused on different planes on the sample. These planes are offset from each other in the height direction and are therefore referred to as height levels. The illumination light of the light source units 1 1 and 12 is guided via beam combination means 15 onto a common beam path. For this purpose, for example, serve a partially transparent mirror. The illumination light is further guided to a coupling element 21, which is arranged in the beam path from the sample 26 to the main camera 28. The coupling element 21 may for example be a mirror, which is either partially transparent or covers only a portion of a lighting light cross section.

If illumination light is radiated onto the sample 26 with the light source units 11 and 12, sample light coming from the sample is likewise deflected by the coupling-in element 21 and passed on to the detector device 30. The Einkop- ele- element 21 may be adjustable to sample light either in the direction of

Main camera 28 or in the direction of the detector device 30 to guide.

With a beam splitter 20 incident illumination light coming from the light source units 1 1 and 12, led to the coupling element 21, while coming from the coupling element 21 sample light is passed to the detector device 30. The beam splitter 20 may be, for example, a partially transparent mirror. In the embodiment of FIG. 1, the detector device 30 comprises a single camera 31 on which an image of a sample plane is generated by imaging means 36. This sample plane can be the focal plane 24, with which the imaged sample plane differs from the two altitude planes, which are illuminated in a focused manner by the light source units 11 and 12.

For example, the two light source units 1 1 and 12 may each successively generate a light spot pattern on the sample 26. This allows the camera 31 to successively record two sample images, with no mechanical movements required. In particular, no height scan is carried out by adjusting the light source device 10, the detector device 30 or optical imaging means 22, 36 between them and the sample 26 for receiving these two sample images.

More generally, the light source device 10, the optical imaging means 22, 36 and the detector device 30 are arranged spatially the same for each of the images of the different sample images.

The following section explains how to obtain altitude information using this setup.

The sample 26 may have an unknown height profile. This may be either the surface of the sample 26, the surface of sample parts or the arrangement of certain sample parts. By incident illumination light sample light is emitted. The sample light may be surface reflected, scattered or diffracted illumination light. Alternatively, the sample light may also be luminescent light emitted from illuminated sample parts.

By the light source units 1 1, 12 a sharp image of a light spot pattern is generated in different height levels. Accordingly, in the respective height plane, light spots of the light spot pattern have minimum cross-sectional dimensions, while as the distance from the respective height plane increases, the cross-sectional dimensions become larger.

The cross-sectional dimensions of the light spots generated on the sample profile therefore depend on how the sample profile extends to a height plane. By imaging light spot patterns in two different height planes, two light spot patterns are generated on the sample profile that differ in the cross-sectional dimensions of their light spots.

In the two recorded sample images, for example, the different cross-sectional dimensions of the light spots can be detected.

Alternatively or additionally, the sample light intensity emanating from a sample point can be determined in the two recorded sample images. The larger the cross-sectional dimensions of a light spot, the lower the sample light intensity reflected back from a sample point within this illuminated area.

In order to be able to make quantitative statements from a calculation of the two sample images, it is crucial that the light spots of different light spot patterns illuminate the same sample areas. Different light spot patterns are thus not laterally offset from one another. Rather, equal lateral areas of the sample are illuminated with both light spot patterns. That is, the lateral area of the sample illuminated by any light spot of a light spot pattern is at least overlapping with the lateral area illuminated by a light spot of the other light spot pattern. Preferably, these lateral areas are concentric. By comparing the measurement signals in the two sample images, which are caused by the same lateral region, it can be determined how the height of this lateral region lies relative to the two height planes and thus relative to the focal plane 24. In particular, it can be determined whether the height of the lateral region lies below or above the focal plane 24. This height ratio can be determined not only qualitatively but also quantitatively.

As an essential feature of this embodiment, light spot patterns are sharply imaged in two height planes, with a spatially unchanged arrangement being used in the meantime. As a result, a speed gain can be achieved and possible errors due to movement inaccuracies are avoided. Another embodiment of the invention is shown in FIG. This corresponds largely to the previous version. However, the light source device 10 here comprises only one light source unit 11. For the generation of the two sample images, the same light spot pattern of the light source unit 11 is imaged in the same height level on the sample. The lighting is therefore identical for both sample images. In the example shown, the light source unit is located in the confocal plane 14, with which the light spot pattern is sharply imaged in the focal plane 14.

However, to accommodate two different sample images, this embodiment differs from the previous one additionally in the detection beam path. After sample light has passed the beam splitter 20, it encounters beam splitting means 33. These may be a partially transmissive mirror, which preferably reflects and transmits sample light in equal proportions. As a result, sample light is forwarded to the detector device 30 on two different beam paths. In the illustrated case, the detector device 30 comprises exactly one camera 31. The two beam paths lead sample light to different detector areas 41, 42 of this camera 31, that is, to different camera elements of the same camera 31. The focal plane 24 is imaged via the two beam paths into two different image planes 34, 35 which are offset relative to each other in the propagation direction of the sample light. Thus, these two image planes 34, 35 are different in the propagation direction of the sample light with respect to a detection plane in which the camera 31 with its two detector regions 41, 42 is arranged. For this purpose, different imaging means 36, 37 or the same imaging means can be arranged at different locations in the two beam paths. To reduce the number of components, optical paths of different lengths can also be used by the beam-splitting means 33 to the detector regions 41, 42.

In the illustrated example, the detection plane of the detector area 41 coincides with the image plane 34. With this detector region 41, therefore, a sample image is recorded which is a sharp image of the focal plane 14. However, because the image plane 35 is different from the detection plane of the detector region 42, the sample image recorded with the detector region 42 is a blurred image of the focal plane. In other words, the detection plane of the detector area 41 is optically conjugate to the focal plane 14, while the detection plane of the detector area 42 is optically conjugate to another altitude plane. Thus, different height levels are imaged onto the detector device 30 in the two sample images. In the two sample images, the image of a lateral area illuminated by a light spot differs depending on the height of this lateral area relative to the focal plane 14. Thus, as described with reference to FIG. 1, a comparison can be made by comparing the sharpness or light intensity in the two sample images. whether and how far the illuminated lateral areas each lie above or below the focal plane 14.

As an important feature, in turn, no shift or change in height of the components used is required to accommodate the two sample images. In addition, the two sample images can be recorded simultaneously. Due to the reduced time required in particular inaccuracies can be avoided by changing the sample.

In the illustrated example, the light source device 30 generates a light spot pattern sharply in the focal plane 14. In general, however, the light spot pattern can also be generated sharply in any height level different from the focal plane 14. Likewise, the detection plane of the detector region 41 does not have to be optically conjugate to the focal plane 14. Rather, it is sufficient that the two detection levels of the two detector areas 41 and 42 are conjugate to two different height levels. In one of these elevation levels, the light spot pattern is preferably sharply imaged, wherein the light spot pattern can, however, in principle also be sharply imaged into another elevation plane. Another embodiment of the invention is shown in FIG. This corresponds to the majority of the embodiment of FIG. 2. Sample light is again divided by beam splitting means 33 into two different beam paths, which lead to two detector areas 41 and 42. In this case, the two detector regions 41 and 42 are also arranged differently with respect to an image plane 34. In contrast to FIG. 2, however, the two detector regions 41 and 42 are not formed by the same camera but by two different cameras 31 and 32. The embodiment of Fig. 2 offers the advantage that only a single camera is required, whereby lower costs and a smaller space requirement are possible. By contrast, the embodiment of FIG. 3 offers the advantage that two smaller camera chips suffice instead of one large camera chip. In addition, the optical axes of the illumination light are perpendicular to the respective camera surface, whereby not distorted images are generated on the camera surfaces.

The embodiment of the invention shown in FIG. 4, in turn, corresponds largely to the embodiment of FIG. 1. Two light source units 1 1 and 12 are arranged in different planes, one of which is located in front of and behind a confocal plane 14. While in the embodiment of FIG. 1 the light source units 1 1 and 12 emit illumination light of the same spectral range, in the embodiment of FIG. 4 the two light source units 1 1 and 12 emit illumination light of different spectral ranges. For example, the light source unit 11 may comprise an array of LEDs or lasers emitting 800nm light while the light source unit 12 comprises an array of LEDs or lasers emitting 850nm light. The arrangements of the LEDs or lasers is the same for both light source units 1 1 and 12 and corresponds to the light spot pattern to be generated.

In Fig. 4, a dichroic optical element is used as the beam combining means 15. This has a cut-off wavelength between transmission and reflection, which lies between the two spectral ranges of the light emitted by the light source units 1 1 and 12 illumination light. As a result, light losses are less than, for example, a semipermeable mirror as beam combining means. Due to the different spectral ranges of the illumination light of the two light source units 11 and 12, sample light with two different spectral ranges is also emitted. While in FIG. 4 the detection beam path is similar to that of FIG. 1, in the embodiment of FIG. 5 the different spectral ranges of the sample light are used. Thus, the illumination beam path of the embodiment of Fig. 5 with the light source units 1 1, 12 is similar to the structure of Fig. 4. In the detection beam path, the sample light, however, with beam splitting means 33 which comprise a dichroic element, divided into two beam paths and guided to different detector areas. The detector areas may be different areas of the same camera 31 (as shown), or may be formed by different cameras (not shown). These designs allow measurements to be made with high light intensity and low light losses, increasing measurement accuracy. In addition, the two sample images can be recorded simultaneously.

Another embodiment will be described below with reference to FIG. As in Fig. 1, two light source units 1 1 and 12 are arranged in different planes here. As beam combining means 15, a polarization beam splitter 15 is used here. This reflects or transmits incident light depending on its polarization. A polarizer 16, 17 is located between the polarization beam splitter 15 and the two light source units 1 1 and 12. The illumination light of the two light source units 1 1 and 12 is polarized differently by the polarizers 16, 17, so that the light from one of the light source units at the polarization beam splitter 1 1 and 12 largely completely transmitted and the light from the other of the two light source units 1 1 and 12 is largely completely reflected. Thus, a combination of the two beam paths is achieved with low light losses.

In the embodiment of FIG. 7, the polarized illumination described with reference to FIG. 6 is used. In addition, a polarization beam splitter is used in the detection beam path as beam splitting means 33. This transmits or reflects incident sample light depending on its polarization. This structure is suitable if the sample light emitted thereon is also polarized by a polarization of illumination light. This is the case, for example, when illumination light is reflected on the sample surface as sample light. Depending on its polarization, the sample light is passed through the polarization beam splitter 33 onto two different beam paths and guided to different detector areas of the detector device 30. In this embodiment, both sample images can be recorded simultaneously with only slight light losses. 8 shows a further embodiment in which two light source units 1 1 and 12 are used, which are arranged in geometrically identical planes 14. The illumination light emitted by the two light sources 1 1 and 12 separates in wavelength and is brought via dichroic beam combination means to a common beam path to the sample 26.

In the foregoing embodiments, the optical imaging means 22, 40 are preferably achromatic. That is, the refractive power and thus imaging are identical for at least two wavelengths. For other wavelengths, differences in the refractive power of the optical imaging means 22, 40 are only small.

On the other hand, in the embodiment of FIG. 8 chromatic optical imaging means 22, 40 are used, preferably a chromatic objective 22. This has a longitudinal chromatic aberration, by means of which illumination light is focused on different height levels as a function of wavelength. Thereby, the light spot patterns of the two light source units 1 1 and 12 are imaged on different height planes 24 and 25, although both light source units 1 1 and 12 are arranged in the same confocal plane 14.

In this embodiment, the detection of the sample light can take place as shown and with reference to FIG. 1 or to FIG. 5. However, it can also be provided that with the beam splitting means 33 the sample light is guided as a function of the wavelength to different beam paths (not shown). Thus, sample light due to the light spot pattern imaged in the elevation plane 24 may be distinguished from the sample light due to the spot pattern imaged in the elevation plane 25. With the sample light of the two different beam paths, an image is respectively generated in two image planes 34 and 35. These two image planes 34 and 35 are different from a detection plane, which is formed in Fig. 8 by a single camera. As a result, different height levels can be examined, as described for FIG. 2. Although 22 light spot pattern in two different height levels 24 and 25 are already generated by the chromatic lens, so that it can be dispensed in principle that different altitude levels are mapped to the two detector areas. However, the two height levels 24 and 25 are usually close to each other, since only a relatively small mutually shifted focal planes can be generated by a longitudinal chromatic aberration. Therefore, it is advantageous if, in addition, different height levels are imaged sharply on the camera 30 by the two detector areas. The differences in the two sample images, from which one Height information is derived, thereby larger, so that the accuracy of the measurement is improved.

Another embodiment of the invention is shown in FIG. The detection of sample light is carried out as described for Fig. 2. On the other hand, the illumination is effected with a light source device 10, which generates a light spot pattern with the aid of lateral scanning means 48, 49. A single illumination light beam of the light source device 10 thus successively generates the light spots of a light spot pattern. The lateral scanning means 48, 49 thus carry out a scan in the lateral direction, that is to say perpendicular to the height direction, and not in the vertical direction. The scan takes place during the recording of the sample images, which are recorded simultaneously in the example shown.

In this embodiment, the imaging means 40 are designed as adjustable focusing means. These can be used to change to which height level a light spot pattern is focused. An adjustment of the focusing means 40 is performed after two sample images have been taken and evaluated. The direction of the adjustment may be selected so that the height level in which a light spot pattern is focused is brought closer to the height of one or more of the lateral areas of the sample. The design of the imaging means 40 as adjustable focusing means can be supplemented in each of the other embodiments.

A final embodiment of the invention is shown in FIG. This is scanned laterally with an illumination light beam, as described for Fig. 9. In addition, sample light is split into two beam paths and guided to different cameras 31, 32, as described for FIG. 3. In the above embodiments, two sample images are generated and evaluated. This should generally be understood as at least two sample images. The two light source units and the division of the sample light into two beam paths to two detector areas or two cameras are also to be understood as "at least two." Thus, the invention includes two sample images in which different altitude information is contained The light microscope according to the invention is spatially identical for the recording of both sample images. Advantageously, no height scan is required for the recording of these images, which is associated with a time and a measurement accuracy advantage.

LIST OF REFERENCE NUMBERS

10 light source device

1 1, 12 light source units

14 confocal plane

15 beam combining means for illumination light

16, 17 polarizers

20 beam splitters

21 coupling element

22 optical imaging agents, lens

24 focus plane, elevation plane

25 height level

26 sample

27 optical imaging means

28 observation equipment, eyepiece, main camera

30 detector device

31, 32 camera

33 beam splitting means for sample light

34, 35 picture plane

36, 37 optical imaging agents

40 optical imaging means, adjustable focusing means

48, 49 Lateral Scanning Agent

41, 42 detector areas

100 light microscope

Claims

light microscope

with a light source device (10) for emitting illumination light in the direction of a sample (26),

with a detector device (30) for recording sample images of the sample (26),

optical imaging means (40, 22, 36) for focusing illumination light as a spot pattern onto a height plane (25) on the sample (26) and for directing sample light from the sample (26) to the detector means (30),

the light source device (10) and the optical imaging means (40, 22, 36) being designed to illuminate a plurality of spaced-apart lateral regions of the sample (26) during a camera integration time of the detector device (30) with the light spot pattern,

characterized ,

in that the light source device (10), the optical imaging means (40, 22, 36) and the detector device (30) are designed to enable taking pictures of at least two sample images in which the light spot patterns are focused on different height planes (25) and / or in which different height levels are imaged on the detector device (30), wherein the light source device (10), the optical imaging means (40, 22, 36) and the detector device (30) are arranged spatially identical for each of the recordings of the different sample images,

wherein in each case the same lateral areas of the sample (26) are illuminated for the at least two sample images, and

in that electronic evaluation means are provided and designed to calculate height information of the sample (26) with the aid of the at least two sample images. Light microscope according to claim 1,

characterized in that the electronic evaluation means are designed to perform an autofocus method,

in which it is calculated from the at least two sample images whether the lateral areas of the sample (26) illuminated by the light spot patterns lie above or below a plane which is imaged sharply on a main camera (28) of the light microscope, and an adjustment direction is thereby determined which is to move the sample (26) relative to an imaging beam path of the sample light to achieve a sharp image on the main camera (28).

Light microscope according to claim 2,

characterized in that a scanning device (40) is provided for shifting a focus of illumination light in its propagation direction or for shifting an imaging plane from the sample light and

in that the electronic evaluation means are designed to control the scanning device (40) in the autofocus method in such a way that the displacement takes place in accordance with the determined adjustment direction in order to achieve a sharp imaging.

Light microscope according to one of claims 1 to 3,

characterized in that for focusing the light spot patterns on different height planes (25), the light source means (10) comprises first and second light source units (11, 12), the first light source unit (11) being arranged in a plane which is optically conjugate to a first height level on the sample (26), and wherein the second light source unit (12) is disposed in a plane that is optically conjugate to a second height level on the sample (26). Light microscope according to claim 4,

characterized in that the illumination light emitted by the at least two light source units (11, 12) differs in wavelength and

in that for combining the illumination light of the at least two light source units (11, 12) onto a common illumination beam path to the sample (26) there is a dichroic element (15) which reflects or transmits light as a function of wavelength.

Light microscope according to claim 4 or 5,

in that polarization means (16, 17) are present, by means of which the illumination light emitted by the at least two light source units (11, 12) is differently polarized, and in that for combining the illumination light of the at least two light source units ( 11, 12) to a common illumination beam path to the sample (26) a polarization beam splitter (15) is present, which reflects incident illumination light depending on its polarization or transmits.

Light microscope according to one of claims 1 to 6,

characterized in that the light source device (10) is adapted to emit illumination light from at least two different spectral regions, and

in that the optical imaging means (22) have a longitudinal chromatic aberration, by means of which illumination light is focused wavelength-dependently on different height planes (25) on the sample (26). Light microscope according to one of claims 1 to 7,

characterized in that between the sample (26) and the detector device (30) beam splitting means (33) are provided, with which sample light is split into at least two spatially different detection beam path,

in that the detector device (30) in each case comprises a detector region (41, 42) for each of the different detection beam paths,

in that different height levels are imaged onto the different detector areas (41, 42).

Light microscope according to claim 8,

Thereby ge ken nzeich n et that the various detector areas (41, 42) are different sections of a common camera (31) of the detector device (30).

0. Light microscope according to one of claims 1 to 9,

characterized in that the light source device (10) comprises at least two light source units (11, 12) whose illumination light differs in wavelength, and in that the beam splitting means (33), with which sample light on at least two spatially different detection beam paths is directed to comprise a dichroic element (33) which reflects or transmits light in a wavelength-dependent manner.

1. Light microscope according to one of claims 1 to 10,

Thereby I net net, that the light source device (10) comprises at least two light source units (11, 12) and polarizing means (16, 17) are provided, through which the illumination light emitted by the at least two light source units (11, 12) is differently polarized in that the beam splitting means (33), with which sample light is conducted onto at least two spatially different detection beam paths, comprise a polarization beam splitter (33) which reflects or transmits incident sample light in dependence on its polarization direction.

12. A method for examining a sample (26) with a light microscope in which illumination light is emitted in the direction of a sample (26) with a light source device (10),

in which sample images of the sample (26) are recorded with a detector device (30),

in which illumination light as light spot pattern is focused onto a height plane (25) on the sample (26) by optical imaging means (40, 22, 36) and sample light is conducted from the sample (26) to the detector device (30), the light source device (10 ) and the optical imaging means (40, 22, 36) are designed to illuminate a plurality of mutually spaced lateral regions of the sample (26) during a camera integration time of the detector device (30) with the light spot pattern;

characterized ,

in that the light source device (10), the optical imaging means (40, 22, 36) and the detector device (30) are arranged spatially equally during recording of at least two sample images, the light spot patterns being focused on different height planes (25) in the at least two sample images and / or different height levels (25) are imaged onto the detector device (30), and wherein in each case the same lateral areas of the sample (26) are illuminated for the at least two sample images, and

height information of the sample (26) can be calculated with the aid of the at least two sample images.

Method according to claim 12,

characterized in that, with beam splitting means (33) which are arranged between the sample (26) and the detector device (30), sample light is split into at least two spatially different detection beam paths,

in that the detector device (30) for each of the different detection beam paths in each case comprises a detector region (41, 42) with which the at least two sample images are recorded simultaneously. 14. The method according to claim 12 or 13,

By this means, a light intensity or a sharpness of image is determined in the at least two sample images for each of the lateral regions

and that height information of the lateral area is obtained by calculating the light intensities or image sharpnesses which were determined in the at least two sample images for the same lateral area.

15. The method according to any one of claims 12 to 14,

characterized in that the detector device (30) is used for capturing images in an autofocusing method,

in that a main camera (28) different from the detector device (30) is used for image acquisition in a normal measuring operation and that a dynamic range of the detector device (30) is greater than a dynamic range of the main camera (28), the dynamic range being the respective range of measurable light intensities indicates.