DE102013016367A1 - Light microscope and method for examining a sample with a light microscope - Google Patents

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 a blanket pattern on a height level at the sample and for directing sample light from the sample to the detector device , 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. The light microscope according to the invention is characterized in that the light source device, the optical imaging means and the detector device are designed to allow images of at least two sample images, in which the light spot patterns are focused on different height levels and / or where different height levels are imaged on 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 in 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 addition, the invention relates to a corresponding method for examining a sample with a light microscope.

Description

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. Autofocussing is particularly advantageous in the case of reflective or light-scattering samples which have an uneven surface. Thus, due to the non-planar surface, a focus adjustment to the height of a sample area of interest is imperative.

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 for example made US 2011/0017902 A1 and US 7,570,795 B2 known and are in the scanning microscope US Pat. No. 7,858,911 B2 stimulated. Another light microscope with an autofocus feature is in US 2010/0033811 A1 described.

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 object of the invention, it can be considered to provide a light microscope and a method of inspecting a specimen with a light microscope, which easily and quickly enable accurate height measurement of the specimen.

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 matter of the dependent claims and are also explained in the following description.

In the light microscope of the above type, the invention Light source means, the optical imaging means and the detector means adapted to allow images of at least two sample images, wherein the light spot patterns are focused on different height levels and / or where different height levels are imaged on the detector means, wherein the light source means, the optical imaging means and the Detector device are arranged spatially identical 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 identical during recordings of at least two sample images, the light spot patterns being focused on different height planes and / or different height planes in the at least two sample images be 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 takes place in conventional light microscopes, in particular by the method of 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 because 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 crucial that the same lateral areas are illuminated for both sample images and measured with the detector device. 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 sample is irradiated by the light spot pattern with a spatially inhomogeneous light intensity distribution. 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 be understood by a height profile and the Sample surface can be considered 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. From a single image, however, it is not yet possible to determine 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 to which the light spot pattern focuses on the first sample image becomes. Therefore, from a comparison of the measurement information obtained on the same lateral area in both sample images, it can be determined whether the height of this lateral area is above or below the first altitude 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 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 on the same lateral area in both sample images, whether the height of this lateral area lies 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 from the sample, in particular reflected or scattered illumination light. However, the sample light can also be luminescent light, ie fluorescence 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 (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 may also have a scanner through which an illuminating light beam successively illuminates different lateral areas 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 region of the sample is illuminated with a light intensity which is suitable for this sample region so as not to bring the detector device to 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 that were determined in the at least two sample images at the same lateral area are then offset against each other to obtain a height information of the lateral area.

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 height levels.

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 divided into at least two spatially different detection beam paths. The detector device comprises for each of the different detection beam paths each have a detector area.

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 merging 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 emit illumination light from at least 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 on the different detection beam paths are low, thus an improved measurement accuracy is achieved.

If there are polarizing 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 or transmits incident sample light depending on its polarization direction , 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 taken, too a height scanning movement are performed, 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, 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 relative to 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. With this, a shifting of a focus of illumination light in its propagation direction or a displacement of an imaging plane from the sample light can be carried out. 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. That is, the sample is moved in the adjustment direction or the focus of the illumination light is shifted opposite to the adjustment.

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, for example, successively record several raw images with exposure times of different lengths and compute them to form 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 is sufficient 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.

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

2 shows a second embodiment of a light microscope according to the invention, in which two different height levels are displayed side by side on a detector device.

3 shows a third embodiment of a light microscope according to the invention, in which two different height levels are mapped to two detector units.

4 shows a fourth 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 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 are shown side by side on a detector device.

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

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 are shown side by side on a detector device.

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

9 shows a ninth 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 side by side imaged onto a detector device.

10 shows a tenth 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 mapped to two detector units.

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

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

This first has observation means 28 which may include an eyepiece and / or a main camera, a lens 22 and other imaging agents 27 on. With these becomes a focal plane 24 on the observation means 28 displayed. In the area of the focal plane 24 can a sample 26 be arranged on, for example, a sample table or other sample holding means.

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

To record altitude information, a light source device 10 and a detector device 30 used. The light source device 10 in this embodiment comprises two light source units 11 and 12 , Each of these emits illuminating light, which as a spot pattern on the sample 26 is directed. The light source units 11 and 12 are arranged in different optical planes, which are not optically conjugate to each other. In 1 is the light source unit 11 behind a confocal plane 14 and the light source unit 12 in front of the confocal plane 14 which one to the focal plane 24 is conjugate level. Thereby, the light spot patterns of the two light source units become 11 and 12 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 11 and 12 is about beam combining agent 15 led to a common beam path. For this purpose, for example, serve a partially transparent mirror. The illumination light continues to be a coupling element 21 guided, which in the beam path of the sample 26 to the main camera 28 is arranged. The coupling element 21 For example, it may be a mirror that is either semitransparent or covers only a portion of an illumination light cross section.

Will illuminate light with the light source units 11 and 12 to the test 26 blasted, so sample light coming from the sample is also with the coupling element 21 distracted and on to the detector device 30 directed. The coupling element 21 can be adjustable to sample light in either direction of the main camera 28 or in the direction of the detector device 30 to lead.

With a beam splitter 20 becomes incident illumination light coming from the light source units 11 and 12 comes to the coupling element 21 passed while from the coupling element 21 incoming sample light to the detector device 30 is directed. The beam splitter 20 may be, for example, a partially transmissive mirror.

In the execution of 1 includes the detector device 30 a single camera 31 on which with imaging agents 36 an image of a sample plane is generated. This sample plane can be the focal plane 24 be, with which the sample plane shown differs from the two altitude levels, which differs from the light source units 11 and 12 be focused focused.

The two light source units 11 and 12 For example, one after the other, one light spot pattern on the sample 26 produce. This allows the camera 31 take two sample images one after the other, with no mechanical Movements are required. In particular, no height scan is performed by adjusting the light source device in order to record these two sample images 10 , the detector device 30 or optical imaging agents 22 . 36 between these and the sample 26 ,

More generally, the light source device 10 , the optical imaging agent 22 . 36 and the detector device 30 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 can either be 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.

Through the light source units 11 . 12 A sharp image of a light spot pattern is generated at different 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 relative to the two height planes and thus relative to the focal plane 24 lies. In particular, it can be determined whether the height of the lateral area is below or above the focal plane 24 lies. 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 in 2 shown. This corresponds largely to the previous version. The light source device 10 here, however, includes 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 mapped to 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 , whereby the light spot pattern sharp in the focal plane 14 is shown.

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

In the example shown, the detection level of the detector area is correct 41 with the picture plane 34 match. With this detector area 41 So a sample image is recorded, which is a sharp image of the focal plane 14 is. But because the picture plane 35 from the detection plane of the detector area 42 is different, that is with the detector area 42 recorded sample image a blurred image of the focal plane.

In other words, the detection plane of the detector area 41 optically conjugate to the focal plane 14 while the detection plane of the detector area 42 is optically conjugated to another altitude level. Thus, in the two sample images different height levels on the detector device 30 displayed.

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 , How to 1 Therefore, by comparing the sharpness or light intensity in the two sample images, a statement can be made as to whether and how far the illuminated lateral regions each above or below the focal plane 14 lie.

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 example shown, the light source device generates 30 a light spot pattern sharp in the focal plane 14 , In general, however, the light spot pattern can also be in any one of the focal plane 14 sharply generated at different altitudes. Similarly, the detection level of the detector area 41 not optically conjugate to the focal plane 14 be. Rather, it is sufficient that the two detection levels of the two detector areas 41 and 42 are conjugated to two different altitude 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 in 3 shown. This corresponds to the majority of the execution of 2 , Sample light again with beam splitters 33 divided into two different beam paths leading to two detector areas 41 and 42 to lead. Here are also the two detector areas 41 and 42 different with respect to an image plane 34 arranged. In contrast to 2 Here are the two detector areas 41 and 42 but not by the same camera, but by two different cameras 31 and 32 educated. The execution of 2 offers the advantage that only a single camera is required, thus lower costs and less space are possible. On the other hand offers the execution of 3 the advantage that two smaller camera chips are sufficient instead of a 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, which in 4 in turn, corresponds largely to the execution of 1 , Two light source units 11 and 12 are arranged in different levels, one of which is in front of and one behind a confocal plane 14 located. While in the execution of 1 the light source units 11 and 12 Send out illumination light of the same spectral range, send when executing 4 the two light source units 11 and 12 Illuminating light of different spectral ranges. For example, the light source unit 11 an array of LEDs or lasers that emits light at 800 nm while the light source unit 12 an array of LEDs or lasers emitting light at 850 nm. The arrangements of the LEDs or lasers are in both light source units 11 and 12 equal and corresponds to the light spot pattern to be generated.

at 4 is used as a beam combining agent 15 a dichroic optical element is used. This has a cut-off wavelength between transmission and reflection, which lies between the two spectral ranges of the light source units 11 and 12 emitted illumination light is located. 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 at 4 the detection beam path of the 1 is similar to when running 5 used the different spectral ranges of the sample light. Thus, the illumination beam path is similar to the embodiment of FIG 5 with the light source units 11 . 12 the construction of 4 , In the detection beam path, the sample light, on the other hand, is provided with beam splitting means 33 , which comprise a dichroic element, divided into two beam paths and guided to different detector areas. The detector areas can be different areas of the same camera 31 be (as shown), or 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 6 described. As in 1 here are two light source units 11 and 12 arranged in different levels. As a beam combining agent 15 here becomes a polarization beam splitter 15 used. This reflects or transmits incident light depending on its polarization. Between the polarization beam splitter 15 and the two light source units 11 and 12 there is one polarizer each 16 . 17 , Through the polarizers 16 . 17 becomes the illumination light of the two light source units 11 and 12 polarized differently, so that at the polarization beam splitter, the light from one of the light source units 11 and 12 largely completely transmitted and the light from the other of the two light source units 11 and 12 is largely completely reflected. Thus, a combination of the two beam paths is achieved with low light losses.

In the execution of 7 becomes the too 6 described polarized illumination used. In addition, in the detection beam path as a beam splitting agent 33 a polarization beam splitter is used. 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. Through the polarization beam splitter 33 Depending on its polarization, the sample light is directed to two different beam paths and to different detector areas of the detector device 30 guided. In this embodiment, both sample images can be recorded simultaneously with only slight light losses.

8th shows a further embodiment in which two light source units 11 and 12 be used in geometrically equal levels 14 are arranged. That of the two light sources 11 and 12 emitted illumination light differs in wavelength and is sent to a common beam path to the sample via dichroic beam combining means 26 brought.

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

On the other hand, in the execution of 8th chromatic optical imaging agents 22 . 40 used, preferably a chromatic lens 22 , This has a color longitudinal error, is focused by which illumination light wavelength dependent on different height level. Thereby, the light spot patterns of the two light source units become 11 and 12 on different altitude levels 24 and 25 although both light source units 11 and 12 in the same confocal plane 14 are arranged.

The detection of the sample light can in this embodiment as shown and to 1 or to 5 described described. But it can also be provided that with beam splitting agent 33 the sample light is guided wavelength-dependent on different beam paths (not shown). Thus, sample light that is on the in the elevation 24 pictured light spot pattern can be distinguished from the sample light that is in the height level 25 pictured light spot pattern goes back. With the sample light of the two different beam paths is in two image planes 34 and 35 each generates an image. These two picture planes 34 and 35 are different to a detection plane, which at 8th formed by a single camera. This allows different altitude levels to be examined, such as 2 described. Although already by the chromatic lens 22 Light spot pattern in two different height levels 24 and 25 generated, so that in principle can be dispensed that different altitude levels are mapped to the two detector areas. The two altitude levels 24 and 25 but are usually close to each other, since only a relatively small mutually shifted focal planes can be generated by a Farblängsfehler. Therefore, it is advantageous if in addition by the two detector areas different heights rally on the camera 30 be imaged. The differences in the two sample images, from which a height information is derived, is thereby greater, so that the accuracy of the measurement is improved.

Another embodiment of the invention is in 9 shown. The detection of sample light takes place here as well 2 described. The lighting, however, is done with a light source device 10 showing a light spot pattern by means of lateral scanning means 48 . 49 generated. A single illumination light beam of the light source device 10 thus successively generates the light spots of a light spot pattern. With the lateral scanners 48 . 49 Thus, a scan in the lateral direction, that is perpendicular to the vertical direction, and not carried out in the height 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 designed as adjustable focusing. These can be used to change to which height level a light spot pattern is focused. An adjustment of the focusing means 40 This is done 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 agent 40 as adjustable focusing means may be supplemented in each of the other embodiments.

A final embodiment of the invention is in 10 displayed. This is scanned laterally with an illumination light beam, such as 9 described. In addition, sample light is split into two beam paths and to different cameras 31 . 32 led, how to 3 described.

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 on two beam paths to two detector areas or two cameras are to be understood as "at least two".

Thus, the invention includes two sample images containing different height information. The arrangement of the components of 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 means

11, 12

Light source units

14

confocal plane

15

Beam combining means for illumination light

16, 17

polarizers

20

beamsplitter

21

coupling element

22

optical imaging means, lens

24

Focus plane, elevation plane

25

height plane

26

sample

27

optical imaging agents

28

Observation equipment, eyepiece, main camera

30

detector device

31, 32

camera

33

Beam splitter for sample light

34, 35

image plane

36, 37

optical imaging agents

40

optical imaging means, adjustable focusing means

48, 49

Lateralscanmittel

41, 42

detector regions

100

light microscope

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 2011/0017902 A1 [0008]
• US 7570795 B2 [0008]
• US 7858911 B2 [0008]
• US 2010/0033811 A1 [0008]

Claims (15)

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 ), with optical imaging means ( 40 . 22 . 36 ) for focusing illumination light as a light spot pattern on a height plane ( 25 ) on the sample ( 26 ) and for conducting sample light from the sample ( 26 ) to the detector device ( 30 ), wherein the light source device ( 10 ) and the optical imaging means ( 40 . 22 . 36 ) are designed during a camera integration time of the detector device ( 30 ) with the light spot pattern a plurality of spaced lateral regions of the sample ( 26 ), characterized in that the light source device ( 10 ), the optical imaging means ( 40 . 22 . 36 ) and the detector device ( 30 ) are designed to allow taking pictures of at least two sample images, in which the light spot patterns on different height levels ( 25 ) are focused and / or where different height levels 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 the same for each of the images of the different sample images, wherein for the at least two sample images in each case the same lateral regions of the sample ( 26 ), and that electronic evaluation means are provided and designed to use the at least two sample images to provide height information of the sample ( 26 ) to calculate. Optical microscope according to claim 1, characterized in that the electronic evaluation means are designed for carrying out an autofocus method in which it is calculated from the at least two sample images whether the lateral areas of the sample illuminated by the light spot patterns ( 26 ) are above or below a plane that is in focus on a main camera ( 28 ) of the light microscope is mapped, and hereby an adjustment direction is determined, in which the sample ( 26 ) is to be moved relative to an imaging beam path of the sample light in order to obtain a sharp image on the main camera ( 28 ) to reach. Light microscope according to claim 2, characterized in that a scanning device ( 40 ) is provided for displacing a focus of illumination light in its propagation direction or for displacing an imaging plane from the sample light, and in that the electronic evaluation means are designed to prevent the scanning device ( 40 ) in the autofocus method so that the displacement takes place in accordance with the determined adjustment direction to achieve a sharp image. Light microscope according to one of claims 1 to 3, characterized in that for focusing the light spot pattern on different height levels ( 25 ) the light source device ( 10 ) a first and a second light source unit ( 11 . 12 ), wherein the first light source unit ( 11 ) is 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 arranged in a plane which is optically conjugate to a second height level on the sample ( 26 ). Light microscope according to claim 4, characterized in that the from the at least two light source units ( 11 . 12 ) differs emitted illumination light in the wavelength and that for merging the illumination light of the at least two light source units ( 11 . 12 ) to a common illumination beam path to the sample ( 26 ) a dichroic element ( 15 ), which reflects or transmits light depending on the wavelength. Optical microscope according to claim 4 or 5, characterized in that polarizing means ( 16 . 17 ) are provided, by which the of the at least two light source units ( 11 . 12 ) is differently polarized, and 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 ), which reflects or transmits incident illumination light depending on its polarization. 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 that the optical imaging means ( 22 ) have a longitudinal chromatic aberration through which illumination light is wavelength-dependent to different levels ( 25 ) on the sample ( 26 ) is focused. 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 divided into at least two spatially different detection beam paths that the detector device ( 30 ) for each of the different detection beam paths each have a detector area ( 41 . 42 ) includes that on the different detector areas ( 41 . 42 ) Different altitude levels are displayed. Light microscope according to claim 8, characterized in that the different detector areas ( 41 . 42 ) different sections of a common camera ( 31 ) of the detector device ( 30 ) are. Light microscope according to one of claims 1 to 9, characterized in that the light source device ( 10 ) 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 is directed to at least two spatially different detection beam paths, a dichroic element ( 33 ) which reflects or transmits light in a wavelength-dependent manner. Light microscope according to one of claims 1 to 10, characterized in that the light source device ( 10 ) at least two light source units ( 11 . 12 ) and polarizing agents ( 16 . 17 ) are provided, by which the of the at least two light source units ( 11 . 12 ) is differently polarized, and that the beam splitting means ( 33 ), with which sample light is directed to at least two spatially different detection beam paths, a polarization beam splitter ( 33 ) which reflects or transmits incident sample light depending on its polarization direction. Method for examining a sample ( 26 ) with a light microscope, in which with a light source device ( 10 ) Illuminating light in the direction of a sample ( 26 ) is emitted, in which with a detector device ( 30 ) Sample images of the sample ( 26 ) in which optical imaging means ( 40 . 22 . 36 ) Illumination light as a light spot pattern on a height level ( 25 ) on the sample ( 26 ) and sample light from the sample ( 26 ) to the detector device ( 30 ), the light source device ( 10 ) and the optical imaging means ( 40 . 22 . 36 ) are designed during a camera integration time of the detector device ( 30 ) with the light spot pattern a plurality of spaced lateral regions of the sample ( 26 ), characterized in that the light source device ( 10 ), the optical imaging means ( 40 . 22 . 36 ) and the detector device ( 30 ) while images of at least two sample images are arranged spatially the same, wherein in the at least two sample images, the light spot patterns on different height levels ( 25 ) and / or different altitude levels ( 25 ) on the detector device ( 30 ) and wherein for the at least two sample images in each case equal lateral regions of the sample ( 26 ) and that with the aid of the at least two sample images height information of the sample ( 26 ) be calculated. Method according to claim 12, characterized in that with beam splitting means ( 33 ), which between the sample ( 26 ) and the detector device ( 30 ), sample light is distributed to at least two spatially different detection beam paths, that the detector device ( 30 ) for each of the different detection beam paths each have a detector area ( 41 . 42 ), with which the at least two sample images are recorded simultaneously. A method according to claim 12 or 13, characterized in that in the at least two sample images to each of the lateral regions a light intensity or image sharpness is determined and that by calculating the light intensities or image sharpness, which were determined in the at least two sample images to the same lateral area, height information of the lateral area. Method according to one of claims 12 to 14, characterized in that the detector device ( 30 ) is used for image recording in an autofocus method, that one of the detector device ( 30 ) different main camera ( 28 ) 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 ), where the dynamic range indicates the respective range of measurable light intensities.

Images