WO2007019895A1

WO2007019895A1 - Light scanning microscope with autofocus mechanism

Info

Publication number: WO2007019895A1
Application number: PCT/EP2006/004433
Authority: WO
Inventors: Peter Westphal; Daniel Bublitz
Original assignee: Carl Zeiss Microimaging Gmbh
Priority date: 2005-05-12
Filing date: 2006-05-11
Publication date: 2007-02-22
Also published as: DE102005022125A1

Abstract

A light scanning microscope is disclosed, with an excitation and a detection beam path, means (9) for raster scanning of an object (2) by displacement of an imaged spot, line or multi-spot region over the object and with a lens (6) for imaging the spot, line or multi-spot region. A focus adjustment mechanism (A) is provided for the lens (6) and an autofocus device (22) is provided for recording a position of the focus plane (F) of the lens (6) which images differing depth ranges on the imaged spot, line or multi-spot region at differing locations on a resolving detector (20).

Description

Scanning microscope with autofocus mechanism

The invention relates to a laser scanning microscope with an autofocus device, in particular a light scanning microscope with an excitation and a detection beam path, means for scanning scanning of an object by moving an imaged spot or multispot area over the object and a spot or multispot area imaging lens, wherein a Fokusverstellmechanismus is provided for the lens.

Light-scanning microscopes are known in the prior art, for this purpose reference is made, for example, to DE 197 02 753 A1 or DE 102 57 237 A1, which describes a light scanning microscope designed as a laser scanning microscope. In this context, it should be noted that the term "light" here means the entire region of the radiation which obeys the laws of optics.

Scanning microscopes typically reach an object image by imaging the aforementioned spot or multispot area with a detection that does not resolve a structure of the spot or the multispots (e.g., confocal detection). Moving the spot or multispot area over the object provides the picture. At the detector there is always only radiation information for the respective spot or multi-spot area, and an electronic combination of this image information taking into account the displacement of the spot or multi-spot area leads to the desired image. Confocal detection is a way to achieve a very high depth resolution. The signal evaluation is then restricted substantially to the focal plane, since areas lying outside the focal plane do not provide any significant signal information in the case of confocal detection; they are displayed in front of or behind the confocal aperture.

Adjusting the focal plane position is thus extremely important for a scanning microscope, especially when working with confocal detection. This is especially true if you have a Sample is used, which is thicker than the depth of field of the lens. It is then necessary to approach the plane which is to be measured before the measurement in the sample. Although the focus adjustment mechanisms are highly precise in conventional scanning microscopes, which would make it possible first to approach a known reference surface and then to set the focal plane to the desired distance from the reference surface, the distance between the current focal plane and the reference surface can be due to thermal effects, vibrations or temporally changed by other disturbances. One would then intermittently repeatedly check the distance to the reference surface, which would be very expensive.

In order to obtain precise images of a sample or a sample section by means of imaging optics in laser scanning microscopy, it is necessary to place the sample exactly in the focal position of the imaging position of the optics and secondly the position of the focal plane in the sample to know. In this context, triangulation methods, imaging methods with contrast evaluation and position determination by means of obliquely arranged convocal slit are known. In Triangulationsverfahren a collimated laser beam is reflected in the pupil plane of an objective and closed from the course of this laser beam relative to the imaging beam path to the z position of the laser light reflected from the sample. When imaging the laser light into different levels of the sample, however, image errors occur so that the autofocus quality varies greatly over a given depth of field. Also, fluctuations are to be determined as to whether the measurement result from the center or at the edge of the sample or the detector used is determined. Usually, a triangulation method is therefore performed iteratively, which is relatively time consuming.

In imaging methods with contrast evaluation, the sample is illuminated with a specific intensity distribution, usually by placing a grid in a field stop plane of an illumination beam path. You take a series of images with different distances between imaging optics and sample and determined in this series, the image with the highest contrast, which is then assigned to the optimal focus distance. The disadvantage of this is that for recording the image series different z-positions must be approached with high accuracy, which in turn is time consuming.

When determining the position by means of an obliquely placed concave slit diaphragm, a slit diaphragm is placed in a field stop plane of the illumination beam path and imaged onto the sample. The light reflected from the specimen is directed to a CCD line inclined relative to the slit and the position on the CCD line where the reflected light has a maximum is determined. This procedure is very fast, though Problems with impurities on the sample or sample surface that can cause intensity fluctuations. Also, a very large Justieraufwand in the image of the gap on the CCD line to apply, because the gap must be very narrow in order to achieve high accuracy can be. An improvement of the position determination by means of obliquely placed convocal slit aperture is described in DE 10319182A1.

All methods have in common that they can find the focal plane while very accurate, but allow the location of this focal plane within the sample, especially with respect to other interfaces, only limited to determine. Often, however, laser scanning microscopy not only seeks to find the measuring plane exactly, but also to determine its position, i. Determine the distance to the reference plane. Reference to a reference plane interface can be accomplished either by using a second autofocus device which is placed on the interface, e.g. a glass / sample interface, is focused. Of course, this increases the optical complexity, and usually you have to reserve a range of detection or illumination aperture for this additional autofocus. Alternatively, it is known in the prior art to interrupt the measurement for a short time and adjust the autofocus device to the desired reference plane (glass / cell interface) by means of a z-adjustment. The distance between the two z-settings then represents the distance of the measurement plane to the reference plane. The disadvantage here is that the measurement must be interrupted for the determination of the distance to the reference plane.

Autofocus approaches known for conventional microscopes are often unusable in light scanning microscopes. This applies to all approaches that make structured illumination of the sample and achieve the focus by means of the structure. However, since a light-scanning microscope displays only one spot or multi-spot area without dissolution of the structure of the individual spot at any time, structured illumination is not possible since the structure could not be resolved. Approaches such as those from US Pat. No. 6,545,765 or US Pat. No. 5,604,344, which effect structured sample illumination for autofocus purposes, are therefore generally unsuitable for light scanning microscopes.

The invention is therefore based on the object of specifying a laser scanning microscope, with which not only the position of the measurement plane can be determined exactly, but at the same time the distance to a reference plane can be detected. In particular, a repeated adjustment of the sample in the z-direction should be avoided for the determination of the reference plane. Also, an accurate determination of the position of the focal plane should be possible with little effort. This object is achieved by a light scanning microscope of the type mentioned above, which has an autofocus device for detecting a position of the focal plane of the objective, which images different depth ranges on the imaged spot or multispot area to different locations of a spatially resolving detector. The light scanning microscope according to the invention thus forms a depth cutout on the spot-resolving detector at each spot, so that the position not only of the focal plane but also of a reference plane, for example a transitional glass / sample material can be determined. The light scanning microscope thus determines the position of the focal plane relative to the reference plane. The Fokusverstellmechanismus is then controlled so that the distance of the focal plane, which represents the measurement plane, is kept constant by the reference plane to a certain extent, or changed according to application-specific specifications.

The invention thus for the first time uses a depth-resolved image of the spot or multispot area for autofocusing. To reproduce the depth resolution, a one-dimensional detector element suffices. The coordinate of the detector element scales the depth information. The intensity of the imaged radiation from the spot or multi-spot region depends on the one spatial coordinate of the detector element, which in turn is assigned taking into account the imaging conditions of the depth coordinate.

In the simplest construction, therefore, a detector line is sufficient, to which the depth-dependent spatially resolved imaging takes place such that different depth ranges are brought to different areas of the detector row for imaging. The center of the detector line is in a plane conjugate to the focal plane rather than the otherwise confocal aperture.

The deep-resolution image can be realized by means of an anamorphic optical system arranged in front of the detector line, which focuses a line focus on the detector line, which lies in one plane with the detector line and intersects the detector line. A tilting of the anamorphic optics or the detector line realizes this geometric arrangement particularly simple. The anamorphic optics can be realized for example as a cylindrical lens, as a toric lens or as a combination of a one-dimensional holographic diffuser with a spherical lens.

In principle, the excitation or the detection beam path can be used in the light-scanning microscope in order to decouple the radiation for the autofocus device. Advantageously, the integration takes place in the illumination beam path. Then, illumination light reflected back from the focal plane of the object or from the reference plane is used in the autofocus device. An increase in radiant power, with the Lighting light is directed to the object is not necessary because the autofocus device has no effect on the radiation intensity in the detection beam path. For structural reasons, it may of course also be appropriate to integrate the autofocus device in the detection beam path. Since the autofocus method relies on reflected light from a reference plane, care should be taken that the spectral range of the excitation light to the autofocus device can enter the detection beam path. In a laser scanning microscope whose detectors are preceded by corresponding excitation filters which block the excitation light, this condition is usually met in the detection beam path up to these excitation filters. The confocal principle suppresses backscattered light from other than the sample plane. However, reflective surfaces such as glass / air, glass / water or even glass / sample produce ghost images which are not confocally suppressed when they are pressed onto the oblique line. These are now used for autofocusing. The reference plane is always a reflective interface.

The decoupling of the radiation for the autofocus device in the beam path between the detector module or excitation module and the scanner, ie when the beam is at rest, advantageously causes the depth information to be averaged over the entire imaged area of the object. Individual object areas in the focal plane, which do not scatter radiation, then do not interfere.

The autofocus device now allows the light scanning microscope to control the focus adjustment mechanism as desired. Therefore, a development is preferred in which a control unit is provided which reads out the signals of the autofocus device and actuates the focus adjustment mechanism.

Expediently, the control unit will use the signals to determine the position of the focal plane with respect to a reference plane. It is therefore preferred that the control unit from the signals determines a measure of a distance between the focal plane of the lens and a reference plane on the object and optionally taken into account in the control of the Fokusverstellmechanismus.

The signals of the autofocus device can be the signal of the detector line in the mentioned embodiments. The signal will usually have at least two intensity maxima: the first maximum corresponds to the position of the current measuring position, ie the position of the focal plane, the second maximum is the position of the reference plane, for example, to assign a glass / sample material interface. The distance between the two maxima provides the distance between focal plane and reference plane, wherein the function with which the depth resolution is transmitted to the spatial resolution of the detector is taken into account. Depending on the embodiment, the angle between the line focus and the longitudinal axis of the detector line, the depth of field of the objective and the image scale can be used to convert the distance between the maxima into the distance between the focal plane and the reference plane. The width of the maxima is usually determined by the depth of field of the lens.

The anamorphic optic produces the mentioned line focus. The intensity distribution along the line focus is rarely constant, or only when considerable effort is driven. It is simpler to consider the intensity distribution along the line focus when determining the maxima. In the case of a cylinder optic, the intensity distribution along the line thereby corresponds to the intensity distribution of the spot illumination with excitation light.

This object is also achieved according to the invention by a laser scanning microscope which directs illumination radiation through an illumination beam path onto a sample and detects radiation emitted on the sample in a detection beam path, wherein the microscope has a beam splitter which can be used in the illumination or detection beam path the beam splitter in the inserted state at the sample reflected illumination radiation from the illumination or detection beam path along an optical axis of the autofocus device decouples, a the beam splitter with respect to the decoupled illumination radiation downstream, anamorphic optical element which focuses the decoupled illumination radiation in a line focus and a the Optical element subordinate line or area detector which has an area sensitive to illumination radiation, which lies in a plane which au of the line focus and the optical axis is biased, wherein the optical element and the detector are arranged to each other and tilted against each other so that the line focus with the sensitive area forms an angle greater than 0 ° and less than 90 °.

Preferably, the autofocus device of the microscope, which can be provided either as a standalone module or permanently installed in the laser scanning microscope, uses reflected or backscattered illumination radiation for the focusing on the sample. In fluorescence microscopy, this illumination radiation is excitation radiation, which is why these terms are also used interchangeably below. The use of the illumination radiation has the advantage that, in the case of a radiation-sensitive sample, there is no additional exposure to radiation of radiation which is only required for autofocus. If you wanted in the prior art this To avoid stress, illumination radiation for the autofocus device would have to be selected spectrally so that it does not damage the specimen. However, this would mean that the chromatic demands on the microscope rise significantly. Both problems avoids the device according to the invention or the microscope according to the invention by the use of the already irradiated for microscopy illumination radiation.

The autofocus device according to the invention couples illuminating radiation returning from the specimen either in the illumination beam path or in the detection beam path and directs it onto a detector line. An anamorphic optical element is used, which bundles the decoupled radiation into a focal line. A detector detects the radiation, wherein the anamorphic element and the detector are aligned in a certain manner to each other such that a radiation-sensitive region of the detector is oblique to the longitudinal extent of the focal line. The detector has a sensitive area that is line or line shaped. This can be realized by using a line detector. Alternatively, of course, an area detector comes into question, which is read in the corresponding line or line-shaped section. Furthermore, the detector allows a spatial resolution along the line-shaped, sensitive area.

The anamorphic element and the beam splitter are adjusted with respect to the arrangement in the illumination or detection beam path so that different levels in the

Sample are mapped to different focal lines, in terms of their

Longitudinal extent lying parallel to each other, the optical axis of the autofocus beam path at different distances to the beam splitter cut. Thus, the intersection of two

Focal lines, which are assigned to spaced planes in the sample, at different longitudinal position of the sensitive detector area and an evaluation of the detector signals provides a direct statement about the distance of the planes in or on the sample, taking into account the geometry and imaging conditions used. In a particularly simple embodiment, the detector will be aligned so that the sensitive area lies in the plane defined by the longitudinal direction of the focal line and the optical axis of the autofocus beam path and at an angle to the optical axis. This construction, in which the

Detector with its line-shaped sensitive area is inclined to the optical axis, is particularly easy to implement.

Of course, conversely, the focal line extend obliquely to the optical axis and the sensitive area is, for example, perpendicular to the optical axis. In both cases, for which there are, of course, intermediate stages with both inclined focal line and inclined sensitive area of the detector, it is achieved that, at least for some focal positions of the microscope objective, the focal line is the sensitive detector area and both lie substantially in the same plane as the optical axis. The point of intersection between the focal line and the sensitive detector area depends, with regard to its position along the sensitive detector area, on the position of the focal line on the optical axis and thus on the position of the associated plane in the sample.

Essential for the anamorphic optical element is that it generates the aforementioned focal line, that is, has a line focus. By way of example, this can be achieved by using a cylindrical lens, a toric lens or a one-dimensional, holographic diffuser.

To determine the distances of several levels in the sample, in particular for determining the distance between the focal plane of the objective and a reference plane, it is expedient that a control device is provided which reads these signals of the detector and determines the layers of intensity maxima along the sensitive area. Intensity maxima are associated with intersections of focal lines, so that taking into account the angle to which the line focus and sensitive range are tilted against each other, the distance of the focal lines along the optical axis of the autofocus device can be determined very simply. Taking into account the reproduction scale of the microscope, the control device thus calculates the distance of the focal plane from a reference plane.

The anamorphic optical element usually generates the focal line with a certain intensity distribution along the line. Of course, this intensity distribution will cause the intensity maxima to vary, depending on which portion of the focal line intersects the sensitive area of the detector. The inhomogeneity of the focal line is therefore expediently taken into account by the control device in determining the positions of the intensity maxima, for example in which a correction function is used.

The autofocus device can be used as a module in the illumination or detection beam path of a laser scanning microscope or can already be provided there in the microscope. In the arrangement in the detection beam path, of course, no blocking of illumination radiation must occur between the sample and the beam splitter, i. It must be provided a color neutral beam splitter and any block filter must be arranged downstream of the beam splitter relative to the sample.

In laser scanning microscopes usually a scanning of the sample is provided. It is favorable to arrange the beam splitter of the autofocus device in the stationary part of the beam path of the illumination or detection beam path. This has the Advantage, that the autofocus device evaluates averaged radiation over an image when the detector integrates over a period of time that is longer than the scanning of at least one section of the sample takes.

As already mentioned, the autofocus device or the microscope according to the invention converts a plane lying in the sample into a focal line, with spaced planes in the sample corresponding to spaced focal lines, which in turn are spaced according to the magnification on the optical axis of the autofocus devices. A particularly large measuring range is obtained by adjusting the beam splitter and the anamorphic optical element so that the focal plane of the objective is conjugate to a focal line which intersects the optical axis in the sensitive region of the detector. The sensitive area of the detector, the optical axis and the focus plane of the objective associated focal line thus ideally intersect as possible in one point. Then there is a symmetrical region before and after the lens focus plane in which reference planes can lie. If one wishes to realize the largest possible search range with respect to the reference plane in one direction, one will adjust the adjustment such that the focal plane associated with the focal plane of the objective intersects the sensitive area of the detector near its left or right edge.

The autofocus device or the microscope according to the invention is particularly suitable, as mentioned, to determine the distance between the current focal plane and a reference plane. As a reference plane, for example, a glass / sample transition is possible, which is present in conventional samples either at the bottom of the sample (i.e., at the slide) or at the top of the sample (on the coverslip). The control device is expediently assigned the positions of the intensity maxima of such interfaces in or on the sample.

The invention will be explained in more detail with reference to the drawings by way of example. Show it:

Fig. 1 is a schematic representation of a light scanning microscope with a

Autofocus device;

2a-c different constructions for the autofocus device of Figure 1,

3 is a simplified sectional view of an object detected by the light scanning microscope of FIG. 1,

Fig. 4 is a simplified representation of a signal waveform, as with the

Autofocus device of the light scanning microscope of Figure 1 is obtained 5 is a schematic diagram of the objective together with the autofocus device of FIG. 1; and FIG. 6 is a schematic diagram for explaining geometrical relationship within FIG

Autofocus device and the resulting detection signals.

FIG. 1 schematically shows a light scanning microscope designed as a laser scanning microscope (LSM) 1. With the laser scanning microscope 1, an object 2 is measured in a manner yet to be explained. The laser scanning microscope 1 is essentially subdivided into an illumination or excitation module 3, a detection module 4 and a microscope module 5. The excitation module 3 provides excitation radiation and feeds it into the microscope module 5, so that it is directed to the object 2 as spot-shaped illumination. The spot-shaped illumination is guided by the microscope module 5 in a scanning manner over the object 2. The spot area illuminated at the object 2 with excitation radiation from the excitation module 3 is confocally detected by the detection module 4 via the microscope module 5, e.g. in the form of a fluorescence analysis.

The microscope module 5 has an objective 6, which can be changed by means of a drive A in a focus adjustment FV with respect to the position of the focal plane in the object 2. This focus adjustment is explained in more detail for example in DE 197 02 753 A1.

The objective 6 is preceded by a tube lens 7. The radiation coming from the excitation module 3 is guided via the objective 2 by means of a scanning optics 8 and a scanner 9 through the tube lens 7 and the objective 6 as a scanning spot. At the same time, the scanner 9 causes a so-called de-scanning in the reverse direction of the beam towards the detection module 4, so that after the scanner 9 in the detection module 4 there is again a stationary beam.

The excitation beam path of the excitation module 3 and the detection beam path of the detection module 4 are combined via a main color splitter HFT.

The effect of the main color splitter HFT is likewise to be found in the already mentioned DE 197 02 753 A1. Instead of a dichroic main color divider, a color-neutral divider can also be used, as described, for example, in DE 102 57 237 A1.

In the detection beam path of the detection module 4, the radiation is divided by further, unspecified color divider into individual detection channels, which are each constructed of a photomultiplier 14 with upstream Pinhole 15 and Pinholeoptik 16. The pinhole 15 narrows the detection area almost completely to the theoretical focal plane; Radiation generated outside this focal plane can not pass through the pinhole 15. The main beam splitter HFT thus transmits the detection radiation, for example due to suitable spectral filter properties or by suitable geometric formation in the form of partial mirroring, as is known in the prior art. In the detection beam path, the radiation is conducted via secondary divider and the Pinholeoptiken 16 and the Pinholeblenden 15 to the PMT detectors. Overall, a confocal image of the illuminated sample spot on the sample 2 on the PMT detectors 14 takes place. The different detection branches differ in their spectral analysis capability, so that the LSM 1 shown here by way of example can have several spectral channels. As indicated by the dot-dash line at 30, even more detection modules can be used. Overall, the LSM 1 is a laser scanning microscope of known design.

The excitation module 3 causes an illumination of the spot with radiation of different wavelengths. For this purpose, different illumination channels are provided, which are each constructed in the embodiment of a laser terminal 10 and 12 for coupling the radiation of a laser and coupling optics 11 and 13 and which are combined via an unspecified mirror staircase. To set the spot diameter, a telescope 17 is provided, which ensures the coupling according to conditioned radiation at the main color divider HFT.

However, the LSM 1 additionally has an autofocus device 22, which is installed in the illustrated embodiment in the illumination beam path. For this purpose, a beam splitter sits as an output coupler 18 in the illumination beam path 2, which decouples radiation backscattered from the object 2 into the excitation beam path of the excitation module 3 at the excitation module 3 and bundles optics 19 formed here as anamorphic optics into a line focus LF. In the case of a point-scanning laser scanning microscope 1, the optic 19 is an anamorphic photon. In a line-scanning microscope, a spherical lens 19 can be used, since this then already provides a line focus. The line focus LF is directed to a detector line 20, which forms an angle α with the optical axis OA and is cut by the line focus LF. The line focus LF and the detector line 20 are thus in one plane. The anamorphic 19 and the detector line 20 form an autofocus device 22, whose operation will be explained with reference to Figures 3 and 4. The present geometry is described below with reference to Figures 5 and 6 in more detail. It is essential here that the anamorphic photodiode 28 produces a linear focus.

A possible embodiment for the optics 19 are shown in FIGS. 2a-c. In addition, the line focus LF or, in FIG. 2c, the longitudinal axis L of the detector line 20 is shown by way of example in FIG. 2b. The optics 19 can be a combination of one-dimensional holographic diffuser 26 with upstream spherical optics 25 (FIG. 2a), toric lens 24 (FIG. 2b) or cylindrical lens 23 (FIG. 2c), if the laser scanning microscope uses a spot spot for scanning.

FIGS. 1 and 2 show an oblique position of the detector line 20 in order to ensure that the line focus LF lies obliquely to the longitudinal axis L of the detector line 20. Of course, this mutual skew can also be achieved without tilting the detector line 20 with respect to the optical axis OA, for example by means of a suitable holographic element or an obliquely arranged cylinder optic. It is therefore essential, as will be explained below, a tilting of the longitudinal direction of the focal line to the longitudinal direction of the detector line 20. This could also be achieved, for example, by the Anamorphot 19 is tilted.

Also, instead of decoupling the reflected radiation from the excitation beam path of the excitation module 3, a decoupling on the detection module 4 can take place when the main color splitter HFT allows backscattered excitation radiation to pass through. A possible attachment point for the autofocus device 22 is indicated in Figure 1 at 30 and indicated by a dashed line.

Figure 3 shows a schematic sectional view through the object 2, which is detected by the laser scanning microscope 1 of Figure 1. The object 2 has a slide 27, on which, covered by a cover glass 28, a cell layer 29 to be microscoped is arranged. Further, by way of example, the position of the focal plane F corresponding to the plane indicated by the confocal condition of the detection module 4, i. is determined by the selective action of Pinholes 15. Above the cell layer 29 there is a transition to the cover glass 28. Such a glass / cell layer transition has a refractive index jump. As is known, radiation is fundamentally reflected at a refractive index jump. The refractive index jump of the transition between cover glass 28 and cell layer 29 can thus be used as a reference plane R.

The imaging of the line focus LF in the autofocus device 22 onto the detector array 20 causes radiation incident on the detector array 20 to be fanned out of different regions along the optical axis OA on which the spot is illuminated. The result of this fanning in the signal of the detector line 20 is shown in FIG. 4.

The reflection at the refractive index jump of the reference plane R leads to an increased radiation intensity at a certain point of the detector line 20. In the signal S of the detector line 20 there is a corresponding peak, which in FIG. 4 is a reference plane peak PR is designated. The structure of the cell layer measured in the measurement plane, ie the focal plane, likewise leads to a reflection which occurs elsewhere on the detector line 20, ie at another x-coordinate in FIG. 4 and likewise leads to an increase in the intensity I (in FIG referred to as focal plane peak PF).

Along the detector line 20, whose corresponding x-coordinate is usually given by the pixel number, there are thus two intensity maxima as peaks, namely focal plane peak PF and reference plane peak PR, which are spaced apart by one pixel spacing d. The pixel spacing d can be converted into the distance D between the focal plane F and the reference plane R in a simple manner. For this purpose, on the one hand, the magnification of the optical image has to be considered. On the other hand, the skew of the detector row 20, i. the angle α between line focus LF and longitudinal plane L of the detector line 20, a roll.

The width of each peak is determined by the depth of field of the lens 6. It can enter into the determination of the center of gravity of the focal plane peak PF and the reference plane peak PR. Furthermore, in the case of the determination of the peak or center of gravity, a basic course of the signal S resulting from the intensity distribution which is fundamentally given in the line focus LF can be taken into account. For example, with a Gaussian illuminated spot, e.g. find this Gaussian distribution also in the line focus LF. The same applies of course to other intensity distributions in the illuminated spot.

The determination of the distance D is made in the embodiment of Figure 1 by a control unit 21, which reads both the signal of the detector row 20, as well as the drive A for focus adjustment of the lens 6 controls accordingly. The controller further determines the peak centroids, the peak distance d and controls the setting of the distance D to a certain extent.

FIG. 5 schematically shows the mode of operation of the autofocus device 22. For the sake of simplicity, only the optical axis OA and the elements 2, 6, anamorphot 19 and detector line 20 lying thereon are shown in FIG. 5. Folds of the optical axis OA, as they occur in particular at the beam splitter 18 in Fig. 1, are not shown in order to keep the figure clear. As can be seen, the objective 6, together with the anamorphic 19, images planes spaced apart in the sample 2 into spaced focal lines. The object focal plane 37 is imaged, for example, in a focal line 38, and a plane 36 located farther in the object 2, ie further away from the objective 6, is imaged into a focal line 39, which is seen along the optical axis OA in the imaging direction in front of the focal line 38, ie closer to Anamorphic 19 is located. Due to the inclination of the Detector line 20 with respect to the optical axis OA cut the focal lines 38 and 39, the sensitive portion of the detector line 20 at various intersections 40, 41, which are spaced along the detector line. Thus, at the points of intersection 40, 41 associated points of the longitudinally of the sensitive area spatially resolving detector line 29 form the already mentioned intensity maxima. This is illustrated in FIG. 6.

FIG. 6 shows in its upper half the detector row 20 and the optical axis OA lying obliquely at an angle α and the focal lines 38, 39 intersecting the detector row 20. Due to the different points of intersection, signal I of detector line 20 as a function of pixel number n of the detector line results in the already mentioned intensity curve S which has peaks as maxima 43 and 44 which correspond to intersection points 40 and 41 of focal line 38 and 39, respectively, with the detector line 29 are assigned. By the corresponding coordinates 45 and 46 of the maxima, as described with reference to FIG. 4, taking into account the angle α, the distance of the focal lines 39 and 38 along the optical axis OA can be easily calculated by the distance d between the coordinates 45 and 46 is multiplied by the cosine of the angle α. Together with the imaging ratio, which is achieved in the microscope module and in particular taking into account the objective 6, one thus obtains the distance D between the planes 37 and 38 in the sample 17.

The two intensity maxima occurring on the detector line 29 are assigned to such excellent planes in the sample 17. The maximum 43 corresponds to the focal plane of the lens 16, d. H. the current measuring position or plane, from which the confocal image in the sample 17 takes place. The second maximum 44 can be assigned as the reference plane of an interface in the sample between the sample material and the substrate (for ease of illustration, the sample 17 in Figures 1, 4 and 5 is not shown structured, so that the interface is not shown, for example, exactly the dashed line 36 would be).

After calculating the distance between the planes, the control unit can set or keep constant the distance of the measurement plane from the interface which serves as the reference plane. Also, simply calibrate the sample stage adjustment mechanism if it exists.

The focal lines 38 and 39 are usually not homogeneous along their longitudinal direction in terms of intensity, since a conventional anamorphic photopotential focus focuses the light on the focal line. The resulting zero curve 47 is shown in Fig. 6 for the intensity. When determining the intensity maxima, it is expedient if this zero curve 47 is considered, for example, subtracted from the signal of the intensity curve S. The illustrated construction shows a laser scanning microscope 1 with punctiform screening. However, the autofocus method can also be used with linear screening, in which case the line shape of the radiation without anamorphic imaging already exists.

Claims

claims

1. Scanning microscope with an excitation and a detection beam path, means (9) for raster scanning of an object (2) by moving an imaged spot,

Line or multi-spot area over the object (2) and a spot, line or multi-spot imaging lens (6), wherein for the lens (6) a Fokusverstellmechanismus (A) is provided, characterized in that a in the excitation or detection beam path (3, 4) coupled autofocus device (22) is provided for detecting a position of the focal plane (F) of the objective (6), the different depth ranges at the imaged spot, line or multi-spot on different locations of a spatially resolving detector (20) maps.

2. Light scanning microscope according to claim 1, characterized in that the autofocus device (22) along a longitudinal axis (L) extending detector line (20) with upstream optics (19), wherein the detector line (20) with its longitudinal axis (L) for optical Axis (OA) is tilted and the optical system (19) focuses the decoupled radiation into a line focus (LF), which is located substantially in the plane defined by the longitudinal axis (L) and the optical axis (OA) plane, so that the line focus ( LF) along the detector line (20).

3. Scanning microscope according to claim 2, characterized in that the light-scanning microscope images a spot and the optics (19) an anamorphic element, in particular a cylindrical lens (23), a toric lens (24) or a combination of a one-dimensional holographic diffuser (26) with a spherical lens (25).

4. Light scanning microscope according to one of the above claims, characterized by a control unit (21), the signals (S) of the autofocus device (22) reads out and controls the Fokusverstellmechanismus (A).

5. light scanning microscope according to claim 4, characterized in that the control unit (21) from the signals (S) is a measure of a distance (D) between the focal plane (F) of the objective (6) and a reference plane (R) on the object ( 2).

6. Scanning microscope according to claim 4 or 5 respectively in combination with claim 2, characterized in that the control unit (21) takes into account the caused by the anamorphic element intensity distribution along the line focus (LF).

7. Light scanning microscope according to claim 5, characterized in that the control unit (21) the focal plane (F) in a constant or specifically set distance (D) for

Reference plane (R) stops.

8. Light scanning microscope according to claim 1, characterized in that the autofocus device (22) comprises: - one in the illumination or detection beam path (3, 4) used beam splitter

(18), the reflected at the object (2) illumination radiation from the lighting or

Detection beam (3, 4) along an optical axis (OA) of the autofocus device (22) decouples, a the beam splitter (18) with respect to the decoupled illumination radiation downstream, anamorphic optical element (19), which decoupled

Illuminating radiation into a line focus (LF) and a line or area detector (20) arranged downstream of the optical element, which has an area sensitive to illumination radiation which lies in a plane which lies on the plane

Line focus (LF) and the optical axis (OA) is spanned, wherein the optical element (19) and the detector (20) to each other are arranged and tilted against each other so that the

Line focus (LF) with the sensitive area an angle (α) greater than 0 ° and less than 90 °.

9. Scanning microscope according to claim 8, characterized in that the anamorphic optical element (19) has a cylindrical lens (23), a toric lens (24) or a one-dimensional, holographic diffuser (26).

10. Light scanning microscope according to one of claims 8 or 9, characterized in that the detector (20) is oblique to the optical axis (OA).

11. Light scanning microscope according to one of the above claims in conjunction with claim 4, characterized in that the control unit (21) reads out the signals of the detector (20) and determines the layers (45, 46) of intensity maxima (43, 44) along the sensitive area of the detector or the detector line.

12. Light scanning microscope according to one of the above claims, characterized in that for scanning the sample (2) a scanning mirror unit (9) is provided, wherein the

Autofocus device is coupled into the stationary beam path of the illumination or detection beam path.

13. A light scanning microscope according to claim 3 or 8, characterized in that the anamorphic element (19) and the detector (20) are adjusted so that the plane of the

Microscope focus (37) is focused into a position of the line focus (LF), which intersects the sensitive area of the detector and the detector line (20) substantially on the optical axis (OA).

14. Microscope according to one of the above claims in conjunction with claim 11, characterized in that the control unit (21) assigns the layers (45, 46) of the intensity maxima (43, 44) interfaces in or on the object (2).