Patent ID: 12216019

DETAILED DESCRIPTION

The present disclosure augments the testing of the confocality of a scanning and to descanning microscope component group known from WO 2020/254 303 A1 with a testing of the axial confocality of the scanning and descanning microscope component group. For this purpose, at least one of a special auxiliary detector and a special auxiliary light source providing auxiliary light is used. The auxiliary detector may have an auxiliary detection aperture which is moveable into a plurality of auxiliary detection aperture positions arranged at distances in direction of the optical axis, or a plurality of auxiliary detection apertures in auxiliary detection aperture positions arranged at distances in direction of the optical axis and laterally with respect to the optical axis. The auxiliary light source may have an auxiliary emission aperture which is moveable into a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis, or a plurality of auxiliary emission apertures in a plurality of auxiliary emission aperture positions arranged at distances in direction of the optical axis and laterally with respect to the optical axis, out of which the auxiliary light emerges.

If the at least one of the auxiliary detection aperture and the auxiliary emission aperture is moveable, this does not require a continues movability. Instead, it is essential that the auxiliary detection or emission aperture positions arranged at distances in the direction of the optical axis may be repeatedly approached precisely.

The at least one of the auxiliary detector and the auxiliary light source is arranged in a focal area of focusing optics of the scanning and descanning microscope component group, wherein, if both the auxiliary detector and the auxiliary light source are arranged in the focal area, the auxiliary detection aperture and the auxiliary emission aperture or the auxiliary detection apertures and the auxiliary emission apertures are concentrically arranged in pairs. This implies that either both the auxiliary detection aperture and the auxiliary emission aperture are moveable or both a plurality of auxiliary detection apertures and a plurality of auxiliary emission apertures are present.

The scanning and descanning microscope component group whose confocality is to be tested comprises a first light source providing first illumination light, focusing optics focusing the first illumination light along and optical axis into a focal area, a first detector comprising a first detection aperture and detecting first light coming out of the focal area, and a scanner scanning laterally with respect to the optical axis, the scanner being arranged between the first light source and the first detector, on the one side, and the focal area, on the other side.

The detector may be a point detector whose detection aperture is defined by the active surface area of its light sensitive element or a pin hole aperture arranged in front thereof. The detector may as well be an array detector comprising a plurality of light sensitive elements. Then, either the active surface area of a central light sensitive element or the totality of the active surface areas of a central group of the light sensitive elements or the totality of the active surface area of all light sensitive elements may be regarded as the detection aperture of the array detector. Generally, each auxiliary detector may also be designed either as a point detector or as an array detector, wherein a design as a point detector is sufficient as a general rule.

For testing both the lateral and the axial confocality of the scanning and descanning microscope component group, the at least one of the auxiliary detector and the auxiliary light source arranged in the focal area are used as follows.

By operating the scanner, the auxiliary detection aperture of the auxiliary detector is or the plurality of auxiliary detection apertures of the auxiliary detector are scanned in the plurality of auxiliary detection aperture positions with the focused first illumination light. In doing so, first intensity distributions of the first illumination light detected by the auxiliary detector are registered over different settings of the scanner.

As an alternative, the first intensity distributions may be registered in that a first further auxiliary detection aperture of a first further auxiliary detector concentrically arranged with respect to a first emission aperture of the first light source is scanned with auxiliary light that emerges through the auxiliary emission aperture or the plurality of auxiliary emission apertures of the auxiliary light source in the plurality of auxiliary emission aperture positions by operating the scanner. Then, the first intensity distribution are those of the auxiliary light, and they are registered by the first further auxiliary detector over the different settings of the scanner. An auxiliary emission aperture of the first and any other light source or auxiliary light source here means the cross-sectional area out of which the illumination light emerges. If the respective light source provides collimated light, each beam cross section of the collimated light may also be regarded as an emission aperture, and it may be used as an emission aperture for the purposes of the present disclosure.

Further, the first detection aperture of the first detector is scanned with the auxiliary light that emerges through the auxiliary emission aperture or the plurality of auxiliary emission apertures of the auxiliary light source in the plurality of auxiliary emission aperture positions by operating the scanner. In doing so, second intensity distributions of the auxiliary light detected by the detector are registered over the different settings of the scanner.

As an alternative, the second intensity distributions may be registered in that the auxiliary detection aperture of the auxiliary detector is or the plurality of auxiliary detection apertures of the auxiliary detector are scanned in the plurality of auxiliary detection aperture positions with first further auxiliary light that emerges through a first further auxiliary emission aperture of a first further auxiliary light source arranged concentrically with respect to the first detection aperture by operating the scanner. In doing so, the second intensity distributions are those of the first further auxiliary light, and they are registered by the auxiliary detector over the different setting of the scanner.

If a plurality of auxiliary detection or emission apertures are provided which are arranged at distances laterally with respect to the optical axis, the plurality of auxiliary detection or emission apertures, in being scanned, should be separated by their lateral distances to such an extent that the first or second intensity distributions, respectively, are registered separately or may at least be separated from one another. However, the lateral distances should also not be much higher than necessary for this purpose.

The intensity distributions are compared to one another. In doing so, a focus position of a focus of the focused first illumination light of the first light source in the focal area in direction of the optical axis is determined from differences between the first intensity distributions, and an image position of an image of the first detection aperture of the first detector in the focal area in direction of the optical axis is determined from differences between the second intensity distributions. For these purposes, it is advantageous, if the diameters of the auxiliary detection and emission apertures, respectively, is about as large as or smaller than the diameter of the focus of the focused first illumination light in the focal area or the diameter of the image of the first detection aperture in the focal area, respectively.

If the focus position and the image position are identical, an axial confocality of the scanning and descanning microscope component group is given. However, the relevant axial confocality is only given if the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the first detector coincide in direction of the optical axis in an object focal area on an object side of an objective. If the wavelengths of the first illumination light and the first light detected by the first detector differ, this, for example due to wavelength dependent properties of the objective, does not need to mean that the focus position and the image position do also coincide in any other focal area, i.e. in the focal area in which the at least one of the auxiliary detector and the auxiliary light source is arranged.

If the focus position and the image position do not coincide in the relevant object focal area, the focus position and the image position may be aligned in the object focal area by means of at least one of a real relative shift and a virtual relative shift of the first detection aperture of the first detector with respect to the first light source. In any focal area, the deviations of the focus position from a target focus position and of the image position from a target image position may be determined from the differences between the first intensity distributions and the differences between the second intensity distributions, respectively. These deviations may then be compensated for by real or virtual shifts of at least one of the first detection aperture of the first detector and of the first light source. The target focus position and the target image position are to be set such that the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the first detector coincide in direction of the optical axis in an object focal area on the object side of an objective. In virtual shifts, the at least one of the first detection aperture of the first detector and the first light source is not shifted as such but optical elements which preferably selectively have an influence on the first light on its way to the first detection aperture or on the first illumination light on its way from the first light source are moved or changed otherwise.

The areas of the two dimensional intensity distributions are of particular importance in the relevant differences between the first intensity distributions and the second intensity distributions, from which the focus position and the image position are determined respectively. These are, for example, the respective smallest areas onto which a certain percentage of the intensity falls upon, or the areas within which a certain threshold value, for example a percentage of the average or maximum intensity of the intensity distribution is exceeded by the intensity. The focus position or image position of interest is found there, where the area of the respective intensity distribution has a minimum along the optical axis. This minimum may be located at one or between several auxiliary detection or emission aperture positions, respectively. If the beam shape of the respective illumination light, auxiliary light or further auxiliary light in the area of the respective detector, auxiliary detector or further auxiliary detector is known so that it is for example known whether it is a Gauß beam having a certain beam waist and Rayleigh length, two different auxiliary detection or emission aperture positions along the optical axis are sufficient. In order to find the minimum without detailed previous knowledge for sure, three different auxiliary detection or emission aperture positions along the optical axis which are also distributed around the focus position or the image position are advantageous. Besides the areas of the intensity distributions, their shapes may also be considered in looking at their differences. To increase the significance of the shapes of the intensity distributions, a distortion, like for example an astigmatism, may purposefully be introduced in the focusing optics as it is generally known to one skilled in the art.

For testing the lateral confocality, one may resort to the teaching of WO 2020/254 303 A1. Thus, the measure of an existing deviation from a desired lateral confocality of the scanning and descanning microscope component group may be deduced from a comparison of one of the first intensity distributions with one of the second intensity distributions, particularly from the comparison of the positions of at least one of their maxima, their centers and their centers of intensity. This conclusion is particularly simple, if the first and second intensity distributions compared to each other correspond to the same auxiliary detection and emission aperture positions. Otherwise, the differences between the auxiliary detection and emission aperture positions on which the first and second intensity distributions are based have to be considered.

In order to set up a laser scanning microscope, the scanning and descanning microscope component group may comprise an objective. Then, the at least one of the auxiliary detector and the auxiliary light source, i.e. the auxiliary detection aperture positions or the auxiliary emission aperture positions or both of them, may be arranged around an object plane of a microscope beam path of such a laser scanning microscope. However, this arrangement in the area of the object plane is only possible temporarily, because the samples to be investigated with the laser scanning microscope are to be arranged here. Further, the auxiliary detection and emission apertures and the distances of their auxiliary detection and emission aperture positions would have to be microscopically small for their arrangement in the area of the object plane. Even with an arrangement of the auxiliary detection aperture positions and auxiliary emission aperture positions around an intermediate image plane of the microscope beam path, it may be necessary to remove the auxiliary detector and the auxiliary light source, respectively, for actually using the laser scanning microscope. If, however, the auxiliary detection aperture positions or the auxiliary emission aperture positions or both of them are arranged around an intermediate image plane in a branch branching off a main beam path of the microscope, the auxiliary detector or the auxiliary light source or both of them may be stationary arranged there.

In order to be for sure able to determine the focus position and the image position from the first and second intensity distributions, respectively, it proves to be advantageous, if the auxiliary detection aperture positions or the auxiliary emission aperture positions or both of them are distributed along the optical axis over a distance from 0.5 mm to 10 mm. This absolute indication of the distance relates to the arrangement of the respective auxiliary detection or emission apertures around an intermediate image plane of the microscope beam path, i.e. on the side of the objective facing away from the object side. Related to an magnification M between the object focal area on the object side of the objective and the intermediate image plane around which the respective auxiliary detection or emission aperture positions are arranged, the magnification M being proportional to the magnification of the objective, the distance over which the respective auxiliary detection or emission aperture positions are preferably distributed along the optical axis is in a range from 0.2 μm×M2to 1 μm×M2.

Advantageously, the plurality of auxiliary detection apertures of the auxiliary detector or the plurality of auxiliary emission apertures of the auxiliary light source or both of them have same dimensions and shapes orthogonal to the optical axis, i.e. when being viewed along the optical axis. Only then, the first and second intensity distributions can be directly compared with one another. Preferably, the auxiliary detection aperture(s) of the auxiliary detector and the auxiliary emission aperture(s) of the light source are not only concentric but also congruent or even identcal in pairs. In order to achieve this, at least one photoelectric component may be used both as a part of the auxiliary light source and as a part of the auxiliary detector. This photoelectric component may, for example, be a photo diode. Alternatively, the auxiliary detection aperture or the auxiliary detection apertures of the auxiliary detector, or the auxiliary emission aperture or the auxiliary emission apertures of the light source, or both of them may be formed with an terminal cross section of an optical fiber. This optical fiber may be branched towards the auxiliary light source, on the one hand, and towards the auxiliary detector, on the other hand. This branching may, for example, be realized by an open beam splitter, a fiber optical beam splitter or a circulator.

In a preferred embodiment, the auxiliary detection apertures of the auxiliary detector or the auxiliary emission apertures of the auxiliary light source or both of them are formed by means of holes in different layers of a layered structure or in different depths of a plate. On the backside of the layered structure or the plate, the beam path may be branched towards the auxiliary light source and the auxiliary detector.

The scanning and descanning microscope component group may further comprise a second light source providing second illumination light, wherein the focusing optics focus the second illumination light along the optical axis into the focal area. If the auxiliary detection aperture of the auxiliary detector is, or the plurality of auxiliary detection apertures of the auxiliary detector are then scanned in the plurality of auxiliary detection aperture positions with the focused second illumination light by operating the scanner, wherein further first intensity distributions of the second illumination light detected by the auxiliary detector are registered over the different positions of the scanner, a further focus position of a focus of the focused second illumination light of the second light source in the focal area in direction of the optical axis may then be determined from differences between the further first intensity distributions. Thus, the axial confocality of the scanning and descanning microscope component group may also be tested with regard to the second illumination light. This second illumination light may, for example, be fluorescence inhibiting light, more particularly so-called STED light.

As an alternative, the further first intensity distributions may be registered in that a second further auxiliary detection aperture of a second further auxiliary detector concentrically arranged with respect to a second emission aperture of the second light source is scanned with auxiliary light that emerges through the auxiliary emission aperture or the plurality of auxiliary emission apertures of the auxiliary light source in the plurality of auxiliary emission aperture positions by operating the scanner. Then, the further first intensity distributions are those of the auxiliary light, and they are registered by means of the separate second auxiliary detector over the different positions of the scanner.

Generally, the light source or any auxiliary light source may comprise at least one of a light emitting diode (LED), a superluminescent diode, and a laser diode.

The scanning and descanning microscope component group may further have a second detector detecting second light coming out of the focal area, the second detector having a second detection aperture. In the normal use of the scanning and descanning microscope component group, this second light may, for example, have another wavelength and may origin from other emitters than the first light. For testing the confocality of the scanning and descanning microscope component group also with respect to the second detector, the second detection aperture of the second detector may be scanned with the auxiliary light that emerges through the auxiliary emission aperture or the plurality of auxiliary emission apertures of the auxiliary light source in the plurality of auxiliary emission aperture positions by operating the scanner, wherein further second intensity distributions of the auxiliary light detected by the second detector are registered over the settings of the scanner. Then, a further image position of an image of the second detection aperture of the second detector in the focal area in direction of the optical axis may be determined from differences between the further second intensity distributions, which allows for conclusions on the axial confocality.

As an alternative, the further second intensity distributions may be registered in that the auxiliary detection aperture is, or the plurality of auxiliary detection apertures of the auxiliary detector are scanned in the plurality of auxiliary detection aperture positions with second further auxiliary light that emerges through a second further auxiliary emission aperture of a second further auxiliary light source concentrically arranged with respect to the second detection aperture by operating the scanner. Here, the further second intensity distributions are those of the second further auxiliary light, and they are registered by means of the auxiliary detector over the settings of the scanner.

Besides the first light source, the focusing optics, the first detector and the scanner, the scanning and descanning microscope component group of the present disclosure comprises at least one of the auxiliary detector already defined above and the auxiliary light source already defined above as well as a testing device which is configured for registering the first and second intensity distributions. The comparison of the intensity distributions may also be executed by the testing device. Further, the testing device may be configured not only for determining deviations of the focus position from a target focus position and of the image position from a target image position but also for compensating these deviations by at least one of real relative shifts and virtual relative shifts of the first detection aperture of the first detector with respect to the first light source. Once again, the target focus position and the target image position are preferably set such that the focus position of the focus of the focused first illumination light of the first light source and the image position of the image of the first detection aperture of the detector, in order to establish the axial confocality, coincide in direction of the optical axis in an object focal area on an object side of an objective added to the scanning and descanning microscope component group for setting up a laser scanning microscope.

For establishing the lateral confocality, the testing device may apply the teaching of WO 2020/254 303 A1 which is incorporated herein by reference.

An auxiliary device according for testing the confocality of a scanning and descanning microscope component group has a connector configured for connection to the scanning and descanning microscope component group in a defined relative position. The optical axis, along which the auxiliary detection aperture positions of the auxiliary detector already defined above and the auxiliary emission aperture positions of the auxiliary light source already defined above, respectively, are arranged as parts of the auxiliary device, runs at a fixed orientation to the connector. With regard to all details, the auxiliary detector and the auxiliary light source may be designed as already explained above. The connector of the auxiliary device may be a standardized connector, and it may, for example, fit to an objective connector of the scanning and descanning microscope component group.

Now referring in greater detail to the drawings, the laser scanning microscope1schematically depicted inFIG.1includes an objective2and further components of a microscope component group3which are enclosed by a dashed line. A first light source4, particularly in form of a laser5, for providing first illumination light23belongs to the microscope component group3. Further, optics27focusing the illumination light23into a focus47in a focal area7around a focal plane6belong to the microscope component group3. InFIG.1, the focal plane6is depicted by a dashed line. InFIG.1, only one lens28is depicted as a component of the focusing optics27. Further, a detector8for first light9coming out of the focal area7belongs to the microscope component group3. A pinhole aperture10and further optics29focusing the light9to be detected by the detector8onto the aperture of the pinhole aperture10are arranged in front of the actual detector8, i.e. its light sensitive area. The aperture of the pinhole aperture10defines a detection aperture11of the detector8which, with a correct adjustment of the microscope component group3, is arranged confocally with respect to the focus47in the focal area7. A scanner12of the microscope component group3is arranged between the light source4and the detector8with the pinhole aperture10, on the one side, and the focal plane6, on the other side. The lens28of the optics27is on that side of the scanner12facing towards the light source4and the detector8. The scanner12includes two rotating mirrors13,14per lateral direction, in which the scanner12is provided for scanning a sample15arranged in front of the objective2with the illumination light23from the light source4. To each rotating mirror13,14, a rotary drive44is assigned. The scanner12does not only serve for scanning the sample15with the illumination light23but also for descanning the light9that is caused by the illumination light23in the sample15such that this light is spatially selectively detected by the detector8. Here, an exact confocality of the microscope component group3, i.e. an exact coincidence of the focus of the illumination light23focused in the sample15with an image of the detection aperture11of the detector8in the sample15is decisive. This confocality is tested in the focal plane6depicted inFIG.1. This focal plane6is located in a branch16branching off the main beam path17of the laser scanning microscope1and corresponds to an intermediate image plane in the main beam path17. A tube lens33is arranged in the main beam path17between the scanner12and the objective2. The branch16branches off the main beam path17at the last rotating mirror14of the scanner12in front of the objective2. If the branch is activated by means of this last rotating mirror14, the other rotating mirrors13,14are usable for scanning the auxiliary device18arranged in the branch16. With respect to the further components of the microscope component group3, the auxiliary device18is in a spatially fixed position around the focal plane6, and it is reachable with the illumination light23by operating the scanner12.

The beam path of the first illumination light23from the first light source4and the beam path of the first light9to be detected by the first detector8are separated by means of a beam splitter26. A further beam splitter36separates the beam path of second illumination light35from a second light source24which typically is also made as a laser5from the beam path of the light9. A beam shaper32is arranged in the beam path of the second light35. The beam shaper32may be provided for forming an intensity distribution of the second illumination light35having a central intensity minimum in the focal area7as it is usual for fluorescence inhibiting light, particularly STED light for resolution enhancement. A further beam splitter31separates a beam path of second light30to be detected from the beam path of the first light9to be detected, wherein the first light9and the second light30may particularly differ in their wavelengths. The second light30is detected by a second detector38whose second detection aperture41is defined by a further pinhole aperture40.

Further, a testing device37is schematically depicted inFIG.1, which is connected to the light sources4,34, the detectors8,38, the scanner12and the auxiliary device18, and which tests the confocality of the scanning and descanning microscope component group3with the aid of these connections.

Except of the additional second light source34, the additional second detector38, the depiction of the testing device37and the only schematic depiction of the auxiliary device18,FIG.1corresponds to FIG. 5 of WO 2020/254 303 A1. The other embodiments of the scanning and descanning microscope component group3disclosed in WO 2020/254 303 A1 and of a laser scanning microscope1set up therewith may be upgraded and used with an auxiliary device18as it will be further explained in the following. Insofar, reference is made to the disclosure of scanning and descanning microscope component groups3and laser scanning microscopes1in WO 2020/254 303 A1.

FIG.2schematically illustrates an embodiment of the auxiliary device18. The focal plane6and the focal area7as well as an optical axis39of the branch16, along which the illumination light23is incident on the auxiliary device18, are depicted. Here, the auxiliary device18comprises a photoelectric component19which is usable both as an auxiliary light source20and as an auxiliary detector24. By means of pinhole apertures not depicted further inFIG.2, three pairs of congruent or identical auxiliary emission apertures21of the auxiliary light source20and auxiliary detection apertures25of the auxiliary detector24are formed. The auxiliary emission apertures21and the auxiliary detection apertures25are arranged at distances in direction of the optical axis39and laterally thereto. A connector73not further specified inFIG.2serves for connecting the auxiliary device18in a defined relative position to the further components of the microscope component group3.

After a first step in which the auxiliary emission apertures21and the auxiliary detection apertures25are arranged in the auxiliary emission and detection aperture positions at axial and lateral distances in the focal area7, the auxiliary detection apertures25of the auxiliary detector24are scanned with the focused first illumination light23by operating the scanner12in a subsequent step in order to register first intensity distributions of the first illumination light23detected by the auxiliary detector24over different settings of the scanner12. Then, in a next step, the photoelectric component19is activated as an auxiliary light source20, and the first detection aperture11of the first detector8is scanned with the auxiliary light22emerging out of the auxiliary emission apertures21by operating the scanner12. In doing so, second intensity distributions of the auxiliary light22detected by the first detector8are registered over the settings of the scanner12. In a subsequent comparison of the light intensity distributions, a focus position of the focus47of the focused first illumination light23of the first light source4in the focal area7in direction of the optical axis39is determined from differences between the first intensity distributions. An image position of an image of the first detection aperture11of the first detector8in the focal area7in direction of the optical axis39is determined from differences between the second intensity distributions. With respect to the differences, the respective areas of the intensity distributions are particularly relevant. From the focus position determined and the image position determined, conclusions may be drawn on the axial confocality of the microscope component group3, wherein the focus position and the image position do not need to coincide at the auxiliary device18but in the sample15. Due to wavelength dependent properties of the objective2and different wavelengths of the illumination light23and the auxiliary light22this may require that the focus position and the image position in the focal area7at the auxiliary device18take target focus and target image positions which are arranged at a distance in the direction of the optical axis39. By means of a comparison of the first and second intensity distributions, particularly in pairs belonging to a respective pair of auxiliary emission aperture21and auxiliary detection aperture25, the lateral confocality of the microscope component group3is tested. This lateral confocality is achieved, if maxima, centers or centers of intensity of the first and second light intensity distributions along the focal plane6coincide in the focal area7.

FIG.3shows an alternative embodiment of the auxiliary device18with only one auxiliary emission aperture21and one auxiliary detection aperture25coinciding therewith in the focal area7around the focal plane6. Both apertures21,25are formed by a terminal cross section46of an optical fiber40. The optical fiber40branches via a circulator41towards a photo diode42serving as the auxiliary detector24and a laser diode43serving as the auxiliary light source20. By means of a traversing unit45at which the connector73also not further specified inFIG.3is provided, the terminal cross section46is movable into different auxiliary emission aperture positions and auxiliary detection aperture positions, respectively. In each of these positions, a first light intensity distribution and a second light intensity distribution may then be registered with the aid of the scanner12according toFIG.1. The traversing unit45may especially be configured for moving the terminal cross section46into a fixed number of discrete auxiliary emission aperture positions and auxiliary detection aperture positions, respectively, for example, into three such discrete positions. The circulator according toFIG.3is only one example of a beam splitter for branching the optical fiber40towards the auxiliary detector24and the auxiliary light source20.

Instead of providing both an auxiliary detector24and an auxiliary light source20in the auxiliary device18, the auxiliary device18may as well only be designed as an auxiliary detector24with a plurality of auxiliary detection apertures25. Then, the first detector8and, if given, also the second detector38may be combined with a further auxiliary light source48, like it is depicted inFIG.4, for registering the second light intensity distributions. The first detection aperture11is formed by an optical fiber49which branches via a circulator50towards a photo diode51serving as the detector8and a laser diode52serving as the further auxiliary light source48. The further auxiliary emission aperture53of the further auxiliary light source48is identical with the first detection aperture11of the first detector8. The auxiliary detection apertures25or the movable auxiliary detection aperture25may be scanned in the different auxiliary detection aperture positions with the further light from the further auxiliary light source48emerging out of the further auxiliary emission aperture63in order to register the second intensity distributions by means of the auxiliary detector24.

Correspondingly, the auxiliary device18may, as an alternative, only comprise an auxiliary light source20with a plurality of auxiliary emission positions of its auxiliary emission aperture(s)21. Then, the first light source4and correspondingly also the second light source34can be combined with a further auxiliary detector54, like this is illustrated inFIG.5, for registering the first intensity distributions. A terminal cross section of an optical fiber55which branches via a circulator59towards a laser diode57serving as the first light source4and a photo diode58serving as the further auxiliary detector54forms both a first emission aperture59of the first light source4and a further auxiliary detection aperture60of the further auxiliary detector54. For registering the first intensity distributions by means of the further auxiliary detector54, this further auxiliary detection aperture60is scanned with auxiliary light coming out of the auxiliary emission apertures21or the auxiliary emission aperture21in the different auxiliary emission aperture positions of the auxiliary light source20. InFIGS.4and5, the optical fibers59and55, respectively, and their branching towards the photo diodes51and58, respectively, and the laser diodes52and57, respectively, are only exemplary. Further ways of implementing the detector8and the further auxiliary light source48with identical detection aperture11and further auxiliary emission aperture53or of the light source4and the further auxiliary detector54with identical emission aperture59and further auxiliary detection aperture60are available to one of ordinary skill in the art.

With regard to the direction of the irradiated or registered light, the diodes42,43,51,52,57and58in theFIGS.3to5are provided with self-designed symbols.

In the embodiment of the auxiliary device18depicted inFIG.6, different optical components are arranged on a base plate61serving as the connector73and in a base body62, which are screwed together with screws63. The auxiliary emission apertures21and auxiliary detection apertures25are formed in pairs by holes64in films65as it will be further explained in connection withFIG.7. Behind the holes64, a beam splitter66branches the beam path towards the photo diode42serving as the auxiliary detector24and the laser diode43serving as the auxiliary light source20. Here, the beam splitter66is made as a50/50beam splitter.

FIG.7shows, how the holes64forming the auxiliary emission apertures21and the auxiliary detection apertures25are provided in the three films65mounted between boundary areas67of the base body63at distances along the optical axis39. In each of the films65, exactly one such hole64is provided. The holes64are arranged at lateral distances with respect to the optical axis39. Along the optical axis39larger holes68are provided in the respective other films65which are aligned with the holes64and which have no influence on the respective auxiliary emission aperture21or auxiliary detection aperture25.

FIG.8shows an alternative design of the holes64at different auxiliary emission or detection aperture positions along and crosswise with respect to the optical axis39. Here, the holes64are the areas of smallest diameter of conical or double-conical holes69in a plate70.

In the embodiment of the auxiliary emission apertures21and the auxiliary detection apertures25by means of the holes64according toFIG.9, blind holes71are formed in the plate70until the plate70has been removed except of thin remainders72which are in different positions along the optical axis39and laterally thereto. One of the holes64has been formed in the center of each of these thin remainders72.

Many variations and modifications may be made to the preferred embodiments of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of the present invention, as defined by the following claims.