DETECTION DEVICE FOR A LASER SCANNING MICROSCOPE

Abstract

The invention relates to a detection device (2) for a laser scanning microscope, the detection device (2) having a light inlet (4), at least one filter module (14) and at least one spatially resolving detector (22) and being configured to guide light from the light inlet (4) to the filter module (14) and from there to the spatially resolving detector (22), at least one filter module (14) being designed as a continuous filter module with two continuously tunable filter elements (16), and at least one compensator element (26) being arranged optically downstream of the continuous filter module (14), by means of which a focal position of light on the spatially resolving detector (22) can be adjusted.

Claims

1. A detection device for a laser scanning microscope, the detection device having a light inlet, at least one filter module and at least one spatially resolving detector and being configured to guide light from the light inlet to the filter module and from there to the spatially resolving detector, wherein the at least one filter module is designed as a continuous filter module with two continuously tunable filter elements, and at least one compensator element is arranged optically downstream of the continuous filter module, by means of which a focal position of light on the spatially resolving detector can be adjusted.

2. The detection device according to claim 1, characterized in that the at least one filter module is part of a filter cascade with at least two filter modules.

3. The detection device according to claim 1, characterized in that the least one compensator element is configured to move the spatially resolving detector.

4. The detection device according to claim 1, characterized in that the at least one compensator element is arranged between the continuous filter module and the spatially resolving detector.

5. The detection device according to claim 4, characterized in that at least one compensator element is designed as a movable mirror, which is preferably mounted such that it can be tilted about at least two tilt axes.

6. The detection device according to claim 1, characterized in that the continuously tunable filter elements are color gradient filters arranged such that they can be displaced in a longitudinal direction.

7. The detection device according to claim 1, characterized in that the detection device comprises multiple detectors, preferably as many detectors as filter modules, wherein multiple detectors are designed as spatially resolved detectors.

8. The detection device according to claim 7, characterized in that all detectors are designed as spatially resolving detectors.

9. The detection device according to claim 7, characterized in that a compensator element (26) is arranged upstream of every spatially resolving element by means of which a focal position of light on the spatially resolving detector can be adjusted.

10. The detection device according to claim 1, characterized in that the detection device has at least one switching element by which the light can be guided from at least one filter module to different detectors and/or by which the light can be guided from multiple filter modules onto a detector, preferably a spatially resolving detector.

11. The detection device according to claim 10, characterized in that at least one switching element is a compensator element.

12. The detection device according to claim 1, characterized in that, except for the last filter module, each filter module has a detector outlet through which light is directed onto a detector and a filter outlet through which light is directed onto another filter module.

13. The detection device according to claim 1, characterized in that the detection device comprises an optical device, preferably a telescope, which is configured in such a way that a waist of an incident Gaussian bundle lies between two adjacent filter modules or within a filter module.

14. The detection device according to claim 1, characterized in that the detection device has an electrical or electronic control unit configured to control the compensator element such that a maximum of the radiation detected by the spatially resolving detector is located at the center on the spatially resolving detector.

15. A laser scanning microscope with a detection device according to claim 1.

16. The laser scanning microscope according to claim 15, characterized in that it comprises a device for checking confocality which includes an auxiliary device with an auxiliary light source and an auxiliary detector aperture arranged together in a focal plane on a common optical axis.

17. The laser scanning microscope according to claim 16, characterized in that the device for checking confocality comprises multiple auxiliary devices, the auxiliary light sources of which emit auxiliary light of different wavelengths.

Description

[0046] In the following, a number of embodiment examples of the invention will be explained in more detail with the aid of the accompanying drawings. They show

[0047] FIG. 1—a schematic representation of a detection device according to a first embodiment example of the present invention,

[0048] FIG. 2—a section of a detection device according to a further embodiment example of the present invention,

[0049] FIG. 3—the section from FIG. 2 in a different setting,

[0050] FIG. 4—a section of a detection device according to a further embodiment example of the present invention and

[0051] FIGS. 5 and 6—a schematic representation of measured intensities.

[0052] FIG. 1 schematically depicts a detection device 2 according to a first embodiment example of the present invention. It has a light inlet 4 through which light previously passed through other optical devices 6, of which only one optical device 6 is shown by way of example, enters the detection device 2. A telescope 8 is schematically depicted. Due to the telescope, the waist of the entering Gaussian bundle shifts to the right between the first two filter modules 14 in the embodiment example shown. The detection device 2 has a filter cascade 12, which in the embodiment example shown has three filter modules 14. Each of the filter modules 14 has two filter elements 16 which are continuously tunable and can be designed, for example, as color gradient filters. The first two filter modules 14 shown each have a detector outlet 18 and a filter outlet 20. The portion of the light beam 10 that passes through the two filter elements 16 in the first filter module 14 is guided from the detector outlet 18 to a spatially resolving detector 22. The portion of the light beam 10 which is reflected at the first of the filter elements 16 in the first filter module 14 is guided out of the filter outlet 20 to the next filter module 14. The last filter module 14, which is shown on the far right in FIG. 1, has two detector outlets 18, since the light emerging in each case is not fed to a further filter module 14, but to a detector designed as a spatially resolving detector 22 and a beam trap 25. Compensator elements 26, not shown in FIG. 1, are provided optically downstream of the filter elements 16 of a filter module 14, wherein said compensator elements can be assigned to the respective detectors 22, 24 and used to change a focal position of the light on a detector 22, 24. For example, a compensator element 26 can be used to displace an imaging lens 32 perpendicular to the beam direction, with the displacement, shown in the figure by shift arrows 27′, adjusting the focus position in the Z direction. Other compensator elements cause displacement, shown in the figure by displacement arrows 27, in the X direction and in the Y direction. For the sake of clarity, only two of the three filter modules 14 are assigned displacement arrows 27, 27′ in FIG. 1. Corresponding compensator elements 26, not shown, are also assigned to the third filter module. In this way, sufficient compensation can be achieved.

[0053] FIG. 2 shows a schematic section of a detection device 2 and depicts three filter modules 14, each with two filter elements 16. Through the detector outlets 18 pointing downwards in FIG. 2, a portion of the light leaves the respective filter module 14 and is guided onto a mirror 28. This mirror 28 reflects the portion of the light onto the compensator element 26, which in the embodiment shown is designed as a movable mirror. It is also a switching element 36.

[0054] In the setting depicted, the switching element 36 guides the light that has left the first filter module 14 through its detector outlet 18 to the spatially resolving detector 22, and guides the light that has left the detector outlets 18 of the second and third filter modules 14 to non-spatially resolving collection detectors 24.

[0055] In one setting, shown in FIG. 3, the switching element 36 guides light that has left the second filter module 14 through its detector outlet 18 onto the spatially resolving detector 22 and guides light that has left the first filter module 14 through its detector outlet 18 into a beam trap 25, while guiding light that has left the third filter module 14 through its detector outlet 18 onto the collection detector 24 adjacent to the spatially resolving detector 22.

[0056] In a third setting, not shown, it guides light that has left the third filter module 14 through its detector outlet 18 onto the spatially resolving detector 22, while guiding remaining light into a beam trap 25, respectively. In this case, the switching element 36 also serves as a compensator element 26. This means that the fixed setting of the switching element 36 in the event of a change in the selected detection wavelength band, i.e. in the event of a change in the setting of the filter module 14 respectively assigned to the spatially resolving detector 24, is such that an error in the focal position of the light on the spatially resolving detector 22 is compensated. An adjustment with respect to the positions of the light on the collecting detectors is not possible, but also not necessary, since their detection apertures are chosen to be sufficiently large. Thus, in the setting shown in the embodiment, the compensator element 26 is arranged in such a way that the light emerging from the detector outlet 18 of the second and third filter modules is guided to the two collection detectors 24, while the light emerging from the detector outlet 18 of the first filter module 14 is guided to the spatially resolving detector 22. The compensator element 26, which is in the form of a movable mirror, also serves as a switching element 36, each of which is used to guide light emerging from a detector outlet of one of the filter modules 14 to the spatially resolving detector 22. In an alternative embodiment, the mirrors 28 may also each be part of a compensation device comprising two compensator elements 26 designed as two mirrors. The respective mirror 28 may then be used, for example, to achieve compensation in a first direction. The second mirror, i.e. preferably the switching element 36, is used in this case to achieve compensation in a second direction, which is preferably perpendicular to the first direction.

[0057] FIG. 3 shows the set-up known from FIG. 2 with the filter modules 14 and their detector outlets 18. The outgoing light is guided to the mirrors 28, from which it is guided to the compensator element 26. This is shown in FIG. 3 in a different position than in FIG. 2. The light from the first filter module is directed into a beam trap 25 and is not available for evaluation. In the beam trap 25, the light is absorbed and thus cannot fall as scattered light on one of the detectors 22, 24. The light from the center filter module 14 is guided to the spatially resolving detector 22 and the light from the right filter module 14 is guided to the collection detector 24. Since the position of incidence of the light on the respective detector 22, 24 is relevant only to the spatially resolving detector 22, a single compensator element 26 is sufficient in the embodiment shown, which has only a single spatially resolving detector 22.

[0058] FIG. 4 shows a different embodiment. This also has three filter modules 14, each with two filter elements 16, each of which is designed to be continuously tunable. These filter modules 14 also each have a detector outlet 18 through which light leaves the respective filter module 14 in a downward direction towards a respective spatially resolving detector 22 in FIG. 4. The light leaving through a detector outlet 18 strikes a compensator element 26 in each case and is guided from there through an imaging lens 32 in each case onto the respective spatially resolving detector 22. A switching element 36 is not necessary. FIGS. 5 and 6 show exemplary measured values. Each field 34 corresponds to a pixel of a spatially resolving detector 22. When rastering over a sample, a plurality of sub-images are recorded by the spatially resolving detector 22, each pixel, i.e. each field 34, detecting the incident photons, i.e., in particular, the incident light intensity. The values shown in FIGS. 5 and 6, which are assigned to the the individual fields 34, correspond to the summed intensities over a plurality of captured partial images. Thus, they are measures of intensities integrated over the plurality of images detected by the respective fields 34. The values have been normalized so that the maximum value is 1.00. In an optimally adjusted device, if a large number of photons are detected and a sufficient area of the sample is scanned, a rotationally symmetrical distribution is obtained on average. Such a distribution is not present in FIG. 5, where the maximum value is shifted to the lower left. In FIG. 6, the maximum value is in the middle field 34, but the distribution of values around it is approximately rotationally symmetrical. Deviations from rotational symmetry can be caused by photon noise, by an insufficiently scanned area of an inhomogeneous sample, and by incomplete compensation. In principle, however, the compensation devices and their compensator elements 26 can be adjusted on the basis of such summed intensities so that errors of the focal positions are compensated. The determination of the compensation can be facilitated if during scanning the sample is not only exposed to excitation light, but also to STED light, which in each case suppresses the emission of fluorescence in outer areas of the excitation focus. The effect of this is that fluorescence is emitted only from the near-axis region of the excitation focus, so that each individual detected intensity distribution is rotationally symmetrical to a better approximation. Inhomogeneities in the sample have less effect. In this way, the system, in particular the microscope, can be adjusted and set without the need for a reference sample or a special device to set the confocality.

REFERENCE LIST

[0059] 2 detection device

[0060] 4 light inlet

[0061] 6 optical device

[0062] 8 telescope

[0063] 10 light beam

[0064] 12 filter cascade

[0065] 14 filter module

[0066] 16 filter element

[0067] 18 detector outlet

[0068] 20 filter outlet

[0069] 22 spatially resolving detector

[0070] 24 collection detector

[0071] 25 beam trap

[0072] 26 compensator element

[0073] 27, 27′ displacement arrow

[0074] 28 mirror

[0075] 30 absorption element

[0076] 32 imaging lens

[0077] 34 field

[0078] 36 switching element