DE102013021182B4 - Device and method for scanning microscopy - Google Patents

Abstract

Device for scanning microscopy, in particular for carrying out the method according to one of claims 15 to 20, with at least one light source (10) for emitting excitation light (12), with optical means (30, 40, 50) for guiding the excitation light (12) in an excitation beam path (A) onto a sample (70) and for guiding detected light (80) emitted by the sample (70) in a detection beam path (D) onto a spectrally resolving detector unit (90), the optical means having at least one scanner (40) for scanning the sample (70) point by point with excitation light (12) and a microscope objective (50) for focusing the excitation light (12) onto or into the sample (70), and with the spectrally resolving detector unit (90) for detecting the detected light ( 80), in each case separately for each sample location (74) illuminated with excitation light (12), characterized in that a separating unit (20) is present for separating the excitation light (12) into a plurality of excitation beam bundles (14 - 17), the switching means ( 200) are available for switching between a single-spot mode, in which the sample (70) is scanned with a single point of the excitation light (12), and a multi-spot mode, in which the sample (70) with several points of the excitation light (12) is scanned, and that a control device (120) is present which interacts at least with the switching means (200), the separating unit (20) and the detector unit (90), the control device (120) being set up to insert the separating unit (20) in the excitation beam path (A) for the multi-spot mode, for removing the separating unit (20) from the excitation beam path (A) for the single-spot mode, and for changing at least parameters of the light source and/or the detection unit (90) when switching between the single-spot mode and the multi-spot mode in such a way that images obtained from one and the same sample area (76) in the single-spot mode on the one hand and in the multi-spot mode on the other hand are the same have image brightness.

Description

In a first aspect, the invention relates to a device for scanning microscopy according to the preamble of claim 1. In a second aspect, the invention relates to a method for scanning microscopy according to the preamble of claim 15.

A generic device is, for example, in DE 10 2009 006 729 A1 and has the following components: at least one light source for emitting excitation light, optics for guiding the excitation light in an excitation beam path onto a sample and for guiding detection light emitted by the sample in a detection beam path onto a spectrally resolving detector unit. The optics have at least one scanner for scanning the sample point by point with excitation light and a microscope objective for focusing the excitation light on or into the sample. In addition, the spectrally resolving detector unit for detecting the detected light is part of the generic device, with the detected light being detected separately for each sample location illuminated with excitation light.

In a generic method, excitation light is focused onto or into a sample using a microscope objective, and the sample is scanned point by point with the excitation light. In addition, the detected light, which is emitted from illuminated sample locations as a reaction to the excitation light, is measured separately and spectrally resolved for each illuminated sample location.

In addition to scanning microscopes in which the sample is scanned with a single focus or illumination point, such scanning microscopes are also known in which a plurality of illumination points, which are also referred to as spots, are guided over a sample. In principle, these microscopy methods, which are also referred to as multi-spot methods, enable shorter acquisition times.

DE 10 2012 203 736 A1 discloses a device for scanning microscopy with an illumination module, which is used in a single-spot mode, in which the sample is scanned with a single point of the excitation light, and a multi-spot mode, in which the sample is scanned with several points of the excitation light is scanned, can be operated.

DE 10 2010 047 353 A1 relates to another laser scanning microscope that can be operated in a single-spot mode and a multi-spot mode.

One object of the invention can be seen as increasing the functionality of a scanning microscope.

This object is achieved by the device having the features of claim 1 and by the method having the features of claim 15.

The device of the above-mentioned type is further developed according to the invention in that a separating unit is present for separating the excitation light into a plurality of excitation beam bundles, that switching means are present for switching between a single-spot mode in which the sample is exposed to a single point of the excitation light is scanned, and a multi-spot mode in which the sample is scanned with multiple points of the excitation light, and that a control device is present which interacts at least with the switching means, the separating unit and the detector unit, the control device being set up for insertion the separating unit in the excitation beam path for the multi-spot mode, for removing the separating unit from the excitation beam path for the single-spot mode, and for changing at least parameters of the light source and/or the detection unit when switching between the single-spot mode and the multi-spot mode in such a way that images obtained from one and the same sample area in the single-spot mode on the one hand and in the multi-spot mode on the other hand have the same image brightness.

The method of the type mentioned above is further developed according to the invention in that between a single-spot mode, in which the sample is scanned with a single point of the excitation light, and a multi-spot mode, in which the sample is scanned with several points of the excitation light is scanned, is switched back and forth, with a separating unit for separating the excitation light into a plurality of excitation beam bundles being inserted into an excitation beam path when switching to the multi-spot mode, and the separating unit being inserted from the excitation beam path when switching over to the single-spot mode is removed and wherein when switching between the single-spot mode and the multi-spot mode, at least parameters of the light source and/or the detection unit are changed in such a way that in the single-spot mode on the one hand and in the multi-spot mode on the other hand images obtained from one and the same sample area have the same image brightness.

A key idea of the present invention can be seen as the ability to switch between a single-spot mode, which is also known as a single-spot mode, in a scanning microscope. Operation can be referred to, and to realize a multi-spot mode, which can also be referred to as multi-spot operation. In concrete terms, this means that the optical components required for multi-spot operation are inserted into the beam path of the microscope when multi-spot operation is desired, and removed from the beam path accordingly when the microscope is in single-spot mode is to be operated. For this purpose, suitable switching means are present according to the invention, which in principle relate to or contain all components that are changed and/or moved when switching between the single-spot mode and the multi-spot mode. Another essential aim of the present invention is to increase the comparability of images that were recorded in single-spot mode on the one hand and in multi-spot mode on the other. For this purpose, the invention provides that a control device is present, which works together at least with the switching means and changes at least parameters of the light source and/or the detection unit when switching between the single-spot mode and the multi-spot mode such that the image brightness of in single-spot operation on the one hand and in multi-spot operation on the other hand is the same for images obtained from one and the same sample area. This enables the images obtained from said sample area by single-spot microscopy on the one hand and multi-spot microscopy on the other hand to be better compared.

A particular advantage of the present invention is that the handling of the microscope is significantly increased due to the very good comparability of images recorded in single-spot mode on the one hand and in multi-spot mode on the other.

For the purposes of the present invention, the term image brightness means the primary signal obtained from a coarse point. This means, for example, that when switching from single-spot mode to multi-spot mode with n light spots (n is a natural number), the intensity of the light source must be increased by a factor of n or, if the intensity of the light source remains the same, the Sensitivity of the detector must be increased by this factor n. In principle, both the light intensity and the detector sensitivity can be increased in order to achieve the same image brightness as a result. In principle, these considerations apply to all color channels, provided that the respective spectral ranges that are imaged on a single detector element remain the same size. In other words, the requirement of the independent claims means that the intensity detected per illumination spot and unit of the wavelength range should be the same in single-spot mode on the one hand and in multi-spot mode on the other.

In principle, in terms of the device, the control device can therefore preferably also interact with the light source to control its intensity.

Preferred exemplary embodiments of the device according to the invention and advantageous variants of the method according to the invention are described below, in particular in connection with the dependent claims and the figures.

In particularly preferred variants of the invention, there is a main beam splitter for separating illumination light and, in particular wavelength-shifted, detection light. In principle, however, arrangements without a main beam splitter are also possible.

Excitation light within the meaning of the invention described here is electromagnetic radiation, meaning in particular the infrared, visible and ultraviolet parts of the spectrum. In principle, all sources that provide the desired electromagnetic radiation with sufficient intensity can be used as light sources. Lasers are usually used for this. In principle, however, light-emitting diodes or other lighting means can also be used.

In principle, all detectors can be used as detectors that detect the light reflected back from the sample sufficiently effectively and with a sufficiently good signal-to-noise ratio. In principle, semiconductor detectors can also be used for this purpose. Because the main application of the present microscopy techniques is fluorescence microscopy, where the count rates are usually comparatively small, photomultipliers are usually used.

Confocal diaphragms are particularly expediently arranged in the detection beam path, so that the device according to the invention is a confocal microscope in multi-spot mode and/or in single-spot mode with all the advantages and properties known in principle.

An aperture is called confocal when it is positioned in or near a confocal plane. A confocal plane denotes a plane of the detection beam path that is optically conjugate to a specimen-side focal plane of the microscope objective. A confocal diaphragm in front of a detector limits the light pickup of that detector to a small target volume at the sample location.

In order to achieve spectral resolution, the detector unit can preferably have at least one unit for spectrally separating the detected light. As actually dispersive elements, the dispersive units can have refractive and/or diffractive optical components.

In principle, the unit or units for spectral splitting of the detected light can have one or more filters for spectral splitting. However, variants are particularly preferred in which the unit or units for the spectral splitting of the detected light has or have a dispersive function. In principle, known dispersive elements, for example diffractive or refractive components such as prisms or gratings, can be used for this purpose.

In principle, one and the same, in particular dispersive, unit can be used both for the single-spot mode and for the multi-spot mode. However, preferred embodiments of the device according to the invention are those in which the detector unit has a first, in particular dispersive, unit for spectral splitting of the detected light in single-spot mode and a second, in particular dispersive, unit for spectral splitting of the detected light in multi-spot mode . A precisely working optical structure can thus be achieved more easily. Means can then expediently be present for selectively inserting and removing the first and/or the second dispersive unit in the detection beam path.

In principle, it is possible to use different detectors for multi-spot operation and single-spot operation. However, the same detector or detectors is/are particularly preferably used for the multi-spot operation and the single-spot operation.

In principle, the different wavelengths of the light emitted by the sample can be measured in chronological succession using one and the same detector. However, the detector unit particularly preferably has a plurality of detectors, which can also be referred to as individual detectors. With this plurality of individual detectors, light of several wavelengths can then be measured or detected simultaneously. The spectral resolution of the detector unit then depends on the size of the spectral section or wavelength range that is directed to the respective individual detector by optical means, in particular a dispersive unit or a plurality of dispersive units. With a higher number of individual detectors, a higher resolution is naturally possible than with a lower number of individual detectors.

In principle, the spectral sections or wavelength ranges that are directed to the individual detectors can be of different sizes.

For example, in a further advantageous embodiment of the device according to the invention, there can be at least one manipulation optics for changing the spatial spectral distribution of the detection light. Specific spectral ranges can then be measured with better resolution than other spectral ranges.

The multi-spot mode is generally used when an image of as large a sample area as possible is to be acquired as quickly as possible. A device according to the invention can therefore expediently be constructed and set up in such a way that the manipulation optics can only be introduced into the detection beam path in single-spot mode.

In principle, in the multi-spot mode, the spectrum of the light emitted from different illuminated sample locations can be measured sequentially with the detector unit. However, the main advantage of the multi-spot mode is achieved when the spectra of the light emitted from different illuminated sample locations are measured simultaneously with the detector unit. If a detector unit with multiple individual detectors is used, this means that due to the limited number of individual detectors, the maximum resolution achievable in multi-spot mode is lower than the maximum resolution possible in single-spot mode.

In principle, the aim of the invention described here is that the images obtained in the single-spot mode on the one hand and in the multi-spot mode on the other hand differ as little as possible.

For example, in an advantageous variant of the method according to the invention, the spectral resolution of the microscopic images is the same in the single-spot mode and in the multi-spot mode. This can be achieved when using a detector unit with a number of individual detectors if the maximum possible resolution is not used in single-spot mode, for example by combining n (n is an integer) of the individual detectors. After switching to a multi-spot mode with n illumination spots, the individual detectors can then be read out individually again, thus achieving the same spectral resolution.

In principle, the switching device or devices include all components that become active or are actuated when the scanning microscope is switched from single-spot mode to multi-spot mode. The switching means can have physical or hardware components as well as control or software components.

Particularly preferably, at least one movable mirror, for example a rotatably mounted mirror, in particular a concave mirror, can be present as part of the switching means.

In principle, the invention can be implemented with a light source, for example a laser, which essentially only emits light of a single wavelength. However, in the device according to the invention, it is particularly preferred to have a plurality of light sources for emitting excitation light with different wavelengths. This enables the examination of samples prepared with several different dyes.

At least one confocal diaphragm is present in the device according to the invention for carrying out confocal microscopy methods. For the multi-spot mode, there can be an aperture with a number of separate confocal apertures corresponding to the number of spots.

In a simple variant of the method according to the invention, parameters of the detector unit are changed when switching from single-spot mode to multi-spot mode and vice versa. For example, when photomultipliers are used as single detectors, the high voltage of the photomultipliers can be changed or adjusted.

The image parameters, which according to the invention should not change when switching from single-spot mode to multi-spot mode and vice versa, can be, for example, the image parameters brightness, contrast and/or spectral distribution of the light on individual detectors of the detection unit act.

The preservation of image parameters when switching from single-spot mode to multi-spot mode and vice versa can be accomplished more easily if, when switching between single-spot mode and multi-spot mode, parameters of the scanner and/or of the light source can be changed.

For example, if the same light source is used for the single-spot mode and the multi-spot mode, the intensity of the excitation light when switching from the single-spot mode to the multi-spot mode is increased by a factor that is in the Essentially corresponds to the number of light spots in multi-spot mode. This factor is preferably greater than the number of light spots in multi-spot mode minus one and less than the number of light spots in multi-spot mode plus one.

A spectrally resolved microscopic image of a sample area can advantageously be assembled from the data measured separately for each sample location.

• 1: eine schematische Darstellung einer erfindungsgemäßen Vorrichtung;
• 2: ein Detail einer erfindungsgemäßen Vorrichtung;
• 3: ein weiteres Detail einer erfindungsgemäßen Vorrichtung;
• 4: eine schematische Darstellung zur Erläuterung des Multi-Spot-Betriebs;
• 5: eine schematische Darstellung einer Reihe von 32 Einzeldetektoren;
• 6: eine schematische Darstellung einer Aufteilung der 32 Einzeldetektoren für den Multi-Spot-Modus und
• 7: ein Flussdiagramm zur Erläuterung des erfindungsgemäßen Verfahrens.

Further advantages and features of the device according to the invention and the method according to the invention are explained below with reference to the figures. It shows:

• 1 : a schematic representation of a device according to the invention;
• 2 : a detail of a device according to the invention;
• 3 : another detail of a device according to the invention;
• 4 : a schematic representation to explain the multi-spot operation;
• 5 : a schematic representation of a row of 32 individual detectors;
• 6 : a schematic representation of a division of the 32 individual detectors for the multi-spot mode and
• 7 : a flowchart to explain the method according to the invention.

An embodiment of a device 100 according to the invention is described with reference to FIG 1 explained. The essential components of this device 100 are a first light source 10 for emitting excitation light 12 and optics 30, 40, 50 for guiding the excitation light 12 in an excitation beam path A onto a sample 70 and for guiding detected light 80 emitted by the sample 70 in a detection beam path D on a spectrally resolving detector unit 90.

The optics include at least one main beam splitter 30 for separating the excitation light 12 and the detection light 80, a scanner 40 for scanning the sample 70 point by point with the excitation light 12, and a microscope objective 50 for focusing the excitation light 12 on or in the sample. The spectrally resolving detector unit 90 is used to detect the detection light 80, this being carried out separately for each sample location.

There is also a separating unit 20 for separating the excitation light 12 into a plurality of excitation beam bundles, which are 1 are not shown in detail. Then, in the device according to the invention, 100 in 1 Switching means 200 present for switching between a single-spot mode, in which the sample 70 is scanned with a single point of the excitation light 12, and a multi-spot mode, in which the sample is scanned with a number of points of the excitation light.

In addition, the device 100 according to the invention comprises a control device 120 which interacts at least with the switching means 200, the separating unit 20 and the detector unit 90. According to the invention, the control device 120 is set up to insert the separating unit 20 into the excitation beam path A to carry out the multi-spot mode and to remove the separating unit 20 from the excitation beam path A for the single-spot mode. For this purpose, the control device 120 is connected via a schematically indicated connection 26 to a movable mirror 22, with which the insertion and removal of the separating unit is accomplished.

in the in 1 In the situation shown schematically, the excitation light 12 is directed in the multi-spot mode via the splitting unit 20 and the movable mirror 22 onto the main color splitter 30 . In the single-spot mode, the separating unit 20 is not in the beam path and the excitation light 12 instead takes an optical path 28 (shown schematically), in which optical components such as lenses or mirrors can also be located, to the movable mirror 22 and is in turn transported by it directed to the main color splitter 30.

To switch the light path between the first light source 10 and the separating unit 20 on the one hand and the optical path 28 on the other hand, there is a switching device 18 which can be a movable mirror, for example.

At the in 1 A second light source 11 for emitting excitation light 13 is also present. Expediently, the excitation light 13 has a different wavelength than the excitation light 12. For example, the excitation light 13 can be non-visible radiation, such as infrared or ultraviolet radiation. Accordingly, the excitation light 12 can be light from the entire visible spectrum from red to blue. As with the first light source 10, the second light source 11 also has a separating unit 21 for separating the excitation light 13 into a plurality of excitation beam bundles and a movable mirror 23, with which the separating unit 21 for the multi-spot mode is inserted into the excitation beam path can and can be removed from the excitation beam path A for the single-spot mode. The movable mirror 23 is connected to the control unit 120 via a schematically indicated connection 27 and can be controlled via the control unit 120 . In the single-spot mode, the excitation light 13 takes a schematically indicated optical path 29.

To switch the light path between the second light source 11 and the separating unit 21 on the one hand and the optical path 29 on the other hand, there is a switching device 19 which can be a movable mirror, for example. The switching devices 18 and 19 can each be controlled via the control unit 120 . this is in 1 represented by the connecting lines between switching devices 18 and 19 and the control unit 120.

The excitation light 13 then reaches a second main color splitter 31 and from there, like the excitation light 12, via the scanner 40, a scanning optics 45 and the microscope lens 50 to the sample 70 to be examined. During operation of the device 100 according to the invention, the user is typically either first light source 10 or the second light source 11 work. For this purpose, the first main color splitter 30 can be inserted into the excitation beam path A with the control unit 21 via a connection 32 if the first light source 10 is to be used and correspondingly removed from the excitation beam path A if the light source 10 is not to be used. The same applies to the second light source 11 and the main color splitter 31, with which excitation light 13 of the second light source 11 can be coupled into the excitation beam path A. The main color splitter 31 can be controlled with the control unit 120 via a line 33 in such a way that the excitation light 13 is either coupled into the excitation beam path A or not.

The scanner 40 can be controlled by the control unit 120 via a connection 34 . The scanner 40 is preferably controlled differently in the multi-spot mode than in the single-spot mode.

The basic situation in single spot mode and multi spot mode is explained with reference to 4 explained. A sample region 76 of the sample 70 to be examined is shown schematically there. Generally, in all figures same or equal Acting components usually marked with the same reference numerals.

In 4 four excitation beam bundles 14, 15, 16, 17 are shown schematically, which illuminate sample locations 71, 72, 73, 74 of the sample 70. In principle, the excitation beam bundles 14 , 15 , 16 , 17 can contain excitation light 12 from the first light source 10 and/or excitation light 13 from the second light source 11 . The sample locations 71, 72, 73, 74 illuminated with the excitation beam bundles 14, 15, 16, 17 emit light 81, 82, 83, 84 as a response or reaction to this irradiation. This can in particular be fluorescent light, which generally has a longer wavelength than the excitation light and which is also radiated non-directionally. At the in 4 The situation shown is the multi-spot situation, because the sample 70 is illuminated simultaneously with a plurality, namely a total of four, excitation beam bundles 14, 15, 16, 17.

In a single-spot situation, only a single sample location would be irradiated with excitation light. Typically, the excitation beam bundles 14, 15, 16, 17 are guided in parallel over the sample 70 in different scan lines. in the in 4 the situation shown is the y-direction.

Back to the consideration of 1 . The light 80 emitted by the sample 70 to be examined is first returned via the same optical components, ie the microscope objective 50, the scanning optics 45, the scanner 40, now on the detection beam path D. In contrast to the excitation light 12 and/or the excitation light 13, the light 80 emitted by the sample 70 is at least partially transmitted by the main color splitters 31 and 30 and is first guided via a pinhole optics 85 to a pinhole diaphragm 86. This pinhole diaphragm has, indicated schematically, four pinholes for four excitation beam bundles. The pinhole aperture 86 can also do what is in 1 is not shown, can be actuated with the control unit 120, for example inserted into the detection beam path D if work is to be carried out in multi-spot mode. For the single-spot mode, the diaphragm 86 with the four confocal diaphragms can then be removed from the beam path or only shifted so that one beam of rays from the pinhole optics 85 actually strikes a pinhole. Via further optics 87, which can be referred to as detection optics, the light 80 emitted by the sample 70 reaches either, namely in single-spot mode via a movable mirror 88, a first dispersive unit 96, which is part of the spectrally resolving detector unit 90 is. The movable mirror 88 can be removed from or inserted into the detection beam path D by the control unit 120, which in this respect is part of the switching means 200, via the connection 89, or suitably redirect the beam path. It is essential that the light 80 emitted by the sample 70 in the multi-spot mode does not reach the first dispersive unit 96 but rather the second dispersive unit 97 .

The light 80 emitted by the sample is spectrally divided in the first dispersive unit 96 and the second dispersive unit 97 . In principle, known components such as prisms or gratings can be present for this purpose. In the case of single-spot operation, the spectrally divided light is guided via a schematically indicated beam path 59 and a concave mirror 24 to a manipulation unit 98, which can optionally be present in the beam path.

Finally, the light 80 emitted by the sample is directed to a detector line 95 with a large number of individual detectors. The spatial distribution of the individual spectral components on the detector line can be changed or manipulated with the manipulation unit 98 . For example, parts of the spectrum can be imaged particularly densely on the detector line, so that the spectral resolution is lower in this area. On the other hand, spectral ranges that are of particular interest can be divided among a particularly large number of individual detectors in the detector row 95 . The spectral resolution is then greater compared to the situation described first.

The manipulation unit 98 can be removed from the detection beam path D or inserted into it with the control unit 120 via the line 57 .

To switch between the multi-spot mode and the single-spot mode, part of the switching means 200 is a movable concave mirror 24 with a control 25 that can be actuated via the control unit 120 via the connection 55 . In the single-spot mode, the movable mirror 88 is positioned such that the light 80 emitted by the sample 70 is directed to the first dispersive unit 96 and from there on the beam path 59 via the concave mirror 24 to the detector line 95. Optionally, the manipulation unit 98 can also be run through here or not.

In the multi-spot mode, the mirror 88 is positioned by the control unit 120 via the connection 89 in such a way that the light 80 emitted by the sample 70 does not reach the first dispersive unit 96 but rather the second dispersive unit 97 . The light 80 emitted by the second dispersive unit 97 is then transmitted to the detector line 95 via a beam path 67 shown schematically directed. For this purpose, on the one hand, the movable concave mirror 24 is suitably positioned and, on the other hand, the manipulation unit 98 is removed from the beam path via the control unit 120 and the connection 57 .

The launching of either a plurality of excitation beams or a single excitation beam is discussed with reference to FIG 2 explained in more detail. First of all, two connections 65, 66 for light sources are shown there, to which, in principle, different light sources are connected. In principle, however, it is also possible for only a single light source to be used, the excitation light 12 of which is expediently supplied to the first connection 65 and/or the second connection 66 with a switching or distribution device (not shown). A separating unit 20 is arranged downstream of the first connection 65 and is used to separate the excitation light 12 into a plurality of excitation beam bundles 14 , 15 , 16 , 17 . The excitation beam bundles 14, 15, 16, 17 are directed onto a movable mirror 22 and from this reach the main color splitter 30 via a diaphragm 64 and a telescope formed by lenses 61 and 62, as well as a mirror 63. This passes through the lenses 61 and 62 The telescope formed can in particular be a zoom telescope and can be used to correct inaccuracies in the detector optics. For the multi-spot mode, the movable mirror 22 can be positioned in such a way that the excitation beams 14, 15, 16, 17 are directed onto the diaphragm 64. On the other hand, for the single-spot mode, the movable mirror 22 is either removed from the beam path or in any case positioned in such a way that a single excitation beam bundle of the excitation light 12 is guided to and through the diaphragm 64 .

The spectral separation of the light emitted by the sample 70 80 is based on 3 explained in more detail. A pinhole diaphragm 86 with a total of four confocal pinholes 41, 42, 43, 44 is shown there. The light 81, 82, 83, 84 emitted by the sample locations 71, 72, 73, 74 (see 4 ) is passed through the pinholes 41, 42, 43, 44 onto a lens 87, which can be referred to as detection optics. The light 80 emitted by the detection optics 87 passes via the mirror 88 to the dispersive unit 97, which is shown schematically as a prism block. The dispersive unit 97 separates the total of four beams of rays into a total of four spectra 181, 182, 183, 184, which are imaged onto a first area 91, a second area 92, a third area 93 and a fourth area 94 of the detector line 95. This is with reference to the 5 and 6 explained further.

In 5 a detector row 95 is shown schematically, which has a total of 32 individual detectors 301...332. In the single-spot mode, the spectrum of the light emitted by the only sample location illuminated with excitation light is directed to these 32 individual detectors 301...332. As explained above in connection with the manipulation unit 98 , the spectral resolution can vary across the detector row 95 . For example, it would be possible to distribute the entire wavelength range between 400 nm and 580 nm to the individual detectors 301 to 310 and also to route the wavelength range from 580 nm to 600 nm to the individual detectors 311 to 332. The individual detectors 301 to 310 then each account for 28 nm of the wavelength spectrum, while the individual detectors 311 to 332 each account for only 0.9 nm. This means that in the area of the individual detectors 301 to 310 the resolution is about 31 times smaller than in the spectral area measured by the individual detectors 311 to 332.

6 shows the same detector line 95 for multi-spot operation. As related to 3 addressed, there the detector line 95 is divided into a total of four areas 91, 92, 93, 94, each comprising eight individual detectors. How out 3 As can be seen, the spectra 181, 182, 183, 184 generated by the dispersive unit 97 are directed or imaged onto the regions 91, 92, 93 and 94, respectively. Suitable optical components such as mirrors and/or lenses can be present for this purpose 3 are not shown. The first area 91 of the detector row 95 includes the individual detectors 301 to 308. The second area 92 includes the individual detectors 309 to 316. The third area 93 includes the individual detectors 317 to 324 and the fourth area includes the individual detectors 325 to 332.

The inventive method is supplemented with reference to the 7 explained. Accordingly, an image is first recorded in the single-spot mode (box a). Various image parameters, such as the image brightness, the contrast and/or the spectral distribution on the individual detectors, can then be determined or ascertained with the aid of software (box b). Irrespective of this, ie at the same time, before or after, a switchover to the multispot mode can be carried out using the switchover means present according to the invention (box c). After this switchover, changes to components of the device according to the invention can be made automatically, for example with the controller 120 . For example, the following parameters can be changed: power of the light source, in particular the laser power, sensitivity of the detector or the detector gain, spectral distribution of the light emitted by the sample on the detectors (box d). Once the adjustments of the various components of the invention After the device has been carried out, image recordings can be carried out in multi-spot mode (box e).

With the device according to the invention, which can also be referred to as a microscope or scanning microscope, a sample can be illuminated both with a single laser spot (single spot) and alternatively with, for example, four spatially offset laser spots (multi-spot mode). A user can therefore use this microscope to view one and the same sample or sample location once in multi-spot mode and immediately afterwards in single-spot mode. The reverse order is of course also possible.

If the same laser is used to generate the majority of the excitation spots for the multi-spot mode as is used to generate the single spot, then the intensity of the single spot in the multi-spot mode is increased by a factor that corresponds to the number of spots in the multi-spot mode. Spot mode corresponds, smaller than that of single spot in single spot mode. In order to obtain comparable images, however, it is advantageous if the intensity of the individual spots in multi-spot mode is the same as the intensity of the individual spots in single-spot mode.

Furthermore, the same detection unit in the same configuration is not used for detecting the detection light emitted from the sample in the multi-spot illumination. In other words, the same detection unit can be used but in a different configuration. In this context, the concept of the configuration of the detector refers to the manner in which the detection light to be detected is guided from the sample locations illuminated with the excitation light to the spectrally resolving detector unit. If the spectrally resolving detector unit has a plurality of individual detectors, the configuration of the detector unit determines how the spectral power density is distributed to the individual detectors. The signal from the individual detectors will change accordingly and these may even be over- or underdriven. This means that the spectrally resolved representation of a sample can change when switching between single-spot mode and multi-spot mode.

However, the aim of the invention is that the graphical representation of the sample, for example on a computer monitor, does not change. This is not always the case, especially in the case of samples that are colored with several dyes, and can therefore be confusing and lead to incorrect evaluations of images.

According to the invention, a method is proposed which ensures that the parameters such as image brightness, contrast and division of the spectral channels are not changed or only minimally changed when switching between the single-spot mode and the multi-spot mode.

In particular, the method can also consist of adjusting the laser intensity. For example, when switching from single-spot mode to multi-spot mode, the total laser power can be changed so that at the end, i.e. after the total power has been divided between the four excitation spots and passing through a more or less different beam path with different optical elements , the laser power in the four spots, considered individually, is equal to the laser power in one spot in single-spot mode.

If further losses are not taken into account, this means that if, for example, four excitation spots are used, the total laser intensity increases by a factor of about four when switching from single-spot mode to multi-spot mode.

In addition, differences in the transmission of the various beam paths can preferably also be compensated for. This leads to corrections to the factor resulting from the number of lighting spots used in multi-spot mode.

In the multi-spot mode, the spectral power density of the detection light can in principle be distributed differently to the individual detectors than in the single-spot mode. Therefore, according to the invention, the detector unit is adjusted accordingly when switching between the single-spot mode and the multi-spot mode.

This can also mean that the amplification or the gain of all individual detectors or just individual detector elements are adjusted. Several detector elements can also be combined into a single channel or separated. Then, by moving optical elements, in particular refractive or diffractive components, the spectral distribution can be changed in such a way that the spectral power distribution on the individual detectors remains the same when switching over or at least changes only very slightly.

Finally, it is proposed that the necessary changes to the recording parameters when switching between single-spot mode and multi-spot mode be carried out automatically by operating software. One can do this Calibration of the relevant parameters, for example intensity of the light source or light sources, detector gain and/or other detector settings and/or insertion and removal of diffracting or refracting components in the beam path, become necessary. This also includes pure software settings, for example combining some detector elements into a single displayed channel. This also includes changes to hardware elements, such as laser intensity, amplification (gain) of the individual detectors and also the position of movable optical elements.

In the method according to the invention and the device according to the invention, at least some operating or device parameters of a scanning microscope are automatically changed when changing between a single-spot mode and a multi-spot mode so that the important for the user, i.e. displayed or otherwise parameters relevant in any way, such as image brightness and contrast, remain almost constant.

Reference List

10

light source

11

light source

12

excitation light

13

excitation light

14

excitation beam

15

excitation beam

16

excitation beam

17

excitation beam

18

switching device

19

switching device

20

ripping unit

21

ripping unit

22

sliding mirror

23

sliding mirror

24

concave mirror

25

Control for concave mirror 24

26

Control connection between control unit 120 and movable mirror 22

27

Control connection between control unit 120 and movable mirror 23

28

Optical components in beam path for single spot mode

29

Optical components in beam path for single spot mode

30

first main beam splitter, moveable or pivotable

31

second main beam splitter, moveable or pivotable

32

Control connection between control unit 120 and first main beam splitter 30

33

Control connection between control unit 120 and second main beam splitter 31

34

Control connection between control unit 120 and scanner 40

40

scanner

41

confocal aperture

42

confocal aperture

43

confocal aperture

44

confocal aperture

45

scanning optics

50

microscope lens

55

Control connection between control unit 120 and mirror control 25

57

Control connection between control unit 120 and manipulation unit 98

59

Beam path in single spot mode

61

telescopic lens

62

telescopic lens

63

motorized adjustable mirror

64

cover

65

Connection for laser

66

Connection for laser

67

Beam path in multi-spot mode

70

sample

71

sample location

72

sample location

73

sample location

74

sample location

76

sample area

80

light emitted from sample 70

81

light emitted from sample site 71

82

light emitted from sample location 72

83

light emitted from sample site 73

84

light emitted from sample location 74

85

Lens, pinhole optics

86

Fourfold pinhole aperture

87

Lens, detection optics

88

controllable, movable mirror

89

Control connection between control unit 120 and movable mirror 88

90

spectrally resolving detector

91

first area of the detector line

92

second area of the detector line

93

third area of the detector line

94

fourth area of the detector line

95

detector line

96

first dispersive unit

97

second dispersive unit

98

manipulation unit

100

microscope

120

control unit

181

Spectrum of the light 81 radiated from the sample location 71

182

Spectrum of the light 82 radiated from the sample location 72

183

Spectrum of the light 83 radiated from the sample location 73

184

Spectrum of the light 84 emitted from the sample location 74

200

switching means

301 to 332

detectors

Claims (20)

Device for scanning microscopy, in particular for carrying out the method according to one of Claims 15 until 20 , having at least one light source (10) for emitting excitation light (12), with optical means (30, 40, 50) for guiding the excitation light (12) in an excitation beam path (A) onto a sample (70) and for guiding from the sample (70) emitted detection light (80) in a detection beam path (D) on a spectrally resolving detector unit (90), wherein the optical means at least one scanner (40) for point-by-point scanning of the sample (70) with excitation light (12) and a microscope objective ( 50) for focusing the excitation light (12) onto or into the sample (70), and with the spectrally resolving detector unit (90) for detecting the detected light (80), separately for each sample location (74) illuminated with excitation light (12) , characterized in that a separating unit (20) is present for separating the excitation light (12) into a plurality of excitation beam bundles (14 - 17), that switching means (200) are present for switching between a single-spot mode in which the Sample (70) is scanned with a single point of the excitation light (12), and a multi-spot mode in which the sample (70) is scanned with multiple points of the excitation light (12), and that a control device (120) is present which interacts at least with the switching means (200), the separating unit (20) and the detector unit (90), the control device (120) being set up to insert the separating unit (20) into the excitation beam path (A) for the multi-spot mode, for removing the separating unit (20) from the excitation beam path (A) for the single-spot mode, and for changing at least parameters of the light source and/or the detection unit (90) when switching between the single-spot mode and the multi-spot mode in such a way that images obtained from one and the same sample area (76) in single-spot mode on the one hand and in multi-spot mode on the other hand have the same image brightness. device after claim 1 , characterized in that at least one main beam splitter is present for separating excitation light and, in particular wavelength-shifted, detected light. device after claim 1 or 2 , characterized in that the detector unit (90) has at least one, in particular dispersive, unit (96) for spectral splitting of the detection light (80). Device according to one of Claims 1 until 3 , characterized in that the detector unit (90) has a first, in particular dispersive, unit (96) for spectral splitting of the detected light (80) in single-spot mode and a second, in particular dispersive, unit (97) for spectral splitting of the detected light (81 - 84) in multi-spot mode and that there are means (24, 25) for selectively inserting and removing the first and/or the second unit in the detection beam path (D). device after claim 3 or 4 , characterized in that the unit or units for spectral splitting of the detected light has or have one or more filters. Device according to one of claims 3 until 5 , characterized in that the dispersive units (96, 97) have refractive and/or diffractive optical components. Device according to one of Claims 1 until 6 , characterized in that the detector unit (90) has a plurality of detectors (301,..,332). Device according to one of Claims 1 until 7 , characterized in that a movable mirror (22 in the excitation beam path and/or a movable mirror (24) in the detection beam path is present as part of the switching means. device after claim 8 , characterized in that at least one of the movable mirrors is a rotatably mounted concave mirror (24). Device according to one of Claims 1 until 9 , characterized in that there are several light sources (10, 11) for emitting excitation light (12, 13) with different wavelengths. Device according to one of Claims 1 until 10 , characterized in that in an excitation beam path (A) there is a telescope (61, 62), in particular a zoom telescope, to compensate for inaccuracies in detector optics (85, 87), in particular pinhole optics. Device according to one of Claims 1 until 11 , characterized in that at least one manipulation optics (98) for changing the spatial spectral distribution of the detection light (80) is present. device after claim 12 , characterized in that the manipulation optics (98) can be introduced into the detection beam path (D) in the single-spot mode. Device according to one of Claims 1 until 13 , characterized in that at least one confocal diaphragm is present for carrying out confocal microscopy. Method for scanning microscopy, in which the excitation light (12) is focused onto or into a sample (70) using a microscope lens (50) and the sample (70) is scanned point by point with the excitation light (12) and in which the detected light (81 - 84), which is emitted from illuminated sample locations (71-74) in response to the excitation light (12), is measured separately and spectrally resolved for each illuminated sample location (71-74), characterized in that between a single-spot mode , in which the sample (70) is scanned with a single point of excitation light (12), and a multi-spot mode in which the sample (70) is scanned with multiple points of excitation light (12), switched back and forth when switching to the multi-spot mode, a separating unit (20) for separating the excitation light (12) into a plurality of excitation beam bundles (14 - 17) is inserted into an excitation beam path (A) and the separating unit (20) when switching over is removed from the excitation beam path (A) into the single-spot mode, and wherein when switching between the single-spot mode and the multi-spot mode, at least parameters of the light source and/or the detection unit (90) are changed in such a way that in single-spot mode on the one hand and in multi-spot mode on the other hand images obtained from one and the same sample area (76) have the same image brightness. procedure after claim 15 , characterized in that when switching from single-spot mode to multi-spot mode and vice versa, the image parameters brightness, contrast and / or spectral distribution of the light on individual detectors of the detection unit (90) do not change. procedure after claim 15 or 16 , characterized in that when switching parameters of the scanner (40) and/or the light source (10) can also be changed between the single-spot mode and the multi-spot mode. Procedure according to one of Claims 15 until 17 , characterized in that the spectral resolution of the microscopic images is the same in the single-spot mode and in the multi-spot mode. Procedure according to one of Claims 15 until 18 , characterized in that the intensity of the excitation light when switching from single-spot mode to multi-spot mode is increased by a factor that corresponds to the number of light spots in multi-spot mode. Procedure according to one of Claims 15 until 19 , characterized in that a spectrally resolved microscopic image of a sample area (76) is assembled from the data measured separately for each sample location (71 - 74).

Images