DE102013021182A1 - Device and method for scanning microscopy - Google Patents

Abstract

The invention relates to an apparatus for scanning microscopy with at least one light source for emitting excitation light, with optical means for directing the excitation light in an excitation beam path to a sample and for directing emitted from the sample detection light in a detection beam path to a spectrally resolving detector unit, wherein the Optics means at least one scanner for pointwise scanning of the sample with excitation light and a microscope objective for focusing the excitation light on or in the sample, and with the spectrally resolving detector unit for detecting the detection light, each separately for each illuminated with excitation light sample location. The device is characterized in that a separation unit is provided for separating the excitation light into a plurality of excitation beam, that switching means are provided for switching 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 a plurality of points of the excitation light, and that a control device is provided, which cooperates with at least the switching means, the separation unit and the detector unit, wherein the control device is adapted to insert the separation unit in the excitation beam path for the multi-spot mode, for removing the separation 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 such that in single-spot M 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. The invention also relates to a method for scanning microscopy.

Description

The invention relates in a first aspect to an apparatus 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 at least one light source for emitting excitation light, optical means for directing the excitation light in an excitation beam path to a sample and for guiding detected light emitted from the sample in a detection beam path to a spectrally resolving detector unit. The optical means have at least one scanner for pointwise scanning of the sample with excitation light and a microscope objective for focusing the excitation light on or in the sample. In addition, the spectrally resolving detector unit for detecting the detection light is part of the generic device, wherein the detection light is detected separately for each illuminated with excitation light sample location.

In a generic method excitation light is focused with a microscope objective on or in a sample and the sample is scanned pointwise with the excitation light. In addition, the detection light emitted from illuminated sample locations in response 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, also such scanning microscopes are known in which a plurality of illumination points, which are also referred to as spots, are guided over a sample known. For example, in DE 2002 10 15 162 an arrangement is described in which the number of illumination spots can be varied. These microscopy methods, which are also referred to as multi-spot method, allow in principle shorter recording times.

As an object of the invention can be considered to increase 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.

According to the invention, the device of the abovementioned type is further developed in that a separation unit is present for separating the excitation light into a plurality of excitation beam bundles, that switching means are provided 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 cooperates with at least the switching means, the separating unit and the detector unit, wherein the control device is adapted for insertion the separation unit in the excitation beam path for the multi-spot mode, for removing the separation 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-sp On the other hand, 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.

The method of the abovementioned type is inventively further developed 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 with multiple points of the excitation light is scanned, is switched back and forth, wherein when switching to the multi-spot mode, a separation unit for separating the excitation light is inserted into a plurality of excitation beam in an excitation beam path and the separation unit when switching to the single-spot mode from the excitation beam path 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 single-spot mode on the one hand and in multi-spot mode on the other have the same image brightness in one and the same sample area.

As a central idea of the present invention, it can be considered, in a scanning microscope, a switchability between a single-spot mode, which can also be referred to as single-spot operation, and a multi-spot mode, which can also be used as a multi-spot mode. Spot operation can be referred to, to realize. Specifically, this means that the optical components necessary for the multi-spot operation are inserted into the beam path of the microscope when the multi-spot operation is desired, and accordingly removed again from the beam path when the microscope in the single spot Mode should be operated. For this purpose are suitable according to the invention Switching means exist that basically involve or include all components that are changed and / or moved when switching between the single-spot mode and the multi-spot mode. Another important aim of the present invention is to increase the comparability of images taken in single-spot mode on the one hand and in multi-spot mode on the other hand. For this purpose, the invention provides that a control device is provided which cooperates with at least the switching means and at least changes parameters of the light source and / or the detection unit when switching between the single-spot mode and the multi-spot mode so that the image brightness of in single-spot operation, on the one hand, and multi-spot operation, on the other, is the same for images obtained from one and the same sample area. This makes it possible to better compare the images obtained from said sample area by single-spot microscopy on the one hand and multi-spot microscopy on the other hand.

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

The term image brightness for the present invention is understood to mean the primary signal obtained from a coarse dot. This means, for example, that when switching from single-spot mode to a 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, with the intensity of the light source remains the same 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. These considerations apply in principle to all color channels, provided that the respective spectral regions which are imaged on a single detector element remain the same size. In other words, therefore, 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 the single-spot mode on the one hand and in the multi-spot mode on the other hand.

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

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

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

Excitation light in the sense of the invention described herein is electromagnetic radiation, in particular the infrared, visible and ultraviolet parts of the spectrum are meant. As sources of light, in principle, all sources can be used which provide the desired electromagnetic radiation in sufficient intensity. Usually, lasers are used for this purpose. In principle, however, light-emitting diodes or other light sources can also be used.

In principle, all detectors can be used as detectors, which 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 as a rule the count rates are comparatively small, photomultipliers are commonly used.

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

Confocal is an aperture when positioned in or near a confocal plane. A confocal plane refers to a plane of the detection beam path which is optically conjugate to a sample-side focal plane of the microscope objective. A confocal aperture in front of a detector limits the light exposure of this detector to a small target volume at the sample site.

To achieve a spectral resolution, the detector unit may preferably have at least one unit for the spectral separation of the detection light. As actually dispersive elements, the dispersive units may comprise refractive and / or diffractive optical components.

In principle, the unit or units for spectrally separating the detection light can have one or more filters for the spectral Have separation. However, variants are particularly preferred in which the unit or units for spectrally separating the detection light has or have a dispersive function. For this purpose, in principle known dispersive elements, such as diffractive or refractive components, such as prisms or grids are used.

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, embodiments of the device according to the invention are preferred in which the detector unit has a first, in particular dispersive, unit for the spectral separation of the detection light in single-spot mode and a second, in particular dispersive, unit for the spectral separation of the detection light in the multi-spot mode , A precise working optical structure can be achieved more easily. Appropriately, then means may 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 the multi-spot operation and the single-spot operation. Particularly preferred is or are used for the multi-spot operation and the single-spot operation of the same or the same detectors.

In principle, the different wavelengths of the light emitted by the sample can be measured successively with one and the same detector. However, the detector unit particularly preferably has a plurality of detectors, which may 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, which are directed by optical means, in particular a dispersive unit or a plurality of dispersive units, to the respective individual detector. With a higher number of individual detectors naturally higher resolution is possible than with a lower number of individual detectors.

In principle, the spectral cut-outs or wavelength ranges, which are each directed to the individual detectors, may be of different sizes.

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

Multi-spot mode is generally used when you want to take a picture of the largest possible sample area as quickly as possible. A device according to the invention can therefore be expediently constructed and set up such that the manipulation optics can be introduced into the detection beam path only in single-spot mode.

In principle, in the multi-spot mode, the spectrum of the light emitted by different lighted sample locations can be measured in succession with the detector unit. However, the main advantage of the multi-spot mode is achieved if the spectra of the light emitted by different lighted sample locations are measured simultaneously with the detector unit. If a detector unit with several individual detectors is used, this means that because of the limited number of individual detectors, the maximum resolution achievable in multi-spot mode is smaller than the maximum resolution that is possible in single-spot mode.

In principle, the aim in the invention described here is that the images obtained in single-spot mode on the one hand and in multi-spot mode on the other hand should 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 single-spot mode and in multi-spot mode. This can be achieved when using a detector unit with a plurality of individual detectors when not working in single-spot mode with the maximum possible resolution, for example, n (n is an integer) of the individual detectors are summarized. After switching to a multi-spot mode with n lighting spots, the individual detectors can then be read out one at a time, achieving the same spectral resolution.

The one or more switching means in principle include all components that are active when switching the scanning microscope from single-spot mode in the multi-spot mode or actuated. The switching means may comprise both physical or hardware components as well as control or software-like components.

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

The invention can in principle be realized with a light source, for example a laser, which essentially emits only light of a single wavelength. In the apparatus according to the invention, however, it is particularly preferable for a plurality of light sources to emit excitation light having different wavelengths. This allows the examination of samples prepared with several different dyes.

For carrying out confocal microscopy methods, at least one confocal diaphragm is present in the device according to the invention. For the multi-spot mode, there may 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, when the single-spot mode is switched to the multi-spot mode and vice versa, parameters of the detector unit are changed. 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, may be, for example, the image parameters brightness, contrast and / or spectral distribution of the light to individual detectors of the detection unit act.

Maintaining image parameters when switching from single-spot mode to multi-spot mode and vice versa can be made easier if, when switching between the single-spot mode and the multi-spot mode, parameters of the scanner and / or the light source to be changed.

For example, when 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 can be increased by a factor that is in the Essentially corresponds to the number of light spots in multi-spot mode. Preferably, this factor is greater than the number of light spots in the multi-spot mode minus one and less than the number of light spots in the multi-spot mode plus one.

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

Further advantages and features of the device according to the invention and of 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 : a further detail of a device according to the invention;

4 : a schematic representation for explaining the multi-spot operation;

5 a schematic representation of a series of 32 single detectors;

6 : a schematic representation of a breakdown of the 32 single detectors for the multi-spot mode and

7 a flow chart for explaining the method according to the invention.

An embodiment of a device according to the invention 100 is related to 1 explained. As essential components, this device 100 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 A on a sample 70 and for conducting the sample 70 radiated detection light 80 in a detection beam D to a spectrally resolving detector unit 90 on.

The optical means comprise at least one main beam splitter 30 for separating the excitation light 12 and the detection light 80 , a scanner 40 for pointwise scanning of the sample 70 with the excitation light 12 as well as a microscope objective 50 for focusing the excitation light 12 on or in the sample. The spectrally resolving detector unit 90 serves to detect the detection light 80 each being done separately for each sample location.

Furthermore, a separation unit 20 present for separating the excitation light 12 into a plurality of excitation beams that are in 1 are not shown in detail. Then are in the inventive device 100 in 1 switching 200 available for switching between a single-spot mode in which the sample 70 with a single point of excitation light 12 is scanned and a multi-spot mode in which the sample is scanned with multiple points of the excitation light.

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

In the in 1 schematically shown situation becomes the excitation light 12 in multi-spot mode via the separation unit 20 and the movable mirror 22 on the main color divider 30 directed. In single-spot mode, the separation unit is located 20 not in the beam path and the excitation light 12 instead takes a schematically shown optical path 28 in which also optical components, such as lenses or mirrors may be located, to the movable mirror 22 and from this turn, the main color splitter 30 directed.

For switching the light path between the first light source 10 and the separation unit 20 on the one hand and the optical path 28 On the other hand, a switching device 18 present, which may be, for example, a movable mirror.

At the in 1 illustrated device according to the invention 100 is also a second light source 11 for emitting excitation light 13 available. Appropriately, in this case, the excitation light 13 a different wavelength than the excitation light 12 , For example, the excitation light may be 13 to be non-visible radiation, such as infrared or ultraviolet radiation act. Accordingly, the excitation light 12 Light from the entire visible spectrum from red to blue. As with the first light source 10 is also for the second light source 11 a separation unit 21 for separating the excitation light 13 into a plurality of excitation beams and a movable mirror 23 present, with which the separation unit 21 for the multi-spot mode can be inserted into the excitation beam path and can be taken out of the excitation beam A for the single-spot mode. The moving mirror 23 is via a schematically indicated connection 27 with the control unit 120 connected and can via the control unit 120 to be controlled. In single-spot mode, the excitation light decreases 13 a schematically indicated optical path 29 ,

For switching the light path between the second light source 11 and the separation unit 21 on the one hand and the optical path 29 On the other hand, a switching device 19 present, which may be, for example, a movable mirror. The switching devices 18 and 19 are each about the control unit 120 controllable. This is in 1 through the connecting lines between switching devices 18 and 19 and the control unit 120 shown.

The excitation light 13 then passes to a second main color divider 31 and from this, like the excitation light 12 over the scanner 40 , a scanning optics 45 and the microscope objective 50 on the sample to be examined 70 , During operation of the device according to the invention 100 Typically, the user is using either the first light source 10 or the second light source 11 work. For this purpose, the first main color divider 30 with the control unit 21 over a connection 32 are inserted into the excitation beam A when the first light source 10 should be used and be removed accordingly from the excitation beam A, when the light source 10 not to be used. The same applies to the second light source 11 and the main color divider 31 , with which excitation light 13 the second light source 11 can be coupled into the excitation beam A. The main color divider 31 can with the control unit 120 over a line 33 be controlled so that the excitation light 13 is either coupled into the excitation beam A or not.

The scanner 40 can from the control unit 120 over a connection 34 be controlled. The scanner is preferred 40 activated differently in multi-spot mode than in single-spot mode.

The basic situation in single-spot mode and in multi-spot mode is related to 4 explained. There is schematically a sample area 76 the sample to be examined 70 shown. In general, identical or equivalent components are generally identified by the same reference numerals in all figures.

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

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

Back to the consideration of 1 , That of the sample to be examined 70 radiated light 80 is first about the same optical components, so the microscope objective 50 , the scanning optics 45 , the scanner 40 , now on the detection beam D, returned. Deviating from the excitation light 12 and / or the excitation light 13 , that will be from the sample 70 radiated light 80 at least partially from the main color dividers 31 and 30 transmits and is first on a Pinholeoptik 85 on a Pinholeblende 86 directed. This Pinholeblende has, schematically indicated, four pinholes for four excitation beam. Also the Pinholeblende 86 can, what in 1 not shown, with the control unit 120 be operated, for example, be inserted into the detection beam D, if you want to work in multi-spot mode. For the single-spot mode, the aperture can then 86 be removed with the four confocal apertures from the beam path or only be shifted so that the one beam of the pinholeoptics 85 actually hits a pinhole. About another optics 87 , which can be referred to as detection optics, gets that from the sample 70 radiated light 80 either, in single-spot mode via a movable mirror 88 on a first dispersive unit 96 , which are part of the spectrally resolving detector unit 90 is. The moving mirror 88 can from the control unit 120 , which in this respect is part of the switching means 200 is, about the connection 89 be removed from the detection beam D or inserted into this, or redirect the beam path suitable. What is essential is that of the sample 70 radiated light 80 in multi-spot mode not on the first dispersive unit 96 but on the second dispersive unit 97 arrives.

In the first dispersive unit 96 and the second dispersive unit 97 becomes the light emitted by the sample 80 divided spectrally. For this purpose, basically known components, such as prisms or grids, may be present. In the case of single-spot operation, the spectrally divided light is transmitted via a schematically indicated beam path 59 and a concave mirror 24 to a manipulation unit 98 passed, which may optionally be present in the beam path.

Eventually, the light emitted by the sample becomes light 80 on a detector line 95 directed with a variety of single detectors. With the manipulation unit 98 The spatial distribution of the individual spectral components on the detector line can be changed or manipulated. For example, parts of the spectrum can be imaged particularly densely on the detector line, so that in this area the spectral resolution is lower. On the other hand, spectral ranges for which one is particularly interested can be found on a particularly large number of individual detectors of the detector line 95 be split. The spectral resolution is then larger in comparison to the situation described first.

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

To toggle between the multi-spot mode and the single-spot mode is as part of the switching means 200 a movable concave mirror 24 with a control 25 present, via the control unit 120 about the connection 55 can be operated. In single-spot mode, on the one hand, there is the movable mirror 88 so that's from the sample 70 radiated light 80 on the first dispersive unit 96 is directed and from this on the beam path 59 over the concave mirror 24 on the detector line 95 arrives. Optionally, here also the manipulation unit 98 to be gone through or not.

In multi-spot mode, the mirror becomes 88 from the control unit 120 about the connection 89 positioned so that from the sample 70 radiated light 80 not on the first dispersive unit 96 but the second dispersive unit 97 arrives. From the second dispersive unit 97 becomes the radiated light 80 then via a schematically illustrated beam path 67 on the detector line 95 directed. For this purpose, on the one hand, the movable concave mirror 24 suitably positioned, on the other hand, the manipulation unit 98 via the control unit 120 and the connection 57 taken out of the beam path.

The coupling of either a plurality of excitation beams or a single excitation beam will be described with reference to FIG 2 explained in more detail. There are initially two connections 65 . 66 for light sources to which in principle different light sources are connected. In principle, however, it is also possible that only a single light source is used, whose excitation light 12 expedient with a switching or distribution device, not shown, the first port 65 and / or the second port 66 is supplied. The first connection 65 is a unraveling unit 20 downstream, which separates the excitation light 12 into a plurality of excitation beam 14 . 15 . 16 . 17 he follows. The excitation beam 14 . 15 . 16 . 17 be on a moving mirror 22 passed and from this via a panel 64 and one through the lenses 61 and 62 formed telescope and a mirror 63 on the main color divider 30 , That through the lenses 61 and 62 In particular, the formed telescope can be a zoom telescope and serve to correct inaccuracies of the detector optics. For the multi-spot mode, the movable mirror 22 be positioned so that the excitation beam 14 . 15 . 16 . 17 on the aperture 64 be directed. On the other hand, for the single-spot mode, the movable mirror becomes 22 either removed from the beam path or anyway positioned so that a single excitation beam of the excitation light 12 to and through the aperture 64 is directed.

The spectral separation of the from the sample 70 emitted light 80 is determined by 3 explained in more detail. Shown there is first a Pinholeblende 86 with a total of four confocal pinholes 41 . 42 . 43 . 44 , That from the sample locations 71 . 72 . 73 . 74 radiated light 81 . 82 . 83 . 84 (please refer 4 ) is through the pinholes 41 . 42 . 43 . 44 on a lens 87 , which can be referred to as detection optics, passed. From the detection optics 87 the radiated light arrives 80 over the mirror 88 on the dispersive unit 97 , which is shown schematically as a prism block. The dispersive unit 97 separates the total of four beams into a total of four spectra 181 . 182 . 183 . 184 on that on a first area 91 , a second area 92 , a third area 93 or a fourth area 94 the detector line 95 be imaged. This will be with reference to the 5 and 6 further explained.

In 5 is schematically a detector line 95 shown, the total of 32 single detectors 301 ... 332 having. In single-spot mode, the spectrum of light emitted from the single sample spot illuminated by excitation light will be reflected on these 32 single detectors 301 ... 332 directed. In this case, as explained above, in connection with the manipulation unit 98 explains the spectral resolution over the detector line 95 vary. For example, it would be possible for the entire wavelength range between 400 nm and 580 nm to the individual detectors 301 to 310 and also the wavelength range of 580 nm to 600 nm on the single detectors 311 to 332 to lead. On the single detectors 301 to 310 then each account for 28 nm of the wavelength spectrum, while on the individual detectors 311 to 332 in each case only 0.9 nm omitted. That means that in the field of single detectors 301 to 310 the resolution is about 31 times smaller than that of the single detectors 311 to 332 measured spectral range.

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

The inventive method will be supplementary with reference to the 7 explained. Accordingly, initially an image recording in single-spot mode (box a). With the aid of software, various image parameters, such as, for example, the image brightness, the contrast and / or the spectral distribution on the individual detectors, can then be determined or established (box b). Regardless, that is, at the same time, before or after, switching to the multi-spot mode can be carried out with the aid of the switching means provided according to the invention (box c). After this switching can be done automatically, for example, with the controller 120 , Changes are made to components of the device according to the invention. 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 to the detectors (box d). Once the adjustments of the various components of the device according to the invention are carried out, image recordings in multi-spot mode can be performed (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 the same sample or sample spot once in multi-spot mode and directly afterwards in single-spot mode. The reverse order is of course possible.

If the same laser is used to generate the plurality of excitation spots for the multi-spot mode as for generating the single spot, then the intensity of the single spot in the multi-spot mode is then a factor that corresponds to the number of spots in the multi-spot mode. Spot mode is less than that of the single spot in single-spot mode. In order to obtain comparable images, it is advantageous if the intensity of each spot in the multi-spot mode is the same as the intensity of each spot in single-spot mode.

Further, for detecting the detection light emitted from the sample in the multi-spot illumination, the same detection unit in the same configuration is not used. In other words, the same detection unit can be used, but in a different configuration. In this context, the term configuration of the detector refers to the manner in which the detection light to be detected is conducted onto the spectrally resolving detector unit by the sample locations illuminated by the excitation light. In the event that 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. Accordingly, the signal of the individual detectors will change and these may even be over or under controlled. Thus, 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. Especially with samples that are colored with several dyes, this is not given easily and therefore can confuse and lead to incorrect ratings of form.

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

The method may in particular also consist in an adjustment of 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, ie after the total power has been split among the four excitation spots and through a more or less different beam path with other optical elements , the laser power in the four spots, each considered individually, is equal to the laser power in a spot in single-spot mode.

If further losses are ignored, for example, using four excitation spots means increasing the total laser intensity by a factor of about four when switching from single-spot mode to multi-spot mode.

In addition, differences in the transmission of the different beam paths may additionally be compensated. This leads to corrections of 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 single detectors or only individual detector elements are adjusted. It is also possible to combine or separate several detector elements into a single channel. Then, by moving optical elements, in particular refractive or diffractive components, the spectral distribution can be changed so that the spectral power distribution to the individual detectors remains the same when switching or in any case changes only very slightly.

Finally, it is proposed that the changes in the recording parameters necessary for switching between single-spot mode and multi-spot mode be carried out automatically by operating software. For this purpose, a calibration of the corresponding parameters, for example intensity of the light source or of the light sources, detector gain and / or further detector settings and / or insertion and removal of diffractive or refractive components into the beam path, may become necessary. This also includes pure software settings, such as combining some detector elements into a single displayed channel. It also refers to changes in elements of the hardware, such as laser intensity, gain of the single detectors and also the position of moving 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 when changing between a single-spot mode and a multi-spot mode are automatically changed so that the important for the user, ie displayed or otherwise in some way relevant parameters, such as image brightness and contrast, remain almost constant.

LIST OF REFERENCE NUMBERS

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

switchover

19

switchover

20

unraveling unit

21

unraveling unit

22

sliding mirror

23

sliding mirror

24

concave mirror

25

Control for concave mirror 24

26

Control connection between control unit 120 and sliding mirror 22

27

Control connection between control unit 120 and sliding 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, displaceable or pivotable

31

second main beam splitter, displaceable 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 scanners 40

40

scanner

41

confocal aperture

42

confocal aperture

43

confocal aperture

44

confocal aperture

45

scan optics

50

microscope objective

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 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

from sample 70 radiated light

81

from sample location 71 radiated light

82

from sample location 72 radiated light

83

from sample location 73 radiated light

84

from sample location 74 radiated light

85

Lens, Pinholeoptik

86

Pinholeblende quadruple

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 row

96

first dispersive unit

97

second dispersive unit

98

manipulation unit

100

microscope

120

control unit

181

Spectrum of the sample site 71 emitted light 81

182

Spectrum of the sample site 72 emitted light 82

183

Spectrum of the sample site 73 emitted light 83

184

Spectrum of the sample site 74 emitted light 84

200

switching

301 to 332

detectors

QUOTES INCLUDE IN THE DESCRIPTION

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

Cited patent literature

• DE 102009006729 A1 [0002]
• DE 20021015162 [0004]

Claims (20)

Device for scanning microscopy, in particular for carrying out the method according to one of Claims 13 to 18, 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 conducting the sample ( 70 ) radiated detection light ( 80 ) in a detection beam path (D) to a spectrally resolving detector unit ( 90 ), wherein the optical means at least one scanner ( 40 ) for pointwise scanning of the sample ( 70 ) with excitation light ( 12 ) and a microscope objective ( 50 ) for focusing the excitation light ( 12 ) on or in the sample ( 70 ), and with the spectrally resolving detector unit ( 90 ) for detecting the detection light ( 80 ), each separately for each with excitation light ( 12 ) illuminated sample location ( 74 ), characterized in that a separation unit ( 20 ) is present for separating the excitation light ( 12 ) into a plurality of excitation beams ( 14 - 17 ) that switching means ( 200 ) are available for switching between a single-spot mode in which the sample ( 70 ) 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 at least with the switching means ( 200 ), the separation unit ( 20 ) and the detector unit ( 90 ), the control device ( 120 ) is set up for inserting the separation unit ( 20 ) in the excitation beam path (A) for the multi-spot mode, for removing the separation 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 such that, in the single-spot mode on the one hand and in the multi-spot mode on the other hand, from one and the same sample area ( 76 ) have the same image brightness. Apparatus according to claim 1, characterized in that for the separation of excitation light and, in particular wavelength-shifted, detection light at least one main beam splitter is present. Apparatus according to claim 1 or 2, characterized in that the detector unit ( 90 ) at least one, in particular dispersive, unit ( 96 ) for the spectral separation of the detection light ( 80 ) having. Device according to one of claims 1 to 3, characterized in that the detector unit ( 90 ) a first, in particular dispersive, unit ( 96 ) for the spectral separation of the detection light ( 80 ) in single-spot mode and a second, in particular dispersive, unit ( 97 ) for the spectral separation of the detection light ( 81 - 84 ) in multi-spot mode and that means ( 24 . 25 ) are provided for selectively inserting and removing the first and / or the second unit in the detection beam path (D). Apparatus according to claim 3 or 4, characterized in that the unit or units for spectrally separating the detection light comprises or comprise one or more filters. Device according to one of claims 1 to 5, characterized in that the detector unit ( 90 ) a plurality of detectors ( 301 , ..., 332 ) having. Device according to one of claims 1 to 6, characterized in that as part of the switching means movable mirror ( 22 . 24 ) available. Device according to one of claims 1 to 7, characterized in that at least one of the movable mirror is a rotatably mounted concave mirror ( 24 ). Device according to one of claims 1 to 8, characterized in that a plurality of light sources ( 10 . 11 ) for emitting excitation light ( 12 . 13 ) are present at different wavelengths. Device according to one of claims 1 to 9, characterized in that in a excitation beam path (A) a telescope ( 61 . 62 ), in particular a zoom telescope, is present to compensate for inaccuracies of a detector optics ( 85 . 87 ), in particular a pinhole optics. Device according to one of claims 1 to 10, characterized in that the dispersive units ( 96 . 97 ) have refractive and / or diffractive optical components. Device according to one of claims 1 to 11, characterized in that at least one manipulation optics ( 98 ) for changing the spatial spectral distribution of the detection light ( 80 ) is available. Device according to one of claims 1 to 12, characterized in that the manipulation optics ( 98 ) in single-spot mode in the detection beam path (D) is insertable. Device according to one of claims 1 to 13, characterized in that for performing confocal microscopy at least one confocal aperture is present. Method for scanning microscopy, in which excitation light ( 12 ) with a microscope objective ( 50 ) on or in a sample ( 70 ) and the sample ( 70 ) pointwise with the excitation light ( 12 ) and the detection light ( 81 - 84 ), of illuminated samples ( 71 - 74 ) in response to the excitation light ( 12 ), for each illuminated sample location ( 71 - 74 ) is measured separately and spectrally resolved, characterized in that between a single-spot mode in which the sample ( 70 ) 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 switched back and forth, wherein when switching to the multi-spot mode, a separation unit ( 20 ) for separating the excitation light ( 12 ) into a plurality of excitation beams ( 14 - 17 ) is inserted into an excitation beam path (A) and the separation unit ( 20 ) is removed from the excitation beam path (A) when switching to the single-spot mode, and at least parameters of the light source and / or of the detection unit (12) during switching between the single-spot mode and the multi-spot mode ( 90 ) 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, from one and the same sample area ( 76 ) have the same image brightness. A method according to claim 15, characterized in that the image parameters brightness, contrast and / or spectral distribution of the light on individual detectors of the detection unit ( 90 ) are. The method of claim 15 or 16, characterized in that when switching between the single-spot mode and the multi-spot mode and parameters of the scanner ( 40 ) and / or the light source ( 10 ) to be changed. Method according to one of claims 15 to 17, characterized in that the spectral resolution of the microscopic images in the single-spot mode and in the multi-spot mode is the same. Method according to one of claims 15 to 18, characterized in that the intensity of the excitation light when switching from single-spot mode to the multi-spot mode is increased by a factor corresponding to the number of light spots in multi-spot mode , Method according to one of claims 15 to 19, characterized in that from the for each sample location ( 71 - 74 ) separately measured data a spectrally resolved microscopic image of a sample area ( 76 ) is composed.

Images