DE102013021222B4 - Microscope and microscopy method - Google Patents

Abstract

Microscope for examining samples by means of light sheet microscopy, with a light source device for emitting at least one illuminating light beam (1), with an illuminating lens (10) for guiding the illuminating light beam (1) as a beam with a focus area extended in the direction of propagation of the beam to a sample, with illuminating scanning means (5) for directing the illuminating light beam (1) successively onto different linear sample areas, so that the linear sample areas illuminated successively with the illuminating light beam (1) define an illuminated sample plane (3) as a light sheet, with optical imaging means (21, 22) for forwarding a first sample light component, which propagates from the sample in the direction of a first detection axis, the first detection axis being transverse to the illuminated sample plane (3), having a first detection device for detecting the first sample light component, the optical imaging means (21, 22) being designed such that the illuminated sample plane (3) is imaged onto the first detection device, characterized in that the optical imaging means (21, 22) are designed to transmit a second sample light component (2), which propagates from the sample in the direction of a second detection axis, wherein the second detection axis extends perpendicularly to the first detection axis, and wherein the second detection axis is within the illuminated sample plane (3) and perpendicular to the direction of propagation of the illuminating light beam (1),that a second detection device (40) is present for detecting the second sample light component (2), that refocusing means (30) are present in a beam path of the second sample light component (2), that a focal plane which is imaged onto the second detection device (40) can be variably adjusted with the refocusing means (30), that electronic control and evaluation means are present and are set up to carry out a scanning movement of the illumination scanning means (5) and an adjustment of the refocusing means (30) simultaneously and in such a way that the focal plane that is imaged on the second detection device (40) is always the one with the illumination light beam (1) at the moment includes illuminated linear sample area.

Description

In a first aspect, the present invention relates to a microscope for examining samples by means of light sheet microscopy according to the preamble of claim 1.

In a second aspect, the invention relates to a microscopy method for examining samples using light sheet microscopy according to the preamble of claim 17.

In the microscopic examination of samples, it is fundamentally advantageous if only one sample plane is illuminated which is also being examined with a detection device. This sample plane can be a few hundred nanometers to a few micrometers thick, for example. Sample areas above and below this sample level should not be illuminated if possible. This can reduce an unwanted amount of scattered light, which generates a background signal and thereby reduces the image quality. In addition, the photostress on the sample is lower, reducing bleaching or other damage to the sample.

Such a sample examination takes place in light sheet microscopy. This is also known as SPIM (single/selective plane illumination microscopy) or LSFM (light sheet fluorescence microscopy).

In principle, with SPIM, the illumination light can be shaped using a cylindrical lens in such a way that the illumination light only illuminates the sample plane. This sample plane is then imaged onto a detection device. Alternatively, the illuminating light can also be radiated linearly into the sample and successively illuminate the different sample areas that form the sample plane.

The second variant is used in generic microscopes for examining samples by means of light sheet microscopy. Such a microscope initially comprises a light source device for emitting at least one illuminating light beam and an illuminating lens for guiding the illuminating light beam as a beam with a focal area extended in the propagation direction of the beam to a sample. The bundle of rays can illuminate a line-shaped sample area due to the extended focus area, with a cross-section of the bundle of rays being approximately constant over the path through the sample. The microscope further comprises illumination scanning means for directing the illumination light beam successively onto different sample areas, the sample areas illuminated successively with the illuminating light beam defining an illuminated sample plane. In addition, the microscope includes optical imaging means for forwarding a first portion of sample light, which propagates from the sample in the direction of a first detection axis. In this case, the first detection axis is transverse, preferably perpendicular, to the illuminated sample plane. The microscope also includes a first detection device for detecting the first sample light component, the optical imaging means being designed in such a way that the illuminated sample plane is imaged onto the first detection device.

The first portion of sample light can be understood to mean the portion of sample light that is guided with the optical imaging means in the direction of the first detection device. Other parts of the sample light are emitted in other directions and do not reach the first detection device.

The sample light to be detected is often fluorescent light. In general, however, the sample light can also be other luminescent light or reflected illumination light, ie in particular scattered or diffracted illumination light.

In a generic microscopy method for examining samples using light sheet microscopy, it is provided that at least one illuminating light beam is guided to a sample with an illuminating lens as a beam with a focus area that is extended in the direction of propagation of the beam of rays, that the illuminating light beam is directed to different sample areas one after the other using illuminating scanning means, the the illuminating light beam successively illuminated sample areas define an illuminated sample plane, and that a first sample light component, which propagates from the sample in the direction of a first detection axis, is detected with a first detection device. The first detection axis is transverse to the illuminated sample plane. In addition, with the aid of optical imaging means, the first sample light component generates an image of the illuminated sample plane on the first detection device.

The position of the sample is usually changed for different image recordings, so that sample images are recorded one after the other from different sample planes. By changing the sample and not the illumination and detection beam path, it is only necessary to adjust once so that the sample plane imaged on the detection device and the illuminated sample plane match one another.

In addition to a translatory height adjustment of the sample, a sample rotation is also often carried out. Because illuminating light is partially absorbed and/or scattered by the sample in the direction of propagation, the light distribution within the light sheet, ie the illuminated sample plane, is not homogeneous and not isotropic. The sample rotation allows illuminating light from a different direction to illuminate the same sample plane. As a result, areas of the sample plane are also illuminated with higher light intensity, in which previously only little illumination light reached. Because sample light is also absorbed and/or scattered within the sample, advantages can also be achieved with regard to the light intensity of the sample light by rotating the sample.

However, sample rotation also has disadvantages. On the one hand, the recording time required overall is lengthened. On the other hand, image registration methods and correlation methods for generating an image overlap of the sequentially measured images or image stacks must be carried out. The sample images, which are recorded at different rotational positions of the sample, must therefore be aligned with one another. One reason for this is that the position of the axis of rotation relative to the field of view of the detection device is not precisely known.

Out of U.S. 2009/0 231 660 A1 the recording of images of a flow field in a wind tunnel or water tunnel is known. DE 10 2010 060 121 A1 describes a microscope for light sheet microscopy, wherein the light sheet is generated by scanning an illuminating light beam. U.S. 7,554,725 B2 describes a method for light sheet microscopy, in which two detection objectives are present and a detection direction is perpendicular to an illumination direction. Also WO 2013 / 150 273 A1 discloses a microscope in which an illumination and detection direction are perpendicular to each other; there is also an additional detection objective. Other microscopes are described in DE 10 2010 013 223 A1 , WO 2009/124 700 A1 and WO 2011/059 826 A2 . A device for optical distance measurement is in DE 101 53 113 A1 described. Out of GB 2422193A a weather measuring device for measuring the speed of drops is known. WO 2013/132 257 A1 relates to an optical tomograph in which an image is recorded transversely or perpendicularly to a propagation direction of fluorescence excitation light.

An object of the invention can be seen as specifying a microscope and a microscopy method which enable a particularly precise sample examination in the shortest possible time.

This object is achieved by the microscope having the features of claim 1 and by the microscopy method having the features of claim 17.

Advantageous variants of the microscopy method according to the invention and the microscope according to the invention are the subject matter of the dependent claims and are also explained in the following description.

In the microscope of the type mentioned above, the optical imaging means are designed according to the invention for forwarding a second sample light component, which propagates from the sample in the direction of a second detection axis, the second detection axis extending perpendicularly to the first detection axis. In addition, a second detection device is present for detecting the second sample light component. Furthermore, refocusing means are present in a beam path of the second sample light component, with a focal plane that is imaged onto the second detection device being variably adjustable with the refocusing means. Electronic control and evaluation means are also present and set up to carry out a scanning movement of the illumination scanning means and an adjustment of the refocusing means simultaneously and in such a way that the focal plane, which is imaged on the second detection device, always includes the sample area currently illuminated with the illuminating light beam .

In the method of the type mentioned above, according to the invention, a second sample light component, which propagates from the sample in the direction of a second detection axis, is passed on to a second detection device using the optical imaging means. The second detection axis extends perpendicular to the first detection axis. A focal plane, which is imaged onto the second detection device, is adjusted with refocusing means, which are arranged in a beam path of the second sample light component. A scanning movement of the illumination scanning means and an adjustment of the refocusing means are carried out simultaneously and in such a way that the focal plane, which is imaged on the second detection device, always encompasses, ie intersects or includes, the sample area currently illuminated with the illuminating light beam.

An essential advantage of the invention can be seen in the fact that two sample images can be recorded from different directions via the two sample light components that are detected simultaneously and separately. It is not necessary to rotate the sample to set different detection directions.

Time is saved with the simultaneous measurement of sample light components that are emitted in different detection directions or axes. In addition, the registration described in the introduction can advantageously be omitted.

A basic idea of the invention for the simultaneous recording of two sample images can be considered that for the recording of a sample image, the sample plane of interest does not have to be completely sharply imaged on the detection plane of the second detection device at one point in time. Rather, it is sufficient if only the sample area of the sample plane that is illuminated at a point in time is imaged sharply on the second detection device. No or hardly any sample light is emitted from the unilluminated sample areas, so that these sample areas also do not have to be imaged sharply on the second detection device.

While the illuminating light beam is guided over the sample plane to be illuminated, refocusing means ensure that the currently illuminated sample area is imaged sharply on the second detection device.

If, with a detection axis that is not perpendicular to the illuminated sample plane, the entire sample plane were to be imaged onto a detection device at a single point in time, either severe imaging errors would occur or disproportionately complex and expensive structures would be required. This problem is avoided in particular by the refocusing means.

The sample light portion of a specific detection axis includes, on the one hand, that part of the sample light that is emitted precisely in this axial direction. However, this also includes sample light that is imaged in the same way as this part of the sample light. The sample light portion of a detection axis thus includes all portions of the sample light that are imaged onto the same area of the detection device together with a portion of the sample light running exactly along this detection axis.

Each detection device can be formed by a separate camera with its own light-sensitive camera chip. Alternatively, two detection devices can also be formed by different sections of the same camera chip.

In principle, a single detection objective can be used in order to forward sample light of the first and the second detection direction separately from one another. In this case, the detection objective merely has to have a sufficiently large numerical aperture. As a result, the total number of components required is advantageously small.

However, it is particularly preferred that the optical imaging means comprise a first detection objective, with which the first sample light component propagating along the first detection axis is forwarded, and a second detection objective, with which the second sample light component propagating along the second detection axis is forwarded. As a result, imaging errors can be kept low with a relatively simple structure and a particularly good image quality can be achieved.

The first detection axis and the second detection axis are perpendicular to each other. This makes it possible for the first detection axis to be perpendicular to the illuminated sample plane and for the second detection axis to be within the sample plane and perpendicular to the direction of propagation of the illuminating light beam.

As a result, the first detection device records a sharp image of the sample plane without changes in the beam path of the first sample light component being necessary during this image recording.

The propagation direction of the illuminating light lies in the focal plane, which is sharply imaged on the second detection device. The illuminating light beam is preferably shifted perpendicular to its direction of propagation with the illuminating scanning means. In order for the shifted illuminating light beam to continue to lie in the focal plane, this now only has to be shifted, but not tilted.

If a sample area or a sample plane is imaged or sharply imaged on a detection device or a section of a detection device, this means that the detection device or its section is arranged optically conjugate to this sample area or the sample plane.

If a sample area and the detection device are not optically conjugate to one another, this should be understood to mean that this sample area is not, ie not sharply, imaged on the detection device.

A detection device preferably includes a two-dimensional camera chip. However, the second detection device can also have a one-dimensional camera chip which comprises a single row of photosensitive elements. Their measurement signals are read out after each adjustment process of the refocusing means and then combined to form a two-dimensional sample image. One can also speak of an associated focal plane in the case of a one-dimensional camera chip. This focal plane forms an optically conjugate plane to the light-sensitive surface of the one-dimensional camera chip.

Several line-shaped sample areas are successively illuminated with the illuminating light beam. A line-shaped illuminated area can have a rotationally symmetrical, in particular circular, cross-section. In the propagation direction of the illuminating light beam, a measurement area that is imaged on the detection devices is larger than a cross-sectional dimension of the illuminating light beam. The second sample light component emanating from a line-shaped sample area is imaged as a line image with the second detection objective. Because only a line-shaped sample area is illuminated by the currently imaged focal plane, the imaging of the focal plane also includes image information in a line-shaped area alone. This image area is referred to as a line image or as an intermediate line image. The line image is now imaged onto a line-shaped detector section of the second detection device with the optical imaging means. In this case, various line-shaped detector sections are arranged optically conjugate to the line-shaped sample regions that are illuminated in succession. That is, at the point in time when sample light from the currently illuminated line-shaped sample area is guided onto a certain line-shaped detector section, this detector section is arranged optically conjugate to the currently illuminated sample area.

In principle, a scanning movement of the illuminating light beam, by which the illuminated sample plane is defined, can take place in any way, as long as it is ensured that the currently illuminated sample area is imaged sharply on each of the detection devices. In order to achieve this easily, a scanning direction in which the illumination light beam is shifted is preferably set perpendicular to the propagation direction of the illumination light beam. For this purpose, the illumination scanning means can be arranged in or in the area of a pupil of the illumination beam path, along which the illumination light beam moves. With a scanning direction perpendicular to the direction of propagation, the focal plane, which is imaged sharply onto the second detection device, advantageously only has to be shifted via the refocusing means, but not tilted.

Basically, the refocusing means can be of any type, as long as they make it possible to change the focal plane, in particular to shift it in the direction of the second detection axis. For example, the refocusing means for variably adjusting the focal plane can have an optical component whose position or shape can be adjusted for variably adjusting the focal plane. This optics component can be formed, for example, by zoom optics, optics within the second detection objective, the second detection objective itself or the second detection device. In the present variant, this can be designed as a line camera with one-dimensionally arranged camera elements, the measurement signals of which are read out after each adjustment process of the refocusing means and then combined to form a two-dimensional sample image.

In a preferred embodiment, the refocusing means comprise detection scanning means with which the second sample light component can be deflected adjustably onto different detector sections of the second detection device. The second detection device is arranged in such a way that its different detector sections are optically conjugate to the different sample areas which are successively illuminated with the illuminating light beam. That is, they are optically conjugate to different illuminated sample areas, which are offset from one another in the direction of the second detection axis. By means of detection scanning means with which a deflection direction can be variably adjusted, an adjustment can advantageously take place very quickly.

In principle, the illumination scanning means and the detection scanning means can each comprise actuating means or a motor, which are controlled simultaneously. However, they preferably each have a light deflection area which is rigidly connected to one another. The rigid arrangement ensures that the light deflection areas are always moved simultaneously. In addition, a vibration or other unwanted movement of the illumination scanning means does not or hardly leads to a worsened measurement result. In this way, the rigid connection to the detection scanning means results in the momentarily illuminated sample area continuing to be imaged on a detector section in the event of an unintentional movement of the illumination scanning means. The light deflection areas can be formed in particular by one or more mirrors or lenses.

For example, the two light deflection areas can be opposing mirror surfaces of a double-sided mirror. This can a moment of inertia of the mirror to be moved must be low, which means that high scanning speeds are possible.

In principle, the different sections can be formed by different camera chips, which are arranged in different optical planes, so that the different sections of the second detection device, to which sample light is successively directed via the detection scanning means, are optically conjugated to different focal planes.

In a cost-effective alternative that nonetheless enables precise measurement results, the second detection device includes a camera chip and the various detector sections are different sections of this camera chip. In this case, a light-sensitive surface of the camera chip can be arranged at an angle deviating from 90° to an impingement direction of the second sample light component, ie to an optical axis of the impinging sample light component. Alternatively, the sample light component can also be routed to the second detection device via an optical component with the detection scanning means, which has a plurality of regions of different refractive power, one region of which is selected by the detection scanning means.

The deflection directions of the second sample light component, which can be selected via the detection scanning means, can define a plane. The optical imaging means are preferably designed in such a way that the line image, which is imaged onto a line-shaped detector section of the second detection device, is aligned perpendicular to this plane. The result of this is that the entire line image is imaged sharply on the second detection device. On the other hand, if the line image were not perpendicular to the aforementioned plane, part of the line image would be imaged out of focus on the second detection device.

In order to achieve the desired alignment of the line image, an image field rotator can be present in particular between the second detection objective and the detection scanning means. With this, an alignment of the line image is rotated about an axis which runs in the direction of propagation of the second sample light component. This rotation can be 90°. The use of an image field rotator can be advantageous in particular if an axis of rotation of the illumination scanning means and an axis of rotation of the detection scanning means coincide with one another or are parallel to one another. On the other hand, an image field rotator can be dispensed with in particular when the axis of rotation of the illumination scanning means and the detection scanning means are perpendicular to one another.

So that even more information about the sample can be obtained, there can be a switching device with which an illuminating light beam from the light source device can be selectively routed either to the illuminating objective or to at least one other objective in order to adjust a propagation direction of the illuminating light beam through the sample. The lenses that can be selected are therefore not lenses that can be selectively placed in a fixed beam path of the illuminating light beam, such as with a lens turret. Rather, these lenses can be spatially fixed, and spatially different beam paths for the illuminating light beam can be selected using these lenses. The at least one other lens that can be selected can be, for example, the first or second detection lens or an additional illumination lens.

With an additional illumination lens, an additional illumination light beam can be guided to the sample as a beam with a focus area that is extended in the propagation direction of the beam. As described above, the additional illuminating light beam can be emitted by the light source device mentioned. Additional illumination scanning means are also present for directing the additional illumination light beam onto different sample areas in succession. In this case, the sample regions illuminated one after the other with the additional illuminating light beam define or form the same sample plane which is illuminated by the illuminating light beam guided via the illuminating objective.

If an illumination light beam is guided into the sample plane via one of the detection lenses or the additional illumination lens, the illumination lens is preferably used for the measurement. For this purpose, a further sample light component coming from the sample can be passed on with the illumination objective. In addition, there is a detection device assigned to the illumination lens for detecting the further sample light component. There are also beam splitting means with which the illuminating light coming from the light source device can be guided to the illuminating objective, provided that the illuminating light is not guided to the additional illuminating objective, and with which the additional sample light component coming from the illuminating objective can be guided in the direction of the detection device, which is assigned to the illuminating objective is.

A third detection objective and an associated detection device for detecting a third sample light component forwarded by the third detection objective can also be present. In this case, the third detection objective is arranges that the illuminated sample plane is imaged onto the detection device belonging to the third detection objective. The first and third detection objective can be arranged on opposite sides of the illuminated sample plane. The arrangements of these detection lenses can in particular be mirrored in relation to one another on the illuminated sample plane. If not two, but only one detection objective is used to forward the sample light components that propagate along the first and second detection axis, only two detection objectives are present together with the third detection objective.

In addition or as an alternative to the third detection objective, an additional detection objective can also be present and arranged in such a way that a detection axis of the additional detection objective is transverse to the detection axes of the first and second detection objective and transverse to the direction of propagation of the illuminating light beam or the additional illuminating light beam. In this case, an additional detection device is present for detecting a sample light component forwarded by the additional detection device. Additional refocusing means are also present in a beam path between the additional detection objective and the additional detection device. A focal plane, which is imaged onto the additional detection device, can be variably adjusted with the additional refocusing means. The electronic control and evaluation means are set up to carry out a scanning movement of the illuminating light beam and an adjustment of the additional refocusing means simultaneously and in such a way that the focal plane, which is imaged on the additional detection device, always includes the sample area illuminated with the illuminating light beam.

In principle, the illumination scanning means can be understood to mean any device with which an illumination light beam can be deflected onto different areas in the sample plane to be illuminated. For example, the illumination scanning means can have one or more adjustable mirrors. Acousto-optical devices can also be used. Furthermore, a mask comprising one or more areas for light transmission can be shifted or rotated. This mask can be a rotatable pinhole disk, for example. The illumination scanning means are preferably formed in such a way that both the illumination scanning means and the refocusing means can be adjusted, in particular moved, with a common actuator or motor device. This is possible, for example, if the illumination scanning means and the refocusing means each include one or more mirror surfaces to be rotated.

A sample holder may be provided to hold the sample. So that the sample can be illuminated and measured from basically any direction, it can be translationally displaceable and/or rotatable. For this purpose, rotation means can be present for rotating the sample holder about an axis that is parallel to the first detection axis. A sample holder that can be rotated about this axis can be structurally simpler than a sample holder used in conventional microscopes whose axis of rotation is perpendicular to the first detection axis.

• 1 zeigt ein Ausführungsbeispiel eines erfindungsgemäßen Mikroskops.

Further advantages and features of the invention are described below with reference to the attached schematic figure.

• 1 shows an embodiment of a microscope according to the invention.

An exemplary embodiment of a microscope 100 according to the invention is shown schematically in 1 shown. This includes, in particular, an illumination objective 10, a first detection objective 21 and illumination scanning means 5. In contrast to known microscopes, a second detection objective 22 and refocusing means 30 are also present.

Illumination light 1, which is emitted by a light source device (not shown), reaches a sample plane via the illumination scanning means 5 and the illumination objective 10. The sample plane represents a plane of a sample to be examined.

By illuminating the sample, it emits sample light, for example fluorescent light. A first sample light component, which propagates in a first detection direction, is passed on via the first detection lens 21 . A second sample light component 2, which propagates in a second detection direction, is passed on via the second detection lens 22. A detection device is assigned to each detection lens 21, 22. Of these, only the detection device 40 of the second detection objective 22 is shown.

In light sheet microscopy, only one sample plane 3 should be illuminated and imaged. No sample areas above and below an imaged sample plane 3 should be illuminated.

For this purpose, the illuminating light beam 1 is guided along the sample plane 3 in such a way that it has an extended focus area in its direction of propagation. So the focus area doesn't have that Shape of a thin sheet or plane perpendicular to the direction of propagation. Rather, the focus area covers a linear sample area, which lies in the sample plane 3 to be illuminated. This can be achieved, for example, by a low numerical aperture of the illumination lens 10, the numerical aperture preferably being less than 0.4 or particularly preferably less than 0.2. A propagation direction of the illumination light beam 1 can be rotated about an axis which is perpendicular to the sample plane 3 with the illumination scanning means 5 . As a result, the illuminating light beam 1 can illuminate the different parts of the sample plane 3 one after the other. During this scanning process, the detection devices can each record a sample image.

The illumination scanning means 5 are preferably arranged in a pupil plane of the illumination beam path, ie in a plane in which the light intensity distribution is determined by a Fourier transformation for the light distribution in the sample area to be illuminated. The advantage of this positioning is that for different deflection angles of the illumination scanning means 5, the illumination light beam 1 is shifted within the sample plane 3 in a scanning direction 3 which is perpendicular to the direction of propagation of the illumination light beam 1. However, there is no inclination of the illuminating light beam within the sample plane 3 that is dependent on the deflection angle.

The first detection objective 21 is arranged in such a way that its detection axis, ie its optical axis, is perpendicular to the illuminated sample plane 3 . In 1 the sample plane 3 lies in the plane of the paper, which is why the detection lens 21 is only shown in outline.

With this arrangement of the first detection objective 21 it can easily be achieved that an associated focal plane, which is imaged sharply onto the detection device of this detection objective 21, is exactly identical to the illuminated sample plane 3.

The detection axis of the second detection objective 22 supplemented according to the invention, on the other hand, lies within the illuminated sample plane 3. Only through a core idea of the invention is it possible to image the sample plane 3 sharply onto the detection device 40 despite such a detection direction. For this purpose, use is made of the fact that at any point in time only the sample area illuminated by the illuminating light beam 1 is sharply imaged on the detection device 40, but not the remaining unilluminated areas of the sample plane 3. Since no or hardly any sample light emanates from such unilluminated sample areas, there is a sharp image of these sample areas also not required.

A focal plane, which is sharply imaged on the second detection device 40, is transverse to the sample plane 3. As an essential idea of the invention, different focal planes are imaged on the second detection device 40 one after the other. The currently imaged focal plane and the sample plane 3 always intersect along the currently illuminated linear sample area.

This is largely achieved with the refocusing means 30 . In the example shown, the refocusing means 30 are formed by detection scanning means 31 and an oblique arrangement of the detection device 40 . This is explained in more detail below. A deflection direction of the impinging second sample light portion 2 can be variably changed with the detection scanning means 31 . The detection scanning means 31 can be formed, for example, as a mirror which can be rotated together with a mirror of the illumination scanning means 5 .

The detection scanning means 31 direct the sample light portion 2 via optical components 32 to the spatially resolving detection device 40. A light-sensitive surface of the detection device 40 is inclined to an optical axis from the detection scanning means 31 to the detection device 40. The oblique orientation of the detection device 40 can also mean that described that an axis of rotation about which the detection device 40 is inclined is parallel to the axis of rotation of the detection scanning means 31 . As a result, an optical path length which the sample light component 2 covers from the detection scanning means 31 to the detection device 40 depends on the deflection direction set with the detection scanning means 31 . Adjacent sections of the detection device are optically conjugate to different sample areas because of the different path lengths. These sample areas are offset in the propagation direction of the sample light portion 2, which runs from an illuminated sample area to the second detection objective 22. Therefore, a rotation of the detection scanning means 31 together with the oblique arrangement of the detection device 40 can cause refocusing.

In other words, the detection device 40 comprises sections which - if they are selected via the detection scanning means 31 - are conjugated to different planes which are mutually offset in the scanning direction 4, i.e. in a direction perpendicular to the direction of propagation of the illuminating light 1 through the sample are offset.

It is important for good image quality that each currently illuminated sample area is imaged completely sharply on the detection device 40 . The alignment of a line image, which is an image of the currently illuminated sample area on the detection device 40, is decisive for this. In 1 an intermediate line image 27 with optical components 25 is first generated in an intermediate image plane 26 . The intermediate image plane 26 is imaged onto the detection device 40 .

The alignment of the line image, ie its longitudinal direction, is preferably parallel to an axis of rotation of the detection scanning means 31. The line image is therefore also parallel to the axis of rotation about which the detection device 40 is inclined. The section of the detection device 40 onto which the line image is imaged is therefore just conjugate to the currently illuminated sample area.

For this alignment of the line image is at 1 a field rotator 28 is present. This is arranged between the intermediate image plane 26 and the detection scanning means 31 . While in the intermediate image plane 26 the alignment of the line intermediate image 27 is still perpendicular to the axis of rotation of the detection scanning means 31, the image field rotator 28 achieves an image field rotation of 90°, whereby the line image is parallel to the axis of rotation about which the detection device 40 is inclined .

In this way, a common axis of rotation of the detection scanning means 31 and the illumination scanning means 5 can be implemented, as shown in FIG 1 shown. In principle, the axes of rotation can also be perpendicular to one another, which means that the field rotator can be dispensed with.

The refocusing means 30 can also be different than in 1 be executed. Thus, the detection scanning means 31 can be dispensed with and the detection device 40 can not be inclined, but can be arranged perpendicularly to an optical axis of the impinging portion of sample light 2 . The only important thing is that a focal plane that is imaged by the second detection objective 22 can be displaced, i.e., seen from the second detection objective 22 , it can be displaced in a vertical direction that can be parallel to an optical axis of the second detection objective 22 . Thus, the refocusing means can comprise an optical component, which is shifted along an optical axis of the sample light portion 2 at the same time as the illumination scanning means 5 is actuated, and thus shifts the focal plane. For example, the second detection objective 22 or a component of a zoom lens can be displaced.

In this embodiment, as well as in the embodiment shown, the essential advantage is that two sample images are recorded from different detection directions without the sample having to be rotated relative to an illumination objective or detection objective. In addition, these sample images are advantageously recorded simultaneously.

In the case of additions to the illustrated embodiment, measurements can also be taken from other detection directions and/or an illumination light beam can be guided into the sample plane 3 to be illuminated from other illumination directions.

A switching device can be present, with which the illuminating light beam 1 is guided either to the illuminating lens 10 (as in 1 shown), or to a further illumination objective.

The further illumination lens can be arranged opposite the illumination lens 10 in relation to the sample plane 3 . This means that an illuminating light beam from the illuminating objective 10 and an illuminating light beam from the further illuminating objective run antiparallel to one another in the sample plane 3 .

Alternatively or additionally, the further illumination lens can also be formed by the detection lens 22 . A color splitter 23 can be provided, with which illumination light can be distinguished from sample light. The color splitter 23 can guide the illuminating light beam from the switching device to the detection objective 22 , while it does not forward a sample light component 2 coming from the detection objective 22 to the switching device but in the direction of the detection device 40 . A color filter 24, which is arranged between the color splitter 23 and the detection device 40, can have a cut-off wavelength between transmission and absorption or reflection, through which only sample light, but not illumination light, is passed on to the detection device 40.

If illuminating light is guided into the sample plane 3 via the detection objective 22 , provision can be made for a sample light component to be detected via the illuminating objective 10 . For this purpose, further refocusing means are arranged in the beam path of the portion of the sample light that runs over the illumination lens 10 . These refocusing means can also be formed by the illumination scanning means 5.

The detection scanning means 31 for the detection objective 22 can also be used as an illumination scanning means when illuminating light is guided via the detection objective 22 . At the arrangement of the color splitter 23 from 1 On the other hand, illumination scanning means that differ from detection scanning means 31 are required for detection lens 22 .

The simultaneous recording of two sample images described above is to be understood as at least two sample images. One or two further detection lenses can also be present. With each of these, a sample image can be recorded from a further detection direction. A further detection objective can be arranged mirrored to the first detection objective 21 on the sample plane 3 . As with the first detection objective 21, no refocusing means are required for a detection objective arranged in this way during the recording of a sample image.

A further detection objective can also be arranged opposite the second detection objective 22 with respect to the sample plane 3 . This means that a detection direction of this further detection objective is antiparallel to the detection direction of the second detection objective 22. This further detection objective can be followed by the same or identical components as the second detection objective, in particular refocusing means 30.

With the invention, even without rotating the sample and without rotating an objective around the sample, sample light can be measured from different detection directions and multiple sample images can thus be recorded simultaneously.

Reference List

1

illumination light beam

2

sample light fraction

3

illuminated sample plane

4

Scanning direction in which the illuminating light beam 1 can be displaced

5

illumination scanning means

10

lighting lens

21

first detection objective

22

second detection lens

23

color divider

24

color filter

25

optical component

26

intermediate image level

27

Line Intermediate Image

28

field rotate

30

refocusing means

31

detection scanning means

32

optical component

40

detection device

100

microscope

Claims (17)

Microscope for examining samples by means of light sheet microscopy, with a light source device for emitting at least one illuminating light beam (1), with an illuminating objective (10) for guiding the illuminating light beam (1) as a beam with a focus area extended in the direction of propagation of the beam to a sample, with illuminating scanning means (5 ) for directing the illuminating light beam (1) successively onto different linear sample areas, so that the linear sample areas illuminated successively with the illuminating light beam (1) define an illuminated sample plane (3) as a light sheet, with optical imaging means (21, 22) for forwarding a first sample light component , which propagates from the sample in the direction of a first detection axis, the first detection axis being transverse to the illuminated sample plane (3), with a first detection device for detecting the first sample light component, the optical imaging means (21, 22) being designed in such a way that the illuminated sample plane (3) is imaged onto the first detection device, characterized in that the optical imaging means (21, 22) are designed to transmit a second sample light component (2), which propagates from the sample in the direction of a second detection axis, wherein the second detection axis extends perpendicularly to the first detection axis, and wherein the second detection axis is within the illuminated sample plane (3) and perpendicular to the direction of propagation of the illuminating light beam (1), that a second detection device (40) is present for detecting the second sample light component (2) that refocusing means (30) are present in a beam path of the second sample light component (2), that a focal plane that is imaged onto the second detection device (40) can be variably adjusted with the refocusing means (30), that electronic control and evaluation means are present and are set up to carry out a scanning movement of the illumination scanning means (5) and an adjustment of the refocusing means (30) simultaneously and in such a way that the focal plane, which is imaged on the second detection device (40), always corresponds to that with the illumination light beam (1) currently illuminated line-shaped sample area includes. microscope after claim 1 , characterized in that the optical imaging means (21, 22) comprise a first detection lens (21) with which the first sample light component propagating along the first detection axis is passed on, and a second detection lens (22) with which the light component propagating along the second Detection axis propagating second sample light component (2) is forwarded. microscope after claim 2 , characterized in that the second sample light component (2) emanating from a line-shaped sample area is imaged as a line image with the second detection lens (22), that the line image is imaged onto a line-shaped detector section of the second detection device (40) with the optical imaging means, and that different line-shaped detector sections are arranged optically conjugate to the successively illuminated line-shaped sample areas. Microscope after one of Claims 1 until 3 , characterized in that the illumination scanning means (5) are arranged in a pupil of the illumination beam path along which the illumination light beam (1) moves. Microscope after one of Claims 1 until 4 , characterized in that the refocusing means (30) have an optical component whose position or shape can be adjusted for variable adjustment of the focal plane. Microscope after one of Claims 1 until 5 , characterized in that the refocusing means (30) comprise detection scanning means (31) with which the second sample light component (2) can be deflected adjustably onto different detector sections of the second detection device (40), and in that the second detection device (40) is arranged in this way that their different detector sections are optically conjugated to the different linear sample areas illuminated one after the other with the illuminating light beam (1). microscope after claim 6 , characterized in that the illumination scanning means (5) and the detection scanning means (31) each have a light deflection area, which are rigidly connected to each other. Microscope after one of Claims 1 until 7 , characterized in that the second detection device (40) comprises a camera chip and the different detector sections are different sections of the camera chip. microscope after claim 8 , characterized in that a light-sensitive surface of the camera chip is arranged at an angle deviating from 90° to an optical axis of the impinging second sample light component (2). Microscope after one of Claims 6 until 9 , so far up claim 6 retrospective, characterized in that the directions of propagation of the second sample light component (2) that can be selected via the detection scanning means (31) define a plane, that the optical imaging means are designed in such a way that the line image, which appears on a line-shaped detector section of the second detection device (40) is imaged is oriented perpendicular to the plane. Microscope after one of Claims 1 until 10 , characterized in that there is a switching device with which an illuminating light beam (1) of the light source device can be selectively routed either to the illuminating lens (10) or to at least one other lens in order to adjust a propagation direction of the illuminating light beam (1) through the sample. Microscope after one of Claims 1 until 11 , characterized in that an additional illumination lens is provided for guiding an additional illumination light beam (1) as a beam with a focus area extended in the direction of propagation of the beam to the sample, that additional illumination scanning means (5) are provided for guiding the additional illumination light beam (1) successively to different sample areas, the sample areas illuminated successively with the additional illuminating light beam (1) forming the same sample plane (3) which is illuminated by the illuminating light beam (1) of the illuminating lens (10). Microscope after one of Claims 1 until 12 , characterized in that a further sample light component coming from the sample can be passed on with the illumination objective (10), that a detection device assigned to the illumination objective (10) is present for detecting the further sample light component, that beam splitting means are present, with which illuminating light coming from the light source device can be guided to the illuminating objective (10) and with which the further sample light component coming from the illuminating objective (10) can be guided in the direction of the detection device, which is assigned to the illuminating objective (10). Microscope after one of Claims 1 until 13 , characterized in that a third detection objective and an associated detection device are present for detecting a third sample light component transmitted by the third detection objective, the third detection objective being arranged in such a way that the illuminated sample plane (3) is imaged onto the detection device associated with the third detection objective. Microscope after one of Claims 1 until 14 , characterized in that an additional detection objective is present and arranged in such a way that a detection axis of the additional detection objective is transverse to the detection axes of the first and second detection objective (21, 22) and transverse to the direction of propagation of the illuminating light beam (1) or the additional illuminating light beam ( 1) is that an additional detection device is present for detecting a sample light component forwarded by the additional detection device, that additional refocusing means are present in a beam path between the additional detection objective and the additional detection device, that the additional refocusing means create a focal plane , which is imaged on the additional detection device, is variably adjustable, in that the electronic control and evaluation means are set up to carry out a scanning movement of the illuminating light beam (1) and an adjustment of the additional refocusing means simultaneously and in such a way that the focal plane, which is on the additional detection device is imaged, always includes the linear sample area illuminated with the illuminating light beam (1). Microscope after one of Claims 1 until 15 , characterized in that there is a sample holder for holding the sample, that rotation means are provided for rotating the sample holder about an axis which is parallel to the first detection axis. Microscopy method for examining samples using light sheet microscopy, in which at least one illuminating light beam (1) is guided to a sample with an illuminating lens (10) as a beam with a focus area that is extended in the direction of propagation of the beam, in which the illuminating light beam (1) is scanned in succession using illuminating scanning means (5). is directed onto different linear sample areas, so that the linear sample areas illuminated one after the other with the illuminating light beam (1) define an illuminated sample plane (3) as a light sheet, in which a first sample light component, which propagates from the sample in the direction of a first detection axis, has a first detection device is detected, the first detection axis being transverse to the illuminated sample plane (3), in which the first sample light component generates an image of the illuminated sample plane (3) on the first detection device with the aid of optical imaging means (21), characterized in that a second sample light component (2), which propagates from the sample in the direction of a second detection axis, is passed on to a second detection device (40) with the optical imaging means (21, 22), the second detection axis extending perpendicularly to the first detection axis, and wherein the second detection axis is within the illuminated sample plane (3) and perpendicular to the direction of propagation of the illuminating light beam (1), that with refocusing means (30), which are arranged in a beam path of the second sample light component (2), a focal plane which is on the second detection device (40) is imaged, is adjusted so that a scanning movement of the illumination scanning means (5) and an adjustment of the refocusing means (30) are carried out simultaneously and in such a way that the focal plane, which is imaged on the second detection device (40), is always the with the illuminating light beam (1) currently illuminated line-shaped sample area.

Images