Patent ID: 12253663

DETAILED DESCRIPTION

Embodiments of the present invention provide a control device for a microscope, a microscope, and a method for controlling a microscope which enables a user to find a sample region to be imaged more easily and faster.

In an embodiment, a control device for a microscope, in particular for a confocal scanning microscope, comprises an operating device configured to be operated by a user to vary focusing and/or positioning of an optical imaging system of the microscope relative to a sample. The control device comprises an actuator configured to adjust an aperture of a detection pinhole which is included in the microscope for eliminating out-of-focus light from detection light which is directed by the optical imaging system onto a detector of the microscope. The control device further comprises a processor configured to detect a predetermined operating condition in response to a user operation of the operating device and to control the actuator to vary, in particular to increase the aperture of the detection pinhole upon detection of the predetermined operating condition.

The control device enables a user to perform a search operation for a suitable target region of the sample to be imaged more easily and faster compared to conventional microscope systems. By increasing the aperture of the detection pinhole using a processor-controlled actuator, it is possible to automatically decrease the confocality of the microscope system and thereby to increase a size of a region along the optical axis from where light from the sample can be detected. In the following this will be described using the expression “depth of detection region”. This can in particular be achieved by decreasing the suppression characteristics for out of focus light to be detected by the detector. This means that light from an increased region above and below the focus plane of the optical imaging system can be detected as well, compared to the size of the region above and below the focus plane which usually is detected by a “true confocal setting”, e.g. with a detection pinhole having a diameter/size of for instance of 1 to 2 Airy units. Thus, it is possible to automatically increase a depth of a detection region from which the detector receives detection light emerging from the sample. Accordingly, an imaged sample region, which can be observed by the user e.g. on a monitor, is correspondingly enlarged when the user actuates an operating device in order to focus the optical imaging system onto the sample and/or to vary a lateral position of the optical imaging system relative to the sample. As a result, the search operation is accelerated, and the user receives a faster visual feedback when observing the continuously updated image on the monitor.

The control device is particularly well-suited for confocal imaging where the depth of detection region is small and an operation for focusing the optical imaging system onto the sample is time-consuming. Thus, when the focal plane of the optical imaging system is not precisely coincident with a target region of the sample including the fluorescent markers or spots, the resulting image on the monitor contains basically only noise and does not even allow a rough estimate of which focus change might be necessary to focus on the fluorescent markers or spots. In such a situation, the control device can be advantageously used to automatically increase the depth of detection region to speed up focusing process to be performed the user.

Although the control device is especially advantageous in terms of focusing, it may also be used to accelerate a search for fluorescent spots in lateral directions perpendicular to the optical axis of the optical imaging system. Thus, increasing the depth of detection region in response to a user operation of the operating device enlarges an axial sample region which is, so to say, laterally scanned when the sample and the optical imaging system are laterally shifted relative to each other during the search operation.

Since the control device enables the user to speed up the search operation, the time during which the sample is exposed to illumination light can be significantly reduced. Accordingly, photobleaching of light sensitive samples can be avoided and/or phototoxity of the illumination light for living samples can be reduced.

The predetermined operating condition may comprise a predetermined focusing mode provided by the operating device. The processor may be configured to control the actuator to increase the aperture of the detection pinhole upon detection of the predetermined focusing mode. According to this embodiment, the processor recognizes an activation of the predetermined focusing mode in response to a user operation of the operating device. The recognition of the predetermined focusing mode can be utilized to trigger a search supporting mode in which the aperture of the detection pinhole is varied, in particular increased compared to an aperture setting which is applied in an operating mode other than the search supporting mode.

In a preferred embodiment, the operating device is configured to selectively vary focusing of the optical imaging system in a coarse focusing mode at a first step size and/or at a first focusing speed and to vary focusing of the optical imaging system in a fine focusing mode at a second step size and/or at a second focusing speed. The first focusing speed may be larger than the second focusing speed, and the first step size may be larger than the second step size. The predetermined operating condition may comprise the coarse focusing mode. According to this embodiment, the search supporting mode may be activated when the user selects the course focusing mode.

Preferably, the processor is configured to control the actuator in the coarse focusing mode to set the aperture of the detection pinhole to a first size and to control the actuator in the fine focusing mode to set the aperture of the detection pinhole to a second size, said first size being larger than said second size. The size of the aperture of the detection pinhole can be adjusted or set in a stepwise manner—for instance, it can be increased or decreased by steps having a factor of e.g. 5% (or 10% or another suitable value) of a smallest or largest aperture value of the detection pinhole—or in a continuous manner where the aperture of the detection pinhole is increased or decreased by e.g. a continuous linear function or any other continuous mathematical function.

The processor may be configured to evaluate image data output from the detector. An evaluation of the image data can be used to support the user in a search operation for a suitable target region. For this purpose, the image data may be evaluated in terms of specific image parameters such as brightness/luminance, contrast, signal-to-noise ratio etc.

According to a preferred embodiment, the processor is configured to detect a target focusing state in which the optical imaging system is focused onto a target region of the sample based on the evaluation of the image data. For Instance, in case that the evaluation of the image data reveals that an image of a sample region exhibits high luminance, this sample region may be identified as the aforementioned target region of the sample.

The processor may be configured to detect the predetermined operating condition based on the evaluation of the image data. For example, in case that the evaluation of the image data reveals that the corresponding image exhibits low luminance, this luminance information may indicate the presence of the predetermined operating condition triggering the search supporting mode because a suitable sample region to be imaged has not yet been found.

The processor may further be configured to control the actuator based on the evaluation of the image data to adjust the aperture of the detection pinhole. Preferably, the evaluation of the image data comprises a luminance evaluation.

In a preferred embodiment, the processor may be configured to detect the predetermined operating condition in accordance with an average luminance represented by the image data. For instance, a low luminance which is averaged over the entire image may be a reliable indicator of the predetermined operating condition.

The processor may be configured to control an intensity of illumination light emitted by a light source of the microscope depending on the size of the aperture of the detection pinhole. This embodiment utilizes the fact that the ratio of the collected photons and the size of the aperture of the detection pinhole is known in advance. Based on this ratio, the detection pinhole can be controlled to avoid luminance variations.

Preferably, the processor is configured to control a scan speed of a scanning device of the microscope depending on the size of the aperture of the detection pinhole. For instance, in a confocal laser scanning microscope such a scanning device is used to direct the illumination and/or detection light such that the illumination light is scanned across the sample and to direct the detection light emerging from the sample onto the detector. In such a configuration, the scan speed can be increased in order to accelerate the scanning process in the search supporting mode so that the visual feedback experienced by the user during the search operation is improved.

The operating device may comprise a focus drive configured to cause relative movement between a microscope stage and the optical imaging system of the microscope along an optical axis thereof. The focus drive may be a motorized drive or manual drive. In addition or as an alternative, the operating device may comprise a motorized or manual X-Y drive which is configured to move a microscope stage holding the sample and the optical imaging system relative to each other in a lateral direction that is perpendicular to the optical axis. In case of a manual drive, the operating device may include a sensor or an encoder which is configured to detect an amount, by which the microscope stage and the optical imaging system are moved relative to each other, and to output a corresponding detection signal to the processor. Further, the operating device may comprise a remote control unit which can be operated by the user to control the respective drives. In any case, the operating device is not limited to the examples mentioned above. Any type of operating device can be used which enables the processor to detect a user operation based on which the detection pinhole is controlled as described herein.

According to another embodiment, a microscope, in particular a confocal microscope is provided. The microscope comprises a detector, an optical imaging system configured to direct detection light from a sample onto the detector, a detection pinhole configured to eliminate out-of-focus light from the detection light directed onto the detector, and a control device as described above.

According to another embodiment, a method for controlling a microscope is provided, wherein the microscope comprises a detector, an optical imaging system configured to direct detection light from a sample onto the detector, a detection pinhole configured to eliminate out-of-focus light from the detection light directed onto the detector, and an operating device configured to be operated by a user to vary focusing and/or positioning of the optical imaging system relative to the sample. The method comprises the following steps: detecting a predetermined operating condition of the microscope in response to a user operation of the operating device; and controlling an actuator to increase the aperture of the detection pinhole upon detection of the predetermined operating condition.

FIG.1shows a schematic diagram of a microscope100which may be configured as a confocal laser scanning microscope (CLSM). Thus, according to the present embodiment, the microscope100serves to perform point-by-point imaging of a sample102resulting in a sample image which is composed of a plurality of image points or pixels.

The confocal laser scanning microscope100comprises a processor104which may be connected to a computer106. Further, a monitor108may be provided which is connected to the computer106and displays an image110of the sample102.

The confocal laser scanning microscope100comprises an optical imaging system112which is configured to image a selected target region of the sample102onto a detector114formed e.g. by a point sensor. Detection light116emerging from one point of the sample102is directed by the optical imaging system112onto the detector114as illustrated by a dashed line inFIG.1. It is to be noted that the optical imaging system112is depicted purely schematically as a single block inFIG.1. However, the optical imaging system112may comprise a plurality of lens elements such as an objective facing the sample102and a tube lens located downstream thereof in an optical detection path.

The confocal laser scanning microscope100may further comprise a laser light source118which emits illumination light120which is guided through the optical imaging system112onto the sample102as illustrated by a solid line inFIG.1. Accordingly, the optical imaging system112serves both to illuminate the sample102with the illumination light120and to direct the detection light116emerging from the illuminated sample102onto the detector114. A beam splitter144may be provided for separating the optical detection path from the illumination path. The beam splitter144is formed e.g. by a dichroic mirror having spectral characteristics to reflect the illumination light120emitted by the laser light source118towards the sample102and to transmit the detection light116towards the detector114.

In order to scan the illumination light120point-by-point through the sample102, the confocal laser scanning microscope100includes a scanning device122. According to the embodiment shown inFIG.1, the scanning device122may comprise one or two tiltable mirrors146which are configured to scan the illumination light120across the sample102along lateral directions X and Y perpendicular to an optical axis O of the optical imaging system112. The scanning device122not only acts on the illumination light120, but the detection light116collected by the optical imaging system112from the sample102also enters the scanning device122when it is directed onto the detector114. Accordingly, the embodiment shown inFIG.1provides for a so-called descanned configuration which allows to use a stationary point sensor in form of the detector114to receive the detection light116, although the illumination light120performs lateral scan movements in X and Y directions across the sample102.

The confocal laser scanning microscope100comprises a detection pinhole124which is located in front of the detector114. The detection pinhole124has an aperture126defining a cross-section of the detection light116falling onto the detector114. As explained below in more detail, the aperture126of the detection pinhole124can be adjusted in order to vary a depth of detection region from which the detection light116is received by the detector114.

Optionally, the confocal laser scanning microscope100further includes an illumination pinhole128having an aperture130which defines a cross-section of the illumination light120emitted from the laser light source118. Thus, the illumination pinhole128may be regarded as a point-type illumination source wherein the detection pinhole124is located in a plane in front of the detector114which is optically corresponding to the illumination pinhole128. It is to be noted that the afore-mentioned illumination point source is not necessarily formed by an illumination pinhole. For example, a suitable optical fiber may be coupled to the laser light source118, a light emitting end thereof forming a point source of illumination.

In order to find a suitable target region of the sample102which is to be observed, a positional relationship between the optical imaging system112and the sample102can be varied in all three spatial directions X, Y, Z, wherein Z is parallel to the optical axis O and X, Y are perpendicular to the optical axis0. For this purpose, the confocal laser scanning microscope100includes an operating device132which can be operated by a user to vary Z focusing and X-Y positioning of the optical imaging system112relative to the sample102. Just as an example, the operating device132may include a remote control which is manually operated by the user to activate an axial Z drive and a lateral X-Y drive. These drives are purely schematically illustrated inFIG.1as double arrows134and136, respectively. The Z drive is operated by the user to perform a focusing operation in which an axial distance between the sample102and the optical imaging system112along the optical axis O is varied. Likewise, the X-Y drive136is operated by the user to perform a lateral positioning operation for shifting the sample102along the directions X and Y relative to the optical imaging system112. The X-Y drive may be integrated with a microscope stage138which is movable perpendicular to the optical axis O along the lateral directions X and Y. The drives134,136may be formed by motorized shafts which are configured to cause the intended movements along X, Y, and Z.

Needless to say that the afore-mentioned focus and lateral positioning mechanisms are to be understood merely as examples. For instance, rather than laterally moving the microscope stage138, it is possible to move the optical imaging system112relative to a stationary microscope stage on which the sample102is located.

The confocal laser scanning microscope100comprises an actuator140which is configured to adjust the aperture126of the detection pinhole124. Optionally, another actuator142may be provided for adjusting the aperture130of the illumination pinhole128. Both actuators140,142may be connected to the processor104

As explained hereinafter, the confocal laser scanning microscope100comprises a control device generally referred to as148inFIG.1, which enables the user to find a sample region to be imaged more easily and faster than in conventional microscope systems. According to the specific embodiment shown inFIG.1, the afore-mentioned control device148may include the processor104, the computer106, the operating device132along with the Z and X-Y drives134,136and the actuator140which can be driven to vary the aperture126of the detection pinhole124. Optionally, the control device148may further include the actuator142for adjusting the size of the aperture130of the illumination pinhole128.

Generally speaking, the processor104is configured to detect a predetermined operating condition in response to a user operation of the operating device132and to control the actuator140to vary, in particular to increase the aperture126of the detection pinhole124upon detection of the predetermined operating condition. When the aperture126of the detection pinhole124is increased, a depth of detection region from which the detection light116is received by the detector114is enlarged at the expense of a lower spatial resolution. As a result, a user operation to find a suitable sample region to be imaged is supported by increasing the depth of detection region at the expense of resolution.

More specifically, the control device148may provide a search supporting mode which is activated in case that the afore-mentioned operating condition is detected by the processor104in response to a user operation of the operating device132wherein the user operation indicates that the user intends to find a suitable sample region while observing the continuously updated image110on the monitor108. The operating condition which triggers an activation of the search supporting mode may be suitably determined in advance such that the operation condition reliably indicates the intention of the user to perform a search operation for a suitable sample region.

According to the specific embodiment shown inFIG.1, the processor104is connected with the operating device132to receive detection information from the operating device132when the latter is operated by the user. For instance, the operating device132may output detection signals to the processor104indicating that the user is actuating the operating device132to perform a focusing operation by means of the Z drive134and/or a positioning operation by means of the X-Y drive136. Thus, the processor104is enabled to recognize the predetermined operating condition of the confocal laser scanning microscope100by detecting a situation where one of the afore-mentioned focusing and positioning operations is performed using the operating device132. Recognition of the predetermined operation then triggers an activation of the supporting mode.

As an example, the operating condition may comprise a predetermined focusing mode which is provided by the operating device132. When the user actuates the operating device132to enter the predetermined focusing mode, a corresponding detection signal is output from the operating device132to the processor104. Based on the detection signal, the processor104recognizes that the predetermined focusing mode is set. Subsequently, the processor104activates the supporting mode. In the supporting mode, the processor104increases the aperture126of the detection pinhole124to a value which is larger than a predefined value that is applied before entering the supporting mode. The predefined value defines a size of the aperture126that allows an imaging operation with relatively short depth of detection region and thus high spatial resolution. By increasing the aperture126of the detection pinhole124, the depth of detection region becomes larger, and the imaged sample region, which can be observed by the user based on the image110displayed on the monitor108, is enlarged accordingly at the expense of a lower resolution. As a result, a search operation for a suitable sample region can be performed faster.

According to a specific embodiment, the control device148may be configured to provide at least two different focusing modes wherein one of these modes defines the operating condition triggering the supporting mode. For example, a coarse focusing mode may be provided in which focusing of the optical imaging system112can be varied at a first step size and/or at a first focusing speed. Further, a fine focusing mode may be provided in which focusing of the optical imaging system112is varied at a second step size and/or at a second focusing speed, wherein the first focusing speed is larger than the second focusing speed and the first step size is larger than the second step size. The processor104is configured to recognize on the basis of the detection signals output from the operating device132whether the coarse focusing mode or the fine focusing mode is selected by the user. In case that the coarse focusing mode is selected, the processor104controls the actuator140to set the aperture126of the detection pinhole124to a first size. On the other hand, in case that the fine focusing mode is selected, the processor104controls the actuator140to set the aperture126of the detection pinhole124to a second size which is smaller than the first size set in the coarse focusing mode. The second size of the aperture126may correspond to the afore-mentioned predefined value. In other words, the processor104controls the detection pinhole124in the coarse focusing mode to enlarge its aperture126so that the depth of detection region increases compared to the fine focusing mode. Thus, the search for a suitable target region to be imaged is facilitated in the coarse focusing mode.

The processor104may further be configured to evaluate image data output from the detector114and to use such an evaluation to support the user in a search operation for a suitable target region. For instance, the processor104may perform a luminance evaluation on the image data and utilize the luminance evaluation for recognizing whether or not the predetermined operating condition triggering the supporting mode is met. Just as an example, the processor104may recognize the predetermined operating condition as fulfilled when an average luminance represented by the image data is low.

An evaluation of the image data may also be used in order to detect a target focusing state in which the optical imaging system112is focused onto a target region of the sample102. For instance, in case that a luminance evaluation is performed on the image data, the processor104can detect the target focusing state by evaluating the average luminance of the current image. A continuous evaluation of the image data enables the aperture126of the detection pinhole124to be continuously adjusted based on the current image data. For example, the processor104may control the actuator140based on the current luminance so that the aperture126of the detection pinhole124is continuously readjusted depending on the luminance. If the luminance is low, the actuator140is controlled to increase the aperture126. In contrast, if the luminance is high, the actuator140is controlled to decrease the aperture126.

Optionally, the processor104is configured to control the laser light source118depending on the size of the aperture126of the detection pinhole124. Thus, it is possible to adjust the intensity of the illumination light120emitted by the laser light source118in accordance with the aperture setting. For instance, the processor104may adjust the intensity of the illumination light120depending on the aperture setting such that luminance variations can be avoided to a large extent. In addition, controlling the intensity of the illumination light120depending on the size of the aperture126may be used to prevent any overexposure from occurring.

Further, the processor104may also be configured to control a speed of the scanning device122in accordance with the size of the aperture126of the detection pinhole124. For instance, the scan speed can be increased in order to accelerate the scanning process in the supporting mode. This accelerates the feedback that is experienced by the user when searching for a suitable sample region by observing the image110on the monitor108.

FIG.2shows a flow diagram illustrating a method for controlling the confocal laser scanning microscope100according to an embodiment.

After starting the control operation ofFIG.2with an initialization process in step S1, in which control parameters of the confocal laser scanning microscope100are initialized in accordance with a default setting, the control operation proceeds with step S2. In S2, the aperture126of the detection pinhole124is set. When S2is executed for the first time, the aperture126is set to a predefined value which is applied to eliminate out-of-focus light in an operating mode other than the supporting mode.

In step S3, a confocal imaging process is performed by scanning the illumination light120across the sample102and receiving the detection light116on the detector114in accordance with a descanned configuration as explained above. Thus, the image110is displayed on the monitor108. It is to be noted that steps S2and S3are depicted inFIG.2as being executed one after the other. However, S2and S3can also be executed simultaneously.

In step S4, a query is made whether or not the imaging process is to be stopped. If imaging is to be stopped, the control operation proceeds with step S5, and the control operation ends. If imaging is to be continued, the control operation proceeds with step S6.

In step S6, the processor104recognizes whether the predetermined operating state, which triggers an activation of the supporting mode in response to a user operation of the operating device132, is present or not. If the predetermined operating state is recognized as not present in step S6, the process returns to step S3, and the confocal imaging process is executed again based on the predefined value of the aperture126. Thus, confocal imaging is continued in accordance with the standard setting of the aperture126until imaging is stopped in step S5or the predetermined operating state is recognized in step S6.

If the predetermined operating state is recognized as present in step S6, the control operation sets the supporting mode and returns to step S2in which the aperture126of the detection pinhole124is changed from the predefined value to a larger value. In step S3, the confocal imaging process is performed in accordance with the increased aperture126. Due to the increased aperture126, the depth of detection region, from which the detection light116is received by the detector114, is correspondingly greater in the supporting mode. Thus, image formation is accelerated in the supporting mode at the expense of a lower spatial resolution. The confocal imaging is continued in accordance with the increased aperture126until imaging is stopped in step S5or until it is recognized in step S6that the predetermined operating state is no longer given.

The embodiments as described above are to be understood merely as examples. For instance, although the above control operation provides only for an operating state-dependent setting of the detection pinhole124, it is also possible to control the illumination pinhole128in accordance with the operating state of the confocal scanning microscope100.

As used herein the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”.

Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

While subject matter of the present disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. Any statement made herein characterizing the invention is also to be considered illustrative or exemplary and not restrictive as the invention is defined by the claims. It will be understood that changes and modifications may be made, by those of ordinary skill in the art, within the scope of the following claims, which may include any combination of features from different embodiments described above.

The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C.

LIST OF REFERENCE SIGNS

100confocal laser scanning microscope102sample104processor106computer108monitor110image112optically imaging system114detector116detection light118laser light source120illumination light122scanning device124detection pinhole126aperture of detection pinhole128illumination pinhole130aperture132operating device134Z drive136X-Y drive138microscope stage140actuator142actuator144beam splitter146mirror148control device