METHOD FOR IMAGING USING A MICROSCOPE AND CORRESPONDING MICROSCOPE

Description

[0001] The present invention relates to a method for generating images using a microscope, wherein a sample to be imaged is located in the object space of the microscope for imaging with predetermined image generation parameters, and to a corresponding microscope. State of the art

[0002] A method according to the preamble of claim 1 is known from EP 2 207 055 A1. In this method, a suitable exposure time for a CCD is calculated within the framework of an automatic exposure control. To shorten the time required in the phase in which the exposure time is successively calculated in several steps, the gain factor of the CCD is increased. After a suitable exposure time has been found, the gain factor is reset, and the actual image acquisition takes place.

[0003] Patent application US 2003 / 016301 A1 discloses an automatic exposure control system in which the movement of a microscope stage is detected. By detecting the stage movement, the exposure time and the gain of an image acquisition element are maintained at previously set values.

[0004] In the publication EP 2 873 998 A1, the speed of a moving microscope stage is recorded and, depending on the stage speed, switching is made between an HDR mode and a normal image generation mode.

[0005] Document US 2012 / 069171 A1 also discloses a method in which the speed of a microscope stage is detected. If the detected speed exceeds a threshold, the frame rate is increased.

[0006] In known imaging techniques using microscopes, the sample to be imaged, or more precisely, an arbitrarily chosen reference point on this sample, is located at a position within the microscope's object space. The imaging process is performed using image generation parameters, which are generally adjusted depending on the sample, the type of illumination, the microscope components used, and, more generally, the chosen microscopy technique. The sample is typically placed on a microscope stage, which can be moved to position the sample in at least the plane perpendicular to the optical axis defined by the microscope objective. The microscope stage plane is usually referred to as the xy-plane, so that the optical axis runs in the z-direction. For focusing, the microscope objective and / or the microscope stage is usually movable in the z-direction.There are also so-called fixed-stage microscopes, in which the microscope assembly moves relative to the stationary microscope stage, which can only be adjusted in height. Within the scope of this application, the term "xy movement of the microscope stage" is intended to always refer to a relative movement of the microscope stage or the sample located on it relative to the optical axis of the microscope objective, thus explicitly including fixed-stage microscopes.

[0007] In the aforementioned microscopes, which are known in principle from the prior art, movement of the microscope stage or objective in the x, y, and / or z directions during image generation results in motion artifacts in the generated image. The microscopes considered within the scope of this application can be broadly divided into wide-field microscopes and confocal microscopes. In wide-field microscopy, the generated image exhibits schlieren (blurring) when such a movement occurs. With a movement in the z-direction, these schlieren are less disruptive; however, at least one full exposure time must be observed to accurately assess the sharpness. Only when no movement has occurred within an exposure time are motion artifacts no longer present in the image. If the movement ceases within an exposure interval, another full exposure time (without movement) must be observed after the end of that interval.

[0008] In confocal microscopy, the sample is illuminated point by point using an xy-scanner, and the resulting image is built up line by line. When the sample moves within the object space, a shift occurs between the lines already acquired, the lines acquired during the movement, and the lines acquired after the movement. The longer a frame, or the sample section within the field of view, is scanned, the longer it takes for all image lines to update and for the sample to be displayed in its new position. As in wide-field microscopy, the correct imaging plane must be determined in confocal microscopy using methods in the z-direction. The resulting artifacts behave similarly to movement in the xy-direction. A full frame rate (refresh rate) is required until all pixels of the generated image represent the current focal plane.

[0009] Therefore, there is a need to eliminate or reduce the problem of motion artifacts arising when the sample to be imaged is shifted or moved within the object space of a microscope. Disclosure of the invention

[0010] To solve this problem, a method for generating images using a microscope and a corresponding microscope according to the independent claims are proposed. Advantageous embodiments are described in the respective dependent claims and the following description.

[0011] In an inventive method for image generation using a microscope, in which a sample to be imaged is located in the object space of the microscope for image generation with predetermined image generation parameters, a movement or an intended movement of the sample is first detected. Subsequently, depending on a measured or predicted movement parameter, at least one image generation parameter is automatically changed during the movement of the sample.

[0012] As mentioned earlier, the sample to be imaged, or more precisely, an arbitrarily chosen reference point on this sample, is usually positioned at a specific location within the microscope's object space to enable imaging of the sample or a section of it. If the sample is moved in any spatial direction (x, y, and / or z) during image acquisition, as also explained above, unwanted motion artifacts will occur. Generally, sample movement can have various causes. Besides the sample displacement already described, the sample itself (e.g., living cells) can move even if the microscope stage remains unchanged, or accidental, unintentional movement can occur, for example, due to a collision with the microscope or the stage.

[0013] To avoid the resulting motion artifacts, the invention first detects any movement of the sample, for example, caused by movement of the microscope stage in the x and / or y direction or by activation of the Z-drive for focusing. According to the invention, an intended movement of the sample, in other words, an impending movement of the sample, can also be detected. This includes cases in which it can be assumed that a movement of the sample is imminent, such as when a user or the microscope system itself has issued corresponding control commands to move the sample, for example, by entering new x, y, and / or z coordinates, i.e., a new position, of the sample or the aforementioned reference point on this sample. From this, the system or the control unit of the microscope can automatically predict corresponding movement parameters, e.g.,The invention determines the speed and / or acceleration at which the new position will be approached and automatically adjusts to changes in image generation parameters. According to the invention, at least one image generation parameter is automatically changed during the movement of the sample. It is provided that the exposure time is reduced as an image generation parameter. In addition, it can be particularly advantageous to increase the frame rate as an image generation parameter. By changing such suitable image generation parameters during the movement of the sample, disruptive motion artifacts in the image can be reduced or completely avoided. A faster image sequence or frame rate leads to a faster refresh of the generated image, while a shorter exposure time (generally, latency) increases the temporal resolution and thus results in imaging adapted to the movement.

[0014] According to the invention, at least one image generation parameter is changed depending on a motion variable during the movement of the sample. This "motion variable" can be one of the following parameters or a combination of such parameters: a movement velocity (absolute or relative change in distance or range per unit time), a movement acceleration (change in movement velocity per unit time), a change in the current coordinates of a reference point on the sample and / or on the microscope stage and / or on the microscope objective. In the case of an impending, i.e.,For the intended movement of the sample, such a movement quantity is determined by using the known device parameters, such as the control curves for the movement of the microscope stage, and the intended displacement of the sample (for example, after entering the new sample position), to determine or predict the corresponding movement quantity (typical movement speed or acceleration of the microscope stage when approaching the new position) in advance.

[0015] Determining such a motion parameter allows the relevant image generation parameter(s) to be modified depending on the determined motion parameter, for example, directly proportionally to it or according to a predefined function depending on the motion parameter. Furthermore, this allows the relevant image generation parameter(s) to be modified only when the motion parameter of the actual movement or the predicted (i.e., intended) movement exceeds a predefined threshold. In this way, minor or slow movements, which consequently lead to only minor motion artifacts, can be disregarded. Conversely, movements over larger distances or rapid movements can be taken into account. This is the case, for example, when the sample is moved partially or completely out of the field of view of a microscope objective or moves out of it on its own.Intrinsic movements of the sample are explicitly included. These occur, for example, in the movement of organelles, movement of single-celled organisms, movement of organs or muscles, such as heartbeat, in small organisms, or movement of the organisms themselves, such as in live cell imaging.

[0016] For example, an intended, i.e., imminent, movement of the sample can be detected when a user input is received to change the sample's coordinates. This user input could include, for example, one or more key presses, touching a touchscreen, gesture input, or voice input. It is also possible to use a separate input device that communicates with the microscope and allows for the selection of a stage movement.

[0017] This can involve a control command for a translational movement of the sample within the field of view and / or for a movement along the optical axis (z-direction). In this case, the system or control unit of the microscope can, for example, also check whether the impending movement of the sample or the corresponding movement magnitude exceeds the predefined threshold. Alternatively, it is possible to operate without a threshold. In this case, the at least one image generation parameter is changed whenever the movement magnitude changes.

[0018] For this purpose, at least one image generation parameter can be changed by a fixed amount or set to a predefined value.

[0019] Alternatively, the change in at least one image generation parameter can be dynamically adjusted in line with the movement of the sample. For this purpose, a function can be stored in the microscope's processing unit that defines a continuous change in at least one image generation parameter depending on the change in the movement amplitude.

[0020] Alternatively, several threshold values ​​can be specified, so that, depending on the magnitude of the movement or the intended movement, certain image generation parameters are changed when the respective threshold values ​​are exceeded and / or a specific image generation parameter is adapted to the magnitude of the movement in different ways.

[0021] This application concerns confocal microscopes. With a confocal microscope, it is advantageous to increase the scanner frequency of the confocal microscope during sample movement as an image generation parameter. Alternatively, the scan resolution of the confocal scanner can be reduced.

[0022] Additionally, for confocal microscopes it can be useful to reduce downstream image processing during sample movement, for example by limiting or stopping the processing of multiple images or post-processing of images.

[0023] Furthermore, it is advantageous to compensate for any light loss or, more generally, any reduction in the amount of light detected or the measured signal strength associated with a change in at least one image generation parameter. In this way, the image quality, in terms of brightness and contrast, can be maintained as much as possible. It is advantageous to compensate for any light loss or the aforementioned signal strength reduction by increasing the gain of a sensor used for imaging, such as the aforementioned light-sensitive imaging detector or the aforementioned camera.Additional or alternative measures include increasing the exposure intensity of a microscope illumination unit used to illuminate the sample to be imaged, enlarging a pinhole used in a confocal microscope, reducing the resolution in the generated image (binning), and / or adjusting the image contrast when displaying the image to a user.

[0024] In a further advantageous embodiment of the method according to the invention, the end of the sample's movement is detected. This is easily achieved, for example, when a command to move the sample is detected, since the movement ends upon reaching the new position. Alternatively, the actual movement is tracked, and after a predetermined period of inactivity, the movement can be considered to have ended. After the movement has ended, the image generation parameters set before the movement are reset. It is advantageous to save the currently set image generation parameters for this purpose. At the very least, the image generation parameters should be saved before any changes occur due to sample movement. Alternatively, new image generation parameters can be set after the sample's movement has ended.

[0025] The invention further relates to a microscope comprising a microscope objective and an image generation device connected downstream of the microscope objective in an observation beam path of the microscope for image generation with predetermined image generation parameters. The microscope further comprises a microscope stage for receiving a sample to be imaged and a positioning unit for positioning the sample (or more precisely, an arbitrarily selectable reference point of the sample) in the object space of the microscope, wherein the positioning unit is in communication with the image generation device such that, after detecting a movement or an intended, i.e., imminent, movement of the sample (depending on an arbitrarily selectable reference point of the sample) in the object space of the microscope, ...The microscope according to the invention includes, in particular, a control unit that communicates with the positioning unit and the image generation unit, i.e., is connected to them via a signal line or wirelessly, and is configured such that, after detecting movement or an intended, i.e., imminent, movement of the sample, at least one image generation parameter is changed during the movement of the sample, depending on a movement parameter.

[0026] Detecting an actual or imminent movement of the sample can be done, for example, via the positioning unit by evaluating control signals from the control unit to the positioning unit or control signals from the positioning unit itself.

[0027] Alternatively or additionally, for example, a signal from a measuring beam reflected by the sample can be evaluated to infer a change in the sample's position. Such position-sensitive measuring beams are known, for example, from autofocus technology.

[0028] Alternatively or additionally, the microscope can be equipped with an image evaluation unit which detects the movement of the sample by evaluating the image and determines a movement parameter, whereby at least one image generation parameter is changed during the movement of the sample, provided that, for example, one of the following conditions is met beforehand: A movement is detected and a corresponding trigger signal is generated; a movement parameter is determined and compared with a threshold value, whereby a trigger signal is generated if the threshold value is exceeded; a movement parameter is determined and a trigger signal is generated that is proportional to the value of the movement parameter; a movement parameter is determined and a trigger signal is generated that is proportional to the value of the movement parameter, whereby if a threshold value is exceeded, the proportionality factor of the trigger signal is changed;

[0029] The microscope objective, together with the microscope's image generation unit, serves to produce an image of the sample or a section of the sample. The microscope stage accommodates the sample to be imaged and is generally movable in a plane perpendicular to the optical axis defined by the microscope objective. The microscope stage and / or the microscope objective are often movable in the direction of the optical axis (z-direction), particularly for adjusting the focus. It should be noted again that all movements are to be interpreted as relative movements of the sample with respect to the optical axis of the microscope objective, thus also including so-called fixed-stage microscopes. The positioning unit of the microscope according to the invention positions the sample in the object space of the microscope, that is, at a reference point on the sample chosen at specific xyz coordinates.

[0030] For the implementation of the method according to the invention, the aforementioned control unit of the microscope according to the invention is advantageously provided. This control unit communicates with the positioning unit on the one hand and with the image generation unit on the other, and is configured such that, depending on a movement parameter, at least one image generation parameter is changed during movement of the sample in the x, y, and / or z direction. The control unit can also represent or include an evaluation unit that detects movement of the sample according to the possibilities described above and / or determines the corresponding movement parameter. For details and advantages of the microscope according to the invention, reference is expressly made to the above explanations in connection with the method according to the invention.

[0031] In an advantageous embodiment, the microscope has an illumination unit for generating an illumination beam path directed onto the sample, wherein the illumination intensity of the illumination unit is adjustable, particularly also via the control unit. In this way, any light losses or reductions in image signal strength that may occur as a result of a change in an image generation parameter can be compensated for by increasing the illumination intensity.

[0032] It is advantageous if the microscope according to the invention includes a storage unit for storing set image generation parameters. In this way, after detection of the end of the sample movement, the image generation parameters set before the sample movement can be reset.

[0033] It is further advantageous if the microscope's control unit is configured to detect an intended movement of the sample when a user input is made to change the sample's coordinates. Reference is made to the above explanations in connection with the method according to the invention.

[0034] Furthermore, it is advantageous if the microscope's control unit is configured such that at least one image generation parameter is only changed during sample movement when the magnitude of the movement or the intended movement exceeds a predetermined threshold. Reference is also made to the above explanations in connection with the method according to the invention regarding this configuration.

[0035] In an alternative embodiment, the microscope's control unit is configured such that at least one image generation parameter is changed proportionally to the magnitude of the movement or the intended movement during the movement of the sample. Reference is also made to the above explanations in connection with the method according to the invention with regard to this embodiment.

[0036] The microscope according to the invention is designed as a confocal microscope and comprises a scanning unit for scanning an illumination beam path across the sample to be imaged, wherein the scanner frequency and / or the scan resolution of the scanning unit is variable. With regard to the aforementioned embodiments of the microscope according to the invention, reference is made to the above descriptions and to the following exemplary embodiments.

[0037] Finally, the invention relates to a computer program with program code, the execution of which on a computing unit, in particular the control unit of the microscope according to the invention, carries out a method according to the invention. Furthermore, the invention relates to a corresponding computer program product with a computer program containing program code stored thereon, the execution of which on a computing unit, in particular the control unit of the microscope according to the invention, carries out a method according to the invention. The computer program can be downloaded or uploaded as such, or stored or cached on a computer program product, or implemented as firmware. Suitable computer program products include volatile or non-volatile storage media, such as a USB flash drive, RAM, or ROM memory of a known type.The aforementioned computing unit can be the control unit of the microscope according to the invention or a part of this control unit.

[0038] Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawing.

[0039] It is understood that the features mentioned above and those to be explained below can be used not only in the combination specified, but also in other combinations or on their own, without leaving the scope of the present invention.

[0040] The invention is schematically illustrated in the drawing using an exemplary embodiment and is described below with reference to the drawing. Character description

[0041] Figure 1 schematically shows the most essential components of a microscope according to the invention in a first embodiment, Figure 2schematically shows the most essential components of a microscope according to the invention in a second embodiment, Figure 3 schematically shows the most essential components of a microscope according to the invention in a third embodiment and Figure 4 shows the sequence of process steps of a method according to the invention in accordance with possible embodiments.

[0042] The figures are treated collectively below; identical reference symbols denote structurally and / or functionally identical elements. The microscopes according to

[0043] Figures 1 to 3 The components shown are merely schematic and are essential for the present invention. Further components of the microscopes discussed therein are known from the prior art.

[0044] Figure 1Figure 1 shows a transmitted light microscope belonging to the class of wide-field microscopes. The microscope 1 includes an illumination unit 17, which is arranged below the microscope stage 13. The microscope objective is designated 11. It defines an optical axis 20 in the usual manner. The illumination unit 17 typically includes illumination optics (not shown) for generating an illumination beam path 18 that illuminates a sample arranged on the microscope stage 13. The sample is imaged by the microscope objective 11 and downstream imaging optics (also not shown), whereby a light-sensitive detector 16, for example a camera, can capture the corresponding image, which is displayed on a display unit 24. The observation beam path is labeled 19. The image-generating device 12 comprises the image-generating optics (not shown) and the light-sensitive detector 16.The microscope stage 13 is an xy-microscope stage that can be moved in the two spatial directions x and y, perpendicular to the optical axis 20 and thus perpendicular to the z-axis, which runs parallel to the optical axis. A positioning unit 14, which controls the corresponding drives of the xy-stage, serves this purpose. A user interface for setting microscope parameters is designated 21. Via the user interface 21, a user can specify the desired microscopy procedure and the parameters to be selected. A control unit 15, which communicates with the user interface 21, then typically takes over the setting of all microscope parameters for the selected microscopic imaging largely automatically.

[0045] As from Figure 1As can be seen, the control unit 15 communicates with the positioning unit 14 and with the image generation unit 12, specifically with the light-sensitive detector or camera 16 of the image generation unit 12. The image is displayed on a display unit 24, which is connected to the control unit 15 and / or the camera 16. If a sample located on the microscope stage 13 is moved in any direction during microscopic imaging, or moves itself in any direction, motion artifacts will occur in the recorded microscope image for the reasons mentioned above. In the following, without loss of generality, only sample movements in the xy-plane will be considered. Figure 1The microscope 1 shown is also equipped for sample movements in the z-direction if the microscope stage 13 shown therein is movable in the z-direction. If only the microscope objective 11 should be movable in the z-direction, i.e., parallel to the optical axis 20, then in this case the positioning unit 14 would either be connected to the z-drive of the objective 11 or the control unit 15 would be in communication with a separately controllable z-drive of the objective 11.

[0046] When the sample is moved in the xy-plane, at least one image generation parameter can be changed immediately after the movement begins in such a way as to minimize or eliminate any resulting motion artifacts. For this purpose, the control unit 15 communicates with the camera 16, particularly to shorten the camera's exposure times. Due to its connection to the microscope stage 13 or the positioning unit 14, the control unit 15 can register the end of the movement. The system then automatically reverts to the image generation parameters set before the movement or sets new ones. These parameters are advantageously stored in a memory 22, which in this case is part of the control unit 15. Of course, this memory 22 can also be implemented independently of the control unit 15.The control unit 15 can also represent or include an evaluation unit that detects movement of the sample according to the possibilities described above and determines the corresponding movement magnitude. In an alternative embodiment, the positioning unit 14 is directly connected to the camera 16 (dashed line). In this way, the control unit 15 can be omitted entirely or partially. The functions necessary for changing the image generation parameters are then implemented via the direct connection between the positioning unit 14 and the camera 16.

[0047] Furthermore, it is advantageous if the image processing of microscopic images, which is usually carried out during the movement of the sample, is reduced in order to better adapt the image generation to the dynamics of the sample movement.

[0048] As from Figure 1As can be seen, the control unit 15 is connected to the illumination unit 17 via a signal line. Naturally, all signal and communication connections shown here can be implemented via wires or wirelessly. The connection to the illumination unit 17 serves the purpose of compensating for the light loss that occurs during imaging with a shortened exposure time. To this end, the control unit 15 increases the illumination intensity of the illumination unit 17 in such a way as to keep the image brightness essentially constant. This function can, in principle, alternatively be implemented via a direct connection between the positioning unit 14 and the illumination unit 17 (dashed line). In this example as well, the original illumination intensity can be restored after the sample movement has ceased.

[0049] In addition to cases where the system itself registers sample movement, there are also conceivable scenarios where the system can detect an intended or imminent sample movement. This occurs, for example, when a user enters a new sample position in the x, y, and / or z direction via the user interface 21, which is then approached by the positioning unit 14. For this purpose, the microscope stage 13 and / or the microscope objective 11 are moved accordingly. As mentioned above, the term "sample movement" encompasses any relative movement between the sample and the objective 11. Thus, if a user selects a new xy position and / or a new focus point via the user interface 21, the control unit 15 can trigger the corresponding change to at least one image generation parameter, so that the changed image generation parameters are set without delay at the start of the movement.Simultaneously, the control unit 15 can save the original settings in memory 22. If a change to the image generation parameters is to be made only when a predefined threshold value of a movement variable is exceeded, the control unit 15 can check in advance whether the upcoming movement exceeds this threshold value or not. A minimum travel distance, a minimum sample movement speed, or a minimum sample movement acceleration could be specified as a threshold value for such a movement variable.

[0050] Figure 2Figure 1 shows another embodiment of a wide-field microscope 2, here in the form of an epifluorescence microscope. Again, only the essential features of such a microscope 2 are shown. This again comprises a microscope stage 13 and a microscope objective 11. A sample is arranged in the object space between the objective 11 and the microscope stage 13. This sample is illuminated by reflected light. An illumination unit 17 serves to generate an illumination beam path 18. The generated illumination beam path 18 is coupled into the optical axis 20 of the microscope 2 via a deflecting element 23 and falls onto the sample via the objective 11. The fluorescence radiation emitted by the sample passes through the objective 11 and the deflecting element 23, which is transparent to this radiation, as well as an image-generating optic (not shown here), to the light-sensitive detector 16, in this case a camera.Such fluorescence microscopes 2 are known from the prior art and will therefore not be explained further here.

[0051] The control unit of the microscope 2 is designated 15. It communicates with both the camera 16 and the positioning unit 14 of the microscope stage 13. Additionally, the control unit 15 can also communicate with the illumination unit 17. Finally, a user interface 21 can be provided, which communicates with the control unit 15.

[0052] Regarding the implementation of different embodiments of the method according to the invention, the following applies to an epifluorescence microscope 2 according to Figure 2 the completely analogous way to Figure 1executed. Again, if sample movement is detected, the exposure time of camera 16 can be reduced to minimize motion artifacts in the image. Simultaneously, the illumination intensity of the lighting unit 17 can be increased, for example, to compensate for the associated light losses. Regarding further developments, such as the detection of an intended or imminent movement, or exceeding a threshold value of a movement parameter, please refer to the explanations in connection with Figure 1 The referenced above applies in a completely analogous manner. The same applies to the alternatively conceivable direct connection of the positioning unit 14 with the camera 16 and / or the lighting unit 17 (dashed lines).

[0053] Figure 3Figure 3 shows a further embodiment of a microscope 3, a confocal microscope with its main elements. The microscope 3 again comprises a microscope stage 13 and a microscope objective 11. An illumination unit 17 generates an illumination beam path 18. This beam is coupled parallel to the optical axis 20 via a deflecting element 23 and focused by the microscope objective 11 onto the sample area to be imaged. Light emanating from this point passes conversely through the microscope objective 11 and the downstream image generation unit 12 to the light-sensitive detector 32, for example, a photomultiplier tube (PMT) or a photon counter (e.g., a hybrid detector). A known scanning unit 31 scans the illumination beam path 18 across the sample area to be imaged in a predefined manner. The image frequency or frame rate is thus predetermined.Therefore, within the scope of the present application, the scanning unit 31 is part of the image generation device 12. It should also be noted that other configurations of a confocal microscope are conceivable, in particular those in which the deflecting element 23 and the scanning unit 31 form a single structural unit, without departing from the basic idea of ​​the present invention.

[0054] In the case of the confocal microscope 3 according to Figure 3 Once sample movement has been detected, a high-quality image can be generated by the control unit 15 being in communication with the scan unit 31 and / or with the light-sensitive detector 32, as well as with the positioning unit 14 and preferably also with the illumination unit 17. Again, the Figure 3The dashed lines illustrate that, alternatively, the positioning unit 14 can be directly connected to the scan unit 31, the illumination unit 17, and / or the detector 32, bypassing or eliminating the control unit. In particular, increasing the scanner frequency of the scan unit 31 increases the frame rate to reduce motion artifacts that occur during sample movement. Furthermore, to compensate for light loss, the gain of the detector 32 can optionally be increased and / or the illumination intensity of the illumination unit 17 can be increased.

[0055] Regarding further embodiments of the example according to Figure 3 Reference is expressly made to the statements in connection with the Figure 1 and 2 referenced, which apply here in a completely analogous way.

[0056] Figure 4Figure 1 shows the sequence of process steps S1 to S7 in different embodiments of a method according to the invention. This method comprises the following steps: S1 denotes the step of continuous imaging or the generation of a live image with a microscope according to the invention, as is the case, for example, with the Figures 1 to 3as described. Step S2 refers to two subcases: case S2a, which involves continuously tracking the relative position of the sample, specifically its xyz coordinates, and case S2b, which involves user input to move the sample to new xyz coordinates. Step S3 describes two subcases: for example, a movement in the xyz direction (S3a), particularly in the case S2a described above, which involves continuously tracking the sample position with continuous changes to the image generation parameters, for example, via a predefined function. Alternatively, the determination of when a threshold value of a motion parameter of this sample movement or displacement is exceeded can be used (step S3b), after which a stepwise or then also continuous change of the image generation parameters takes place. In step S4, the control unit of the microscope triggers the change of the at least one image generation parameter according to the method of the invention, as illustrated by the exemplary embodiments shown. Figures 1 to 3This was described as an example (S4b). To be able to restore the original settings at a later time, the image generation parameters can be saved in a preliminary step S4a, as they were when sample movement was detected. In step S5, image generation continues with the modified parameters while the sample is moving. In step S6, the end of the sample movement is detected / predicted. Subsequently, in step S7, the control unit can either determine new image generation parameters for further imaging (step S7a) or restore the image generation parameters saved in step S4a (step S7b ). The imaging process is then resumed, which corresponds to step S1 at the beginning of the procedure.

[0057] After an actual or imminent movement or displacement is detected in step S2 and one of the procedures according to S3a or S3b is selected, whereby steps S2a and S3a can generally also be carried out in parallel, the control unit of the microscope triggers the change of the at least one image generation parameter in step S4b according to the method according to the invention, as illustrated by the exemplary embodiments shown. Figures 1 to 3This has been described as an example. To be able to restore the original settings at a later time, the image generation parameters can be saved in a preliminary step S4a, as they were when sample movement was detected. Instead of detecting sample movement or displacement with continuous changes to the image generation parameters in step S3a, the determination of whether a threshold value of this sample movement or displacement is exceeded can alternatively be performed (step S3b). In this case, it can be determined, for example, whether the sample movement exceeds a certain minimum speed or not. Only if a certain minimum speed is exceeded are the image generation parameters changed.

[0058] After changing at least one image generation parameter in step S4, image generation continues with the changed parameters during sample movement (step S5). The resulting image provides better images without motion artifacts compared to the prior art with unchanged image generation parameters. In step S6, the end of sample movement is detected / anticipated. The control unit can then either determine new image generation parameters for further imaging (step S7a) or restore the image generation parameters stored in step S4a (step S7b). Imaging then resumes with these image generation parameters, corresponding to step S1 at the beginning of the process.

[0059] In the event of user input for sample movement or displacement to new coordinates in step S2b, the system recognizes an intended, imminent movement of the sample. Subsequently, the image generation parameters present before the movement can be saved immediately (S4a), and the change of at least one image generation parameter can be triggered by the control unit (S4b). Both steps S4a and S4b can be triggered immediately upon commencement of the sample movement. The steps S5, S6, and S7, already described above, then follow. In this case as well, a change of at least one image generation parameter can only be made when a predetermined threshold of a movement variable is exceeded (S3b instead of S3a). The further course of the process corresponds to the embodiments already described in detail above. Reference symbol list

[0060] 1, 2, 3 Microscope 11 Microscope objective 12 Image generation unit 13 Microscope stage 14 Positioning unit 15 Control unit 16 Detector, camera 17 Illumination unit 18 Illumination beam path 19 Observation beam path 20 Optical axis 21 User interface 22 Memory 23 Deflection element 24 Display unit 31Scanning unit 32Detector x, y, zSpatial direction S1,S2,S2a,S2b,S3,S3a,S3b,S4,S4a,S4b,S5,S6,S7a,S7bProcedure steps

Claims

1. Method for image generation by means of a microscope (3), wherein, for image generation with predefined image generation parameters, a sample to be imaged is located in the object space of the microscope, wherein first a movement or an intended movement of the sample is detected and thereupon, depending on a movement variable, at least one image generation parameter is automatically changed during the movement of the sample, characterized in that a confocal microscope (3) is used as microscope and, as image generation parameter, a scanner frequency of a scanning unit (31) of the confocal microscope (3) is increased or a scan resolution of the scanning unit (31) is reduced.

2. Method according to claim 1, characterized in that at least one image generation parameter is automatically changed during the movement of the sample when the movement variable of the movement or of the intended movement exceeds a predefined threshold value.

3. Method according to claim 1 or 2, characterized in that an intended movement of the sample is detected when a user input for changing the position of the sample is made.

4. Method according to one of claims 1 to 3, characterized in that, as image generation parameter, the image frequency is increased and / or the exposure time is reduced.

5. Method according to one of claims 1 to 4, characterized in that a reduction, accompanying a change of said at least one image generation parameter, of an amount of light detected for image generation or of signal strength used for image generation is compensated for.

6. Method according to claim 5, characterized in that the compensation of the reduction of the measured amount of light is carried out by an increase of the gain of a sensor (16, 32) used for image generation, an increase of the illumination intensity of an illumination unit (17) used for illumination, an enlargement of a pinhole used in a confocal microscope (3), a reduction of the resolution and / or an adaptation of the image contrast in the generated image.

7. Method according to any one of the preceding claims, characterized in that the change of said at least one image generation parameter is carried out dynamically.

8. Method according to any one of the preceding claims, characterized in that an end of the movement of the sample is detected and, after the end of the movement, the image generation parameters set before the movement of the sample are set again or new image generation parameters are set.

9. Microscope (3) comprising a microscope objective (11) and an image generation device (12), downstream of the microscope objective (11) in an observation beam path (19) of the microscope, for image generation with adjustable image generation parameters, a microscope stage (13) for receiving a sample to be imaged, a positioning unit (14) for positioning the sample in the object space of the microscope, wherein the positioning unit (14) is in communication connection with the image generation device (12) such that, after detection of a movement or of an intended movement of the sample, depending on a movement variable, at least one image generation parameter is changed during the movement of the sample, characterized in that the microscope is configured as a confocal microscope (3) and comprises a scanning unit (31) for scanning an illumination beam path (18) over the sample to be imaged, wherein the scanner frequency or the scan resolution of the scanning unit (31) can be changed, and in that, as image generation parameter, the scanner frequency of the scanning unit (31) is increased or the scan resolution of the scanning unit (31) is reduced.

10. Microscope according to claim 9, comprising a control unit (15), which is in communication connection with the positioning unit (14) and the image generation device (12), wherein the control unit (15) is configured such that, after detection of a movement or of an intended movement of the sample, depending on a movement variable, at least one image generation parameter is changed during the movement of the sample.

11. Microscope according to claim 9 or 10, wherein, for detecting a movement of the sample, an evaluation unit is present, which is configured such that control signals for the positioning unit (14) are evaluated and / or a position-sensitive measurement beam signal of a measurement beam reflected by the sample is evaluated and / or an image of the sample is evaluated by means of image processing.

12. Microscope according to one of claims 9 to 11, wherein the microscope comprises an illumination unit (17) for generating an illumination beam path (18) directed onto the sample, wherein the illumination intensity is adjustable.

13. Microscope according to one of claims 9 to 12, wherein the microscope comprises a storage unit (22) for storing set image generation parameters.

14. Microscope according to one of claims 9 to 13, wherein the control unit (15) of the microscope is configured such that an intended movement of the sample is detected when a user input for changing the position of the sample is made via a user interface (21).

15. Microscope according to one of claims 9 to 14, wherein the control unit (15) of the microscope is configured such that said at least one image generation parameter is changed during the movement of the sample when the movement variable of the movement or of the intended movement of the sample exceeds a predefined threshold value.

16. Computer program with program code for carrying out all method steps of a method according to one of claims 1 to 8 when the computer program is executed on a computing unit or a control unit (15) of a microscope (3) according to one of claims 9 to 15.

17. Computer program product with a computer program with program code for carrying out all method steps of a method according to one of claims 1 to 8 when the computer program is executed on a computing unit or a control unit (15) of a microscope (3) according to one of claims 9 to 15.