Method of controlling microscopic imaging, corresponding microscope control device, and microscope

Abstract

To provide an improved method for controlling microscopic imaging, a corresponding microscope control device for controlling microscopic imaging, and a corresponding microscope comprising such a microscope control device.SOLUTION: A method of controlling microscopic imaging of a microscope is provided, comprising providing a microscope control device configured to receive a focusing request and sample information on a sample to be imaged (S1). Responding to receipt of the focusing request after receiving the sample information (S2), the microscope control device activates a predefined focusing setting depending on the received sample information to control focusing of the microscope for microscopic imaging of the sample (S3).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for controlling microscope imaging of a microscope, a microscope control device for controlling microscope imaging of a microscope, and a microscope including the microscope control device. More specifically, microscope imaging requires focusing control to ensure that the acquired image is in focus.

Background Art

[0002] In microscope imaging, the user defines appropriate microscope imaging parameters for imaging a sample and requires advanced expertise to maintain the focusing of the sample. The microscope imaging parameters vary depending on the microscope imaging mode (e.g., wide field or confocal) and the type of sample being imaged. In addition, during the inspection of the sample, the focus and / or the sample itself may drift over time. Therefore, in recent years, specialized knowledge has often been required for microscope imaging.

[0003] In microscopy, an autofocusing method based on the basic method of triangulation autofocusing is widely used, for example, described in German Patent Application Publication No. 102010030430, U.S. Patent No. 5136149, and German Patent Application Publication No. 19537376.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The problem of the present invention is to provide an improved method for controlling microscope imaging, a microscope control device for controlling corresponding microscope imaging, and a corresponding microscope including the microscope control device.

Means for Solving the Problems

[0005] This problem is solved by a method for controlling microscopic imaging of a microscope according to claim 1. The method includes providing a microscope control device configured to receive focusing requests and sample information relating to a sample to be imaged. The microscope control device, in response to receiving a focusing request after receiving sample information, activates a predefined focusing setting depending on the received sample information to control the focusing of the microscope for microscopic imaging of the sample. The microscope control device may include a graphical user interface (GUI) or other arbitrary interface adapted to receive focusing requests and sample information relating to a sample to be imaged. In the microscope control device, the corresponding request or sample information may be received directly by the user or indirectly by another device, for example, triggered by the user. For example, sample information relating to a sample to be imaged may be received from an automatic sample detection unit implemented in the microscope. In this case, in response to receiving a focusing request, the microscope control device will request and / or receive sample information from the automatic sample detection unit. In this context, it should be noted that the microscope control device may receive sample information before, simultaneously with, or after receiving a focusing request. Next, the microscope control unit activates predefined focusing settings based on the received sample information. Focusing requests are typically entered by the user and can be received by the microscope control unit directly or indirectly.

[0006] "Activation of a predefined focus setting" means that a predefined focusing setting adapted to the sample being imaged is applied to the microscope's focusing device so that the acquired image is in focus without requiring further user input. This activation of a predefined focusing setting is also an embodiment of the present invention and is performed by a microscope control device operably coupled to such a focusing device of the microscope in order to realize and control the setting of all parameters of the components of the microscope focusing device adapted to the sample being imaged during microscope imaging.

[0007] Instead of prompting the user to decide what focusing strategy to use (e.g., whether to create a focus map or perform focus correction during image acquisition), what focusing mechanism to use (e.g., image-based or reflection-based), and / or what capture range to define for image-based autofocusing, and / or how many focuses to set on or within the sample for focus map generation, and / or how to correct for focus drift, the microscope control unit initiates the decision by activating predefined focusing settings. Thus, embodiments of the present invention enable users without prior knowledge of the concept of microscope focusing to acquire a focused and / or z-stacked microscope image around the focal plane simply by defining the type of sample to be imaged, and thereby have all necessary focusing settings automatically made by the microscope control unit. However, this process does not mean that the possibility of the user overriding the settings by entering settings into a settings menu and / or the possibility of the user further adjusting the predefined focusing settings according to their own knowledge and needs is excluded.

[0008] In one embodiment, the sample information received by the microscope control device relates to one or more sample characteristics in the three-dimensional sample space and / or the type of sample carrier supporting the sample and / or the material of the sample carrier supporting the sample and / or the sample staining typically used in fluorescence microscopy. The sample characteristics in the three-dimensional sample space particularly relate to the constant or changing elongation of the sample in the focal direction, the sample attached to or separated from the sample carrier, the sample adhered to the sample carrier, the sample that can move particularly in the focal direction, or the sample having a surface topology that changes over time. The type of sample carrier supporting the sample relates particularly to slides, Petri dishes, well plates, multi-chambers, lab-on-a-chips, etc. Regarding the refractive index, the material of the sample carrier also has an effect, with typical materials being glass or plastics such as PMMA. Regarding sample characteristics, it may also be useful to identify whether the sample is alive or immobilized. Knowledge of sample staining can be important when selecting the desired emission wavelength for image acquisition.

[0009] In one embodiment, the microscope control unit does not need to receive additional information, particularly user input, to activate a predefined focusing setting. For example, when a user activates focusing, the microscope control unit, in response to receiving the corresponding focusing request, activates a predefined focusing setting that matches a defined sample type, defined by user input and / or sample information received from the automatic sample detection unit. No further information from the user is required to activate the predefined focusing setting. This dramatically improves the user-friendly operability of the microscope, allowing even inexperienced users to operate it.

[0010] In one embodiment, a predefined focusing setting includes the automatic, system-based determination of one or more focal points at defined x and y positions of the sample.

[0011] For attached samples that are directly attached to the bottom of the sample carrier or have a specific surface topology, a single focus may suffice. The focus must be maintained constant, and focusing must be maintained over a defined x and y range.

[0012] A focus map containing one or more such foci across a defined xy range is typically applied to separated samples.

[0013] In the case of dynamic samples, because the sample is in motion, it is virtually impossible to define a focus map in which the focus, particularly its z-value, depends on the x and y positions of the sample at a given time point in time. Therefore, the focus cannot be determined in advance. Methods for defining focusing settings for various sample types will be described in more detail in the detailed explanation section.

[0014] In another embodiment, one or more of the one or more focal points can be overridden by the user according to their own knowledge or experience and needs gained during imaging of a particular sample.

[0015] In further embodiments, one or more focal points are determined by an autofocusing method that depends on a predefined and activated focusing setting. In one embodiment, the autofocusing method includes at least one of a triangulation autofocusing method and an image-based autofocusing method that evaluates an image stack of a sample in which each image is captured at a different focal position. Autofocusing methods are described in more detail in the detailed description section.

[0016] In one embodiment, the image-based autofocusing method includes the steps of generating a first image stack of a sample by capturing each image at a different focal position using a first focus step size over a first range of focal positions, and generating a second image stack of the sample using a second focus step size over a second range of focal positions, wherein the second focus step size is smaller than the first focus step size. By applying the first "coarse" focus step size over the first range, a first "coarse" focus level can be obtained in a short time. Starting from this, the optimal focus level can be determined with high accuracy by applying a smaller second focus step size over a smaller second range around the first "coarse" focus level.

[0017] In one embodiment, a predefined focusing setting is corrected for focal drift over time. In particular, focal drift is corrected in at least one of the following cases: after a predetermined number of image acquisition positions at different x and y positions of the sample; after a predetermined number of image acquisition points; after a predetermined time interval; and when there are two or more foci for a predetermined number of foci. Further details on drift correction are provided in the detailed description section.

[0018] In one embodiment, sample information relating to the sample to be imaged is pre-categorized into at least two different sample specifications, particularly predefined sample specifications that can be selected by the user. This improves user convenience when defining sample information for the sample to be imaged.

[0019] With respect to predefined sample specifications, particularly sample properties in three-dimensional sample space, a sample may have at least one of the following characteristics: attached to a sample carrier supporting the sample ("attached" sample), separated from a sample carrier supporting the sample and / or elongating while changing in the focal direction ("separated" sample), or capable of moving particularly in the focal direction and / or having a surface topology that changes over time ("dynamic" sample).

[0020] In further embodiments, if the sample is "adhered," autofocusing control based on triangulation autofocusing is used to determine and / or maintain the focus level used for sample imaging.

[0021] In another embodiment, if the sample is “separated”, a focus map of one or more foci is defined. In one embodiment, the focus map is defined by a predefined or selectable number of foci and / or foci density within a region of interest (“ROI”).

[0022] Furthermore, the focus map can be determined by an image-based autofocusing method that evaluates the image stack of the sample, where each image is captured at a different focal point. Such an image-based autofocusing method may be reapplied during the imaging of the sample. After determining the focus map or during the imaging of the sample, autofocusing control based on triangulation autofocusing may be used to maintain focus in a time-saving manner. Further details are provided in the detailed description section.

[0023] In another embodiment, if the sample is "dynamic," each image is captured at a different focal position, and autofocusing control based on an image-based autofocusing method that evaluates the image stack of the sample is used to find the optimal focus for imaging the sample at a given time.

[0024] In another embodiment, the microscope control device is configured to enable different microscope imaging modes that are selectable independently of a predefined activated focusing setting. Such microscope imaging modes can include wide-field, confocal or light-sheet microscopes. In the case of fluorescence microscope imaging, the two different microscope imaging modes can be wide-field microscopy and confocal microscopy. What is particularly useful in this embodiment is that since the predefined focusing setting remains unchanged or is automatically adapted by the microscope control device, the user does not need to specify different focusing settings when changing from one microscope imaging mode to another.

[0025] In another aspect of the present invention, a microscope control device is provided. The microscope control device includes one or more processors and is adapted to control microscope imaging of a microscope. The microscope control device is configured to receive sample information about a sample to be imaged and receive a focusing request. The microscope control device is further configured to activate a predefined focusing setting depending on the received sample information in order to control the focusing of the microscope for imaging the sample in response to receiving the focusing request.

[0026] In one embodiment, the microscope control device is configured to execute the method according to the present invention.

[0027] In one embodiment, the microscope control device comprises a graphical user interface including a control widget configured to receive user input. The control widget includes at least one focusing control widget for the user to receive a focusing request and at least one sample information widget for the user to receive sample information.

[0028] In another aspect of the present invention, a microscope for microscopic imaging of a sample is provided. The microscope includes a focusing device and a microscope control device, and the focusing device is operably coupled to the microscope control device.

[0029] It should also be noted that the description of the method and method embodiments shows the description of the microscope control device and microscope according to other aspects of the present invention and corresponding embodiments.

[0030] It should be noted that the above features of each embodiment can be combined in whole or in part to realize other embodiments that still fall within the scope of the concept of the present invention defined in the appended claims.

[0031] 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 " / ".

[0032] Further embodiments and their advantages will be described below in connection with the following figures. [[ID=IS]]

Brief Description of the Drawings

[0033] [Figure 1] It is a flowchart for controlling the microscopic imaging of a microscope. [Figure 2] It is a diagram showing a graphical user interface configured to receive sample information of a sample to be imaged. [Figure 3] It is a diagram showing a graphical user interface configured to receive a focusing request. [Figure 4] It is a diagram showing a graphical user interface corresponding to a first sample specification. [Figure 5] It is a diagram showing a graphical user interface corresponding to a second sample specification. [Figure 6]This figure shows a graphical user interface corresponding to the third sample specification. [Figure 7] This is a schematic diagram of a microscope used for microscopic imaging of a sample. [Modes for carrying out the invention]

[0034] Each figure is comprehensive, and the same reference number indicates the same or at least functionally identical element.

[0035] Figure 1 is a flowchart that can be adapted according to embodiments of the present invention. Microscopic imaging can be performed in different microscopic imaging modes, and the present invention has proven to be highly advantageous when using a microscope capable of activating different microscopic imaging modes. Microscopic imaging modes may include bright-field or transmitted light broad-field imaging modes and confocal imaging modes, or light-sheet imaging modes. In embodiments of the present invention, different microscopic imaging modes, particularly broad-field imaging modes and confocal imaging modes, can be selected on the same microscope, especially a fluorescence microscope used to image various different sample types.

[0036] In the first step, step S0, the user initiates microscopy imaging. For fluorescence imaging, the user can select the fluorescence channel, i.e., the emission wavelength of the stained sample to be observed. The user can also select the microscopy imaging mode.

[0037] In the next step S1, the sample information of the sample to be imaged must be input by either the user or the automated sample detection unit. In the embodiment shown in Figure 1, it is assumed that there are three different types of sample information S11, S12, and S13, or that the input sample information can be assigned to three different types of samples, or that one of three different types of predefined sample specifications can be selected. Note that the number of sample specifications can preferably be two or more, as will be described in more detail in relation to Figure 2.

[0038] In the next step, S2, the microscope control unit receives a focusing request, either directly or indirectly, from the user, or as a direct result of having fully received the necessary sample information.

[0039] In the next step, S3, a predefined focusing setting is activated depending on the received sample information S11, S12, or S13. Various available settings are described below in relation to the following diagram.

[0040] In one embodiment, steps S1 and S2 are both performed by user input. When the user sends a focusing request in S2 to initiate focusing, a predefined focusing setting matching the sample type defined by the user in S1 is activated.

[0041] It should be noted that steps S1 and S2 may be swapped so that the user (or the automated sample detection unit) is prompted to define the type of sample to be imaged, a focusing request is sent before the necessary sample information is received, and then a predefined focusing setting is selected.

[0042] In one embodiment, in step S4, the user may select a microscopy imaging mode for microscopic imaging of the sample. Furthermore, in step S5, the user may specify that z-stack imaging be applied, that is, multiple images of the sample are generated by capturing each image at a different focal position to obtain a substantially parallel image stack in the focal direction, from which a three-dimensional image can be generated.

[0043] It should be noted that steps S4 and S5 do not affect the process of selecting a predefined focusing setting that depends on the received sample information. On the other hand, in embodiments of the present invention, for convenience, the user can override a specific setting of the predefined focusing setting.

[0044] In one embodiment, the sample information relates to one or more sample characteristics in a three-dimensional sample space and / or the type and / or material of the sample carrier supporting the sample and / or the staining of the sample for fluorescence microscopy. To improve user convenience, one of a number of sample characteristics / specifications and / or one of various sample carriers can be selected by the user through a graphical user interface. This embodiment will be described in more detail with reference to Figure 2.

[0045] Since the predefined focusing settings are activated in step S3 based on the sample information received in step S1, and no further information needs to be received from the user, the user does not need extensive experience as a microscopy user other than their basic knowledge of the sample being imaged.

[0046] Figure 2 is a schematic diagram of the graphical user interface 200, and more specifically, a part of the menu of the graphical user interface used in a microscope for microscopy imaging according to an embodiment of the present invention. This part of the graphical user interface mainly concerns inputting sample information necessary to select predefined focusing settings. In this embodiment, the sample information consists of the type of sample carrier and / or the material / carrier type of the sample carrier, specific sample characteristics in a three-dimensional sample space that are pre-categorized by predefined sample specifications / characteristics, and sample staining characteristics in the case of fluorescence microscopy.

[0047] The graphical user interface 200 shown in Figure 2 has three menu bars 210, 220, and 230. The first menu bar 210 and the second menu bar 220 help the user select a carrier type and sample characteristics that match the sample being imaged. Menu bar 210 consists of a number of different types of carriers, which can be exemplary slides 212, petri dishes 214, well plates 216, and multi-chambers 218. The user can also specify the material of the sample carrier by selecting a menu point 217 assigned to each carrier type. Based on the selection of the carrier type and material, the software can determine the appropriate focal distribution or appropriate sample range for focusing settings to be used.

[0048] The menu bar 220 allows the user to select from predefined sample specifications / characteristics, which in this embodiment are exemplified by "adhered" 222, "separated" 224, "dynamic" 226, in addition to "fixed" 228 and "living" 229.

[0049] Specification "Adhered" 222 is characterized by substantially flat samples that are substantially topologically nonexistent or substantially constant in the focal direction, such as two-dimensional cell cultures on the bottom surface of a coverslip or well plate. Typically, "adhered" samples are directly attached to a sample carrier.

[0050] The “separated” sample 224 is characterized by being a sample that is elongating and / or separated from the bottom of the sample carrier, changing in the direction of the focal point, such as tissue placed on a slide without a coverslip. Both effects cause the focal point to change across the sample surface. As used herein, the “separated” sample does not move over time.

[0051] In contrast, the "dynamic" sample 226 is characterized by being a sample that moves over time, particularly approaching or moving away from the bottom of the sample carrier, or by having a topology on its surface that changes over time.

[0052] In addition to the three sample specifications 222, 224, and 226, the user can select whether the sample is "fixed" 228 or "living" 229. This specification is used by the software and can be switched to a different focusing setting where appropriate. For example, if the user selects the "separated" sample feature 224 for a living sample 229, the software may apply a predefined focusing setting for the "dynamic" sample feature 226 instead of setting the "separated" sample 224.

[0053] The menu bar 230 allows the user to select the type of sample staining. The user can select a dye by specifically choosing one of the sample staining types 232, 234, 236, or 238. This allows the user to select a specific fluorescence channel or emission wavelength that represents details of the sample of interest. For example, the cell nucleus or cell wall may be imaged with different fluorescence channels or emission wavelengths.

[0054] Figure 3 is a schematic diagram showing an embodiment of the graphical user interface 300, and more specifically, a part of the graphical user interface related to initiating focusing. The graphical user interface 300 in Figure 3 is provided with "F", which is a menu point or widget 310 for requesting a user-selectable focusing. If the sample definition according to the embodiment in Figure 2 has not been completed, in response to selecting the focusing request 310, the user will be prompted to define the sample. Selecting the menu point 310 corresponds to sending the focusing request by step S2 in Figure 1.

[0055] In embodiments of a fluorescence microscope, the user may further select or has already selected a microscope imaging mode, such as wide-field imaging or confocal imaging. Furthermore, the user may select three-dimensional imaging by selecting the menu point "3D Imaging" 320 in the graphical user interface 300 in Figure 3. When menu point 320 is selected, z-stacked images in the focal direction around the optimal focal level are acquired, and these images are further processed to generate a three-dimensional image. Step S5 in Figure 1 corresponds to the user selecting menu point 320 in Figure 3.

[0056] Any menu points or widgets depicted in Figures 2 to 6 but not described herein may not be relevant to the present invention as described herein, even if they are broadly related to image acquisition.

[0057] Figure 4 is a schematic diagram showing an embodiment of the graphical user interface 400, and more specifically, a part of the graphical user interface related to predefined focusing settings for the selected sample specification "adhered" 222 (see Figure 2). As already mentioned above, the user can override or adjust the predefined focusing settings according to their experience and needs. In the illustrated example, the user can optionally open the "adhered" type focus menu by selecting the widget 410 of the graphical user interface 400. In the illustrated example, the user can edit the default settings related to drift correction. Before discussing the drift correction options in more detail, the predefined focusing settings for "adhered" samples will be described.

[0058] The predefined focusing settings for "adhered" samples (hereinafter referred to as "adhered focusing settings") are as follows: Since "adhered" samples do not change topology in the focal direction, a single focal point at the x and y positions of the sample is determined, for example, by a triangulation autofocusing method, and this focal point can be used as the focus level for imaging the entire sample or different parts of the sample. The triangulation autofocusing method can find and maintain the focal point of an adhered sample so that the microscopic image of the sample is in focus, as described, for example, in German Patent Application Publication No. 102010030430. For details of the design, structure, operation and function of such a triangulation autofocusing method and device, please refer explicitly to German Patent Application Publication No. 102010030430. The autofocusing detector of such a triangulation autofocusing device can detect any deviation of the sample from the selected (optimal) focal / level in the focal direction. Feedback control allows for the reversal of any deviation from the (optimal) focus / level by responding to changes in the distance between the sample / sample carrier and the microscope objective lens. For simplicity of notation, this type of triangulation autofocus control will be abbreviated as "AFC" below.

[0059] By clicking / selecting widget 410, the user can open the attached focus menu and edit the default settings related to drift correction. Focus drift can occur due to external influences such as temperature changes, movement, or vibration, or due to the motion of the sample carrier. In microscopy imaging, image acquisition is typically repeated at predefined time intervals (e.g., intervals from 0.1 seconds to 5 minutes), depending on the experiment. The image acquisition time interval is either predefined by default or defined by the user. Drift correction can be performed every nth time point. By default, n can be 1, but other settings are possible. By selecting menu point 420, the user can also increase the number of n to reduce the frequency of drift correction during imaging. Furthermore, drift can usually be corrected every nth position, such as every image acquisition position (n=1) or every second image acquisition position. In this embodiment, the default value is to correct drift every position, but this value can be edited by the user via menu point 430.

[0060] Drift can be effectively corrected by focus-holding devices such as AFC devices. These devices are fast and do not irradiate living samples with light that could damage or whiten them. For drift correction, a reference z-level (where the z-direction corresponds to the focal direction) must be defined, which is typically the focus level of the sample. When acquiring a z-stack of a 3D imaging image of the sample, the upper and lower z-limits of the z-stack are referenced to the defined focus level. During image acquisition, the AFC adjusts the predefined focus level to correct for drift. Because the upper and lower z-limits of the z-stack are referenced to the focus level, a drift-corrected z-stack can be acquired.

[0061] Figure 5 is a schematic diagram of an embodiment of the graphical user interface 500, and more specifically, a part of the graphical user interface related to predefined focusing settings (hereinafter referred to as "separation-type focusing settings") for "separation" samples. As already mentioned above, users can override or adjust the focusing settings according to their experience or needs. In the illustrated example, the user can optionally open the separation-type focus menu by selecting the "separation-type" menu point 510 of the graphical user interface 500. Before discussing the user options resulting from selecting menu point 510, we will describe the predefined separation-type focusing settings for "separation" samples.

[0062] As described above, the "separated" sample extends while changing in the focal direction and / or separates from the bottom of the sample carrier, and therefore a change in topology in the focal direction must be assumed. In one embodiment, a focus map is generated for such a sample, and the focus map contains one or more foci across the xy region of the sample, thereby the focus map attempts to approximate the sample topology using particularly high-density foci. The foci of the focus map can be determined by an image-based autofocusing method. This autofocusing method evaluates the image stack of the sample by capturing each image at a different focal position. A metric is applied to each image in the image stack, calculating a metric value for each image, and each different metric value is associated with a different image sharpness or blur of the corresponding image. There are a considerable number of such metrics suitable for autofocusing, including metrics based on gradient methods, wavelet decomposition methods, contrast methods, autocorrelation methods, etc. Such an image-based autofocusing method can determine the optimal focus at a given xy position of the sample.

[0063] To save time and processing power, image-based autofocusing can also be combined with AFC, which allows for the definition of an appropriate starting point or interval by determining the focus found by AFC as the starting point, and an image stack for image-based autofocusing is acquired in the z-region around the starting point.

[0064] Additionally or alternatively, the image-based autofocusing method includes the steps of generating a first image stack of a sample by capturing each image at a different focal position using a first focus step size over a first range of focal positions, and generating a second image stack of the sample using a second focus step size over a second range of focal positions, wherein the second focus step size is smaller than the first focus step size. In this embodiment, a first "coarse" focus measurement cycle is performed to determine a first "coarse" focus / level, and then a second focus measurement cycle with a smaller step size is performed in the z region of the first focus / level to obtain an optimal focus / level with greater accuracy. The second range is preferably smaller than the first range.

[0065] A predefined separation focusing setting determines a predetermined number of focus maps or focal density using the image-based autofocus method described above. This method can also be combined with AFC as described above. By selecting menu point 510 of the graphical user interface 500, the user can edit the default settings. For example, the user can select from different focus map densities such as "Low" 520, "Medium" 522, "High" 524, and "Very High" 526.

[0066] Furthermore, users can change the default settings related to drift correction. Here again, by default, drift correction is performed at each image acquisition point. This setting can be changed so that drift correction is performed at every nth point by selecting menu point 530 as appropriate. Additionally, in this example, drift correction can be performed (by default) at each focus of the focus map, or (by user selection in menu point 532) at every second, third, or usually only at the nth focus. Selecting n>1 can save image acquisition time, especially when drift is minimal.

[0067] Furthermore, by selecting menu point 540 of the graphical user interface 500 shown in Figure 5, the user can select the emission wavelength to be used for the separation focusing setting, according to the dye selected in the menu bar 230 of the graphical user interface 200 shown in Figure 2.

[0068] Finally, menu bar 550 displays all the focal points used to create the focus map, using their x and y positions. All single focal points, for example, the second focal point 552, can be overridden by the user.

[0069] Another option provided by the graphical user interface 500 in Figure 5 is to either automatically create a focus map at the start of the experiment (default: "On Experiment Start") or create a focus map upon user request ("Now").

[0070] Figure 6 is a schematic diagram of an embodiment of the graphical user interface 600, and more specifically, a part of the graphical user interface related to predefined focusing settings (hereinafter referred to as "dynamic focusing settings") for "dynamic" samples. As already mentioned above, users can override or adjust the focusing settings according to their experience or needs. In the illustrated example, the user can optionally open the dynamic focus menu by selecting the "dynamic" menu point 610 of the graphical user interface 600. Before discussing the user options resulting from selecting menu point 610, the predefined dynamic focusing settings will be described.

[0071] As mentioned above, "dynamic" samples are those that may move in the direction of focus and / or whose topology changes over time. Therefore, the optimal focus must be determined at any point in image acquisition. Focusing is performed by image-based autofocusing methods, particularly fast methods. Image-based autofocusing methods are described above in relation to the separation focusing settings.

[0072] By selecting menu point 610, the user can edit the default settings for dynamic focusing. For example, the user can select menu point 620 to change the selected fluorescence channel, i.e., to switch to a different fluorescence emission wavelength and observe the sample. Furthermore, by selecting menu point 630, the user can change the default settings, and the image-based autofocus method will be executed accordingly at each image acquisition point. "Software autofocusing" specifies the implemented high-speed image-based autofocusing method. When the sample's motion is low, it is effective to make n greater than 1 and execute software autofocus only at the nth time point.

[0073] By selecting menu point 640, the implemented software autofocus will be performed at predefined positions for each region of interest (ROI) selected by the user for image acquisition. In contrast, by selecting menu point 650 and choosing a value for n (n=4 in this example), software autofocus can be performed at the nth position of each ROI.

[0074] Figure 7 is a schematic diagram of a microscope 700 for microscopic imaging of a sample according to an embodiment of the present invention. Figure 7 shows a fluorescence microscope 700 having two detection units, namely a first detection unit 780a and a second detection unit 780b. By a switchable or displaceable mirror 782 (illustrated by a bidirectional arrow) or any other switching means, the observation light can be selectively coupled to either the first detection unit 780a or the second detection unit 780b. In the position illustrated in Figure 7, the observation light is coupled to the second detection unit 780b on the right via the mirror 782. When the mirror 782 is moved from the illustrated position to position 782', the optical path travels straight without deflection, so the observation light, illustrated as a dotted ray, is coupled to the first detection unit 780a. Optical lenses in the illumination and detection beam paths are not specifically shown.

[0075] The first detection unit 780a is a wide-field detection unit, with its image plane indicated as 784a in the example shown in Figure 7. By using lenses not specifically shown, the observation light is collimated and irradiated onto a detector device 786a within the first detection unit 780a. The detector device 786a may be configured to split the observation light into different detection channels. With regard to the detector device 786a usable in the first detection unit 780a, refer to European Patent No. 3721279, in particular in relation to the detection unit 10 shown in Figure 3 and the corresponding description therein, and its disclosure is incorporated herein by reference. The wide-field detection illumination unit in the first detection unit 780a is shown in a simplified form as 790a. Its light can be coupled to the illumination beam path of the fluorescence microscope 700 in any manner known in the art of microscopy illumination, such as by using a dichroic mirror 791a and a barrier filter 705.

[0076] The second detection unit 780b is a confocal detection unit whose image plane is indicated as 784b in the example shown in Figure 7. A point light source 790b may be provided for confocal detection using the confocal detection unit 780b. The point light source 790b may be, in particular, a (single) pinhole aperture from which laser light can be focused, or the end of a light guide or optical fiber from which light is emitted in a point manner. The point light source 790b is conjugate at the intermediate image plane 784b and the objective plane 721 of the sample 720, and the illumination light can be focused in a scanning manner to a point on the objective plane 721 using an X / Y scanner 795b which may be positioned in a telecentric plane or a plane conjugate thereto, via a dichroic mirror 791b and an illumination optical system not specifically shown. This allows the sample 720 to be scanned on the sample plane 721 as is generally known. The pinhole is indicated as 797b. Again, further details are specifically referenced in relation to European Patent No. 3721279, particularly in connection with the detection unit 20 shown in Figure 3 of that specification and the corresponding description, and its disclosure is incorporated herein. The same applies to detector device 786b, which may be configured to split the observation light into different detection channels. Specific references to European Patent No. 3721279 are also made to detector device 786b, which may be used in the second detection unit 780b.

[0077] In the microscope 700, the user can select either (at least) two different microscope operating modes or microscope imaging modes, namely wide-field imaging and confocal imaging. Of course, the focusing concepts described herein can also be applied to microscopes that are provided with only one microscope imaging mode, such as wide-field imaging only. In such microscopes that are provided with two or more imaging modes, the operating concepts in each operating mode are substantially different from one another. Settings that affect the image obtained in these operating modes include, for example, the illumination settings for scanning light sources and the components of their respective illumination beam paths, as well as the detection settings for area detectors, including the components of their respective detection beam paths, and the detection and focusing settings for components such as line detectors or point detectors, as described above. Each of these components usually has to be carefully adjusted individually, as it can affect the image result if adjustable. According to embodiments of the present invention, at least the focusing settings are automatically determined depending on the type of sample without further interaction with the user.

[0078] Therefore, particularly for users unfamiliar with any of the operating modes, or in more stressful observation situations such as observing moving samples, embodiments of the present invention overcome considerable obstacles and reduce the likelihood of distraction during the actual task of observing and examining samples. As stated above, embodiments of the present invention are not limited to wide-field operation and confocal operation, even if described herein.

[0079] In conventional concepts for operating the fluorescence microscope 700, the user still needs to have detailed knowledge of the technical background and the results achieved in determining and maintaining focus during image acquisition in each operating mode. As mentioned above, in conventional devices, the user may be required to "switch" conceptually and mentally between various general operating and interaction concepts. This problem is overcome by embodiments of the present invention, as described above, in which focus control based on the sample type of the microscope is realized in a variety of selectable microscope operating modes.

[0080] Figure 7 also shows a focusing apparatus including an autofocusing control unit 745 operably coupled to a microscope control device 760. In the illustrated example, the AFC is implemented as described, for example, in U.S. Patent No. 5,136,149 or German Patent Application Publication No. 102010030430. For a more detailed description of the AFC, explicit references to these documents are made. According to the basic scheme of the underlying triangulation autofocusing, an autofocusing measurement beam 732 is generated by an autofocusing light source 730, deflected by a deflection element 736 to pass through a microscope objective lens 750, and directed toward the sample 720. The reflected beam 734 passes through the microscope objective lens 750 and returns to the deflection element 736, and is directed toward an autofocusing detector 740 which is position-sensitive with respect to the position of the beam reflected on the detection surface of the autofocusing detector 740. Therefore, the output signal of the detector 740 correlates with the position of the reflected beam 734 relative to a predefined position on the detection surface of the autofocusing detector 740. The position of the reflected beam 734 changes when the distance of the sample 720 relative to the microscope objective lens 750 changes. This distance variation can be caused by a change in the topology of the sample toward the focal point, or by the sample moving toward the focal point, or by the focal point drifting, as described above.

[0081] The autofocusing detector 740 is operably connected to the autofocusing control unit 745 and maintains a constant distance between the sample 720 (or reference plane) and the microscope objective lens 750 by moving the microscope stage, including the microscope objective lens 750 and / or objective plane 721, in the direction of focus (z) (illustrated by a small arrow next to the objective plane 721) to equalize out-of-focus motion / drift.

[0082] In this embodiment, only examples of AFC control are illustrated. Other additional or alternative autofocusing methods, in particular image-based autofocusing methods, can and should be implemented according to the embodiments of the present invention as described above. Such image-based autofocusing methods can also be controlled by the autofocusing control unit 745 without limiting the possibility of having other autofocusing control units, which are illustrated herein by a general concept of the autofocusing control unit 745. According to this embodiment, a microscope control device 760 operably coupled to the autofocusing control unit 745 has one or more processors, is adapted to control microscopic imaging of the microscope 700, is further configured to receive sample information relating to the sample 720 to be imaged, receives focusing requests, and, in response to receiving a focusing request, is further configured to activate a predefined focusing setting depending on the received sample information to control the focusing of the microscope 700 for microscopic imaging of the sample. [Explanation of symbols]

[0083] Steps of the S0, S1, S11, S12, S13, S2, S3, S4, S5 methods 200 Graphical User Interfaces 210, 220, 230 Menu Bar 212, 214, 216, 217, 218 Menu points, widgets 222,224,226,228,229 Menu points, widgets 232,234,236,238 Menu points, widgets 300 Graphical User Interfaces 310,320 menu points, widgets 400 Graphical User Interfaces 410, 420, 430 Menu points, widgets 500 Graphical User Interfaces 510, 520, 522, 524, 526 Menu points, widgets 530, 532, 540, 550, 552 Menu points, widgets 600 Graphical User Interfaces 610, 620, 630, 640, 650 Menu points, widgets 700 Microscopes 705 Barrier Filter 720 samples 721 Objective surface 730 Autofocusing Light Source 732 Autofocusing measurement beam 734 Reflected beam 736 Deflection element 740 Autofocusing Detector 745 Autofocusing Control Unit 750 Microscope Objective Lens 760 Microscope Control Device 780a First detection unit 780b Second detection unit 782 Miller 782' Miller 784a image plane 784b intermediate image plane 786a Detector device 786b Detector device 790a Lighting Unit 790b point light source 791a Dichroic mirror 791b Dichroic Mirror 795b Scanner 797b Pinhole

Claims

1. A method for controlling (S0) microscopic imaging of a microscope (700), said method comprising: providing a microscope controller (760) configured to receive focusing requests and to receive sample information regarding the sample (720) to be imaged; the microscope control device (760), in response to receiving a focusing request (S2) after receiving sample information (S1), activates (S3) a predefined focusing setting depending on the received sample information to control focusing of the microscope (700) for microscopic imaging of the sample (720); method.

2. the sample information relates to one or more sample properties in a three-dimensional sample space and / or to the type of sample carrier carrying the sample and / or to the material of the sample carrier carrying the sample and / or to a sample stain; The method of claim 1.

3. No further user input information needs to be received by the microscope control device to activate (S3) the predefined focusing setting.

3. The method according to claim 1 or 2.

4. the predefined focusing settings include automatic determination of one or more focal points at defined x-y positions of the sample; The method of claim 1.

5. one or more of the one or more focal points are overridable by a user; The method of claim 4.

6. the one or more focal points are determined by an autofocusing method that depends on predefined and activated focusing settings; 6. The method according to claim 4 or 5.

7. the autofocusing method comprises at least one of a triangulation autofocusing method and an image-based autofocusing method that evaluates an image stack of the sample, each image being captured at a different focus position; The method of claim 6.

8. The image-based autofocusing method includes capturing each image at a different focus position using a first focus step size over a first range of focus positions to generate a first image stack of the sample, and generating a second image stack of the sample using a second focus step size over a second range of focus positions, the second focus step size being smaller than the first focus step size. The method of claim 7.

9. the predefined focusing settings are corrected for focus drift over time; The method of claim 1.

10. The focus drift is After a predetermined number of image acquisition positions at different x-y positions of the sample, After a predetermined number of time points of image acquisition, After a predetermined time interval, and the predefined focusing settings include automatic determination of one or more foci at defined x-y positions of the sample, where there are more than one foci for a predefined number of foci; is corrected in at least one of the following cases:

10. The method of claim 9.

11. the sample information about the sample to be imaged is pre-categorized into at least two different pre-defined sample specifications, each of which is selectable by a user; The method of claim 1.

12. The predefined sample specifications include: The sample is attached to a sample carrier carrying the sample (S11); the sample is separated from the sample carrier carrying the sample (S12); The sample extends while changing in the focus direction (S12). The sample is allowed to move (S13). the sample has a surface topology that changes over time (S13); having at least one of: The method of claim 11.

13. When the sample is attached to the sample carrier carrying the sample (S11), an autofocusing control based on a triangulation autofocusing method is used to determine and / or maintain a focus used for sample imaging.

13. The method of claim 12.

14. a focus map of one or more foci is defined when the sample is separated from the sample carrier carrying the sample (S12) or when the sample is elongated with a change in focus direction; 13. The method of claim 12.

15. The focus map is defined by a predefined or selectable number of foci and / or densities of foci.

15. The method of claim 14.

16. the focus map is determined by an image-based autofocusing method that evaluates an image stack of the sample, each image being captured at a different focus position.

16. The method according to claim 14 or 15.

17. maintaining focus after determining the focus map and during imaging of the sample using autofocusing control based on a triangulation autofocusing method; 17. The method of claim 16.

18. If the sample is movable (S13), an autofocusing control based on an image-based autofocusing method is used to image the sample, where each image is captured at a different focus position and an image stack of the sample is evaluated.

13. The method of claim 12.

19. The microscope control device is configured to activate (S0; S4) different microscope imaging modes that are independently selectable from predefined activated focusing settings. The method of claim 1.

20. A microscope control device (760), the microscope controller (760) comprises one or more processors and is configured to control microscopic imaging of the microscope (700), the microscope controller (760) being configured to receive sample information about a sample to be imaged and to receive focusing requests; the microscope control device (760) is further configured, in response to receiving the focusing request, to activate a predefined focusing setting in dependence on the received sample information to control focusing of the microscope for microscopic imaging of the sample (720). Microscope control device (760).

21. The microscope control device (760) is configured to perform the method of claim 1. The microscope control device (760) of claim 20.

22. the microscope control device (760) comprises a graphical user interface (200, 300) including control widgets configured to receive user input, the control widgets including at least one focusing control widget (310) through which the user receives the focusing request, and at least one sample information widget (212, 214, 216, 218; 222, 224, 226, 228, 229; 232, 234, 236, 238) through which the user receives the sample information; The microscope control device (760) of claim 20.

23. A microscope (700) for microscopic imaging of a sample (720), comprising: The microscope (700) comprises an autofocusing control unit (745) and a microscope control device (760) according to claim 20, the autofocus control unit (745) is operably coupled to the microscope control device (760); Microscope (700).

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