Objective lens unit for microscope, microscope comprising objective lens unit, and method for setting object plane of microscope

Abstract

An objective unit (6) for a microscope comprises at least one optically detectable target (48) located on at least one optical component of the objective unit. The at least one optically detectable target has a defined position in each case with respect to the distal front end (62) of the housing of the objective unit (6) or the distal end of the microscope. An adjustable lens (65) of the objective unit is adjusted to clearly image at least one of the at least one optically detectable target, e.g., on a sensor attached to a camera of the microscope. Thus, the setting of the adjustable lens for the reference object plane is determined. The adjustable lens can then be adjusted to move the object plane by a distance in a distal direction to position the object plane in the sample volume (100) at a desired position distally from a distal front end (62) of the housing of the objective unit (6) or from a distal end of the microscope, respectively, by a distance (s).

Description

Technical Field

[0001] The subject matter claimed herein generally relates to instruments and methods suitable for microscopy. More specifically, the invention relates to the subject matter set forth in the claims. Background Technology

[0002] In certain applications of microscopy, it is necessary to set the object plane of the microscope (i.e., the plane in which the object is clearly imaged) at a certain distance in front of or from the front optical aperture of the microscope, without any reference object present during the object plane setting. This is common in, but not limited to, inline and in-situ microscopy, particularly when the object to be observed traverses the sample volume. In such cases, for example, there is no stationary object that can be focused upon. A typical (but again non-limiting) example is cytology, particularly imaging cytology of bioprocesses.

[0003] Bioprocessing is the process of obtaining a desired product using intact, living biological cells or components thereof (such as bacteria, enzymes, or chloroplasts). Bioprocessing typically takes place in a bioreactor, i.e., a process vessel, preferably a reusable and therefore sterilizable tank or disposable bag. The biological cells, as part of the bioprocess, are dispensed into a liquid culture medium, thereby establishing a suitable environment for the desired process. Typically, the culture medium contains nutrients, such as those for the cells discussed, as well as gases they require, such as O2 and CO2. In most cases, it is crucial to ensure that no cells other than those involved in the bioprocess are present in the bioreactor; this means that a sterile barrier is preferably maintained between the process and the external environment for as long as possible, thereby minimizing the frequency and duration of unwanted cell contamination. Bioprocessing typically uses devices to continuously mix the cells and the culture medium. Examples of such devices are stirrers arranged inside the bioreactor or vibrators that move the entire bioreactor. Thus, cells suspended in the culture medium typically move within the bioreactor. In many cases, the culture medium is closely monitored to ensure that the desired conditions are maintained. For this type of monitoring, a variety of optical, photochemical, and electrochemical sensors are currently available and in use, capable of reliably measuring, online or in situ, values ​​such as pH or dissolved oxygen levels. However, there is currently no similar reliable measuring device available for direct cell monitoring.

[0004] Online or in-situ measurements—in the case of processes, particularly bioprocesses—are measurements performed directly in the reactor, specifically in a bioreactor, or in the pipeline transporting the fluid to be monitored. In particular, the fluid to be monitored is a liquid in a bioprocess containing culture medium and biological cells.

[0005] Cytology is used to characterize living or dead biological cells, and preferably, for the imaging cytometer of the present invention at hand, the cells to be characterized are provided in a liquid. For example, the density, size, and morphology of biological cells can be determined by microscopy. In the absence of reliable and quantitative online measurement methods, bioengineers must collect samples to perform cytology using offline measurement devices. This requires removing sample volumes from the bioreactor at specific time intervals, which increases the risk of contamination. Furthermore, this is time-consuming, and due to the sparse time intervals, the operator cannot obtain sufficient statistical data and real-time information about the cytology. Compared to offline methods, online cytometers for characterizing biological cells that are typically cultured in a liquid environment will significantly reduce complexity and will allow process controllers to monitor cell counts and cell characteristics (such as those mentioned above) in near real-time.

[0006] In some applications, the volume of a sample in which an object is clearly imaged can be defined optically: In the first dimension, the sample volume can be optically defined by the depth of field of the microscope. Preferably, the depth of field of the microscope is determined by the objective lens unit. In the two dimensions perpendicular to the first dimension, the sample volume is optically defined by the imaging region: this region is determined, for example, by the field of view of the microscope, which is defined, for example, by the size of the optical sensor attached to the microscope or by the aperture (e.g., the front optical aperture) arranged in the optical path. The imaging region can also be defined after the image is acquired, for example, by appropriate cropping or by selecting appropriate sub-regions in the acquired image accordingly.

[0007] An objective lens unit includes an immersion lens (e.g., a solid immersion lens) to collect light through a front optical aperture, providing a depth of field of only a few micrometers. However, the axial position of the sample volume along the direction of microscope observation may be of interest, but may not be reliably determined in the absence of any reference in the sample. It may also be desirable to scan the sample, i.e., record frames at different axial positions.

[0008] WO 2019 / 081333 discloses a microscope that includes autofocus control with electrically adjustable lenses. However, this autofocus control relies on monitoring the reflection of the light beam at the coverslip-sample interface, and is therefore unsuitable for online and in-situ applications that do not use coverslips, such as online cytology. Summary of the Invention

[0009] The invention claimed herein is defined by the appended claims.

[0010] The purpose of this disclosure is to address the subject matter initially mentioned. In one aspect, the subject matter disclosed herein should enable the object plane in which an object is clearly imaged to be referenced accordingly to the structure of a microscope, such as the distal front optical aperture of the microscope or the objective lens unit of the microscope.

[0011] This is achieved through the subject matter set forth in the appended claims. Further effects and advantages of the disclosed subject matter, whether expressly mentioned or not, will become apparent from the disclosure provided below.

[0012] Therefore, an objective lens unit for a microscope is disclosed.

[0013] The objective lens unit includes a housing having a distal front end. The distal front end may also be referred to hereinafter as the front end. The front optical aperture of the objective lens unit is disposed in the distal front end of the housing. The objective lens unit also includes an objective lens system disposed within the housing and proximal to the front optical aperture of the objective lens unit. The objective lens system is configured to collect light received through the front optical aperture of the objective lens unit. The objective lens system includes an adjustable lens. The objective lens unit also includes at least one optical component located distal to the adjustable lens. For example, the at least one optical component may be located between the optical lens system and the front optical aperture, or within the front optical aperture. Preferably, the at least one optical component is the most distal optical component of the objective lens unit. At least one optically detectable target is disposed on the at least one optical component.

[0014] The adjustable lens is specifically configured to correct the effective focal length of the objective lens system. Examples of adjustable lenses are described below. Typically, the adjustable lens may include, for example, a lens with a variable focal length, an axially displaceable lens, and / or an axially displaceable lens with a variable focal length.

[0015] The at least one optically detectable target allows adjustment of the at least one adjustable lens such that at least one of the at least one optically detectable target is clearly imaged, for example, clearly imaged on an image captured by the optical sensor of the microscope using the objective lens unit.

[0016] As used in this application, the object plane is a plane in which an object positioned is clearly imaged on, for example, a sensor of a camera connected to the objective lens unit. The effective focal length of the objective lens system affects the axial position of the object plane. An object located in the object plane produces a clear image on the image plane of the objective lens system. This image plane of the objective lens system can be the object plane of an eyepiece or camera connected to the objective lens system, or an optical sensor can be located at the image plane. In all cases, correcting the effective focal length can be used to adjust the axial position of the object plane of the objective lens system while maintaining its image plane, meaning that neither the sensor nor (if present) the eyepiece or camera needs to be adjusted or moved. Furthermore, if the effective focal length of the objective lens system or the entire objective lens unit changes over time—for example, possibly due to mechanical or thermal stress during equipment use and / or cleaning—it can be corrected by using an adjustable lens to correct the effective focal length of the objective lens system. For ease of reference, the effective focal length will be referred to as the "focal length" below.

[0017] The at least one object plane defined by the at least one optically detectable target is referred to below as the “reference object plane”: the reference object plane is the object plane in which at least one feature portion of the optically detectable target is located.

[0018] The axial position of the reference plane is called the "reference position".

[0019] "Axial position" and "axial distance" refer to the axial coordinates in a coordinate system using a cylindrical coordinate system that defines the axial direction with the optical axis. Therefore, for example, a target can have the same axial position as its reference plane, regardless of whether it is positioned on or off the axis. Similarly, the axial distance between two targets can be the difference between their axial positions.

[0020] The adjustment range of an adjustable lens can be understood as the physical parameters applied to adjust the adjustable lens to shift the object plane. Depending on the design of the adjustable lens and the actuation method used to adjust it, the adjustment range can be defined by different physical parameters. Those skilled in the art will readily determine what changes in the physical parameters and / or which physical parameters represent the adjustment range of the adjustable lens. The adjustment range of an adjustable lens can be, for example, the magnitude of axial displacement of an axially displaceable lens, or a control signal provided to the adjustable lens, for example, with a given intensity or duration, or a combination of both.

[0021] The characteristic portion of an optically detectable target used to define a reference plane is also referred to below as a "focusing target".

[0022] In a preferred embodiment, the optically detectable target is a focusing target; however, in some embodiments, a single target may include multiple focusing targets, as explained in more detail below. It goes without saying that this implicitly requires at least one of the at least one optical target to be arranged at an axial position within an object plane that can be clearly imaged by an objective lens unit with adjustable lenses. Furthermore, the optical target needs to be identifiable in the image. Preferably, therefore, the optical target is sufficiently distinguishable from visually similar structures in planar images located above and below the plane including the at least one optically detectable target. In embodiments, the optically detectable target is specifically manufactured for this purpose, for example by machining, cutting, etching, printing, or otherwise altering the at least one optical component.

[0023] In an embodiment, the optically detectable target is a geometric shape, such as a cross, a circle, a dot, a square, or a triangle, or a combination of these shapes in two-dimensional or three-dimensional space.

[0024] In some embodiments, the field of view of the objective lens unit or the microscope using the objective lens unit is larger than a segment of the resulting image being evaluated for measurement purposes. In these embodiments, the optically detectable target is preferably positioned in an area not evaluated for measurement purposes, thus avoiding any obstruction of the measurement by the target. In other embodiments, however, the target is located in the imaging area to be used for measurement: since the target is typically in a strongly defocused state during measurement, its impact on the final image is negligible.

[0025] Preferably, the targets are a set of crosses: these targets are easily identified by automated image processing and allow for the detection of sharpness. Furthermore, these targets are easy to manufacture and significantly different in shape from biological cells.

[0026] In an embodiment, in addition to the adjustable lens, the objective lens system may also include at least one fixed lens with a fixed focal length. The lens is spatially fixed relative to the housing of the objective lens unit and has a fixed focal length. The at least one fixed lens with a fixed focal length may specifically include at least one aspherical lens.

[0027] It should be noted that, within the framework of this disclosure, the use of the indefinite article "a" or "one" does not imply a singularity, nor does it preclude the existence of multiple named components or features. Therefore, it should be understood as "at least one" or "one or more".

[0028] The adjustable lens can be a motorless, automatically adjusting lens. Motorless systems offer several advantages. For example, autoclaving-compatible motorless systems can be provided. Furthermore, automatic adjustment of the adjustable lens can be achieved without causing vibration. In other aspects, the motorless, automatically adjusting lens can be housed in a compact objective lens unit and / or used over a wide temperature range.

[0029] The motorless, automatically adjusting lens may include one of an adjustable lens and a lens axially displaced along the optical axis of the objective lens unit by a piezoelectric actuator. The adjustable lens is commercially available. The adjustable lens is configured to controllably change the radius of curvature of at least one of its light-refracting surfaces, thereby changing its focal length. Common adjustable lenses are autoclaved. Lenses displaceable by a piezoelectric actuator may include either a fixed-focal-length lens or an adjustable lens. As will be understood, the piezoelectric actuator is also compact and operates without vibration.

[0030] At least one of the at least one optically detectable target may be disposed on a planar surface extending perpendicular to the optical axis of the objective lens unit. As will be understood, the planar surface may define a reference object plane, i.e., an object plane in which the optically detectable target can be clearly imaged.

[0031] In embodiments where the target is or includes a geometric shape, the geometric shape is preferably a geometric shape extending on the planar surface. In this case, if the object plane is selected to coincide with the reference object plane, the geometric shape will be fully in focus, making it easier to detect the geometric shape in the captured image.

[0032] An immersion lens can be positioned distal to the adjustable lens and configured to receive light through the front optical aperture. When using an immersion lens, the relatively narrow depth of field (typically only a few micrometers measured along the axial or observation direction) and the field of view captured by a suitable sensor, for example, 150 μm × 150 μm, optically define a narrow sample volume. By using an adjustable lens, manufacturing tolerances and tolerances or misalignments that occur during use (e.g., when the objective unit is subjected to autoclaving or exposure to thermal or mechanical stress) can be corrected. The adjustable lens allows the actual object plane of the objective unit or the microscope to be moved to coincide with the intended object plane, thereby placing the sample volume at the desired position relative to the objective unit or the microscope.

[0033] The immersion lens can be a solid immersion lens (so-called SIL), but in other cases it can include a liquid immersion lens. In the case of a liquid immersion lens, the front optical aperture of the objective unit can be closed by a window serving as the farthest optical element. In this case, the liquid immersion lens has a gap located between the window and the liquid immersion lens. The gap is filled with liquid. The materials of the window, liquid, and immersion lens are chosen such that the refractive index matches a well-defined relationship, wherein the shape and focal length of the immersion lens can be further considered. Those skilled in the art of microscopy and / or optical engineering are very familiar with the appropriate selection of materials and designs. Solid immersion lenses typically have a truncated spherical shape, wherein the solid immersion lens most commonly comprises a hemisphere (so-called hemispherical SIL), or more than a hemisphere (so-called Weierstrass SIL), also called a super-hemispherical SIL or super SIL. In this case, the front optical aperture of the objective unit can be closed by the SIL serving as the farthest optical element. In some embodiments, the solid immersion lens can be bonded or pressed against the window that closes the front optical aperture. Such a window can be optimized to withstand the conditions of the liquid being observed, cleaning procedures commonly used in bioprocessing, and to delay the adhesion of contaminants and / or bubbles. For example, the window can be made of sapphire. In some embodiments, a coating is applied to the SIL to improve durability and / or observation conditions, similar to the window. The coating can be an anti-reflective coating. In this case, the front optical aperture of the objective unit can be closed by a combination of coated SIL or SIL window as the farthest optical component. Those skilled in the art of microscopy and / or optical engineering are familiar with the appropriate selection of materials and designs to ensure that the optical properties of the SIL-window or SIL-coating combination remain those of the SIL, i.e., an enhanced numerical aperture, achieved by ensuring that the refractive index of the combination remains greater than that of standard glass, preferably greater than 1.5.

[0034] Solid immersion lenses offer high numerical apertures with relatively small entrance areas, which in turn reduces noise and improves resolution. Therefore, objectives employing immersion lenses as front lenses have narrow depths of field. Typically, the depth of field that can be clearly imaged on an optical sensor is a few micrometers. For example, the field of view captured by a suitable sensor can be 150 μm × 150 μm. Thus, a very narrow sample volume can be optically defined without mechanical definition. In the context of cytology, the narrow depth of field is primarily used to avoid overlapping cells in the line-of-sight being captured in the image, thereby preventing interference from overlapping cells.

[0035] In another, non-limiting embodiment, at least one of the at least one optically detectable target is disposed on the most distal component of the objective lens unit. As will be understood, the intended object plane during data collection is correspondingly located distal to the distal front end of the objective lens unit housing or distal to the front optical aperture. The sample volume is typically centered in the axial direction at a desired distance from the intended object plane. Therefore, the choice of the intended object plane determines the axial position of the sample volume. Those skilled in the art will understand the benefit of using a reference object plane that is as close as possible in the axial direction as possible to the intended object plane (i.e., the object plane required for data collection and / or measurement). Alternatively, in other words, it is advantageous to use a reference position close to the axial position of the sample volume. This proximity reduces errors caused by uncertainties in the adjustment of the adjustable lens on the one hand, and reduces the magnitude of axial distance variation required by the adjustable lens to achieve both the intended object plane and the reference object plane on the other.

[0036] The distal optical component of the objective lens unit can be either an immersion lens or a window that closes the front optical aperture. If the distal optical component is an immersion lens, it is preferably a SIL, coated SIL, or a combination of SIL and a window. This avoids the need for additional optical components, thus facilitating production. Furthermore, by placing an optically detectable target on the distal optical component, it is possible to compensate for any offsets that may occur near the distal optical component.

[0037] At least one of the at least one optically detectable targets may have a three-dimensional shape. Thus, the target extends in an axial direction, for example, parallel to or along the optical axis of the objective lens unit. Due to its three-dimensional shape, such an optically detectable target provides two focusing targets located at different axial positions and having a known axial distance between them. For example, a three-dimensional optical target may include two sets of edges offset from each other axially, such as the edges of two sides of a cube. As explained in more detail below, the two focusing targets can be used to calibrate the relationship between the amplitude of the adjustable lens adjustment and the associated axial displacement of the object plane. The amplitude of the adjustable lens adjustment may be, for example, the amplitude or duration of a drive signal, or a set value of the adjustable lens. Each focusing target can be used to define a reference object plane.

[0038] In some embodiments, the target is or comprises a two-dimensional geometry extending in a plane at an angle of less than 90° relative to the optical axis. In these embodiments, only a portion of the geometry is in sharp focus at the actual object plane under a given setting of the adjustable lens or a drive signal.

[0039] In other embodiments, the objective lens unit includes at least one first optically detectable target and at least one second optically detectable target, wherein the at least one first optically detectable target and the at least one second optically detectable target are positioned at different axial locations. Preferably, the at least one first optically detectable target and / or the at least one second optically detectable target extends perpendicular to or across the optical axis. The at least one first optically detectable target and the at least one second optically detectable target provide two focusing targets at different axial locations and therefore in different reference object planes. The axial distance between the two reference object planes is preferably known. The axial distance can be used to calibrate the relationship between the adjustment amplitude of the adjustable lens and the relevant axial displacement of the object plane. The meaning of the term adjustment amplitude has been explained in more detail above. Additional optically detectable targets may be provided at other axial locations. This can thus define additional focusing targets and additional reference object planes. For example, this may be useful for calibrating the nonlinear relationship between the adjustment amplitude of the adjustable lens and the relevant axial displacement of the object plane. At least two optically detectable targets positioned at different axial locations can have different shapes, such that when the corresponding optically detectable target is clearly imaged, the corresponding reference plane can be identified.

[0040] In other embodiments, the housing may be specified as comprising a cap and a sleeve, wherein the objective lens system is disposed within the sleeve. The cap includes lateral sheaths, a front wall, and a rear port. Furthermore, the cap includes the at least one optical component. The front optical aperture of the objective lens unit is disposed in the front wall of the cap. At least one of the at least one optically detectable targets is disposed on the at least one optical component of the cap. The sleeve is at least partially received within the cap, particularly through the rear port of the cap. The cap is at least partially received within the sleeve, particularly the lateral sheaths of the cap are at least partially received in the front end of the sleeve, while the objective lens system can at least partially extend through the rear port. The front wall of the cap forms the front end of the housing, particularly when the sleeve is received within the cap. In an embodiment, the sleeve may be threadedly received within the cap. In an embodiment, the cap may be threadedly received within the sleeve. A seal, for example, provided by an O-ring, may be disposed between the cap and the sleeve. In other embodiments, the sleeve can be simply inserted into the cap, wherein, of course, the sleeve and the cap can be equipped with an alignment device such that when the sleeve is received inside the cap, the optical axis of any optical component disposed in the cap coincides with the optical axis of the objective lens system. Such embodiments are possible due to the adjustability of the motorless automatic adjustment lens: the relative displacement between at least one optical component of the cap and the objective lens system resulting from mounting the cap to the sleeve can be corrected by the adjustable lens.

[0041] The cap can, for example, be fixed to a bioreactor and extend through a port of the bioreactor, wherein the front wall of the cap is located inside the bioreactor, while the rear port of the cap is accessible from the outside of the bioreactor. Preferably, the distal front section of the sleeve is received in the portion of the cap located inside the bioreactor in such a way that the cap forms a liquid-tight boundary between the inside and outside of the bioreactor. Such an embodiment has the advantage that only the cap needs to be sterilized and / or cleaned, since only the cap comes into contact with the contents of the bioreactor, while the sleeve can be removed for SIP, CIP, or sterilization. This reduces the requirements on the sleeve and the objective system mounted therein, for example, by eliminating the need to withstand large temperature changes. The sleeve can be continuously inserted into different caps for use in different bioreactors or at different locations within a single bioreactor.

[0042] In this regard, a cap is also proposed, configured as part of an objective lens unit having a housing including a sleeve and a cap, as described above. The cap includes a rear port, a front wall, and a lateral sheath extending axially from the front wall. A front optical aperture is formed in the front wall. The cap also includes at least one optical element, wherein the most distal optical element of the at least one optical element closes the front optical aperture, and wherein the at least one optically detectable target is disposed on at least one of the optical elements. The lateral sheath is configured to receive the sleeve therein, or to be received within the sleeve. The cap is configured to either receive the sleeve in the axial direction through the rear port, or to be received in the axial direction by the sleeve, wherein the lateral sheath of the cap is inserted into the sleeve. The rear port is located at the rear end of the cap. The advantages and embodiments of the proposed type of cap have been summarized above in conjunction with an objective lens unit having a housing including a sleeve and a cap.

[0043] In a more specific, non-limiting embodiment, at least one of the at least optically detectable targets may be disposed on the most distal optical component of at least one optical component of the cap. This has the advantage that at least one of the optically detectable targets is adapted to provide the information needed to compensate for the misalignment between the objective lens system in the sleeve and the optical components of the cap. Furthermore, the target is positioned as close as possible to the intended sample volume. In most cases, there is only a small distance between the intended object plane and the reference object plane defined by the at least one optically detectable target disposed on the most distal optical component of the at least one optical component of the cap.

[0044] A microscope is further disclosed comprising an objective lens unit of any of the types described above and an optical sensor functionally coupled to the objective lens unit to receive light transmitted from the front end of the objective lens unit and through the objective lens system. In a particular aspect, the microscope can be configured for attachment to a bioreactor including a standard port, wherein the microscope is further configured such that at least a distal segment of the objective lens unit (i.e., particularly the segment including the front optical aperture) can be positioned inside the bioreactor, while the microscope head can be located outside the bioreactor. The microscope may also include a sample illumination unit comprising an illumination source and means for coupling light from the illumination source into the objective lens unit. The sample illumination unit may, for example, be arranged in the microscope head or in the objective lens unit, as described above. The optical path of light emitted from the sample illumination unit coincides with the optical path of light collected from the sample at least in a corresponding segment of the optical path. The optical sensor may particularly include an image sensor, and more particularly a CCD or CMOS image sensor. The optical sensor may be arranged in the microscope head or may be integrated into the objective lens unit. The microscope may also include a control unit functionally coupled to the objective lens unit and controlling the adjustable lens. The microscope may also include control hardware configured to control the sample illumination unit and optical sensors, as well as other means configured to evaluate and / or transmit optical data collected and recorded by the optical sensors. Specifically, the means configured to evaluate the optical data may include computer hardware and software to detect a focusing target on the image and induce adjustment of the adjustable lens by adjusting the amplitude of the adjustable lens, thereby moving the object plane to a desired object plane. In a preferred embodiment, the device configured to evaluate optical data may include computer hardware and software to: additionally detect different focused targets at different reference distances on the image; preferably correlate the distances between the detected focused targets; evaluate or read the corresponding adjustment calibration difference; use information about the distances and the adjustment calibration difference to determine the adjustable lens adjustment amplitude required to set the object plane to the desired object plane position; and induce adjustment of the adjustable lens by the required adjustable lens adjustment amplitude and by further adjustable lens adjustment amplitude, thereby moving the object plane to the desired object plane and the reference object plane required to determine the adjustment calibration difference.

[0045] A further aspect of the subject matter disclosed herein relates to a method for setting the object plane of a microscope. The microscope includes at least one optical sensor and at least one adjustable lens. The at least one adjustable lens is configured to axially shift the object plane of the microscope relative to the distal end of the microscope. The object plane is intended to refer to the plane in which an object is clearly imaged on the at least one optical sensor. Furthermore, at least one optically detectable target is disposed in the region imaged on the optical sensor and located at an axial position defined relative to the distal end of the microscope. Specifically, the axial position is defined along the optical axis of the microscope. For non-limiting examples, the microscope may include any of the types of objective lens units described above. The method includes defining a desired object plane position relative to the distal end of the microscope, the desired object plane position being located at a desired distance from the distal end of the microscope from the distal side. The method further includes adjusting the at least one adjustable lens until the at least one optically detectable target is clearly imaged on the optical sensor, whereby the object plane includes the at least one optically detectable target. In this configuration, the actual object plane coincides with a reference object plane. Since the at least one optically detectable target is located at the determined axial position, the reference object plane is also located at the determined axial position relative to the distal end of the microscope. The object plane is thus positioned at the determined axial position relative to the distal end of the microscope. Since both the determined axial position and the desired object plane position are known relative to the distal end of the microscope, the axial distance between the two positions is also known. The method further includes adjusting the at least one adjustable lens to axially move the object plane to the desired object plane position along the optical axis of the microscope.

[0046] Those skilled in the art, with a full understanding of the optical system used and the characteristics of the adjustable lens or its adjustment actuator, will be able to readily determine the magnitude of the adjustable lens adjustment required to move the object plane axially a specific distance in a particular direction. Therefore, once the object plane is positioned at a well-defined reference location by adjusting the adjustable lens, those skilled in the art can easily move the axial position of the object plane to the desired object plane by a defined distance. The determined axial position is an example of a well-defined reference location.

[0047] Preferably, the method is performed by a control unit of the microscope, the control unit being functionally coupled to control the adjustable lens and including means configured to evaluate and / or transmit optical data collected and recorded by the optical sensor. In an embodiment, the microscope is coupled to an external computing device to perform the method. In this embodiment, the microscope includes a control unit functionally coupled to control the adjustable lens and adapted to communicate with the external computing device. The external computing device receives image data from the microscope and includes means configured to evaluate the optical data collected and recorded by the optical sensor. In this embodiment, the external computing device is configured to evaluate the received data and issue control signals to the microscope to perform the method described above.

[0048] The distal end of the microscope can preferably be the distal front end of the objective lens unit, which is part of the microscope.

[0049] Alternatively, it can be specified that the two focusing targets are positioned at different axial locations. The two focusing targets are positioned in the region imaged on the optical sensor and have a known axial distance between them. The two focusing targets are provided by at least one of the following options: According to the first option, at least one of the at least optically detectable targets has a three-dimensional shape. The target with the three-dimensional shape includes at least two optically distinguishable features, which have a known axial distance from each other. The two optically distinguishable features form the two focusing targets. The target with the three-dimensional shape can extend along the optical axis of the microscope, wherein the two focusing targets are positioned at different axial locations. Preferably, the optically distinguishable features are axially offset edges of the optically detectable target with the three-dimensional shape.

[0050] According to the second option, at least one first optically detectable target is disposed at a first defined axial position, and at least one second optically detectable target is disposed at a second defined axial position. The first and second optically detectable targets have a known axial distance from each other. The first and second optically detectable targets form the two focusing targets.

[0051] The two focusing targets can be positioned in the region imaged on the optical sensor. Preferably, the focusing targets are provided by using an objective lens unit comprising at least one optically detectable target having a three-dimensional shape and / or using an objective lens unit comprising at least first and second optically detectable targets positioned at different axial locations.

[0052] In this configuration of the microscope, the method may include performing a first calibration adjustment by adjusting the at least one adjustable lens until the first focusing target of the focusing targets is clearly imaged on the optical sensor. Thus, the actual object plane has reached a first axial reference position. Subsequently, a second calibration adjustment may be performed by adjusting the at least one adjustable lens until the second focusing target of the focusing targets is clearly imaged on the optical sensor. Thus, the actual object plane has reached a second axial reference position. The calibration difference is determined as the magnitude of the adjustable lens adjustment applied to move the object plane from the first focusing target to the second focusing target.

[0053] The required adjustment amplitude of the adjustable lens can then be determined. The required adjustment amplitude is the amplitude of adjustment that needs to be applied to the adjustable lens to set the object plane from its actual position to the desired object plane position. The required adjustment amplitude is determined based on the adjustment calibration difference, the axial distance between the first and second focusing targets, and the axial distance between the first or second focusing target and the desired object plane position. Finally, the at least one adjustable lens is adjusted by applying the required adjustment amplitude to set the object plane to the desired object plane position.

[0054] For example, in simple cases where the relationship between the adjustable lens adjustment amplitude and the axial displacement of the object plane is known or at least assumed to be linear, a simple proportionality rule can be applied. As an example, to determine the adjustable lens adjustment amplitude required to set the object plane from the second axial reference position to the desired object plane position, the following calculation can be performed: divide the previously determined adjustment calibration difference by the axial distance between the two focusing targets, and then multiply by the axial distance between the second axial reference position and the desired object plane position. Of course, the direction of the corresponding object plane offset needs to be considered when determining the sign of the result. If the relationship between the adjustable lens adjustment amplitude and the axial displacement of the object plane is not linear, the calculation may become more complex; however, those skilled in the art are familiar with such calculations.

[0055] It can be provided that more than two focusing targets are arranged in the region imaged on the optical sensor and at different axial positions. This will also make it possible to determine parameters of the nonlinear relationship between the adjustable lens adjustment amplitude and the axial displacement of the object plane.

[0056] It should be understood that determining whether an optically detectable target or a focused target is clearly imaged on a sensor can be performed using known or readily available software algorithms. In other embodiments, an operator may view frames recorded from the sensor and determine when a clear image is obtained, inputting this information into a user interface. Other methods for determining whether a focused target, or more specifically an optically detectable target, is clearly imaged on a sensor will readily be apparent to those skilled in the art. The shape and appearance of the optically detectable target can be selected to enable easy identification by automated means. Furthermore, masks, filters, and / or correlation functions can be applied, if desired, to further facilitate and accelerate identification.

[0057] At least the distal or front section of the objective system (including the front optical aperture) can be adapted to be immersed in a liquid. Furthermore, at least the objective system, and in some embodiments the entire objective unit, can be autoclaved or able to remain mounted in the bioreactor during CIP or SIP procedures. In view of the latter requirement, the auto-adjusting lens is motorless.

[0058] The circumferential outer wall of the aforementioned objective lens unit can be cylindrical, with an outer diameter of 25 mm or less, particularly 12 mm or less. This allows the objective lens unit to be inserted into the bioreactor via standard ports (e.g., Ingold standard access ports and Eldon-James ports).

[0059] It should be understood that the features and embodiments disclosed above can be combined with each other. It should be understood that, within the scope of this disclosure and the claimed subject matter, those skilled in the art will readily conceive of other embodiments based on the content of this disclosure. Attached Figure Description

[0060] Figure 1 A microscope is set up for in situ cytology inside the bioreactor; Figure 2 This document proposes a first exemplary embodiment of an objective lens unit of the type described herein; Figure 3 A second exemplary embodiment of the objective lens unit of the type proposed herein; Figure 4 This document provides details of an exemplary embodiment of the arrangement of the immersion lens and the front optical aperture of the proposed objective unit. Figure 5 Another embodiment of the objective lens unit of the type proposed herein includes a sample illumination unit and a mirror arranged to be located far from the front optical aperture; Figure 6 Another possible arrangement of the mirror located on the far side of the front optical aperture; Figure 7An exemplary embodiment of an objective lens unit with a different tip geometry; Figure 8 A view along the optical axis of the objective lens unit with the focusing target.

[0061] Figure 9a A perspective view of a planar window as an example of the farthest optical components of two targets at different axial positions.

[0062] Figure 9b A perspective view of a planar window as an example of the farthest optical component of a three-dimensional target including two focusing targets at different axial positions.

[0063] It should be understood that the accompanying drawings are only partial schematic diagrams, and unnecessary details in the description may have been omitted for ease of understanding and depiction. It should also be understood that the drawings only show selected exemplary embodiments, and embodiments not shown may still be fully within the scope of the subject matter disclosed and / or claimed herein. Detailed Implementation

[0064] Figure 1A microscope 1 is shown, which is mounted to the wall 21 of a container 2 and extends through the wall 21 into the container 2 and into the sample liquid 3 contained within the container 2. The microscope 1 is attached to the wall 21 of the container 2 via a flange 4. The head 5 of the microscope 1 is disposed outside the container 2. A camera 51 is mounted at the proximal end of the microscope 1 and provides an optical sensor 52. The distal end 11 of the microscope 1 is provided by an objective lens unit 6. The objective lens unit 6 and the head 5 are connected by a hollow shaft 7. O-ring seals 71 and 72 are respectively provided at the corresponding connection between the shaft 7 and the head 5 and between the shaft 7 and the objective lens unit 6. Light collected by the objective lens unit 6 at the distal end 11 of the microscope 1 travels along the optical path through the hollow shaft 7 and is projected onto the optical sensor 52 of the camera 51. The optical sensor 52 and the camera 51 are configured to capture or record an image of light or an object projected onto the optical sensor 52. An illumination source 53 is mounted laterally to the head 5, facing the optical path. In the optical path from objective lens unit 6 to optical sensor 52, a beam splitter 54 is arranged inside the head 5. The beam splitter 54 acts as a device for coupling light from illumination source 53 into objective lens unit 6 and deflecting a portion of the light from illumination source 53 toward objective lens unit 6, where the light exits microscope 1 at distal end 11 and illuminates the sample volume of liquid 3. The light from the sample volume of liquid 3, thus illuminated, enters microscope 1 at distal end 11 and travels through objective lens unit 6 and hollow shaft 7 toward optical sensor 52. The returning light is partially deflected by beam splitter 54, while another portion of the light from the sample volume propagates to camera 51 and is captured by optical sensor 52. Therefore, a magnified view of the object in liquid 3 can be imaged, and the image is recorded by camera 51. The illumination optical path and the image optical path coincide in most sections of the device, i.e., in the current illustration, below beam splitter 54. The container 2 can be a bioreactor. Therefore, liquid 3 may contain biological cells. The cells can be observed in situ using the microscope 1 shown. Microscope 1 is therefore capable of in situ cytology. Illumination source 53 can be pulsed. Illumination source 53 can emit a single pulse, typically tens of microseconds, during each frame captured by camera 51. Therefore, even if the object is moving, clear and sharp images of the object, even at strong magnification, can be obtained. On the other hand, longer illumination times or multiple illumination pulses per frame recorded by camera 51 can help determine the flow rate and direction through the sample volume.

[0065] Light source 53 can emit monochromatic light or narrow-bandwidth light to avoid chromatic aberration effects. Alternatively, a bandpass optical filter can be arranged in the illumination and / or imaging optical path. In other embodiments, the sample volume can be illuminated by a broadband or multicolor light source, and the optical sensor 52 can receive light of different wavelengths. Due to chromatic aberration, the distance from the object plane receiving a clear image on the optical sensor 52 to the distal end 11 of the microscope 1 is different for different wavelengths or colors. Therefore, if light with sufficiently large wavelength differences is used, some 3D information about the object can be obtained.

[0066] Figure 2 A more detailed view of the objective lens unit 6 is shown. An exemplary embodiment of the objective lens unit 6 shown includes a housing 61, which includes optical components 64, 65, and 66. A front optical aperture 63 is correspondingly positioned through the distal front end 62 of the housing 61 of the objective lens unit 6 or the distal end 11 of the microscope 1. Typically, if the objective lens unit 6 is disposed in a microscope, the distal front end 62 of the housing of the objective lens unit 6 is preferably used to define the distal end 11 of the microscope 1. Light from the illumination source 53 can exit the objective lens unit 6 through the front optical aperture 63, and light from the sample volume can enter the objective lens unit 6 through the front optical aperture 63. A solid immersion lens 64 is... Figure 2 In the illustrated embodiment, the solid immersion lens 64 is arranged inside the front optical aperture 63 to receive light passing through it. The solid immersion lens 64 serves multiple purposes. On one hand, the solid immersion lens provides a high numerical aperture with a relatively small entrance area, which in turn reduces noise. On the other hand, an objective lens employing an immersion lens as the front lens has a narrow depth of field. Typically, the depth of field that can be clearly imaged on an optical sensor is a few micrometers. For example, the field of view captured by a suitable sensor can be 150 μm × 150 μm. Thus, a very narrow sample volume is optically defined without mechanical definition. This is achieved by appropriately adjusting the adjustable lens to a desired object plane that determines the axial position of the sample volume. The narrow depth of field primarily serves to prevent cells that overlap in the line of sight from being captured in the same image, thus avoiding interference from overlapping cells. Furthermore, having a known sample volume allows for estimation of cell density. Since cell diameters are typically in the range of a few micrometers, the depth of field is of a similar order of magnitude. Optical components 65 and 66 together form an objective lens system, which is arranged to collect light passing through the front optical aperture 63 and received by the immersion lens 64.

[0067] The disadvantage of using an immersion lens at the front end 62 of the objective lens unit 6 is that the positional tolerances of the optical components 65 and 66 relative to the immersion lens translate into significant variations in the position of the object plane for clear imaging on the optical sensor, and thus, significant variations in the sample volume. For example, DE 10 2015 014110 specifies a requirement of 30 micrometers of precision to define the object plane with an accuracy of approximately 3 micrometers. However, this makes the manufacture of such an objective lens unit with an immersion lens unit very expensive. Furthermore, it is difficult to maintain such precision throughout the microscope's lifespan, especially if the microscope is subjected to thermal and / or mechanical stress.

[0068] The proposed solution alleviates this problem. Lens 65 is a motorless, automatically adjusting lens. In embodiments, such as in the illustrated example, an adjustable lens is used. It has been shown that by using readily available adjustable lenses, manufacturing and assembly tolerances of 1 mm or more can be compensated by changing the focal length of the adjustable lens. In other embodiments, the adjustable lens can be displaced along the axis of the objective lens unit 6 via a piezoelectric actuator. Both exemplary solutions can be implemented within a compact housing of the objective lens unit 6, are autoclaved resistant, and can operate without vibration. The adjustable lens 65 is disposed adjacent to the immersion lens 64, with a small air gap 69 between the adjustable lens 65 and the immersion lens 64, such that deformation of the adjustable lens surface is not impeded by the immersion lens 64. Lens 66 is disposed near the adjustable lens 65 and is, for example, an aspherical lens. Lenses 65 and 66 are held in place by a threaded sleeve 67 screwed into the housing 61, and a distance sleeve 68 used to maintain the distance between the aspherical lens 66 and the adjustable lens 65. The distal or front surface of the immersion lens 64 is flush with the outer surface of the distal front end of the housing 61. Therefore, less dirt and air bubbles can accumulate, and the objective lens unit 6 is easier to clean.

[0069] exist Figure 3 In another embodiment shown, housing 61 includes a cap 611 and sleeve 612. Cap 611 includes a distal front wall and a lateral sheath. Flange 613 extends radially outward at the proximal rear end of the lateral sheath. An objective lens system including an adjustable lens 65 and an aspherical lens 66 is disposed within sleeve 612. Typically, the objective lens system is assembled and joined... Figure 2 Similar to the overview. The sleeve 612, which houses the optical lens system, can be inserted into the cap 611 through its rear port. The front optical aperture 63 is located in the distal front wall of the cap 611. (Turn) Figure 4Detail IV will be discussed. A planar window 641 remotely closes the front optical aperture 63 and is flush with the remote outer surface of the front wall of the cap 611. A liquid immersion lens 642 is disposed near the window 641, wherein a gap 643 is provided between the liquid immersion lens 642 and the window 641. The gap 643 is filled with an immersion liquid, such as oil, water, silicon, etc. The refractive indices of the window 641, the liquid immersion lens 642, and the immersion liquid within the gap 643, as well as the geometry of the liquid immersion lens 642, are chosen to match well-defined relationships known to those skilled in the art of microscopy and / or optical engineering. In other embodiments, a solid immersion lens may be bonded to a window disposed at the remote end, which remotely closes the front optical aperture. It should be noted that these exemplary immersion lens assemblies can of course also be used with... Figure 2 The outer casing shown is used in combination.

[0070] For example Figure 4 As shown, the air gap 69 is positioned near the immersion lens 642. Turning again... Figure 3 As can be seen, the adjustable lens 65 is positioned adjacent to the immersion lens 642. The sleeve 612 with the objective lens system can be functionally connected to an optical sensor at its proximal end, allowing the image transmitted through the objective lens unit 6 to be captured by the optical sensor. In a non-limiting example, the proximal end of the sleeve 612 can be connected to a shaft 7, which in turn is connected to the microscope head 5, as shown below. Figure 1 As shown. The distal section of the cap 611 can be inserted into the bioreactor through the port of the bioreactor and can be immersed in the sample liquid. The flange 613 remains outside the bioreactor and is used to secure the cap 611 to the bioreactor. Therefore, the rear port of the cap 611 is located outside the bioreactor. Except for the rear port, the cap 611 is liquid-tight. A sleeve 612 with an optical sensor functionally connected thereto can be inserted into the cap 611 from outside the bioreactor to collectively form a microscope suitable for in situ cytology. The objective system and all components attached thereto are protected from contact with the liquid inside the bioreactor.

[0071] Another exemplary embodiment of the objective lens unit 6 is in Figure 5As shown in the diagram. An illumination unit is provided in the objective lens unit 6, which includes an illumination source 44 and a beam splitter 45 as a means for coupling light from the illumination source 44 into the objective lens unit 6. The beam splitter 45 is arranged in the optical path of the objective lens unit 6, while the illumination source 44 is arranged laterally to the optical path. As will be understood, the beam splitter 45 deflects a portion of the light from the illumination source 44 toward the forward or distal end of the objective lens unit 6 and toward the immersion lens 64. The light is emitted through the front optical aperture 63 in the forward or distal direction of the objective lens unit 6. The objective lens unit 6 is adjusted by the adjustable lens 65 such that the object plane is located at a distal end at a desired distance s from the distal front end of the objective lens unit 6 housing. Therefore, the sample volume 100 is clearly imaged on an optical sensor functionally coupled to and located proximal to the objective lens unit 6. The typical dimensions of the sample volume 100 have been discussed above. However, due to the small size of the biological cells in the sample volume and the small difference in refractive index between the cells and their suspension, backscattering from the biological cells in the sample volume is generally likely to be low. Therefore, a reflector 621 (e.g., a mirror or retroreflector) is cantilevered from the housing 61 of the optical lens unit 6 and arranged on the optical axis of the objective lens unit 6. The reflector 621 is correspondingly arranged at the distal end of the housing of the objective lens unit 6 or at the distal end of the microscope 1. It should be understood that the reflector 621, together with the cantilever beam, can be threaded or otherwise mounted to the housing 61 of the optical lens unit 6. The reflector 621 is arranged such that at least a portion of the light emitted through the front optical aperture 63 is reflected back into the front optical aperture 63. The reflected light passes through the sample volume 100. Therefore, the biological cells contained in the sample volume 100 are illuminated from the distal end. Therefore, forward-scattered light from biological cells or other particles in sample volume 100 is received by optical lens unit 6 and imaged onto an optical sensor functionally connected to the rear or near end of objective lens unit 6. The intensity of forward-scattered light can be several orders of magnitude higher than that of backscattered light. Because forward-scattered light, rather than backscattered light, is imaged onto the optical sensor, the quality of cytology is improved.

[0072] like Figure 6 As shown, in another non-limiting example, reflector 621 can also be disposed on a U-shaped carrier. In any embodiment, the distance between the front optical aperture 63 and reflector 621 can also be specified to be variable.

[0073] Figure 7 One embodiment is shown in which the distal front end 62 of the housing of the objective lens unit 6 is not flat (e.g., Figures 2 to 6 (as shown in the example), but is generally convex, for example, conical. For other non-limiting examples, the front wall of the distal end 62 of the housing of the objective lens unit 6 may be circular, for example, dome-shaped.

[0074] Figure 8 A top view of the distal or front end 62 of the housing of an exemplary objective lens unit 6 is shown. One or more optically detectable targets 48 are disposed on the distal surface of an optical component for closing the front optical aperture 63 at the distal end. The optical component is, for example, a... Figure 2 , 5 6 and Figure 7 The solid immersion lens 64 in the middle, or is Figure 3 and Figure 4 Window 641 in the illustrated embodiment. These are all focusing targets. In the operating mode of the microscope including the objective unit 6, the adjustable lens 64 or other adjustable lens can be adjusted to generate a clear image of these targets 48 on the optical sensor. Here, the actual object plane is a reference object plane located at a reference position, i.e., a clearly defined or determined axial position relative to the objective unit 6 or the microscope 1, respectively. In an exemplary embodiment, the reference position is the same as the position of the distal front end 62 of the housing of the objective unit 6 or the position of the distal end 11 of the microscope 1, respectively. In other embodiments, the reference position may be proximal to the front end, but at a clearly defined distance from the front end. Therefore, when the focusing targets are clearly imaged on the sensor, the position of the object plane relative to the objective unit 6 or the microscope 1 and their respective distal ends 11 or distal front ends 62 is known. If the expected position where the sample volume should be is defined relative to the same end or component, the distance required to move the object plane from the expected distance from the distal front end 62 of the housing of the objective unit 6 or relative to the distal end 11 of the microscope 1 is also known. Therefore, the adjustable lens 64 or another automatically adjustable lens can then be adjusted to move the object plane by the distance, thereby setting the object plane at a predetermined distance from the front optical aperture 63 from the far side, so that objects (e.g., cells or particles) in the sample volume located at the predetermined distance from the front optical aperture 63 from the far side are clearly imaged on the optical sensor.

[0075] Figure 9aA perspective view of a planar window 641, an example of the farthest optical component of objective lens unit 6, is depicted. The planar window 641 includes two targets 48a and 48b at different axial positions. Both targets 48a and 48b are focusing targets. They are arranged at different axial positions. The axial distance D between them is known: in this example, it is the thickness of the planar window 641 on which the targets are disposed. Therefore, the planar window 641 in the depicted example provides two reference positions separated from each other by an axial distance D. Since the planar window 641 shown is the farthest optical component of objective lens unit 6, and assuming the microscope does not include any additional farthest components, the farthest target 48a, arranged on the far side of window 641, has a defined axial position of zero relative to the far end of the microscope. Due to the knowledge of the axial distance D, the axial position of the near-side target 48b is known. If the microscope includes additional components, their relative positions are known, and therefore the defined axial positions of targets 48a and 48b are also known.

[0076] In the example shown, the planar window 641 is the shape of a straight cylinder with a circular bottom. The optical axis extends parallel to the normal vector at the bottom of the cylinder, and therefore is parallel to or coincides with the rotational symmetry axis of the cylinder. This is in Figure 9a The arrows indicate the target. In the illustrated embodiment, one of the targets 48b is a smaller circle arranged proximally on the window 641, while the other target 48a is a larger circle arranged distally on the window 641. Targets 48b define a proximal reference plane at the proximal reference position. Target 48a defines a distal reference plane at the distal reference position. Sample volume 100 is shown at a predetermined distance s from the front end. In this example, the predetermined distance s can be selected by the user or preset in the controls of the microscope. Sample volume 100 is the volume of sample liquid 3. Particles and cells located within sample volume 100 are clearly imaged by the microscope using the depicted planar window 641.

[0077] In the method according to the invention, an adjustable lens of the microscope is used to move the object plane, thereby enabling a clear image of the proximal target 48b. In this configuration, the object plane is a proximal reference object plane. In one embodiment, the amplitude of the adjustable lens adjustment applied to set the object plane to the proximal reference object plane is stored. In another embodiment, once the object plane is on the proximal reference object plane, a counter adapted to capture changes in the amplitude of the adjustable lens adjustment is reset. The adjustable lens is then used to move the object plane, enabling a clear image of the distal target 48a. In this configuration, the object plane is a distal reference object plane. In one embodiment, the amplitude of the adjustable lens adjustment applied to set the object plane to the distal reference object plane is compared with the amplitude of the adjustable lens adjustment applied to set the object plane to the proximal reference object plane. In another embodiment, the comparison is the result of a measurement by an appropriate counter used to measure the change in the amplitude of the adjustable lens adjustment applied to move the object plane from the proximal reference object plane to the distal reference object plane. The result of this comparison is also referred to as the adjustment calibration difference. The adjustment calibration distance and the axial distance D between the near and far reference positions can be used to determine or confirm the required adjustable lens adjustment range. The required adjustable lens adjustment range is the amount of adjustable lens adjustment required to move the object plane from the far reference object plane (i.e., the position where the far target 48a is clearly imaged) to the expected object plane position at the expected distance s (i.e., the position where cells or particles in the sample liquid can be clearly imaged). The position of the sample volume 100 is defined by the expected distance s and the optical axis. The size of the sample volume 100 is defined by the imaging area and depth of field of the microscope.

[0078] Figure 9b Another example of a planar window as the farthest optical component is shown. In this case, the window includes a three-dimensional target with two focusing targets 48a and 48b at different axial positions. In this example, the target is in the shape of a triangular pyramid, providing a near-side focusing target 48b in the form of a point and a far-side focusing target 48a in the form of a triangle. On the object plane located between the near-side and far-side reference object planes defined by the focusing targets 48a and 48b, a set of three points (i.e., the vertices of triangles of different sizes) are in a focused state. The distance between these points can be used as an indication of the actual object plane position. Therefore, such a three-dimensional target can help improve the adjustment speed and / or accuracy of the adjustable lens. Furthermore, an additional reference object plane can be defined to establish or confirm a non-linear relationship between the magnitude of the adjustable lens adjustment and the resulting object plane offset.

[0079] While the subject matter of this disclosure has been explained with the aid of exemplary embodiments, it should be understood that these exemplary embodiments are in no way intended to limit the scope of the claimed invention. It should be understood that the claims cover embodiments not expressly shown or disclosed herein, and embodiments deviating from the disclosed exemplary modes of implementing the teachings of the invention will still be covered by the claims.

[0080] List of reference numerals

Claims

1. An objective lens unit (6) for a microscope (1). The objective lens unit (6) includes a housing having a distal front end (62). The front optical aperture (63) of the objective lens unit is located in the distal front end (62) of the housing. The objective lens unit (6) further includes an objective lens system (65, 66), which is arranged inside the housing (61) and located near the front optical aperture (63) of the objective lens unit. in, The objective lens system (65, 66) is configured to collect light received through the front optical aperture (63) of the objective lens unit. The objective lens system (65, 66) includes an adjustable lens (65). The objective lens unit (6) further includes at least one optical component (64, 641, 642) located on the distal side of the adjustable lens, wherein at least one optically detectable target (48) is disposed on the at least one optical component (64).

2. The objective lens unit (6) according to the preceding claim, wherein, The adjustable lens (65) is a motorless, automatically adjusting lens. Specifically, the motorless automatic adjustment lens (65) includes one of an adjustable lens and a lens that can be axially shifted along the optical axis of the objective lens unit (6) by means of a piezoelectric actuator.

3. The objective lens unit (6) according to any one of the preceding claims, wherein, The at least one optically detectable target (48) is disposed on a planar surface extending perpendicular to the optical axis of the objective lens unit (6).

4. The objective lens unit (6) according to any one of the preceding claims, wherein, Immersion lenses (64, 642) are disposed on the far side of the adjustable lens (65) and are configured to receive light through the front optical aperture (63).

5. The objective lens unit (6) according to any one of the preceding claims, wherein, At least one of the at least one optically detectable target (48) is disposed on the farthest part (64, 641) of the objective lens unit (6).

6. The objective lens unit (6) according to the preceding claim, wherein, The farthest optical component (64, 641) of the objective lens unit (6) is one of the immersion lens and the window that closes the front optical aperture.

7. The objective lens unit (6) according to any one of the preceding claims, wherein, At least one of the at least optically detectable targets (48) has a three-dimensional shape.

8. The objective lens unit (6) according to any one of the preceding claims, wherein, The objective lens unit (6) includes at least one first optically detectable target and at least one second optically detectable target, wherein the at least one first optically detectable target and the at least one second optically detectable target are disposed at different axial positions.

9. The objective lens unit (6) according to any one of the preceding claims, wherein, The outer casing (61) includes a cap (611) and a sleeve (612). - The objective lens system (65, 66) is disposed inside the sleeve. - The cap includes a side sleeve, a front wall, a rear port, and the at least one optical component (641, 642). - Wherein, the front optical aperture (63) of the objective lens unit (6) is disposed in the front wall of the cap, and - The at least one optically detectable target (48) is disposed on at least one optical component (641, 642) of the cap. - wherein the sleeve (612) is at least partially received inside the cap (611), or wherein the cap (611) is at least partially received inside the sleeve (612).

10. A cap (611) configured for use in the objective lens unit (6) according to the preceding claim, wherein, - The cap includes a rear port, a front wall, and a lateral sheath extending axially from the front wall. - Wherein, the front optical aperture (63) is formed in the front wall, - The lateral sleeve is configured to receive a sleeve (612) therein, wherein the sleeve is axially insertable through the rear port of the cap, or the lateral sleeve is configured to be received in the sleeve (612), wherein the lateral sleeve is axially insertable into the sleeve. - The cap includes at least one optical component, and wherein the farthest optical component of the at least one optical component closes the front optical aperture (63), and - Wherein, at least one optically detectable target (48) is disposed on at least one of the optical components.

11. The cap (611) according to the preceding claim, wherein, At least one of the at least one optically detectable target (48) is disposed on the farthest optical component of the at least one optical component.

12. A microscope comprising an objective lens unit (6) according to any one of claims 1 to 9 and an optical sensor (52), wherein, The optical sensor (52) is functionally coupled to the objective lens unit (6) to receive light transmitted from the front end of the objective lens unit (6) and through the objective lens system (65, 66).

13. A method for setting the object plane of a microscope (1), - The microscope includes at least one optical sensor (52) and at least one adjustable lens (65). - in, The at least one adjustable lens (65) is configured to axially shift the object plane of the microscope relative to the distal end (11) of the microscope, wherein the object plane is the plane in which the object is clearly imaged on the at least one optical sensor (52). Furthermore, at least one optically detectable target (48) is disposed in the region imaged on the optical sensor (52) and located at an axial position defined relative to the distal end (11) of the microscope. - The method includes the following steps: a. Define a desired object plane position relative to the distal end (11) of the microscope, the desired object plane position being located at a desired distance from the distal end (11) of the microscope from the distal side. b. Adjust the at least one adjustable lens (65) until the at least one optically detectable target (48) is clearly imaged on the optical sensor (52), and c. Adjust the at least one adjustable lens to move the object plane axially to the desired object plane position along the optical axis of the microscope.

14. The method according to the preceding claim, wherein, a. Using two focusing targets positioned at different axial locations, the two focusing targets being positioned in the region imaged on the optical sensor and having a known axial distance (D) between them. b. Among them, The two focusing targets are provided in the following manner: i. At least one of the at least two optically detectable targets (48) has a three-dimensional shape and includes at least two optically distinguishable features that are axially distanced from each other, each of these features forming one of the at least two focusing targets. and / or ii. At least one first optically detectable target (48) is located at a first axial position, and at least one second optically detectable target (48) is located at a second axial position, wherein the first axial position and the second axial position are separated by a known axial distance from each other, and the first optically detectable target (48) and the second optically detectable target (48) each form one of the two focusing targets. c. The method includes the following steps: i. A first calibration adjustment is performed by adjusting the at least one adjustable lens (65) until the first of the focusing targets is clearly imaged on the optical sensor (52). ii. A second calibration adjustment is performed by adjusting the at least one adjustable lens (65) until the second focusing target of the focusing targets is clearly imaged on the optical sensor (52). iii. The adjustment calibration difference is defined as the magnitude of the adjustable lens adjustment applied when moving the object plane from the first focusing target to the second focusing target. iv. Determine the adjustable lens adjustment range required to position the object plane at the desired object plane position based on the following parameters: • The adjustment calibration difference, • The axial distance between the first and second focusing targets in the focusing target set, and • The axial distance between the first or second focusing target and the position of the target plane. v. and adjusting the at least one adjustable lens by applying the desired adjustable lens adjustment amplitude to set the object plane to the desired object plane position.

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