Objective lens unit for microscope, microscope comprising objective lens unit, and method of performing cytometry

Abstract

An objective lens unit (6) for a microscope is disclosed. The objective lens unit is adapted for use when at least partially immersed in a liquid sample. The objective unit includes an immersion lens (64, 642) positioned adjacent a distal front end (62) of a housing of the objective unit. The immersion lens is arranged and configured to receive light through a front end optical aperture (63). An objective lens system (65, 66) is disposed inside the housing (61) and proximal to the immersion lens (64, 642). The objective lens system is configured to collect light received by the immersion lens (64, 642) through the front end optical aperture (63). The objective lens system comprises a motorized auto-adjustment lens (65), in particular an adjustable lens, configured to be able to correct the focal length of the objective lens system with respect to the immersion lens.

Description

Technical Field

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

[0002] Cytology is used to characterize living or dead biological cells. Imaging cytometers do this by analyzing images of the cells. The objective unit according to the invention is preferably used in imaging cytometers. Preferably, for cytology performed using the objective unit according to the invention, the cells are in a liquid. For example, cell density, size, and morphology 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.

[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, either in a bioreactor or in the pipeline transporting the fluid to be monitored. In particular, the fluid to be monitored can be a liquid in a bioprocess containing culture media and biological cells.

[0005] Attempts have been made to construct sensors capable of in-situ cytometry by building in-situ microscopes and recording real-time images of biological cells. However, to obtain cell counts per unit volume, a volume must be defined in the process. Initial commercial attempts attempted to mechanically limit this small volume by opening and closing mechanical chambers within the bioprocess. The mechanical movement and the limited precision of defining the volume hinder the sensor's accurate and reliable operation under real process conditions. Another approach uses objectives comprising a spherical solid immersion lens (so-called SIL) and aspherical lenses for imaging, with the SIL in direct contact with the process liquid. The measurement volume is thus defined by a very narrow depth-of-field optics, a few micrometers close to the SIL. However, maintaining a sharp focus near the SIL surface depends entirely on the alignment of the SIL and aspherical lenses, with tolerances along the optical axis of only a few micrometers. These tolerances are impractical for industrial-scale products. Furthermore, the thermal and mechanical stresses, as well as aging effects, experienced by in-line bioprocess sensors during their lifespan can cause minute displacements of different components relative to each other, thus limiting the lifespan of existing solutions.

[0006] As an example, DE 10 2015 014 110 (DE'110) teaches the use of in-situ microscopy in liquids. DE'110 teaches in more detail how to fix the distance from the objective lens to the SIL with an accuracy of 30 μm, and according to DE'110, it fixes the position of the object plane with an accuracy of approximately 3 μm. Furthermore, it is noted that the entire optical system requires no further adjustments during operation, thus making it suitable as a durable microscope sensor with a long service life.

[0007] In addition to the difficulties mentioned above, even rigid optical designs have been observed to become inaccurate over time, especially when subjected to temperature variations. It may be desirable to set the object plane with higher precision for capturing sharp images, to have more flexibility in the choice of object plane, and in some applications, to change the object plane. Summary of the Invention

[0008] The invention claimed herein is defined by the appended claims.

[0009] The purpose of this disclosure is to present the subject matter initially mentioned. In one aspect, the subject matter disclosed herein will enable in-situ microscopic examination in liquids. In another aspect, the subject matter disclosed herein will overcome at least some of the disadvantages of the prior art.

[0010] 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.

[0011] An objective lens unit for a microscope is disclosed, suitable for use when at least partially immersed in a liquid sample. The proposed objective lens unit includes a housing and certain optical elements disposed within the housing. An immersion lens is positioned adjacent to the distal front end of the housing. The front end includes a front optical aperture of the objective lens unit. The immersion lens is arranged and configured to receive light through the front optical aperture. The objective lens unit also includes an objective lens system disposed within the housing and proximal to the immersion lens, wherein the objective lens system is configured to collect light received by the immersion lens through the front optical aperture. The objective lens system includes a motorless auto-adjusting lens. Specifically, the motorless auto-adjusting lens is configured to correct the effective focal length of the objective lens system. Specifically, the motorless auto-adjusting lens is configured to axially move the focal point of the objective lens system relative to the immersion lens. In particular, since the effective focal length of the objective lens unit is affected by the effective focal length of the objective lens system, the motorless automatic adjustment lens can shift the axial position of the object plane, thereby causing the measurement volume that can be observed through the objective lens unit to move relative to the immersion lens.

[0012] With immersion lenses, the relatively narrow depth of field (typically only a few micrometers measured axially) 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, the axial position of the object plane, in which the object is clearly imaged on a sensor arranged in a functional relationship with the objective unit for image recording, can be adjusted. Similarly, by using an adjustable lens, manufacturing tolerances and 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. In other words, this means that the object plane can be defined more precisely with larger acceptable manufacturing and / or assembly tolerances compared to existing technologies, such as the DE'110. Furthermore, the lifespan and robustness of the objective unit and microscope are improved because misalignments can be corrected using an adjustable lens. The adjustable lens is motorless, meaning size limitations are a minor issue, and no vibration is generated when adjusting the adjustable lens. Below is an example of a motorless, automatically adjusting lens.

[0013] 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, which serves as the farthest optical component. In this case, a gap exists between the window and the liquid immersion lens. This gap is filled with liquid. The materials of the window, the liquid, and the immersion lens are chosen such that their refractive indices are matched to 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 design.

[0014] Solid-state immersion lenses (SILs) typically have a truncated spherical shape, most commonly comprising a hemisphere (so-called hemispherical SIL), or more than a hemisphere (so-called Weierstrass SIL), also known as a super-hemispherical SIL or super SIL. In this case, the front optical aperture of the objective unit can be closed by the SIL as the farthest optical component. In some embodiments, the SIL can be bonded or pressed against a window that closes the front optical aperture. This window can be optimized to withstand the conditions of the liquid being observed, cleaning procedures commonly used in biological processes, 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 a coated SIL or a 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., the enhanced numerical aperture, which is achieved by ensuring that the refractive index of the combination remains greater than that of standard glass, preferably greater than 1.5.

[0015] Solid immersion lenses, or SILs, offer high numerical apertures with relatively small entrance areas, which in turn reduces noise and improves resolution. Therefore, objectives employing immersion lenses as the front lens have 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 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.

[0016] In an embodiment, in addition to the motorless automatic adjustment lens, the objective lens system may also include at least one fixed lens with a fixed focal length. The at least one fixed lens with a fixed focal length may specifically include at least one aspherical lens.

[0017] As described above, the objective lens unit described herein is adapted for use when at least partially immersed in a liquid sample. At least a distal section of the objective lens unit, including the front optical aperture, is adapted to be immersed in the liquid. Preferably, this distal section is the front end of the housing.

[0018] Furthermore, at least the housing of the objective unit is sterilizable and SIP and / or CIP compatible. In some embodiments, the housing is a sterilizable, SIP and / or CIP compatible shell. In some embodiments, the shell in which the objective system is internally mounted is sterilizable and SIP and / or CIP compatible. In some embodiments, the housing includes a cap and a sleeve, and at least the cap is sterilizable and SIP and / or CIP compatible. In some embodiments, the housing includes a cap and a sleeve, and at least the cap of the sleeve, which is mounted to the objective system disposed in the sleeve, is sterilizable and SIP and / or CIP compatible. In embodiments, the entire objective unit is sterilizable and SIP and / or CIP compatible. In particular, the use of motorless automatic adjustment lenses makes the entire objective unit or components of the objective unit including the objective system sterilizable and / or SIP and / or CIP compatible. This eliminates the need for disassembly of the objective unit, making sterilization, CIP, and SIP faster and easier.

[0019] Common cleaning procedures in bioprocessing include "SIP" (Sterilization in Place) and "CIP" (Cleaning in Place). SIP refers to sterilizing production equipment without prior disassembly. Sterilization is typically performed using superheated steam, for example, at 120°C and 2 bar for approximately 60 to 70 minutes, although the exact parameters can vary depending on the application and system. CIP usually precedes SIP and involves cleaning the surfaces to be sterilized. The production equipment is cleaned in a cyclical or continuous process without prior disassembly and typically includes a series of cleaning steps, which usually include rinsing with water, using a cleaning agent, and using an acid.

[0020] A component is preferably sterilizable if it can withstand autoclaving and / or exposure to ionizing radiation such as X-rays or gamma radiation without losing its functionality. A component is preferably SIP and / or CIP compatible if it can undergo SIP and / or CIP procedures in its intended configuration for use in the process (i.e., in a reactor used for measurement or in its installed piping) without losing its functionality.

[0021] In the preferred option where at least a portion of the objective unit or its housing (e.g., a shell or cap) is SIP and / or CIP compatible, the objective unit is particularly comfortable to use in bioprocesses, especially when using reusable bioreactors: during measurements, the opening through which the microscope using the objective unit is inserted in the process remains closed by at least a portion of the objective unit's housing (e.g., a shell or cap) during the SIP or CIP process, which is simultaneously cleaned and sterilized along with the rest of the apparatus. This preferred option, where at least a portion of the objective unit or its housing (e.g., a shell or cap) is sterilizable, allows for the provision of objective units that can be comfortably used with disposable bags: in these embodiments, at least a portion of the objective unit or its housing (e.g., a shell or cap) can be attached to a disposable bag or suitable adapter, and the entire assembly can be sterilized together.

[0022] 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".

[0023] The housing of the objective lens unit may be a shell, wherein the objective lens system is mounted inside the shell. In this embodiment, the shell has a front surface. In this embodiment, the front optical aperture of the objective lens unit is disposed in the front surface of the shell. This front surface of the shell defines the front end of the shell. In this embodiment, the immersion lens is arranged adjacent to the front optical aperture. In particular, the immersion lens is mounted to the shell, most preferably such that the immersion lens forms part of the hydrophobic seal of the front optical aperture.

[0024] In other non-limiting embodiments, the housing may include a sleeve and a cap. In these embodiments, the objective lens system is disposed within the sleeve. The cap includes a lateral sheath, a front wall, and a rear port. The front optical aperture of the objective lens unit is disposed in the front wall of the cap, and the immersion lens is attached to and particularly disposed within the cap. The sleeve is at least partially housed within the cap, particularly through the rear port of the cap. In particular, the front wall of the cap forms the front end of the housing, especially when the sleeve is housed within the cap. Preferably, the immersion lens is mounted to the cap such that the immersion lens forms part of a hydrophobic closure of the front optical aperture.

[0025] In one embodiment, the sleeve may be threadedly received inside the cap. For example, a seal provided by an O-ring may be provided between the cap and the sleeve. In other embodiments, the sleeve may simply be inserted into the cap. The sleeve and the cap are preferably equipped with an alignment device such that when the sleeve is received inside the cap, the optical axis of the immersion lens 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 the immersion lens and the objective lens system resulting from mounting the cap to the sleeve can be corrected by the adjustable lens.

[0026] 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, and the rear port of the cap is located outside the bioreactor. Preferably, the distal front section of the sleeve is accommodated within the portion of the cap located inside 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 variations. Furthermore, the sleeve can be continuously inserted into different caps for use in different bioreactors or at different locations within a single bioreactor.

[0027] Preferably, at least the distal section of the objective lens unit is liquid-resistant. This makes the objective lens unit suitable for use when at least partially immersed in a liquid sample. In a more specific embodiment, the objective lens unit includes a proximal rear connector interface, wherein at least one section of the objective lens unit other than the rear connector interface is liquid-resistant. Preferably, the housing and the distal optical element (preferably a window or immersion lens) of the objective lens unit together form a liquid-tight barrier enclosing the space for housing the objective lens system. Preferably, a seal is provided between the housing (preferably a shell or cap) and the distal optical element (preferably a window or immersion lens). Preferably, this seal is achieved by molding, by using a suitable adhesive, by interference fit, or by arranging a sealing structure (whether or not a sealing material such as an O-ring) between the housing and the distal optical element. Preferably, the distal optical element is a SIL. In some embodiments, the SIL and at least a portion of the housing are made of the same material and form a single structure. In embodiments where a cap and sleeve are used as the housing, the cap forms the outer surface of the distal section of the objective lens unit, or the cap and sleeve can be connected in a liquid-resistant manner such that when the cap is installed as intended, the combination of the cap and sleeve forms the liquid-resistant distal section of the objective lens unit.

[0028] The motorless, automatically adjusting lens can be an adjustable lens. Adjustable lenses are 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. Adjustable lenses require minimal space, can be used over a wide temperature range, and operate without vibration.

[0029] In other embodiments, the motorless automatic adjustment lens may be specified to include a piezoelectric actuator and a lens axially displaceable by the piezoelectric actuator along the optical axis of the objective lens unit. The lens displaceable by the piezoelectric actuator may include a fixed-focus lens or an adjustable lens. As will be understood, the piezoelectric actuator requires minimal space, can be used over a wide temperature range, and operates without vibration.

[0030] The motorless auto-adjusting lens can be positioned between the immersion lens and the stationary lens in the objective lens system. The term "stationary lens" refers to a lens in the objective lens system that has a fixed (i.e., static) position and a fixed focal length. More specifically, the motorless auto-adjusting lens can be the next lens located near the immersion lens.

[0031] The farthest optical element of the objective lens unit (e.g., particularly a window or immersion lens) can be flush with the far outer surface of the front end of the housing. Preferably, the far outer surface is correspondingly the far outer surface of the front surface of the housing or the far outer surface of the front wall of the cap. This makes the far tip of the objective lens unit smooth, thereby improving the robustness of the objective lens unit, significantly reducing the risk of dirt accumulation, and reducing eddies around the far tip of the objective lens unit when placed in a liquid flow.

[0032] In this respect, the front or distal tip of the shell can be flat or convex. While a flat front requires less space, a convex front may generate less disturbance in the liquid flow, thereby reducing the risk of air bubbles adhering to it.

[0033] The circumferential outer wall of the objective lens unit may 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).

[0034] Other exemplary embodiments of the objective lens unit described herein include at least two motorless auto-adjusting lenses arranged laterally offset from each other and capable of being controlled independently of each other. These motorless auto-adjusting lenses can be operated to simultaneously image objects in different object planes on the sensor. This operation, for example, enables the acquisition of images from a single biological cell from different perspectives, i.e., performing cell tomography. This operation also enables the acquisition of images of a statistically significant number of cells, even at low cell densities.

[0035] Alternatively, a broadband or multicolor light source can be used to illuminate the sample, and a beam splitter can be used, for example, to separate the light from the sample volume along the optical path from the front optical aperture to the sensor. Elements (e.g., but not limited to filters) that allow selection of different narrow wavelength ranges can be arranged in the resulting different optical paths. Therefore, images at different wavelengths can be processed. If the different wavelengths are sufficiently far apart, the images at different wavelengths will originate from different object planes due to chromatic aberration. In another embodiment, a beam splitter can be used, for example, to divide the optical path, and different lenses can be arranged in the different optical paths to generate different object planes that image on the sensor through the different optical paths.

[0036] In another embodiment, the objective lens unit proposed herein may include a sample illumination unit. The sample illumination unit includes an illumination source and means for coupling light from the illumination source into the objective lens unit. The means for coupling light from the illumination source into the objective lens unit is configured to project the light in a proximal-to-distal direction toward the immersion lens. In an embodiment, the means for coupling light from the illumination source into the objective lens unit may be arranged between the immersion lens and a motorless auto-adjusting lens. Light used to illuminate the sample may then exit from the front optical aperture of the objective lens unit. The means for coupling light from the illumination source into the objective lens unit may be a beam splitter. The illumination source, such as an LED, may be functionally coupled to the means for coupling light from the illumination source into the objective lens unit and may be positioned laterally toward the optical path of the objective lens unit. However, in other embodiments, for example, an optical fiber may be used to couple light from the illumination source into the objective lens unit or to the means for coupling light from the illumination source into the objective lens unit.

[0037] In another embodiment, the mirror may be positioned distal to the front end of the housing, and the mirror is located on the extension of the optical axis of the objective lens unit. In this embodiment, the adjustable lens is configured such that the object plane, which is clearly imaged on the sensor, is closer to the front end of the housing than the mirror. In these cases, when the mirror is positioned distal to the front end, the biological cells in the sample volume are illuminated by light emitted from the front end of the housing and light reflected from the mirror. Thus, the object is essentially observed by two types of microscopes simultaneously: reflected light microscopy along the path from the light source to the mirror, and transmitted light microscopy along the path from the mirror to the detector. This creates two images simultaneously: this situation is generally considered unfavorable and corrected by filtering out one of the two images using a polarization filter or similar device. However, it has been recognized that biological cells scatter significantly more light forward than backward, such that, for cytological purposes, the image produced by reflected light does not affect the evaluation of the dominant transmitted light image. Therefore, as described above, for cytological measurements of cells using in-situ microscopy, robust and space-saving illumination with a mirror is preferred.

[0038] On the other hand, a cap is proposed as part of an objective lens unit having a housing including a sleeve and a cap, as described above. The cap includes a front end and lateral sheaths extending axially from the front end. A front end optical aperture is formed in the front end, and an immersion lens is attached to the front end in a manner functionally related to the front end optical aperture, such that light from the front side of the cap and located outside the cap passes through the front end optical aperture and then through the immersion lens into the interior of the cap. The lateral sheaths are configured to accommodate the sleeve therein, wherein the cap is configured to receive the sleeve axially from 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.

[0039] In an embodiment, one or more optically detectable targets may be positioned on the farthest optical element of the objective unit. This optical element may be an immersion lens, particularly the cap or the solid immersion lens of the objective unit, or a window disposed distal to or directly on the immersion lens. These targets allow the assembled microscope system to determine the state in which the motorless auto-adjusting lens positions the object plane on the far-end surface defined by the target. For this purpose, in addition to the objective unit, the microscope system includes an optical sensor, a control unit configured to control the motorless auto-adjusting lens, and other means configured to evaluate and / or transmit the optical data collected and recorded by the optical sensor. In this embodiment, these other means include an image processing unit configured to detect targets in the image captured by the optical sensor. Such means for determining, at least indirectly, the distance between the farthest optical element and the objective system is particularly suitable when the user arranges the two components relative to each other, for example, when using the cap, or after events such as cleaning, SIP, or CIP cycles, which may cause components of the objective unit to move relative to each other due to thermal or mechanical stress.

[0040] In another aspect, a microscope for in-situ application within a bioreactor is disclosed. The microscope includes any of the aforementioned types of objective lens units and optical sensors. Preferably, the distal end of the microscope is formed by the front end of the housing. However, in some embodiments, the microscope is also equipped with a protective shield or illumination device that extends distally beyond the front end of the housing. The optical sensor is functionally coupled to the objective lens unit to receive light transmitted from the front side of the objective lens unit and passing through it. In particular, the optical sensor is functionally coupled to the objective lens unit to receive light transmitted through the immersion lens and through the objective lens system. For example, the optical sensor may be coupled to the rear connector interface of the objective lens unit. In a particular aspect, the microscope may be configured for attachment to a bioreactor including standard ports, wherein the microscope is further configured such that at least a distal segment of the objective lens unit (particularly the segment including the front optical aperture and the immersion lens) can be positioned inside the bioreactor, while the microscope head and / or the rear connector interface can be located outside the bioreactor. Preferably, the microscope head or the rear connector interface includes an interface for receiving and transmitting data and / or power. The microscope may also include a sample illumination unit comprising an illumination source and means for coupling light from the illumination source to the objective lens unit. The sample illumination unit may be disposed, for example, in the microscope head or the objective lens unit, as described above, or coupled to the rear connector interface. The optical path of the light emitted from the sample illumination unit preferably coincides with the optical path of the light collected from the sample at least in segments of the respective optical paths. The optical sensor may particularly include an image sensor, and more particularly, a CCD or CMOS image sensor. The optical sensor may be disposed in the microscope head, coupled to the rear connector interface, or integrated into the objective lens unit.

[0041] The microscope may further include a control unit functionally coupled to the objective lens unit and configured to control the motorless auto-adjusting lens. The microscope may also include control hardware configured to control the sample illumination unit and optical sensors, and other means configured to evaluate and / or transmit optical data collected and recorded by the optical sensors. Preferably, the control unit, the control hardware, and / or the other means configured to evaluate and / or transmit optical data are disposed in the microscope head, coupled to a rear connector interface, or are part of the rear connector interface.

[0042] In a further aspect, a method for performing cytometry is proposed. In one aspect, the method includes using a microscope as described above. The method further includes inserting at least a distal segment of the objective lens unit through a port of a bioreactor and immersing at least a portion of the distal segment of the objective lens unit, including its front end and a front optical aperture, in a sample liquid contained within the bioreactor. The motorless auto-adjusting lens is operated to adjust the object plane for clear imaging onto the optical sensor. Once the object plane is correctly set, at least one image generated by the objective lens unit is recorded using the optical sensor.

[0043] On the other hand, using a microscope as described above, wherein the objective unit includes a cap and a sleeve, as described above. The method includes inserting at least a distal section of the cap through a port of a bioreactor and sealing the port of the bioreactor with the cap. The rear port of the cap remains outside the bioreactor. At least a portion of the distal section of the cap, including a front wall with a front optical aperture, is immersed in the sample liquid inside the bioreactor. A sleeve including the objective system is inserted into the cap. The motorless auto-adjusting lens is operated to adjust the object plane for clear imaging on the optical sensor, and at least one image generated by the objective unit is recorded using the optical sensor.

[0044] The sample illumination unit can operate intermittently. The illumination source can emit light pulses with a duration of 20 μm or less, and in a more specific embodiment, emit light pulses with a duration of 10 μm or less. The resulting short illumination time allows for the capture of clear images even when cells are moving within the sample volume. The pulsed operation of the illumination source, with its short microsecond-level pulse duration, enables the acquisition of the desired stroboscopic effect independent of the sensor's frame rate. On the other hand, the blurring of the image during longer illumination times can be used to determine the flow rate through the sample volume.

[0045] Capturing monochrome images or images using light within a narrow wavelength range can be used to avoid chromatic aberration, thereby further improving image quality. This can be achieved by emitting monochromatic or low-bandwidth light from the sample illumination source, using filters, or a combination thereof. However, as mentioned above, in some applications, capturing images at at least two sufficiently different wavelengths may be useful, thereby utilizing chromatic aberration to simultaneously acquire images from two different object planes.

[0046] In some other applications, the objective lens unit and the control unit for controlling the motorless auto-adjusting lens can be configured to automatically adjust to the object plane in which a clear image is received by the optical sensor. For this purpose, at least one optically detectable reference structure can be provided in the optical path of the objective lens unit. The control unit for controlling the motorless auto-adjusting lens can then first adjust the motorless auto-adjusting lens to obtain a clear image of at least one reference structure on the optical sensor. The motorless auto-adjusting lens can then be adjusted to obtain a clear image of an object at a desired distance from the reference structure or from the front end of the housing.

[0047] 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

[0048]

[0049] 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

[0050] 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 from 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.

[0051] 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.

[0052] 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 comprising optical components 64, 65, and 66. A front optical aperture 63 is disposed through either the front end 62 of the housing 61 of the objective lens unit 6 or 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 disposed within 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 defined optically without mechanical definition. A narrow depth of field is primarily used to prevent cells that overlap in the line of sight from being captured in the image, thus avoiding interference from overlapping cells. Optical components 65 and 66 together form an objective lens system arranged to collect light passing through the front optical aperture 63 and received by the immersion lens 64. A 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 large variations in the position of the object plane for clear imaging on the optical sensor, and thus, large variations in sample volume. For example, DE 10 2015 014110 indicates a requirement of 30 micrometers of precision to define the object plane with an accuracy of approximately 3 micrometers. However, this makes manufacturing such an objective lens unit with an immersion lens unit expensive and makes this existing objective lens unit sensitive to changing conditions such as aging and temperature variations.

[0053] The proposed solutions alleviate these problems. 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. Furthermore, the adjustable lens is typically autoclaved. In other embodiments, the adjustable lens can be displaced along the axis of the objective lens unit via a piezoelectric actuator. Both exemplary solutions can be implemented within a compact housing of the objective lens unit 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 (the farthest optical element in the illustrated embodiment) is flush with the outer surface of the distal front end of the housing 61. Therefore, localized turbulence in the liquid flow is avoided during measurement, dirt buildup is reduced, and the objective lens unit 6 is easier to clean.

[0054] exist Figure 3 In another embodiment shown, housing 61 includes a cap 611 and sleeve 612. Cap 611 includes a distal front end and a lateral sheath. In the illustrated embodiment, 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 description. The sleeve 612, in which an optical lens system is provided, can be inserted into the cap 611 through its rear port. In some embodiments, the cap 611 and the sleeve 612 are connected via threads. This can be similar to... Figure 1This is accomplished in embodiments where the objective lens unit 6 is mounted to the hollow shaft 7. In some embodiments, the cap 611 is preferably directly connected to the hollow shaft 7 via threads, while a sleeve 612 is arranged between the cap 611 and the hollow shaft 7. In these cases, the sleeve 612 can essentially serve as a carrier and mounting device for the objective lens system, and can be attached to either the cap 611 or the hollow shaft 7 before they are connected to each other; preferably, the sleeve 612 is first mounted to the hollow shaft 7, as this allows the connection required for a motorless adjustable lens to be established. In particular, if the cap 611 is connected to the hollow shaft 7 directly or via the sleeve 612, the cap 611 typically does not include a flange, but instead includes seals and mounting devices (e.g., O-rings and threads) to ensure a preferred fluid-resistant connection between the cap 611 and the sleeve 612 or the hollow shaft 7. In some embodiments, a front optical aperture 63 is disposed in the distal front end of the cap 611.

[0055] Turn Figure 4 Detail 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 end of the cap 611. A liquid immersion lens 642 is disposed near the window 641, with a gap 643 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 or pushed against the window that remotely closes the front optical aperture. In these cases, the gap 643 is not present. Instead of a window, the solid immersion lens may be coated. The window and coating may be selected to protect the immersion lens or to improve measurement conditions by delaying contamination or reducing the likelihood of air bubbles adhering to the most distal optical element. It should be noted that these exemplary immersion lens assemblies can of course also be used with, for example, Figure 2 The outer casing shown is used in combination.

[0056] 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 1As 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-proof. A sleeve 612 with an optical sensor functionally attached 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.

[0057] 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 is adjusted by the adjustable lens 65 such that the object plane is located at a distance s from the front optical aperture 63 at the distal end. Therefore, the sample volume 100 is clearly imaged on an optical sensor functionally coupled to and located proximal to the objective lens unit 6. Typical dimensions of the sample volume 100 have been discussed above. However, due to the small size of biological cells in the sample volume and the small difference in refractive index between the cells and their suspension, backscattering from biological cells in the sample volume is typically low. Therefore, mirror 621 is cantilevered from the housing 61 of the optical lens unit 6 and arranged on the optical axis of the objective lens unit 6. It should be understood that mirror 621, together with the cantilever beam, can be threaded or otherwise mounted to the housing 61 of the optical lens unit 6. Mirror 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. Thus, forward-scattered light from biological cells or other particles in the sample volume 100 is received by the optical lens unit 6 and imaged onto an optical sensor functionally connected to the rear or proximal end of the objective lens unit 6. The intensity of the forward-scattered light can be several orders of magnitude higher than the intensity of the backscattered light. Therefore, the forward-scattered light dominates the image on the optical sensor relative to the backscattered light. This allows for improved cytology quality using the strong signals obtained through transmission microscopy. Simultaneously, it eliminates the need for light sources outside the objective unit and inside the liquid being observed, as well as the need for devices (such as polarization filters) to separate the reflected image from the transmitted image. This results in a particularly robust and compact illumination system and microscope.

[0058] like Figure 6 As shown, in another non-limiting embodiment, mirror 621 can also be mounted on a U-shaped carrier. This minimizes the impact of the flow of the liquid being observed. In any embodiment, the distance between the front optical aperture 63 and mirror 621 can also be specified as varying.

[0059] Figure 7 One embodiment is shown in which the 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 end 62 of the housing of objective unit 6 can be circular, for example, dome-shaped. Dome or convex shapes can help minimize the effects of the flow of the liquid being observed and can help reduce the likelihood of air bubbles adhering to the front end 62.

[0060] Figure 8 A top view of the distal or front end 62 of the housing of the objective lens unit 6 is shown. In embodiments of the subject matter disclosed herein, one or more optically detectable targets 48 may be disposed on the distal surface of an optical component for closing the front optical aperture 63 at the distal end, the optical component being, for example, Figure 2 , 5 6 and Figure 7 Solid immersion lens 64, or Figure 3 and Figure 4 Window 641 in the illustrated embodiment. In the operating mode of the microscope including the objective lens unit, the adjustable lens 64 or other motorless auto-adjusting lens can be adjusted to generate a clear image of the target object 48 on the optical sensor. Then, the adjustable lens 64 or other motorless auto-adjusting lens is adjusted to position the object plane at a predetermined distance from the front optical aperture 63, so that the sample volume located at the distance from the front optical aperture 63 is clearly imaged on the optical sensor. Therefore, autofocus operation can be achieved.

[0061] 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.

[0062] List of reference numerals

Claims

1. An objective lens unit (6) for a microscope (1), the objective lens unit being adapted for use when at least partially immersed in a liquid sample (3), the objective lens unit comprising a housing (61). The objective lens unit includes an immersion lens (64, 642) positioned at the distal front end (62) adjacent to the housing (61), the front end including the front optical aperture (63) of the objective lens unit, and the immersion lens being arranged and configured to receive light through the front optical aperture. The objective lens unit further includes objective lens systems (65, 66), which are arranged inside the housing (61) and near the immersion lenses (64, 642), wherein, The objective lens system is configured to collect light received by the immersion lenses (64, 642) through the front optical aperture (63). The objective lens system includes a motorless automatic adjustment lens (65).

2. The objective lens unit according to the preceding claim, wherein, The housing (61) is a housing in which the objective lens system (65, 66) is mounted. The housing has a front surface, wherein the front optical aperture (63) of the objective lens unit is disposed in the front surface of the housing, and the immersion lens (64, 642) is arranged adjacent to the front optical aperture (63).

3. The objective lens unit according to claim 1, wherein the outer shell (61) comprises a cap (611) and a sleeve (612), wherein, An objective lens system (65, 66) is disposed inside the sleeve, the cap including a lateral sheath, a front wall and a rear port, wherein the front optical aperture (63) of the objective lens unit is disposed in the front wall of the cap, and an immersion lens (64, 642) is attached to the cap (611), wherein the sleeve (612) is at least partially housed inside the cap (611).

4. The objective lens unit according to any one of the preceding claims, wherein, At least the distal section of the objective lens unit is liquid-resistant.

5. The objective lens unit according to any one of the preceding claims, wherein, The motorless automatic adjustment lens (65) is an adjustable lens.

6. The objective lens unit according to any one of the preceding claims, wherein, The motorless automatic adjustment lens (65) is a lens that can be axially shifted along the optical axis of the objective lens unit (6) by means of a piezoelectric actuator.

7. The objective lens unit according to any one of the preceding claims, wherein, The motorless automatic adjustment lens (65) is arranged between the immersion lens (64, 642) and the fixed lens (66) of the objective lens system, wherein, in particular, the motorless automatic adjustment lens is the next lens located near the immersion lens.

8. The objective lens unit according to any one of the preceding claims, wherein, The farthest optical element (64, 641) of the objective lens unit is flush with the far outer surface of the front end (62) of the housing.

9. The objective lens unit according to any one of the preceding claims, wherein, The front end (62) of the outer shell is either a flat shape or a convex shape.

10. The objective lens unit according to any one of the preceding claims, wherein, The objective lens unit includes a sample illumination unit, wherein the sample illumination unit includes an illumination source (44) and a means (45) for coupling light from the illumination source (44) into the objective lens unit (6), wherein the means for coupling light from the illumination source into the objective lens unit is configured to project the light in the proximal-to-distal direction of the objective lens unit toward the immersion lenses (64, 642), and the means is preferably arranged between the immersion lenses (64, 642) and the motorless automatic adjustment lens (65).

11. A cap (611) configured for use in an objective lens unit according to claim 3 or any one of claims 4 to 10 when claim 3 is referenced, wherein, The cap includes a front wall and a lateral sheath extending axially from the front wall, wherein a front optical aperture (63) is formed in the front wall, and immersion lenses (64, 642) are attached to the front wall in a manner functionally related to the front optical aperture (63) such that light from the front side of the cap and located outside the cap passes through the front optical aperture and enters the interior of the cap through the immersion lenses, and wherein the lateral sheath is configured to accommodate a sleeve (612) therein, wherein the sleeve is axially insertable from the rear end of the cap.

12. A microscope (1) for in-situ application inside a bioreactor, the microscope comprising an objective lens unit (6) according to any one of claims 1 to 10 and an optical sensor (52), wherein, The optical sensor is functionally coupled to the objective lens unit to receive light transmitted from the front side of the objective lens unit and passing through the objective lens unit.

13. The microscope according to the preceding claim, wherein, The microscope includes a control unit that is functionally coupled to the objective lens unit (6) and configured to control the motorless auto-adjusting lens (65).

14. A method for performing cytology using a microscope according to any one of claims 12 and 13, wherein, The method includes inserting at least a distal segment of the objective lens unit (6) through a port of the bioreactor (21) and immersing at least a portion of the distal segment of the objective lens unit (6), including the front end (62) and the front end optical aperture (63), in the sample liquid (3) contained inside the bioreactor, operating a motorless automatic adjustment lens (65) to adjust the object plane to be clearly imaged on the optical sensor (52), and using the optical sensor to record at least one image generated by the objective lens unit (6).

15. A method for performing cytology using a microscope (1) according to any one of claims 12 or 13, wherein, The objective unit (6) is an objective unit according to claim 3 or any one of claims 4 to 10 when claim 3 is referenced. The method includes inserting at least a distal section of the cap (611) through a port of the bioreactor and sealing the port of the bioreactor with the cap, leaving the rear port of the cap (611) outside the bioreactor, immersing at least a portion of the distal section of the cap (611) including the front wall with the front optical aperture (63) in the sample liquid (3) inside the bioreactor, inserting a sleeve (612) including the objective system (65, 66) into the cap (611), operating the motorless auto-adjusting lens (65) to adjust the object plane to be clearly imaged on the optical sensor (52), and using the optical sensor to record at least one image generated by the objective unit.

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