Device for guiding illumination, instrument comprising said device, and method of performing cytometry

Abstract

An apparatus for guiding illumination for a sample volume in microscopy, in particular in in situ cytometry. The device includes a body (611) having a distal front end (62) and an optical aperture (63) disposed in the front end, and a reflector (621). And a reflector disposed distal of the front end at a non-zero distance from the optical aperture, the reflector configured to receive light emitted through the front end and to reflect at least a portion of the light received from the front end back into the optical aperture. The reflector is connected to the body by at least one cantilevered strut. The proposed device may be part of an objective unit (6).

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] Cytometry is used to characterize living or dead biological cells. Preferably, for cytometry performed using the apparatus according to the invention, the cells are in a liquid. For example, the density, number density, size, and morphology of biological cells can be determined by microscopy. In the absence of reliable and quantitative online and in-situ measurement methods, bioengineers must collect samples to perform cytometry using offline measurement devices. This requires removing a certain volume of sample contents 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 cytometry. Compared to offline methods, in-situ applicable online cytometers significantly reduce complexity and allow process controllers to monitor cell counts and cell status in near real-time.

[0003] Online imaging cytometry requires high-quality images of biological cells. A key requirement for obtaining high-quality images is proper illumination of the object being observed. Since cells within a bioreactor typically move rapidly due to agitation, short light pulses of a few microseconds are preferred to avoid image trailing and blurring. This significantly limits the light energy collected per frame. Furthermore, biological cells typically have a refractive index very close to that of the surrounding medium, which further reduces interaction with light and thus decreases the object's contrast. For biological cells in liquid suspensions, backscattered light can be observed to be several orders of magnitude smaller than forward scattered light. Therefore, transmission microscopy (i.e., illuminating the object from outside the object while viewing it through a microscope objective, or in other words, using an illumination source that emits light in a proximal direction and guides said light through the sample volume toward the microscope aperture) significantly improves image quality compared to pure backscattered light produced by illuminating the object with light propagating distally. However, for in situ cytometry in bioprocessing, placing the light source below the object or sample volume can introduce certain disadvantages.

[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 can be a liquid in a bioprocess containing culture media and biological cells.

[0005] US 6,809,862 discloses a microscope comprising a microscope apparatus having a sample area between a slide body and a lens cover glass, the lens cover glass closing the microscope in a distal direction. An illumination device is positioned distal to the sample area and covered by the slide body, illuminating the sample area through it, wherein the illumination is directed to the microscope lens. Thus, the sample is observed in transmitted light. A large unit including a light source, a power supply for the light source, and collimating optics is arranged in front of or distal to the microscope and within a reactor, which, when applied to in-situ microscopy, affects the flow patterns within the reactor.

[0006] DE 40 32 002 teaches the application of transmission microscopy inside a bioreactor. For this purpose, a microscope is positioned outside the bioreactor and separated from its interior by a window. A curved tube containing optical fibers is inserted into the bioreactor and guided to pass through the sample volume inside the bioreactor and accordingly emit light toward the window or microscope. Insertion of the curved tube requires an additional dedicated port on the bioreactor.

[0007] US 4,515,445 discloses an optical system for transmitted light microscopy with incident illumination. The proposed apparatus includes a general-purpose upright reflected light microscope positioned on one side of an object, and a retroreflection device comprising an optical system that images the object itself in a non-reversed and upright manner from the other side of the object. The object is placed on a microscope slide of the upright microscope. The purpose of the subject matter is to observe light passing through the object twice. US 4,515,445 does not provide any indication of its application in in situ cytology, and the retroreflection device, which needs to be placed on the far side of the volume of the object or sample being observed, is quite large and therefore unsuitable for in situ cytology.

[0008] GB 784,822 teaches the emission of plane-polarized light having a first polarization plane to illuminate a sample, and the observation of light polarized in a polarization plane perpendicular to the first polarization plane. The sample is placed on a microscope slide. A quarter-wave plate is rotatably arranged near the microscope slide, i.e., correspondingly arranged in the optical path between the light source and the microscope slide or sample. A depolarizing reflector is arranged far from the microscope slide. GB 784,822 essentially discloses an upright microscope. No tips or suggestions are given on how to apply the teachings described therein to in situ cytology.

[0009] US 2003 / 0011881 describes a confocal microscope that uses a Nipkow disk to scan a sample. A deflection device is positioned distal to the sample to be scanned, allowing the sample to also be illuminated in the direction of the objective lens. Scanning the sample naturally implies that the sample needs to remain in place during at least one scan. This makes the teaching unsuitable for online and in situ cytology.

[0010] An instrument is needed that can perform in situ cytology using in situ transmission microscopy. Summary of the Invention

[0011] The invention claimed herein is defined by the appended claims.

[0012] The purpose of this disclosure is to address the subject matter initially mentioned. In one aspect, the subject matter disclosed herein should support the provision of transmitted illumination for objects in in situ microscopy, particularly for objects suspended in sample liquids (e.g., liquid culture media in bioprocesses). In another aspect, the subject matter disclosed herein will overcome at least some of the disadvantages of the prior art.

[0013] 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. In a more specific sense, the subject matter disclosed herein enables transmission microscopy of the interiors of liquids.

[0014] Therefore, a device for guiding illumination is disclosed, particularly for sample volumes in microscopy, especially in in situ cytology. In the case of cytology, the object to be observed is a biological cell. The device includes a body having a distal front end and an optical aperture disposed in the front end. Furthermore, the device includes a reflector connected to the body and disposed distally on the front end and at a non-zero distance from the optical aperture. The reflector is arranged and configured to receive light emitted through the front end of the body and to reflect at least a portion of the light received from the front end back into the optical aperture. This achieves a particularly robust and compact structure, and, as explained in more detail below, the scattering properties of numerous cells suspended in a liquid allow for the simultaneous observation of forward and backward images.

[0015] Preferably, the light received by the reflector is emitted through the optical aperture. However, the reflector may also be illuminated from a direction tilted relative to the optical axis defined by the optical aperture and the reflector. For example, the reflector may be a curved mirror, which is illuminated by an annular light source surrounding the optical aperture and tilted such that the reflected light is collimated. In some embodiments, the aperture through which the emitted light exits the body is the same as the optical aperture. In some embodiments, the front end includes one or more additional apertures through which the emitted light can exit the body. The additional apertures may be provided, for example, by a light guide embedded in the material of the body.

[0016] In an embodiment, the device allows a sample liquid containing the object to be observed to continuously flow in and out of the space between the reflector and the optical aperture. The reflector requires very little space and, when connected to the body of the device, no additional support structure is needed, such as the support structure typically used in tabletop microscopes designed for observing objects on a slide.

[0017] The sample volume located in the space between the optical aperture and the reflector can be observed through the optical aperture. In some cases, illumination of the sample volume can be provided through the optical aperture. Illumination light emitted through the optical aperture is reflected by the reflector and returns towards the optical aperture, thus passing through the sample volume. The illumination light is scattered by objects (e.g., particles or cells) in the sample volume, and the resulting forward-scattered light propagates through the optical aperture toward the observer or receiver. In some cases, the intensity of the forward-scattered light can be several orders of magnitude higher than the intensity of the backscattered light. Therefore, the signal received from the object is significantly greater than the signal that could be received if only the backscattered signal produced by the illumination light propagating directly from the optical aperture and passing through the sample volume were observed.

[0018] In particular, when the light initially emitted from the optical aperture returns from the reflector to the optical aperture along the same path as the light emitted from the optical aperture to the reflector, it should be understood that the light passes through the sample volume twice, and backscattered light and forward scattered light from the sample volume are observed through the optical aperture.

[0019] Depending on the distance between the observed object plane and the optical aperture, in some cases, it is also possible to observe forward-scattered light as illumination light propagates from the optical aperture to the reflector and back-scattered light as illumination light propagates from the reflector to the optical aperture. For example, the distance between the object plane and the optical aperture can be set by adjusting the focal length of the objective lens or objective lens unit of a microscope having a field of view that passes through the optical aperture.

[0020] For example, if an immersion lens, and more specifically a solid immersion lens (so-called SIL), is used to observe the sample volume, the depth of field is relatively narrow, typically measuring only a few micrometers along the axial or proximal-to-distal direction or the line-of-sight direction. The field of view captured by a suitable sensor is, for example, 150 μm × 150 μm. This depth of field and the field of view optically define a narrow sample volume. An adjustable lens can be used to adjust the position of the object plane, in which the object is clearly imaged onto a sensor arranged functionally associated with an objective lens unit that extends through or has a field of view oriented through the optical aperture of the body for recording an image along the line-of-sight direction. The object plane can be selected to lie between the optical aperture and the reflector.

[0021] 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".

[0022] The reflector can be a mirror or a retroreflector. In particular, using a retroreflector in combination with light emitted through an optical aperture offers the advantages of being a robust component and having tolerance relative to alignment. Mirrors can be manufactured by applying a suitable coating and / or polishing a suitable material, thus avoiding the need to mount the reflector to, for example, a part of the body or a cantilever or similar device for attaching it to the body. This makes the device more robust and prevents it from getting dirty.

[0023] In one embodiment, the reflector is connected to the body via at least one support. The support may be a cantilever support. Preferably, the total circumferential range of the at least one support does not exceed 180°. In another embodiment, the total circumferential range of the at least one support may not exceed 90°, 60°, or 30°. This allows substantially unimpeded flow or fluid exchange in the space between the reflector and the optical aperture. A single support may be provided. The at least one support may be releasably connected to the body. This, for example, would allow a microscopic apparatus containing the means for guiding illumination claimed herein to be used with or without a reflector. The reflector may be a reflective element directly attached to the cantilever support, or it may be an additional structure (e.g., a bubble shield) capable of being attached to the support and including or holding the reflector.

[0024] The distance between the reflector and the optical aperture can be adjusted, which will further increase the versatility of the subject matter presented in this paper.

[0025] In this embodiment, the cross-section of the body steadily increases along the optical aperture and the optical axis of the reflector in a direction from the far side to the near side. The cross-section increases from the optical aperture to the position of the maximum cross-section of the body. The cross-section is perpendicular to the optical axis.

[0026] Specifically, the body may include at least two regions with positive curvature separated by a region with negative curvature. Preferably, the distal region with positive curvature is adjacent to or includes the optical aperture, while the proximal region with positive curvature is adjacent to or includes the segment of the body with the largest cross-section. This shape provides space between the distal front end and the vicinity of the reflector, allowing fluid carrying the object to be observed to easily enter the sample volume. Simultaneously, this shape provides sufficient internal volume to accommodate at least some optical elements (e.g., components of lenses and illumination units), thereby allowing the device to be used as part of a microscope housing. Preferably, the curvature is Gaussian curvature.

[0027] In an embodiment, the device for guiding illumination is adapted to be arranged in the flowing liquid to be measured and equipped with a bubble shield. The bubble shield preferably comprises a blunt body. The cross-section of the bubble shield is larger than the cross-section of the optical aperture. The bubble shield is arranged on the distal side of the distal front end of the body. Preferably, the reflector is part of the bubble shield. The bubble shield preferably has a positive curvature on the side facing the flow direction or on the side facing away from the optical aperture.

[0028] In a preferred embodiment, the flowing liquid to be measured has a flow direction, and the bubble shield generates a wake when inserted into the flowing liquid. The device can be configured such that the optical aperture is positioned within the wake during the intended operating conditions.

[0029] Many biological processes generate gases. In other cases, gases are introduced into the liquid to be observed through mixing or intentional means. These gases can form bubbles that adhere to the walls of the container holding the liquid. Bubbles adhering to optical apertures and / or reflectors can interfere with observation. Therefore, it is beneficial to prevent bubbles from entering the volume between the optical aperture and the reflector. Bubbles typically move upwards with the fluid flow, but due to their large volume, they generally do not follow small-scale local flows. A bubble shield can create a local flow field that can entrain the object to be observed without entraining the bubbles, thereby reducing the risk of bubble interference with observation.

[0030] In a preferred embodiment, the cross-section of the optical aperture is smaller than the cross-section of the bubble shield. More preferably, this is also true for the effective cross-section, which ensures that the bubble shield effectively protects the entire optical aperture. The effective cross-section is preferably a cross-section perpendicular to the flow direction during the expected operating conditions.

[0031] In a preferred embodiment, the cross-section of the body is larger than the cross-section of the optical aperture and substantially equal to the cross-section of the bubble shield. More preferably, this also applies to the effective cross-section as defined above. This embodiment ensures that the bubble shield effectively protects the entire optical aperture while avoiding a total cross-section of the device larger than the cross-section of the body. This, for example, facilitates the mounting of microscopes using the device. Furthermore, this embodiment can be combined with a body having a cross-section that steadily increases along the optical axis of the optical aperture and reflector in a direction from the distal to the proximal side, which helps to provide space for liquids to easily enter the sample volume and can generate desired localized flow.

[0032] In a preferred embodiment, the bubble shield includes a flow divider extending toward the optical aperture. Preferably, the reflector is mounted to the flow divider. In an embodiment, the flow divider extends in the direction of flow extension in the wake of the distal portion of the bubble shield under the expected operating conditions. In an embodiment, the flow divider extends along the optical axis. During operation, when the bubble shield generates a wake, the flow divider is able to split the wake, thereby generating a time-stabilized flow and reducing the total drag of the bubble shield.

[0033] In a preferred embodiment, the diverter has a conical shape, its radial extension decreasing in the distal-proximal direction. The diverter reduces the volume of locally very low velocities in the wake, thereby ensuring the desired flow of the particles to be observed within the field of view of the microscope using the device. The conical shape supports desired localized eddies that bring the fluid to be measured into the sampling area. Furthermore, the diverter provides a convenient location for mounting a reflector: the reflector is preferably located at the apex of the conical shape, i.e., at its closest end.

[0034] In an embodiment, the bubble shield, preferably comprising a blunt body and / or a splitter, is rotationally symmetric, wherein the optical axis is the axis of symmetry. This symmetry may be disrupted by the supports used to connect the bubble shield to the body of the device.

[0035] The device for guiding illumination presented herein can be used with an objective lens unit, such as an objective lens unit for a microscope. The device and the objective lens unit can form an assembly. The objective lens unit includes a housing having a distal front end. A front optical aperture of the objective lens unit is disposed in the distal front end of the objective lens unit. 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 device for guiding illumination is attached to the objective lens unit. The device for guiding illumination is arranged such that a reflector is positioned distal to the distal front end of the objective lens unit and at a non-zero distance from the front optical aperture of the objective lens unit. The device for guiding illumination is arranged to reflect at least a portion of the light received from the front end of the body back into the front optical aperture of the objective lens unit. Preferably, the light received by the reflector is emitted through the front optical aperture of the objective lens unit and through the front end of the body. Those skilled in the art will understand that the reflector is implicitly located within the field of view of the objective lens unit. The objective lens unit allows observation of the sample volume located between the front optical aperture of the objective lens unit and the reflector, and in certain embodiments, observation is made through the optical aperture of the body of the device used for guiding illumination.

[0036] The objective lens unit is particularly suitable for use when at least partially immersed in a liquid, such as in a sample liquid containing suspended biological cells for cytology. Specifically, the device for guiding illumination and / or the objective lens unit, or a combination of both, can be liquid-resistant. This makes the assembly suitable for use when at least partially immersed in a liquid. For example, a ring-shaped body can be attached to a liquid-resistant objective lens unit, or a liquid-resistant cap can serve as a body while simultaneously covering at least a portion of a non-liquid-resistant objective lens unit, thereby making the combination liquid-resistant. In another embodiment, the liquid-resistant body can be mounted onto a container (e.g., a bioreactor) of the liquid to be observed, thereby forming a liquid-resistant barrier between the interior of the bioreactor and the objective lens system.

[0037] In another aspect, an objective lens unit for a microscope is disclosed. This objective lens unit is particularly suitable for use when at least partially immersed in a liquid. Preferably, the liquid is a sample liquid comprising an object to be observed. The objective lens unit includes a housing. At least a portion of the housing is formed by the body of a device for guiding illumination of the type described above. The front end of the body of the device for guiding illumination is configured and arranged to provide a distal front end of the housing. The optical aperture of the body of the device for guiding illumination provides the front optical aperture of the objective lens unit. A reflector of the device for guiding illumination is connected to the housing. The reflector is arranged distally to the front end of the housing and at a non-zero distance from the front optical aperture of the objective lens unit. The reflector is configured to receive light emitted through the distal front end of the housing and to reflect at least a portion of the light received from the distal front end back into the front optical aperture. The objective lens unit also includes an objective lens system disposed inside the housing and proximal to the front optical aperture of the objective lens unit, the objective lens system being configured to collect light received through the front optical aperture.

[0038] In a more specific embodiment of the objective lens unit described above, the housing includes a cap and a sleeve, wherein the objective lens system is disposed within the sleeve. The cap is provided by the body of the device for guiding illumination. 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. The sleeve is preferably received at least partially inside the cap, particularly through the rear port of the cap. In an embodiment, the cap is received at least partially inside the sleeve, particularly the objective lens system is received through the rear port of the cap, while the lateral sheath contacts the interior of the housing defining the exterior of the sleeve. In an embodiment, the sleeve may be threadedly received inside the cap, or the cap may be threadedly received inside the sleeve. For example, a seal provided by an O-ring may be disposed between the cap and the sleeve. In other embodiments, the sleeve may be simply inserted into the cap, wherein the sleeve and the cap may be equipped with an alignment device such that when the sleeve is received inside the cap, the optical axis of the front optical aperture of the objective lens unit coincides with the optical axis of the objective lens system. The cap can, for example, be fixed to a bioreactor and extend through a port of the bioreactor, wherein the distal front section of the lateral sheath and the front wall are located inside the bioreactor, while the rear port of the cap is accessible from the outside of the bioreactor. The sleeve can be continuously inserted into different caps for use in different bioreactors or at different locations within a single bioreactor. The rear port of the cap and the sleeve can be equipped with structures that allow the sleeve to be secured to the cap. For example, the cap can be equipped with threads, radial pins, or slots, while the sleeve can include a ring clamp and a lock nut with corresponding threads, slots, or radial pins, thereby allowing for threaded connections or bayonet mounting structures.

[0039] Typically, the objective lens unit or the body may include an immersion lens arranged and configured to collect light entering the objective lens unit through a front optical aperture of the objective lens unit or through an optical aperture of the body. In one embodiment, the immersion lens may be arranged inside and close the corresponding optical aperture. However, in other embodiments, the immersion lens may be arranged proximal to the optical aperture, i.e., between the optical aperture and the objective lens system in the assembled state. The immersion lens may be a solid immersion lens (so-called SIL), but in other cases may include a liquid immersion lens. The front optical aperture may be closed by a window as the farthest optical component. 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 selected such that the refractive index matches a well-defined relationship, wherein the shape and focal length of the immersion lens may 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 known as a super-hemispherical SIL or super SIL.

[0040] Objective units containing immersion lenses have a relatively narrow depth of field, typically measuring only a few micrometers along the axial direction or the line of sight. The position of the object plane—that is, the position on the sensor functionally associated with the objective unit and used to record the image where the object is clearly imaged—varies significantly along the line of sight with the tolerances of the objective unit, particularly with the relative positions of the immersion lens and the objective system. If the manufacturing and assembly tolerances of the objective unit are not limited to the micrometer range, it may be found that the positional tolerance of the object plane is unsuitable, resulting in high manufacturing costs and making it unsuitable for mass production. Furthermore, objective units may be exposed to thermal or mechanical stresses during use. For example, objective units used in bioprocesses may require autoclaving or “sterilization in place” (SIP) or “clean-in-place” (CIP) procedures. Sterilization is typically performed, for example, by means of superheated steam, such as steam at 120°C and 2 bar for a period of about 60 to 70 minutes, where the exact parameters can vary depending on the application and system. Such treatments, along with stresses caused by aging and during expected use, can lead to minor deformations and relative displacements of the components. Therefore, it may be advantageous if the objective system includes at least one adjustable lens. The adjustable lens may be the most distant optical element of the objective system, i.e., the one closest to the immersion lens. For several reasons, including avoiding vibration and autoclaving capability, the adjustable lens may be motorless. In one embodiment, the adjustable lens can be axially displaced relative to the immersion lens via a piezoelectric actuator. In other embodiments, the adjustable lens may be a tunable lens, i.e., a lens whose focal length can be controllably changed, for example, by changing the curvature of an optically active surface. Such adjustable lenses are commercially available. Therefore, the position of the object plane can be adjusted. The position of the object plane can be selected to be between the optical aperture of the body or the objective unit and the reflector.

[0041] In embodiments, the front optical aperture of the objective lens unit or the optical aperture of the body of the illumination guiding device can be closed by one of a window and an immersion lens, and furthermore, can be closed in a liquid-proof manner. Therefore, in these embodiments, the objective lens unit, the device or assembly including the objective lens unit, is adapted for use when at least partially immersed in a liquid. The liquid is preferably a sample liquid in which the object to be observed is suspended. Thus, at least the distal or frontal section of the objective lens system including the front optical aperture, or at least the distal front end of the body including the optical aperture, is adapted to be immersed in a liquid. Furthermore, at least the objective lens system or the illumination guiding device, and in embodiments the entire objective lens unit or the assembly including the objective lens unit, can be autoclaved or adapted to undergo "SIP" or "CIP" procedures. To make the objective lens unit or the assembly including the objective lens unit suitable for use when at least partially immersed in a liquid, at least the distal section of the objective lens unit or the body can be liquid-proof.

[0042] In a more specific embodiment, the objective lens unit or the body includes a proximal rear connector interface, wherein at least one section of the objective lens unit or the body, excluding the rear connector interface, is liquid-resistant. In a specific embodiment, the body is a cap, the rear connector interface is the rear port of the cap, the lateral sheath and the front wall form a liquid-resistant container, the only opening of which is the rear port. In a more specific embodiment, the objective lens unit includes an interface to which an illumination source and / or optical sensor may be functionally coupled, and the interface is the rear connector interface. Preferably, the optical sensor is a two-dimensional optical sensor, such as a CCD or CMOS sensor, and most preferably is provided as part of a camera.

[0043] In a specific embodiment, an illumination source and / or optical sensor are integrated into the objective lens unit, and the objective lens unit includes an interface that allows for the reception and transmission of data and / or power. Preferably, such an objective lens unit is liquid-resistant. For example, data can be transmitted wirelessly, and power can be transmitted via induction. In other examples, a cable connector couples electrical and / or optical transmission lines to the objective lens unit. In other embodiments, the interface that allows for the reception and transmission of data and / or power is a rear connector interface.

[0044] The farthest optical element of the objective lens unit or the body (e.g., particularly a window or immersion lens that closes the front optical aperture of the objective lens unit or the front of the body) can be flush with the far outer surface of the far front end of the objective lens unit, the housing, or the body. This makes the far tip of the objective lens unit or the body smooth, thereby improving the robustness of the objective lens unit and significantly reducing the risk of dirt or biofouling buildup, and reducing eddies around the far tip of the objective lens unit when placed in a liquid flow. The far front end or tip of the objective lens unit or the far front end of the body can be flat or convex in this respect.

[0045] The objective lens unit, the component including the objective lens unit, or the circumferential outer wall of the body may be cylindrical, with an outer diameter of 25 mm or less, particularly 12 mm or less. Preferably, the device for guiding illumination is shaped such that it can be surrounded by a cylinder with an outer diameter of 25 mm or less, particularly 12 mm or less, in at least one orientation. This allows the objective lens unit to be inserted into a bioreactor or container via standard ports (e.g., Ingold standard access ports and Eldon-James ports). In embodiments, the distal portion of the objective lens unit, the component, and / or the body is adapted in at least one direction to fit within the volume of a cylinder with an outer diameter of 25 mm or less, particularly 12 mm or less, while the proximal portion extends at least partially beyond the cylinder in the radial direction. This proximal portion may include an annular device or mounting device that facilitates the installation of other components (e.g., cables or data transmitters) and prevents the objective lens unit, the component, and / or the body from being inserted into the bioreactor or container to an unintended depth.

[0046] The device, component, or objective lens unit described above for guiding illumination may further include a sample illumination unit. The sample illumination unit includes an illumination source. The sample illumination unit also includes means for coupling light from the illumination source into the objective lens unit or the body.

[0047] In a particular embodiment, the means for coupling light from the illumination source into the objective lens unit is arranged on the far side of the objective lens system and on the near side of the front optical aperture of the objective lens unit, and is further configured to project the light onto the reflector through the front optical aperture in a near-far direction along the objective lens unit.

[0048] In embodiments that include an immersion lens, means for coupling light from an illumination source into the objective lens unit or body may be arranged on the far side of the objective lens system and on the near side of the immersion lens.

[0049] The illumination source may be functionally coupled to the means for coupling light from the illumination source into the objective lens unit or the body, and may be positioned laterally to the optical path of the objective lens unit. The means for coupling light from the illumination source into the objective lens unit may be a beam splitter arranged and configured to reflect a portion of the light from a laterally positioned illumination source. For example, light from an illumination source arranged off-axis relative to the optical axis of the objective lens unit and located outside the detection optical path of the objective lens system may be coupled to the distal direction of the objective lens unit, toward and through the front optical aperture of the objective lens unit and / or through the optical aperture of the body.

[0050] In other embodiments, the illumination source is arranged in an additional aperture disposed in the objective lens unit or the body, or light from the illumination source is guided to such an additional aperture, for example, through a light guide, optical fiber, or suitable optical element (such as a reflective surface). In other embodiments, however, optical fibers or other light guides may be used alone or in combination with other optical elements to couple light from the illumination source into the objective lens unit or the body.

[0051] The illumination source may be an LED or include LEDs. In embodiments, the illumination source may be a monochromatic light source or a light source with a narrow half-power bandwidth (e.g., 50 nm or less, 20 nm or less, or 10 nm or less). Monochromatic or narrow-bandwidth imaging allows for the avoidance of chromatic aberration when imaging objects, thereby further improving image quality. This can be achieved by emitting monochromatic or low-bandwidth light from the illumination source, using filters, or a combination thereof. In other cases, a bandpass filter may be used at a suitable location within the instrument.

[0052] However, in some applications, capturing images at at least two sufficiently different wavelengths can be useful, thereby utilizing chromatic aberration to simultaneously acquire images from at least two different object planes. Therefore, broadband or multi-band light sources, or multiple light sources emitting light with different spectra, can be used, for example. As a non-limiting example, broadband LEDs or multiple LEDs emitting light with different peak wavelengths can be used.

[0053] In some aspects, the reflector is part of a reflector assembly. The reflector assembly includes a reflector attached to at least one support post. The at least one support post is configured to be mounted to the housing of the objective lens unit, for example, to a cap adapted for attachment to a sleeve of the objective lens unit. The size and shape of the reflector assembly are designed such that the reflector is positioned at a non-zero distance from the front end of the objective lens unit housing from the distal side, and such that the reflector is positioned and configured to receive light emitted through the front end of the objective lens unit and to reflect at least a portion of the received light back into the optical aperture when the reflector assembly is attached to the housing of the objective lens unit. Preferably, the light received by the reflector is emitted through a front optical aperture disposed in the front end of the objective lens unit. The support post may be a cantilever post, to which the reflector, as a reflecting element, is directly mounted. Additional structures, such as bubble shields, may be attached to the support post and include or retain the reflector. In this case, the reflector assembly includes the reflector, the support post, and the additional structure.

[0054] In another aspect, a microscope for in-situ applications is disclosed, preferably for measurements inside a bioreactor. The microscope includes an objective lens unit or an assembly including an objective lens unit of any kind as described above, and an optical sensor. 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 through the objective lens unit. Specifically, the optical sensor is functionally coupled to the objective lens unit to receive light transmitted through the front optical aperture and through the objective lens system. In a particular aspect, the microscope can be configured for attachment to a container (e.g., a bioreactor) including a standard port, wherein the microscope is further configured such that at least a distal segment of the objective lens unit or the body (particularly the segment including the optical aperture) can be positioned inside the container (e.g., 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 optical sensor may particularly include an image sensor, and more particularly a CCD or CMOS image sensor. The optical sensor can be placed in the microscope head or integrated into the objective lens unit.

[0055] The microscope may further 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 arranged, for example, proximal to the objective lens unit, such as in the microscope head, and / or may be integrated into the objective lens unit as described above, or coupled to the rear connector interface. It is recommended that the sample illumination unit be arranged in the objective lens unit, and that light from the illumination source be coupled to the objective lens unit located distal to the objective lens system, to provide the most efficient utilization of the light from the illumination source. The optical path of the light emitted from the sample illumination unit may coincide with the optical path of the light collected from the object in the sample volume, at least in segments of the respective optical paths. The possible spectral characteristics of the illumination source and their effects have been discussed above. The microscope may provide means for splitting the light along the optical path from the front optical aperture to the sensor, such as a beam splitter. Elements (e.g., but not limited to bandpass 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. When a sample volume is illuminated by a broadband or multicolor light source, and the difference between different wavelengths is sufficiently large, images of different wavelengths 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 different optical paths to image different object planes on the sensor through different optical paths.

[0056] In a further aspect, a method for performing cytology is proposed. One method includes using a microscope as described above. The method further includes immersing at least the most distal anterior segment of the objective lens unit, including the distal front end and the front optical aperture, or the distal front end of the body, including the optical aperture, into a sample liquid. Preferably, the sample liquid is contained within a bioreactor. Light is emitted through the front end of the objective lens unit or the body and directed toward a reflector. At least a portion of the light striking the reflector is reflected back to the front optical aperture of the objective lens unit and / or the optical aperture of the body. The light passes through a sample volume of the sample liquid. At least one image generated by the objective lens unit is recorded using an optical sensor. Preferably, the at least one image includes forward-scattered and back-scattered light from objects (e.g., biological cells) contained in the sample volume, under specific illumination by light propagating from the front optical aperture of the objective lens unit to the reflector and by light reflected back from the reflector to the front optical aperture of the objective lens unit. A similar situation occurs when the illumination light arrives at the reflector at a small angle relative to the optical axis. This small angle preferably occurs when light from the illumination source passes through an additional aperture that is close to but not necessarily the same as the optical aperture, or when light from the illumination source does not follow the path of the reflected light.

[0057] Preferably, the method further includes the step of inserting at least a distal segment of the objective lens unit or at least a distal segment of an assembly including the objective lens unit through a port of a container (e.g., a bioreactor).

[0058] The method may further include emitting light in pulses, wherein one pulse is emitted for each frame recorded by the optical sensor, and wherein the pulse duration is particularly 20 μs or less. The duration of the light pulse may be 10 μs or less. The resulting short illumination time allows for the capture of clear images even when cells are moving within the sample volume. On the other hand, the blurring of the image under longer illumination times can be used to determine the flow rate through the sample volume.

[0059] The method may further include emitting light in a pulsed manner, wherein the pulse duration is 20 μs or less, preferably 10 μs or less. The pulsed operation of the illumination source has a short microsecond-level pulse duration, enabling the desired flicker effect to be obtained independently of the frame rate recorded by the sensor.

[0060] In another embodiment, each frame recorded by the optical sensor emits multiple pulses, and if the pulse rate is appropriately selected based on the particle velocity and the recorded field of view, multiple images displaying the same object on the same frame are generated. This allows for the measurement of the velocity and, if the object is rotating, imaging from different sides.

[0061] The method may further include the step of setting the focal length of the objective lens unit such that an object in the sample volume located at a desired position between the front optical aperture of the objective lens unit and the reflector is clearly imaged on the optical sensor. This may involve actuating an adjustable lens, particularly a tunable lens as described above. The desired position can be defined relative to a target positioned on an optical element arranged on the optical axis: for example, adjusting the adjustable lens so that the target is clearly imaged, and then adjusting the adjustable lens by a predetermined adjustment range, thereby moving the position of the volume of the object clearly imaged to the desired position.

[0062] If the objective lens unit or the assembly including the objective lens unit is an objective lens having a housing including a cap and a sleeve as described above, the method may further include inserting at least a front section of the cap through a port of the bioreactor and sealing the port of the bioreactor with the cap. The rear port of the cap remains outside the bioreactor. At least the farthest front section of the cap, including a front wall with an optical aperture, is immersed in the sample liquid inside the bioreactor. A sleeve including the objective lens system is inserted into the cap.

[0063] The method may further include the step of setting the focal length of the objective lens unit such that the reflector is imaged onto an optical sensor. In this embodiment, a warning is issued if the observed reflectance is lower than expected and / or if the image displays unexpected structures. A decrease in reflectance or structures appearing over time can indicate biofouling, dirt, or optical surface degradation. To minimize the risk of false warnings, the method may further include the steps of evaluating observations of the sample volume before and / or after imaging the reflector, estimating object or cell density based on these images, and taking into account the observed appearance and / or density of the object or cells when setting the expected values. Furthermore, a warning may only be issued if multiple images of the reflector show a consistent trend indicating a decline in image quality (e.g., biofouling, dirt, or aging effects).

[0064] 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

[0065]

[0066] 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

[0067] 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. In an embodiment, the hollow shaft 7 may be part of the housing of the objective lens unit 6. 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. In this embodiment, an illumination source 53 is mounted laterally to the head 5, facing the optical path. In the optical path from the objective lens unit 6 to the optical sensor 52, a beam splitter 54 is disposed inside the head 5. The beam splitter 54 serves as a means of coupling light from the illumination source 53 into the objective lens unit 6 and deflecting a portion of the light from the illumination source 53 toward the objective lens unit 6, wherein the light exits the microscope 1 at the distal end 11 and illuminates the sample volume of the liquid 3. In another embodiment, a light guide, such as an optical fiber, is coupled to the illumination source 53 and guides the light such that the light exits the microscope 1 at the distal end 11 and illuminates the sample volume of the liquid 3. In these embodiments, the beam splitter 54 may be omitted. The light from the sample volume of the liquid 3 thus illuminated enters the microscope 1 at the distal end 11 and travels through the objective lens unit 6 and the hollow shaft 7 toward the optical sensor 52. The returning light (if present) is partially deflected by the beam splitter 54, while another portion of the light from the sample volume propagates to and is captured by the optical sensor 52. Therefore, a magnified view of the object in the liquid 3 can be imaged, and the image can be recorded by the camera 51. In the illustrated embodiment, the illumination and imaging optical paths overlap in most sections of the device, specifically below the beam splitter 54 in the current illustration. In other embodiments, the illumination and imaging optical paths are separate or overlap only near the distal end 11. The vessel 2 may be a bioreactor. Therefore, the liquid 3 may contain biological cells as the object to be observed. The cells can be observed in situ using the microscope 1 shown. The microscope 1 thus enables in situ cytology. The illumination source 53 may be pulsed. The illumination source 53 can emit a single pulse, typically tens of microseconds, during each frame captured by the camera 51. Therefore, even if the object moves, a clear and sharp image 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 the camera 51 can help determine the flow rate and direction through the sample volume.

[0068] 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.

[0069] 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. An optical aperture 63 is disposed via the housing 61 at the distal front end 62 of the objective lens unit 6, or at the distal end 11 of the microscope 1. Light from the illumination source 53 can exit the objective lens unit 6 through the optical aperture 63, and light from the sample volume can enter the objective lens unit 6 through the optical aperture 63. A solid immersion lens 64 is disposed inside the optical aperture 63 to receive light passing through the front optical aperture 63. 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 64 translate into larger variations in the position of the object plane for clear imaging on the optical sensor, and therefore, larger variations in the position of the 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 such prior art solutions sensitive to varying conditions such as aging and temperature changes.

[0070] The proposed solutions alleviate these problems. Lens 65 can be a motorless, automatically adjusting lens. In embodiments, such as in the illustrated example, an adjustable lens 65 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 65. In other embodiments, the adjustable lens can be displaced along the axis of the objective unit via a piezoelectric actuator. Both exemplary solutions can be implemented within a compact housing of the objective unit 6, are autoclaved and / or withstand SIP or CIP processes, and can operate without vibration. The adjustable lens 65 is positioned 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 arranged close to the adjustable lens 65 and is, for example, an aspherical lens. Lenses 65 and 66 are held in place by a threaded sleeve 67, which is screwed into the housing 61. The distance sleeve 68 is shown as a device for maintaining the distance between the aspherical lens 66 and the adjustable lens 65. The distal end or front surface of the immersion lens 64 is flush with the outer surface of the distal front wall of the housing 61. Therefore, eddies in the liquid flow are avoided, the accumulation of dirt and biofouling is reduced, and the objective lens unit 6 is easier to clean.

[0071] exist Figure 3 In another embodiment shown, the housing 61 includes a cap 611 and a sleeve 612. The cap 611 includes a distal front wall and a lateral sheath. A flange 613 extends radially outward at the proximal rear end of the lateral sheath. An objective lens system 6, including an adjustable lens 65 and an aspherical lens 66, is disposed within the sleeve 612. Typically, the objective lens system is assembled and joined... Figure 2 Similar to the one described above. The sleeve 612, which houses the optical lens system, can be inserted into the cap 611 through its rear port. The optical aperture 63 is located in the distal front wall of the cap 611. (Turn) Figure 4 Detail IV will be discussed. A planar window 641 closes the optical aperture 63 at its distal end and is flush with the distal outer surface of the front wall of the cap 611. A liquid immersion lens 642 is disposed proximal to 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 its distal end, which closes the optical aperture at its distal end. 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.

[0072] 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. In other examples, the microscope head 5 can be directly attached to the proximal end of the sleeve 612. The distal section of the cap 611 can be inserted into the container (such as tank 2, e.g., a bioreactor) through a port 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 container, e.g., 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. The sleeve 612, with optical sensors functionally attached thereto, can be inserted into the cap 611 from outside the container (e.g., 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 container (e.g., the bioreactor). Therefore, the objective system and all components attached thereto can be replaced and / or removed without affecting the contents inside the container.

[0073] Figure 5The diagram illustrates an objective lens unit 6 incorporating features of the invention claimed herein. An illumination unit is provided within the objective lens unit 6, comprising 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 direction of the objective lens unit 6 and toward the immersion lens 64. The light is emitted along the forward or distal direction of the objective lens unit 6 through a front optical aperture 63. 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 from the distal side. 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 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 typically likely to be low. Therefore, reflector 621 is cantilevered on cap 611, which surrounds the sleeve 612 of the optical lens unit 6. Reflector 621 is arranged on the optical axis of the objective lens unit 6. Reflector 621 can be, for example, a mirror or a retroreflector. In the illustrated embodiment, reflector 621, cap 611, and cantilever support form a reflector assembly attachable to the objective lens unit 6. It should be understood that reflector 621, together with the cantilever support, can also be a reflector assembly that can be threaded or otherwise mounted to the housing 61 of the optical lens unit 6. 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 objects (e.g., biological cells or other particles) in sample volume 100 is received by objective unit 6 and imaged onto an optical sensor functionally connected to the rear or proximal end of objective unit 6. The intensity of forward-scattered light can be several orders of magnitude higher than that of backscattered light. Therefore, forward-scattered light dominates these images on the optical sensor relative to backscattered light. This allows for improved cytology quality using the strong signal obtained through transmission microscopy. Simultaneously, the elimination of the need for a light source or devices for removing reflected images, such as polarization filters, within the liquid being observed provides a particularly robust and compact illumination system and microscope.

[0074] like Figure 6 As shown, in another non-limiting example, reflector 621 can also be disposed on a U-shaped carrier. This minimizes the influence of the flow of the liquid to be observed. In any embodiment, it can also be specified that the distance between the forward optical aperture 63 and reflector 621 can vary.

[0075] Figure 7 One embodiment is shown in which the distal end 62 of the objective lens unit 6 is not flat (e.g., Figures 2 to 6 (as shown in the example), but is generally convex, such as conical. For other non-limiting examples, the distal end 62 of the objective lens unit 6 may be circular, such as 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 distal end 62.

[0076] Figure 8 A top view of the distal end 62 or front end 62 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 end 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 48 on the optical sensor. Then, the adjustable lens 64 or other motorless auto-adjusting lens is adjusted to set the object plane at a predetermined distance from the front optical aperture 63, such that the sample volume located at this distance from the front optical aperture 63 is clearly imaged on the optical sensor. Therefore, autofocus operation can be achieved.

[0077] Figure 9a and Figure 9b First and second examples of objective lens units including a reflector 621 and a bubble shield 623 are disclosed. The housing 61 of the objective lens unit forms the body of a device for guiding illumination. An optical aperture 63 is disposed in the form of the housing 61 at the distal front end 62 of the body. In the depicted embodiment, a solid immersion lens 64 is used to close the optical aperture 63. Inside the housing 61, there is an objective lens system consisting of two lenses 65 and 66, wherein lens 65 may be an adjustable lens. The objective lens system is configured to collect light received through the optical aperture 63. The reflector 621 is arranged on the side of the bubble shield 623 facing the distal front end 62 of the body 61. The optical axes 84 of the objective lens systems 65 and 66 and the optical aperture and reflector 621 coincide.

[0078] The bubble shield 623 is arranged such that the intended flow direction 80 of the liquid in which the objective lens unit is intended to be used is parallel to the optical axis 84. In the example shown, the bubble shield 623 is a blunt body. The bubble shield has a positive curvature on the side facing the flow direction 80 and is rotationally symmetric, wherein the optical axis is the axis of symmetry. Under the intended operating conditions, the optical aperture 63 is located in the wake of the bubble shield 623. The effective cross-section is the cross-section perpendicular to the flow direction 80. The cross-section and effective cross-section of the optical aperture 63 are smaller than the cross-section and effective cross-section of the bubble shield 623, respectively. The circumferential outer wall of the objective lens unit is cylindrical. As shown by the dashed lines, the distal portion of the objective lens unit, including the bubble shield 623 and the reflector 621, is housed within the volume of a cylinder 81 with an outer diameter of 12 mm in this example. The bubble shield 623 is connected to the body 61 by a support 622. The support 622 has a circumferential range of approximately 10° around the flow direction 80 (i.e., with the axis of symmetry of the cylinder 81 as the axis), thereby ensuring sufficient mechanical stability while minimizing the impact on liquid flow. The support 622, the bubble shield 623, and the reflector 621 form a reflector assembly.

[0079] exist Figure 9a and 9b In this embodiment, and along the optical axis 84, in the direction from the distal to the proximal side, the body 61 has a steadily increasing cross-section: the body has its minimum at the optical aperture 63. The region of the optical aperture 63 forms a first plateau, which steadily increases in the distal-proximal direction. Starting from the maximum cross-section (here, the cross-section of the cylinder 81), the cross-section steadily decreases in the proximal-distal direction. The increase and the decrease are matched to achieve a smooth and stable transition. In the depicted example, the regions of increasing and decreasing cross-section of the body have positive curvature, while the regions where the transition occurs have negative curvature. Such a shape, while exposing the optical aperture 63, still provides sufficient volume within the body or housing 61 adjacent to the optical aperture 63, thereby enabling the objective lens system to be positioned close to the optical aperture 63.

[0080] exist Figure 9a In the illustrated embodiment, body 61 includes an additional aperture 82 through which light from illumination source 44 is emitted onto reflector 621. Reflector 621 is a reflective coating applied to the proximal side of bubble shield 623. In the illustrated example, illumination source 44 is an LED disposed within body 61 or housing. In other embodiments, the illumination source may also be disposed in different locations, and light may be guided to additional aperture 82 via a light guide such as an optical fiber. Additional aperture 82 is closed by a window or lens. In other embodiments, additional aperture 82 may also be formed directly from illumination source 44 or a suitable light guide.

[0081] exist Figure 9b In the illustrated embodiment, the lighting is similar to that regarding Figure 5 The illumination discussed herein 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 direction of the objective lens unit 6 and toward the immersion lens 64. The bubble shield 623 of this embodiment also includes a diffuser 624. A reflector 621 (e.g., a cat's-eye retroreflector) is arranged in the tip of the diffuser 624. The bubble shield 623 with the diffuser 624 is rotationally symmetric, wherein the optical axis is the axis of symmetry. The diffuser 624 has a tapered shape, its radial extension decreasing in the distal-proximal direction. Figure 9b An example of the connecting device 625 for the reflector is further depicted: the support 622 is received in the recess of the body 61 and locked in the recess of the body 61.

[0082] Figure 10a and Figure 10bAnother example of a reflector assembly is shown: the reflector assembly includes a cubic base in which a connecting device 625 is arranged. The cubic base has a thickness perpendicular to the base surface. A top surface is arranged parallel to the base surface at a distance equal to the thickness. In the depicted example, the connecting device 625 is a recess for receiving a screw. The recess extends in the thickness direction of the base. The diameter of the recess is larger at the top surface than at the base surface. A support 622 extends from one side of the base in the thickness direction. The support has a height measured from the base surface in the thickness direction, the height being approximately twice the thickness of the base. The support 622 includes a lower portion and an upper portion, the lower portion having a triangular prism shape, one side of which is equal to the side of the cubic base at its origin, and the upper portion resembling a second cube from which two cylindrical segments are cut, thereby forming a cantilever structure. The cubic base and the second cube are arranged parallel to each other but offset such that the section including the cut is not above the base, and the section without the cut contacts the second side of the triangular prism. The thickness of the second cube is approximately the same as the thickness of the cubic base. A reflector 621 is embedded in the tip of the cantilever formed by the cut. The reflector is arranged on the lower side and reflects light downward in the thickness direction, i.e., along the direction from the top surface to the base surface. This reflector assembly can be manufactured efficiently, can be easily mounted onto the body, and provides sufficient stability. To provide a smooth surface when in contact with the measurement medium, the assembly using the reflector in the microscope or objective lens unit or therein may include a cover for covering the connection device and similar small-scale structures.

[0083] 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.

[0084] List of reference numerals

Claims

1. A device for guiding lighting, the device comprising: Body (61, 611), the body having a distal front end (62) and an optical aperture (63) disposed in the front end; And a reflector (621) connected to the body and disposed on the far side of the front end and at a non-zero distance from the optical aperture, the reflector being configured to receive light emitted through the front end and to reflect at least a portion of the light received from the front end back into the optical aperture.

2. The apparatus according to any one of the preceding claims, wherein, The reflector is connected to the body via at least one support (622).

3. The apparatus according to any one of the preceding claims, wherein the apparatus is adapted to be arranged in the flowing liquid to be measured, wherein, The device is equipped with a bubble shield, wherein the cross-section of the bubble shield is larger than the cross-section of the optical aperture, the bubble shield is disposed on the far side of the far front end of the body, and wherein the reflector is preferably part of the bubble shield.

4. The apparatus according to claim 3, wherein, The bubble shield has a positive curvature on the side facing the flow direction or on the side away from the optical aperture.

5. The apparatus according to any one of claims 3 to 4, wherein, The bubble shield includes a diffuser extending toward the optical aperture, and wherein the reflector is preferably mounted to the diffuser.

6. A component comprising: a. Objective lens unit (6), and b. A device for guiding lighting according to any one of the preceding claims, c. The objective lens unit includes: i. A housing having a distal front end, ii. The front optical aperture (63) of the objective lens unit is disposed in the distal front end (62) of the objective lens unit. iii. The objective lens unit further includes objective lens systems (65, 66) arranged inside the housing (61) and located near the front optical aperture (63) of the objective lens unit. iv. Wherein, the objective lens system is configured to collect light received through the front optical aperture (63) of the objective lens unit, d. Wherein, the device for guiding illumination is attached to the objective lens unit, i. Wherein, the means for guiding illumination is arranged such that the reflector (621) is positioned distal to the front end (62) of the objective lens unit and at a non-zero distance from the front end optical aperture (63) of the objective lens unit, and the reflector is arranged to reflect at least a portion of the light received from the front end of the body back into the optical aperture of the objective lens unit. e. Wherein, the device for guiding illumination and / or the objective lens unit or a combination thereof is preferably liquid-resistant, such that the components are suitable for use when at least partially immersed in a liquid.

7. An objective lens unit (6), the objective lens unit comprising: a. Outer shell (61), i. wherein at least a portion of the housing (61) is formed of the body (611) of the device for guiding illumination according to any one of claims 1 to 5, ii. Wherein, the front end of the body of the device for guiding lighting is the distal front end (62) of the housing, and iii. The optical aperture of the body of the device for guiding illumination is the front optical aperture (63) of the objective lens unit, and iv. Wherein, the reflector (621) of the device for guiding illumination is connected to the housing (61), and b. The objective lens unit further includes an objective lens system (65, 66) disposed inside the housing (61) and near the front optical aperture (63) of the objective lens unit, wherein the objective lens system is configured to collect light received through the front optical aperture (63). c. Wherein, the objective lens unit is preferably adapted for use when at least partially immersed in a liquid.

8. The objective lens unit according to the preceding claim, wherein the housing (61) comprises a cap (611) and a sleeve (612), wherein, The objective lens system (65, 66) is disposed inside the sleeve, wherein the cap (611) is provided by the body of the device for guiding illumination, the cap (611) 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 (611), wherein the sleeve (612) is preferably received at least partially inside the cap (611).

9. The apparatus, component, or objective lens unit according to any one of the preceding claims, wherein, The objective lens unit (6) or the body (6) includes a proximal rear connector interface, wherein at least a portion of the objective lens unit (6) or the body (6) other than the rear connector interface is liquid-resistant.

10. The apparatus, component, or objective lens unit according to any one of the preceding claims, wherein the apparatus, component, or objective lens unit includes a sample illumination unit, wherein, The sample illumination unit includes: a. Lighting source (44), and b. A device (45) for coupling light from the illumination source (44) into the objective lens unit (6) and / or the body. c. In particular, the means for coupling light from the illumination source into the objective lens unit is arranged on the far side of the objective lens system (65, 66) and on the near side of the front optical aperture (63) of the objective lens unit, and is further configured to project the light onto the reflector (621) through the front optical aperture in the near-far direction of the objective lens unit.

11. A reflector assembly (621) comprising a reflector and at least one support, wherein, The reflector is attached to at least one support post, and the at least one support post is configured to be mounted on the housing of the objective lens unit according to any one of claims 7 or 8, wherein the size and shape of the reflector assembly are designed such that the reflector is positioned at a non-zero distance from the front end of the housing of the objective lens unit from the distal side, and such that the reflector is positioned and configured to receive light emitted through the front end of the objective lens unit when the reflector assembly is attached to the housing of the objective lens unit and to reflect at least a portion of the received light back into the optical aperture.

12. A microscope (1) for in-situ applications, the microscope comprising a component or objective lens unit (6) according to any one of claims 6 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 (1) for in-situ application according to the preceding claim, wherein, The microscope (1) includes a sample illumination unit, which includes an illumination source (44) and a device (45) for coupling light from the illumination source (44) into the objective lens unit (6), wherein the device for coupling light from the illumination source into the objective lens unit is preferably arranged near the objective lens unit.

14. A method of performing cytology using a microscope according to any one of claims 12 or 13, the method comprising: The objective lens unit (6), including at least the farthest front section of the objective lens unit (6) including the distal front end (62) and the front optical aperture (63) of the objective lens unit, or the far front end (62) of the body including the optical aperture (63) and the reflector, are immersed in the sample liquid (3), which is preferably contained inside the bioreactor. Light is emitted through the front end of the objective lens unit or through the front end of the body, and the emitted light is guided toward the reflector (621). At least a portion of the light striking the reflector is reflected back to the front optical aperture (63) of the objective lens unit and / or the optical aperture of the body and passes through the sample volume (100) of the sample liquid. At least one image generated by the objective lens unit (6) is recorded using an optical sensor (52). Preferably, the at least one image includes forward-scattered light and back-scattered light from objects contained in the sample volume.

15. The method according to the preceding claim, wherein, The method includes: setting the focal length of the objective lens unit such that the reflector is imaged on the optical sensor; and issuing a warning if the observed reflectivity is lower than expected and / or if the image displays an unexpected structure.

Images