US-20260126638-A1 - OBJECTIVE LENS UNIT FOR A MICROSCOPE, MICROSCOPE COMPRISING THE OBJECTIVE LENS UNIT, AND METHODS OF PERFORMING CYTOMETRY

US20260126638A1US 20260126638 A1US20260126638 A1US 20260126638A1US-20260126638-A1

Abstract

An objective lens unit for a microscope is disclosed. The objective lens unit is suitable to be used when at least partially submerged in a liquid sample. The objective lens unit includes an immersion lens positioned adjacent a distal front end of the enclosure of the objective lens unit. The immersion lens is arranged and configured to receive light through a front end optical aperture. An objective lens system is arranged inside an enclosure and proximal from the immersion lens. The objective lens system is configured to collect light received by the immersion lens through the front end optical aperture. The objective lens system includes a motorless automated adjustable lens, in particular a tuneable lens, configured to amend the focal length of the objective lens system relative to the immersion lens.

Inventors

Sandro Link
Rudolf Gati

Assignees

METTLER-TOLEDO GMBH

Dates

Publication Date: 20260507
Application Date: 20250905
Priority Date: 20241104

Claims (17)

1 . An objective lens unit for a microscope, where the objective lens unit is suitable to be used when at least partially submerged in a liquid sample, the objective lens unit comprising: an enclosure; an immersion lens positioned adjacent a distal front end of the enclosure, the distal front end comprising a front end optical aperture, wherein the immersion lens is arranged and configured to receive light through the front end optical aperture; and an objective lens system arranged inside the enclosure and proximal from the immersion lens; wherein the objective lens system is configured to collect light received by the immersion lens through the front end optical aperture; wherein the objective lens system comprises a motorless automated adjustable lens.
2 . The objective lens unit of claim 1 , wherein: the enclosure is a housing; the objective lens system is mounted inside the housing; the housing has a front face; the front end optical aperture of the objective lens unit is provided in the front face of the housing; and the immersion lens is arranged adjacent the front end optical aperture.
3 . The objective lens unit of claim 1 , wherein: the enclosure comprises a cap and a sleeve; the objective lens system is provided inside the sleeve; the cap comprises a lateral sheath, a front wall, and a rear port; the front end optical aperture of the objective lens unit is provided in the front wall of the cap and the immersion lens is attached to the cap; and the sleeve is at least partially received inside the cap.
4 . The objective lens unit of claim 1 , wherein: at least a distal section of the objective lens unit is liquid-proof.
5 . The objective lens unit of claim 1 , wherein: the motorless automated adjustable lens is a tuneable lens.
6 . The objective lens unit of claim 1 , wherein: the motorless automated adjustable lens is a lens axially displaceable along an optical axis of the objective lens unit by a piezo actuator.
7 . The objective lens unit of claim 1 , wherein: the motorless automated adjustable lens is arranged between the immersion lens and a fixed lens of the objective lens system.
8 . The objective lens unit of claim 7 , wherein: the motorless automated adjustable lens is the next lens proximal from the immersion lens.
9 . The objective lens unit of claim 1 , wherein: the most distal optical element of the objective lens unit is flush with a distal outer surface of the front end of the enclosure.
10 . The objective lens unit of claim 1 , wherein: the front end of the enclosure is flat and convexly shaped.
11 . The objective lens unit of claim 1 , further comprising: a sample illumination unit, wherein the sample illumination unit comprises an illumination source and a means for coupling light from the illumination source into the objective lens unit, wherein the means for coupling light from the illumination source into the objective lens unit is configured to project said light into a proximal-distal direction of the objective lens unit and towards the immersion lens.
12 . The objective lens unit of claim 11 , wherein: the means for coupling light from the illumination source into the objective lens unit is arranged between the immersion lens and the motorless automated adjustable lens.
13 . A cap configured for the objective lens unit of claim 3 , wherein: the cap comprises a front wall and a lateral sheath extending axially from the front wall; a front end optical aperture is formed in the front wall and an immersion lens is attached to the front wall in a functional relationship with the front end optical aperture such that light from the front side of the cap and outside the cap passing through the front end optical aperture and into the interior of the cap passes through the immersion lens; and the lateral sheath is configured to receive a sleeve therein, the sleeve being insertable in an axial direction from a back end of the cap.
14 . A microscope for in-situ application inside a bioreactor, the microscope comprising: the objective lens unit of claim 1 ; and an optical sensor, wherein the optical sensor is functionally coupled to the objective lens unit to receive light transmitted from a front side of the objective lens unit and through the objective lens unit.
15 . The microscope of claim 14 , wherein: the microscope comprises a control unit functionally coupled to the objective lens unit and configured to control the motorless automated adjustable lens.
16 . A method of performing cytometry using the microscope of claim 14 , the method comprising: inserting at least a distal section of the objective lens unit through a port of a bioreactor and submerging at least a part of the distal section of the objective lens unit including the front end and the front end optical aperture in a sample liquid contained inside the bioreactor; operating the motorless automated adjustable lens to adjust an object plane sharply imaged on the optical sensor; and recording at least one image generated by the objective lens unit using the optical sensor.
17 . A method of performing cytometry using the microscope of claim 14 wherein the enclosure comprises a cap and a sleeve, the objective lens system is provided inside the sleeve, the cap comprises a lateral sheath, a front wall, and a rear port, the front end optical aperture of the objective lens unit is provided in the front wall of the cap and the immersion lens is attached to the cap, and the sleeve is at least partially received inside the cap, the method comprising: inserting at least a distal section of the cap through the port of a bioreactor and sealing the port of the bioreactor with the cap; maintaining the rear port of the cap outside the bioreactor; submerging at least a part of the distal section of the cap including the front wall with the front end optical aperture in a sample liquid inside the bioreactor; inserting the sleeve comprising the objective lens system into the cap; operating the motorless automated adjustable lens to adjust an object plane which is sharply imaged on the optical sensor; and recording at least one image generated by the objective lens unit using the optical sensor.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 24210649.0 filed Nov. 4, 2024, the disclosures of which are hereby incorporated by reference as if fully restated herein. TECHNICAL FIELD The herein claimed subject matter relates generally to instruments and methods applicable for in-situ cytometry. More in particular, it relates to the subject matter set forth in the claims. BACKGROUND ART Cytometry is applied to characterize biological cells, living or death. Image cytometers do so by analyzing images of cells. The objective lens unit according to the invention is preferably used in an image cytometer. Preferably, for the cytometry using the objective lens unit according to this invention, the cells are in a liquid. For instance, density, size and morphology of cells may be determined by microscopy. In the absence of reliable and quantitative in-line measurement methods, bioengineers have to take samples to perform cytometry with off-line measurement devices. This requires taking a sample volume of the bioreactor within certain intervals, which increases the risk of contamination. In addition, it is time-consuming and due to the sparse interval, little statistics and no live information about cytometry is available to the operator. An in-line cytometer, characterizing the biological cells in the, typically liquid, environment in which they are cultivated, would drastically reduce the complexity compared to off-line methods and would allow the process controller to monitor the cell count and cell characteristics, such as the ones named above, close to real-time. Bioprocesses are processes which use complete living, biological cells or their components such as for example bacteria, enzymes or chloroplasts, to obtain desired products. A bioprocess is typically done in a bioreactor, i.e. a process vessel which is preferably either reusable and therefore sterilizable tank or a single-use bag. The biological cells being part of the bioprocesses are dispensed in a liquid medium, establishing thereby the suitable environment for the desired process. Typically, the medium comprises for example nutrients for the cells in question as well as gases needed by them such as O2 and CO2. In most cases, it is important to ensure that no cells other than the ones involved in the bioprocess are present in the bioreactor, implying that it is preferred to maintain a sterile barrier between the process and the outside as long as possible thereby minimizing the number and duration of occasions where a contamination with unwanted cells can happen. Often, the bioprocess uses a means to mix the cells and the medium constantly. Examples of such means are stirrers arranged on the inside of the bioreactor or shakers which move the whole bioreactor. Therefore, in general, the cells suspended in the media are moving inside the bioreactor. In many cases, the medium is closely monitored to ensure that the desired conditions are maintained. For this monitoring, there are today different optical, opto-chemical and electrochemical sensors available and in use which can measure for example the pH-value or the amount of dissolved oxygen in-line or in-situ in a reliable manner. There is, however, currently no similar reliable measurement device available to monitor the cells directly. In-line or in-situ measurements are-in the case of a process, in particular a bioprocess-measurements which take place directly in the reactor respectively in the bioreactor or in a tube transporting the fluid to be monitored. In particular the fluid to be monitored can be the liquid of the bioprocess comprising the medium and the biological cells. There have been attempts to build a sensor capable of in-line cytometry by building an in-situ microscope and recording live images of biological cells. However, to get a cell count per volume, one must define a volume within the process. First commercial attempts tried to mechanically confine a small volume by opening and closing a mechanical chamber within the bio process. The mechanical movement and the limited precision in defining the volume hindered the sensor to perform accurate and reliable under real process conditions. Another approach used an objective that includes a spherical solid immersion lens, so-called SIL, that is directly in contact with the process liquid and an aspheric lens to shape the image. The measurement volume is hereby optically defined by a very narrow depth of field of some μmin close proximity to the SIL. However, keeping a sharp focus close to the surface of the SIL entirely depends on alignment of the SIL and the aspheric lens with tolerances along the optical axis of only some μm. These tolerances are impractical for an industrial series product. In addition, thermal and mechanical stresses to which in particular an in-line bio-process sensor is subjected during his lifetime as well as aging effects can cause small displacem