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CN-121995612-A - Objective lens unit for a microscope, microscope comprising an objective lens unit, and method of performing a cytometry

CN121995612ACN 121995612 ACN121995612 ACN 121995612ACN-121995612-A

Abstract

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

Inventors

  • S. Link
  • R. Garty

Assignees

  • 梅特勒-托莱多有限公司

Dates

Publication Date
20260508
Application Date
20251017
Priority Date
20241104

Claims (15)

  1. 1. An objective unit (6) for a microscope (1), the objective unit being adapted for use when at least partially immersed in a liquid sample (3), the objective unit comprising a housing (61), The objective unit comprising an immersion lens (64, 642) positioned adjacent a distal front end (62) of a housing (61), the front end comprising a front optical aperture (63) of the objective unit, and the immersion lens being arranged and configured to be able to receive light through the front optical aperture, The objective unit further comprises an objective system (65, 66), the objective system (65, 66) being arranged inside the housing (61) and proximal to the immersion lens (64, 642), wherein the objective system is configured to collect light received by the immersion lens (64, 642) through the front optical aperture (63), Wherein the objective system comprises a motorized auto-adjust lens (65).
  2. 2. Objective unit according to the preceding claim, wherein the housing (61) is a shell inside which the objective system (65, 66) is mounted, the shell having a front surface, wherein a front optical aperture (63) of the objective unit is provided in the front surface of the shell, and the immersion lens (64, 642) is arranged adjacent to the front optical aperture (63).
  3. 3. The objective unit according to claim 1, the housing (61) comprising a cap (611) and a sleeve (612), wherein an objective system (65, 66) is provided inside the sleeve, the cap comprising a lateral sheath, a front wall and a rear port, wherein a front optical aperture (63) of the objective unit is provided in the front wall of the cap, and an immersion lens (64, 642) is attached to the cap (611), wherein the sleeve (612) is at least partially accommodated inside the cap (611).
  4. 4. Objective unit according to any one of the preceding claims, wherein at least a distal section of the objective unit is liquid-proof.
  5. 5. Objective unit according to one of the preceding claims, wherein the motor-free automatic adjusting lens (65) is an adjustable lens.
  6. 6. Objective unit according to one of the preceding claims, wherein the motor-free automatic adjusting lens (65) is a lens axially displaceable along an optical axis of the objective unit (6) by a piezoelectric actuator.
  7. 7. Objective unit according to one of the preceding claims, wherein the motor-free auto-adjustment lens (65) is arranged between the immersion lens (64, 642) and a fixed lens (66) of the objective system, wherein in particular the motor-free auto-adjustment lens is the next lens located proximal to the immersion lens.
  8. 8. Objective unit according to any one of the preceding claims, wherein the most distal optical element (64, 641) of the objective unit is flush with the distal outer surface of the front end (62) of the housing.
  9. 9. Objective unit according to one of the preceding claims, wherein the front end (62) of the housing is one of a flat shape and a convex shape.
  10. 10. Objective unit according to one of the preceding claims, wherein the objective unit comprises a sample illumination unit, wherein the sample illumination unit comprises an illumination source (44) and means (45) for coupling light from the illumination source (44) into the objective unit (6), wherein the means for coupling light from the illumination source into the objective unit are configured to be able to project the light in a proximal-distal direction of the objective unit and towards an immersion lens (64, 642), and the means are preferably arranged between the immersion lens (64, 642) and a motorized automatic adjustment lens (65).
  11. 11. A cap (611) configured for an objective lens unit according to claim 3 or any one of claims 4 to 10 when dependent on claim 3, wherein the cap comprises a front wall and a lateral sheath extending axially from the front wall, wherein a front optical aperture (63) is formed in the front wall, an immersion lens (64, 642) being attached to the front wall in functional relation to the front optical aperture (63) such that light from the front side of the cap and located outside the cap passes through the immersion lens when entering the interior of the cap through the front optical aperture, and wherein the lateral sheath is configured to be able to accommodate a sleeve (612) therein, wherein the sleeve is able to be inserted in an axial direction from the rear end of the cap.
  12. 12. A microscope (1) for in situ application inside a bioreactor, the microscope comprising an objective unit (6) according to any one of claims 1 to 10 and an optical sensor (52), wherein the optical sensor is functionally coupled to the objective unit to receive light transmitted from a front side of the objective unit and passing through the objective unit.
  13. 13. The microscope according to the preceding claim, wherein the microscope comprises a control unit functionally coupled to the objective lens unit (6) and configured to be able to control the motorized automatic adjustment lens (65).
  14. 14. A method of performing a cytometry using a microscope according to any one of claims 12 and 13, wherein the method comprises inserting at least a distal section of the objective unit (6) through a port of a bioreactor (21) and immersing at least a portion of the distal section of the objective unit (6) comprising a front end (62) and a front end optical aperture (63) in a sample liquid (3) contained inside the bioreactor, operating a motor-free automatic adjustment lens (65) to adjust an object plane clearly imaged on an optical sensor (52), and recording at least one image generated by the objective unit (6) using the optical sensor.
  15. 15. A method of performing a cytometry using a microscope (1) according to any one of claims 12 or 13, wherein the objective lens unit (6) is an objective lens unit according to claim 3 or any one of claims 4 to 10 when dependent on claim 3, the method comprising inserting at least a distal section of the cap (611) through a port of a bioreactor and sealing the port of the bioreactor with the cap, leaving a rear port of the cap (611) outside the bioreactor, immersing at least a portion of a distal section of the cap (611) comprising a front wall with a front optical aperture (63) in a sample liquid (3) inside the bioreactor, inserting a sleeve (612) comprising an objective lens system (65, 66) in the cap (611), operating the motorized automatic adjustment lens (65) to adjust an object plane clearly imaged on an optical sensor (52), and recording at least one image generated by the objective lens unit using the optical sensor.

Description

Objective lens unit for a microscope, microscope comprising an objective lens unit, and method of performing a cytometry Technical Field The subject matter claimed herein relates generally to instruments and methods suitable for in situ cytometry. More particularly, the invention relates to the subject matter set forth in the claims. Background Cytometry is used to characterize living or dead biological cells. Image cytometry does this by analyzing the image of the cells. The objective unit according to the invention is preferably used in an image cytometer. Preferably, for a cytometry performed using an objective lens unit according to the present invention, the cells are in a liquid. For example, the density, size and morphology of cells can be determined by microscopy. In the absence of a reliable and quantitative on-line measurement method, a bioengineering must collect samples to perform cytometry using an off-line measurement device. This requires that sample volumes be taken 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 statistics and real-time information about the cytometry. An online cytometer that characterizes biological cells typically cultured in a liquid environment would significantly reduce complexity compared to an offline method, and would allow a process controller to monitor cell counts and cell characteristics (such as those mentioned above) in near real-time. Biological processes are processes that use whole living biological cells or components thereof (e.g., bacteria, enzymes, or chloroplasts) to obtain a desired product. The biological process is usually carried out in a bioreactor, i.e. a process vessel, which is preferably a reusable and thus sterilizable tank or a disposable bag. Biological cells as part of the biological process are distributed in a liquid medium to create a suitable environment for the desired process. Typically, the culture medium contains nutrients for e.g. the cells in question and the gases they require, e.g. O2 and CO2. In most cases it is important to ensure that no other cells than those involved in the biological process are present in the bioreactor, which means that it is preferable to maintain a sterile barrier as long as possible between the process and the outside, thereby minimizing the number and duration of instances where unwanted cell contamination may occur. Generally, biological processes use devices to mix cells and culture medium continuously. Examples of such means are a stirrer arranged inside the bioreactor or a vibrator moving the whole bioreactor. Thus, in general, cells suspended in a medium move within a bioreactor. In many cases, the culture medium is closely monitored to ensure that the desired conditions are maintained. For such monitoring, a variety of optical, photochemical and electrochemical sensors are currently available and in use, which are capable of measuring in a reliable manner, for example, pH or dissolved oxygen content, on-line or in situ. However, there is currently no similar reliable measurement device available for directly monitoring cells. In-line or in-situ measurements-in the case of processes, in particular biological processes-are measurements which are carried out directly in the reactor, respectively in the bioreactor or in the line conveying the fluid to be monitored. In particular, the fluid to be monitored may be a liquid of a biological process comprising a culture medium and biological cells. Attempts have been made to construct sensors capable of performing on-line cytometry by constructing an in situ microscope and recording real-time images of biological cells. However, in order to obtain a cell count per unit volume, a volume must be defined in the process. The first commercial attempts have attempted to mechanically limit the small volume by opening and closing the mechanical chamber in the bioprocess. The limited precision of the mechanical movement and the defined volume prevents the sensor from performing accurately and reliably under real process conditions. Another approach uses an objective lens comprising a spherical solid immersion lens (so-called SIL) in direct contact with the process liquid and an aspherical lens for imaging. The measurement volume is thus defined optically by a very narrow depth of field of a few microns close to the SIL. However, maintaining a clear focus near the SIL surface is entirely dependent on the alignment of the SIL and the aspherical lens, with a tolerance along the optical axis of only a few microns. These tolerances are impractical for industrial series products. Furthermore, thermal and mechanical stresses and ageing effects experienced by in-line bio-process sensors, in particular during their lifetime, may cause small displacements of the different components relative to each other, limiting th