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US-20260126639-A1 - DEVICE FOR DIRECTING ILLUMINATION, INSTRUMENTS COMPRISING SAID DEVICE, AND METHODS OF PERFORMING CYTOMETRY

US20260126639A1US 20260126639 A1US20260126639 A1US 20260126639A1US-20260126639-A1

Abstract

A device for directing illumination for a sample volume in microscopy, such as in-situ cytometry. The device includes a body having a distal front end with an optical aperture provided therein, and a reflector. The reflector is arranged distal from the front end and at a non-zero distance from the optical aperture and is configured to receive light emitted through the front end and reflect at least part of the light received from the front end back into the optical aperture. The reflector is connected to the body by at least one cantilevering strut. The device may be part of an objective lens unit.

Inventors

  • Sandro Link
  • Rudolf Gati
  • Nicolas Sobanski

Assignees

  • METTLER-TOLEDO GMBH

Dates

Publication Date
20260507
Application Date
20251020
Priority Date
20241104

Claims (20)

  1. 1 . A device for directing illumination, the device comprising: a body, the body having a distal front end and an optical aperture provided in the front end; and a reflector connected to the body and arranged distal from the front end at a non-zero distance from the optical aperture and configured to receive light emitted through the front end and to reflect at least part of the light received from the front end back into the optical aperture.
  2. 2 . The device of claim 1 , wherein: the reflector is connected to the body by at least one strut.
  3. 3 . The device of claim 1 wherein: the device is configured to be arranged in a streaming liquid to be measured; the device comprises a bubble shield; and the bubble shield has a cross section greater than the one of the optical aperture and is arranged distal of the distal front end of the body.
  4. 4 . The device of claim 3 wherein: the reflector is part of the bubble shield.
  5. 5 . The device of claim 3 , wherein: the bubble shield has a positive curvature on the side facing the flow direction or the side facing away from the optical aperture.
  6. 6 . The device of claim 3 , wherein: the bubble shield comprises a flow divider; and the flow divider extends towards the optical aperture.
  7. 7 . The device of claim 6 , wherein: the reflector is mounted to said flow divider.
  8. 8 . An assembly comprising: the device for directing illumination of claim 1 ; and an objective lens unit, wherein the objective lens unit comprises: an enclosure, the enclosure having a distal front end; a front end optical aperture provided in the distal front end; and an objective lens system arranged inside the enclosure and proximal from the front end optical aperture; wherein the objective lens system is configured to collect light received through the front end optical aperture of the objective lens unit; wherein the device for directing illumination is attached to the objective lens unit; wherein the device for directing illumination is arranged such that the reflector is arranged distal from the front end of the objective lens unit at a non-zero distance from the front end optical aperture of the objective lens unit and is arranged to reflect at least part of the light received from the front end of the body back into the optical aperture of the objective lens unit; wherein the device for directing illumination and/or the objective lens unit is configured to be used when at least partially submerged in a liquid.
  9. 9 . The assembly of claim 8 , wherein: the device for directing illumination and/or the objective lens are liquid-proof.
  10. 10 . An objective lens unit, the objective lens unit comprising: an enclosure, wherein at least a part of the enclosure is formed by a body for directing illumination, the body having a distal front end and an optical aperture provided in the front end, wherein the front end of the body of the device for directing illumination is a distal front end of the enclosure, the optical aperture of the body of the device for directing illumination is a front end optical aperture of the objective lens unit, and the reflector of the device for directing illumination is connected to the enclosure; and an objective lens system arranged inside the enclosure and proximal from the front end optical aperture of the objective lens unit, wherein the objective lens system is configured to collect light received through the front end optical aperture.
  11. 11 . The objective lens unit of claim 10 , wherein: the objective lens unit is suitable to be used when at least partially submerged in a liquid.
  12. 12 . The objective lens unit of claim 10 , wherein: the enclosure comprises a cap and a sleeve; the objective lens system is provided inside the sleeve; the cap is provided by the body of the device for directing illumination; the cap comprises a lateral sheath, a front wall, and a rear port; and the front end optical aperture of the objective lens unit is provided in the front wall of the cap.
  13. 13 . The objective lens unit of claim 12 , wherein: the sleeve is at least partially received inside the cap.
  14. 14 . The objective lens unit of claim 10 , wherein: the objective lens unit comprises a proximal rear connector interface, wherein at least a section of the objective lens unit excluding the proximal rear connector interface is liquid-proof.
  15. 15 . The objective lens unit of claim 10 , 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 arranged distal from the objective lens system and proximal from the front end optical aperture of the objective lens unit and is configured to project said light into a proximal-distal direction of the objective lens unit through the front end optical aperture and onto the reflector.
  16. 16 . A reflector assembly, comprising: a reflector; and at least one strut, whereby the reflector is attached to at least one strut, and the at least one strut is configured to be mounted to the enclosure of the objective lens unit of claim 10 ; wherein the reflector assembly is sized and shaped such that the reflector is positioned in a non-zero distance distal from a front end of the enclosure of the objective lens unit and is positioned and configured to receive light emitted through the front end of the objective lens unit and to reflect at least part of the light received back into the optical aperture when the reflector assembly is attached to the enclosure of said objective lens unit.
  17. 17 . A microscope for in-situ application, the microscope comprising: the objective lens unit of claim 10 ; 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.
  18. 18 . The microscope of claim 17 , wherein: the microscope comprises a sample illumination unit comprising an illumination source and a means for coupling light from the illumination source into the objective lens unit; and said means for coupling light from the illumination source into the objective lens unit is arranged proximal from the objective lens unit.
  19. 19 . A method of performing cytometry using the microscope of claim 17 , the method comprising: submerging at least a distalmost front section of the objective lens unit including the distal front end and the front end optical aperture of the objective lens unit or the distal front end of the body including the optical aperture and the reflector in a sample liquid; emitting light through the front end of the objective lens unit or through the front end of the body, said emitted light directed in a direction towards the reflector; reflecting at least a part of the light that hits the reflector, back to the front end optical aperture of the objective lens unit and/or the optical aperture of the body and through a sample volume of the sample liquid; and recording at least one image generated by the objective lens unit using the optical sensor.
  20. 20 . The method of claim 19 , the method comprising: setting a focal length of the objective lens unit such that the reflector is imaged on the optical sensor and issuing a warning where the observed reflectivity is lower than an expected value and/or where the image shows unexpected structures.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Application No. 24210649.0 filed Nov. 4, 2024, European Application No. 25163723.7 filed Mar. 14, 2025, and European Application No. 25170204.9 filed Apr. 11, 2025, the disclosures of each 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 Cytometry is applied to characterize biological cells, alive or death. Preferably, also for cytometry using the device according to the invention, the cells are in a liquid. For instance, density, number density, size and morphology of biological cells may be determined by microscopy. In the absence of reliable and quantitative in-line and in-situ measurement methods, bioengineers have to take samples to perform cytometry with off-line measurement devices. This requires taking a sample volume of content from 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-situ applicable in-line cytometer would drastically reduce the complexity compared to off-line methods and would allow the process controller to monitor the cell count and cell condition close to real-time. In-line imaging cytometry requires high-quality images of the biological cells. A key requirement for images with high quality is appropriate illumination of the objects to be observed. Since the cells within the bioreactor are typically moving fast due to the stirring of the bioreactor, the use of short light pulses of a few microseconds is preferred to avoid smearing and blurring of the image. This strongly limits the light energy collected per frame taken. In addition, the biological cells typically have a refractive index very close to the surrounding medium, further reducing the interaction with the light and therefore the object contrast. For biological cells in a liquid suspension, it was observed that back-scattered light can be several orders of magnitude smaller than light scattered in forward direction. Therefore, transmission microscopy, i.e., illuminating the objects to be observed from beyond the object when seen from the microscope objective, or, in still other words, with an illumination source emitting the light in a proximal direction and directing the light through the sample volume and towards the aperture of the microscope, results in drastically enhanced image quality compared to purely back-scattered light with the objects illuminated by distally travelling light. However, placing a light source underneath the objects respectively the sample volume for an in-situ cytometer within a bioprocess may yield certain drawbacks. 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. U.S. Pat. No. 6,809,862 suggests a microscope including a microscope arrangement having a specimen zone between a slide glass body and a lens cover glass, the lens cover glass closing the microscope in a distal direction. An illumination arrangement is provided distal from the sample zone and covered by the slide glass body and illuminates the sample zone through the sample zone with the illumination directed towards the microscope lens. Thus, the sample is viewed in transmitted light. A bulky unit comprising a light source, a power supply for the light source, and a collimator optics is arranged in front of, or distal from, the microscope and inside the reactor and influences flow patterns inside the reactor when applied for in-situ microscopy. DE 40 32 002 teaches to apply transmission microscopy inside a bioreactor. For that purpose, a microscope is arranged outside the bioreactor and is separated from the interior of the bioreactor by a window. A bent tube containing an optical fiber is inserted into the bioreactor and is directed to emit light inside the bioreactor through a sample volume and towards the window, or the microscope, respectively. The insertion of the bent tube requires an additional dedicated port of the bioreactor. U.S. Pat. No. 4,515,445 discloses an optical system for transmitted-light microscopy with incident illumination. The therein suggested device comprises a common upright reflected light microscope provided on one side of an object and a retroreflection device which contains an optical system which images the object unreversed and upright on itself on the other side of the object. The obj