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CN-121995614-A - Device for guiding illumination, instrument comprising said device, and method for performing a cytometry

CN121995614ACN 121995614 ACN121995614 ACN 121995614ACN-121995614-A

Abstract

A device for directing illumination for a sample volume in microscopy, in particular 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). The reflector is disposed distally of the front end and a non-zero distance from the optical aperture, the reflector being configured to receive light emitted through the front end and 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 cantilever strut. The proposed device may be part of an objective unit (6).

Inventors

  • S. Link
  • R. Garty
  • N. Sobanski

Assignees

  • 梅特勒-托莱多有限公司

Dates

Publication Date
20260508
Application Date
20251104
Priority Date
20241104

Claims (15)

  1. 1. An apparatus for guiding illumination, the apparatus comprising a body (61, 611) 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 distally of the front end and at a non-zero distance from the optical aperture, the reflector being configured to be capable of receiving 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. 2. The device of any of the preceding claims, wherein the reflector is connected to the body by at least one strut (622).
  3. 3. Device according to any of the preceding claims, which is adapted to be arranged in a flowing liquid to be measured, wherein the device is provided with a bubble shield, wherein the bubble shield has a cross-section which is larger than the cross-section of the optical aperture, which bubble shield is arranged distally of the distal front end of the body, and wherein the reflector is preferably part of the bubble shield.
  4. 4. A device according to claim 3, wherein the bubble shield has a positive curvature on the side facing the flow direction or on the side facing away from the optical aperture.
  5. 5. The device of any one of claims 3 to 4, wherein the bubble shield comprises a diverter extending towards the optical aperture, and wherein the reflector is preferably mounted to the diverter.
  6. 6. An assembly, comprising: a. an objective lens unit (6), and B. The device for guiding illumination according to any of the preceding claims, C. the objective lens unit includes: i. A housing having a distal front end, The front optical aperture (63) of the objective unit is arranged in the distal front end (62) of the objective unit, The objective unit further comprises an objective system (65, 66), the objective system (65, 66) being arranged inside the housing (61) and being located proximally of a front optical aperture (63) of the objective unit, Wherein the objective system is configured to collect light received through a front end optical aperture (63) of the objective unit, D. Wherein the means for guiding illumination is attached to the objective unit, I. Wherein the means for directing illumination is arranged such that the reflector (621) is arranged distally of the front end (62) of the objective unit and at a non-zero distance from the front end optical aperture (63) of the objective unit, and the reflector is arranged to be able to reflect at least part of the light received from the front end of the body back into the optical aperture of the objective unit, E. Wherein the means for guiding illumination and/or the objective unit or a combination of both are preferably liquid proof such that the assembly is adapted for use when at least partially submerged in liquid.
  7. 7. An objective unit (6), the objective unit comprising: a. a housing (61), I. wherein at least a portion of the housing (61) is formed by a body (611) of the device for guiding illumination according to any one of claims 1 to 5, Wherein the front end of the body of the means for directing illumination is the distal front end (62) of the housing, and The optical aperture of the body of the device for guiding illumination is the front optical aperture (63) of the objective unit, and Wherein the reflector (621) of the means for guiding illumination is connected to the housing (61), and B. The objective unit further comprises an objective system (65, 66), the objective system (65, 66) being arranged inside the housing (61) and being located proximally of a front optical aperture (63) of the objective unit, wherein the objective system is configured to collect light received through the front optical aperture (63), C. Wherein the objective unit is preferably adapted for use when at least partially immersed in a liquid.
  8. 8. Objective unit according to the preceding claim, the housing (61) comprising a cap (611) and a sleeve (612), wherein the objective system (65, 66) is arranged inside the sleeve, wherein the cap (611) is provided by the body of the means for guiding illumination, the cap (611) comprising a lateral sheath, a front wall and a rear port, wherein a front optical aperture (63) of the objective unit is arranged in the front wall of the cap (611), wherein the sleeve (612) is preferably at least partially received inside the cap (611).
  9. 9. Device, assembly or objective unit according to any one of the preceding claims, wherein the objective unit (6) or the body (6) comprises a proximal rear connector interface, wherein at least a portion of the objective unit (6) or the body (6) other than the rear connector interface is liquid-proof.
  10. 10. The device, assembly or objective unit according to any one of the preceding claims, comprising a sample illumination unit, wherein the sample illumination unit comprises: a. an illumination source (44), and B. means (45) for coupling light from the illumination source (44) into the objective unit (6) and/or into the body, C. Wherein in particular the means for coupling light from the illumination source into the objective unit is arranged distally of the objective system (65, 66) and proximally of a front optical aperture (63) of the objective unit, and is further configured to be able to project the light through the front optical aperture onto a reflector (621) in a proximal-distal direction of the objective unit.
  11. 11. A reflector assembly (621) comprising a reflector and at least one post, wherein the reflector is attached to the at least one post and the at least one post is configured to be mountable to a housing of an objective unit according to any of claims 7 or 8, wherein the reflector assembly is sized and shaped such that the reflector is positioned distally at a non-zero distance from a front end of the housing of the objective unit and such that the reflector is positioned and configured to be capable of receiving light emitted through the front end of the objective 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 unit.
  12. 12. A microscope (1) for in situ applications, comprising an assembly or objective 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 unit to receive light transmitted from the front side of the objective unit and through the objective unit.
  13. 13. Microscope (1) for in situ applications according to the preceding claim, wherein the microscope (1) comprises a sample illumination unit comprising 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 is preferably arranged proximally of the objective unit.
  14. 14. A method of performing a cytometry using the microscope of any one of claims 12 or 13, the method comprising: Immersing at least the most distal front section of the objective unit (6) comprising the distal front end (62) and a front optical aperture (63) of the objective unit, or the distal front end (62) of the body comprising the optical aperture (63) and the reflector, in a sample liquid (3), preferably contained inside the bioreactor, Light is emitted through the front end of the objective unit or through the front end of the body, the emitted light being directed in the direction of a reflector (621), Reflecting at least a part of the light hitting the reflector back to a front optical aperture (63) of the objective unit and/or an optical aperture of the body and through a sample volume (100) of the sample liquid, and At least one image generated by the objective unit (6) is recorded using an optical sensor (52), Wherein preferably the at least one image comprises forward and back scattered light from an object contained in the sample volume.
  15. 15. Method according to the preceding claim, wherein the method comprises setting a 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 below an expected value and/or if the image shows an unexpected structure.

Description

Device for guiding illumination, instrument comprising said device, and method for 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. Preferably, for a cytometry performed using a device 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 on-line and in-situ measurement methods, biological engineers must collect samples to perform cytometry using an off-line measurement device. This requires that a volume of sample content be removed 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. In-situ applicable online cytometry will significantly reduce complexity compared to offline methods and allow the process controller to monitor cell counts and cell conditions in near real-time. In-line imaging cytometry requires high quality images of biological cells. A key requirement for obtaining high quality images is proper illumination of the object to be observed. Since cells within a bioreactor are typically moved rapidly due to agitation of the bioreactor, short light pulses of several microseconds are preferably used to avoid smearing and blurring of the image. This greatly limits the light energy collected per frame. Furthermore, biological cells typically have a refractive index very close to the surrounding medium, which further reduces the interaction with light and thus reduces the contrast of the object. For biological cells in liquid suspension, it is observed that the back-scattered light can be several orders of magnitude smaller than the forward-scattered light. Thus, transmission microscopy (i.e., illuminating an object to be observed from outside the object when viewed from the microscope objective, or in other words, using an illumination source that emits light in the proximal direction and directs the light through the sample volume and toward the microscope aperture) can significantly improve image quality compared to pure back-scattered light produced by illuminating the object with distally propagating light. However, for in situ cytometry in biological processes, placing the light source below the object or sample volume may present certain drawbacks. 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, correspondingly 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. US 6,809,862 proposes a microscope comprising a microscope device having a sample region between a slide body and a lens cover glass which encloses the microscope in a distal direction. An illumination device is disposed distal to the sample region and covered by the slide body, the illumination device illuminating the sample region through the sample region, wherein the illumination is directed to the microscope lens. Thus, the sample was observed in transmitted light. A bulky unit comprising a light source, a power supply for said light source and collimating optics is arranged in front of or distally of the microscope and inside the reactor, which affects the flow pattern inside the reactor when applied in situ microscopy. DE 40 32 002 teaches the use of transmission microscopy inside a bioreactor. For this purpose, the microscope is arranged outside the bioreactor and is separated from the interior of the bioreactor by a window. A curved tube containing an optical fiber is inserted into the bioreactor and directed to emit light inside the bioreactor through the sample volume and toward the window or microscope, respectively. The insertion of the curved tube requires an additional dedicated port of the bioreactor. US 4,515,445 discloses an optical system for transmitted light microscopy with incident illumination. The proposed device comprises a generic vertical reflected light microscope arranged on one side of the object, and a retro-reflective device comprising an optical system that images the object itself on the other side of the object, non-inverted and vertical. The object is placed on a microscope slide of a vertical microscope. The purpose of the taught subject matter is to observe light passing through an object twice. US 4,515,445 does not give any indication of application to in situ cytometry and the retro-reflective means required to be pla