EP-4737974-A1 - DEVICE FOR DIRECTING ILLUMINATION, INSTRUMENTS COMPRISING SAID DEVICE, AND METHODS OF PERFORMING CYTOMETRY

EP4737974A1EP 4737974 A1EP4737974 A1EP 4737974A1EP-4737974-A1

Abstract

A device for directing illumination for a sample volume in microscopy, in particular in in-situ cytometry. The device comprises a body (611), the body having a distal front end (62) and an optical aperture provided (63) in the front end, and a reflector (621). 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 to 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 suggested device may be part of an objective lens unit (6).

Inventors

LINK, Sandro
GATI, RUDOLF
Sobanski, Nicolas

Assignees

Mettler-Toledo GmbH

Dates

Publication Date: 20260506
Application Date: 20250411

Claims (15)

Device for directing illumination, the device comprising a body (61, 611), the body having a distal front end (62) and an optical aperture provided (63) in the front end, and a reflector (621) connected to the body and arranged distal from the front end and a non-zero distance from the optical aperture and is 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.
Device according to any preceding claim, wherein the reflector is connected to the body by at least one strut (622).
Device according to any preceding claim suitable to be arranged in a streaming liquid to be measured, whereby the device is equipped with a bubble shield, whereby 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 and whereby the reflector is preferably part of the bubble shield.
Device according to claim 3, whereby the bubble shield has a positive curvature on the side facing the flow direction or the side facing away from the optical aperture.
Device according to any one of claims 3 to 4, whereby the bubble shield comprises a flow divider, which extends towards the optical aperture, and whereby the reflector is preferably mounted to said flow divider.
An assembly comprising a. an objective lens unit (6) and b. a device for directing illumination according to any of the preceding claims, c. the objective lens unit comprising i. an enclosure, the enclosure having a distal front end, ii. a front end optical aperture (63) of the objective lens unit provided in the distal front end (62) of the objective lens unit, iii. the objective lens unit further comprising an objective lens system (65, 66) arranged inside the enclosure (61) and proximal from the front end optical aperture (63) of the objective lens unit, iv. wherein the objective lens system is configured to collect light received through the front end optical aperture (63) of the objective lens unit, d. wherein the device for directing illumination is attached to the objective lens unit, i. wherein the device for directing illumination is arranged such that the reflector (621) is arranged distal from the front end (62) of the objective lens unit and a non-zero distance from the front end optical aperture (63) 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 e. whereby device for directing illumination and/or the objective lens unit or the combination of both is preferably liquid-proof, such that the assembly is suitable to be used when at least partially submerged in a liquid.
An objective lens unit (6), the objective lens unit comprising a. an enclosure (61), i. wherein at least a part of the enclosure (61) is formed by the body (611) of a device for directing illumination according to any of claims 1 through 5, ii. wherein the front end of the body of the device for directing illumination is a distal front end (62) of the enclosure and iii. the optical aperture of the body of the device for directing illumination is a front end optical aperture (63) of the objective lens unit, and iv. whereby the reflector (621) of the device for directing illumination is connected to the enclosure (61), and b. the objective lens unit further comprising an objective lens system (65, 66) arranged inside the enclosure (61) and proximal from the front end optical aperture (63) of the objective lens unit and wherein the objective lens system is configured to collect light received through the front end optical aperture (63) c. whereby the objective lens unit is preferably suitable to be used when at least partially submerged in a liquid.
Objective lens unit according to the preceding claim, the enclosure (61) comprising a cap (611) and a sleeve (612), wherein the objective lens system (65, 66) is provided inside the sleeve, wherein the cap (611) is provided by the body of the device for directing illumination, the cap (611) comprising a lateral sheath, a front wall and a rear port, wherein the front end optical aperture (63) of the objective lens unit is provided in the front wall of the cap (611), wherein the sleeve (612) is preferably at least partially received inside the cap (611).
Device, assembly or objective lens unit according to any of the preceding claims, wherein the objective lens unit (6) or the body (6) comprises a proximal rear connector interface, wherein at least a section of the objective lens unit (6) or the body (6) excluding the rear connector interface is liquid-proof.
Device, assembly or objective lens unit according to any of the preceding claims, comprising a sample illumination unit, wherein the sample illumination unit comprises a. an illumination source (44) and b. a means (45) for coupling light from the illumination source (44) into the objective lens unit (6) and/or into the body, c. wherein in particular the means for coupling light from the illumination source into the objective lens unit is arranged distal from the objective lens system (65, 66) and proximal from the front end optical aperture (63) of the objective lens unit and is further 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 (621).
A reflector assembly (621), 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 an objective lens unit according to any one of claims 7 or 8, 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.
Microscope (1) for in-situ application, the microscope comprising an assembly or an objective lens unit (6) according to any of claims 6 through 10 and an optical sensor (52), 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.
Microscope (1) for in-situ application according to the preceding claim, wherein the microscope (1) comprises a sample illumination unit comprising an illumination source (44) and a means (45) for coupling light from the illumination source (44) into the objective lens unit (6), wherein said means for coupling light from the illumination source into the objective lens unit is preferably arranged proximal from the objective lens unit.
Method of performing cytometry using a microscope according to any of claims 12 or 13, the method comprising submerging at least a distalmost front section of the objective lens unit (6) including the distal front end (21) and the front end optical aperture (63) of the objective lens unit or the distal front end (62) of the body including the optical aperture (63) and the reflector in a sample liquid (3), preferably contained inside the bioreactor, 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 (621), reflecting at least a part of the light that hits the reflector, back to the front end optical aperture (63) of the objective lens unit and/or the optical aperture of the body and through a sample volume (100) of the sample liquid, and recording at least one image generated by the objective lens unit (6) using the optical sensor (52), wherein preferably the at least one image includes forward scattered light and backward scattered light from objects contained in the sample volume.
Method according to the preceding claim, 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, if the observed reflectivity is lower than an expected value and/or if the image shows unexpected structures.

Description

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, 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. US 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. US 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 object is placed on a microscope slide of the upright microscope. The purpose of the taught subject matter is to observe light which passes an object twice. US 4,515,445 gives no hint for application in in-situ cytometry, and the retroreflection device which needs to be placed distal from the object, or a sample volume, to be observed is rather bulky and thus unsuitab