FR3146734A1 - Compact reflecting microscope - Google Patents

Abstract

The invention relates to a reflection microscope (2) used to observe/detect an object (O), said microscope comprising several components: A light source adapted to emit light radiation (EXC_2), called excitation radiation, to the object (O) to illuminate it, A reading device (24) for radiation, called emission radiation (REF_2), coming from the object (O), generated after illumination, An objective (22) and a tube lens (23) associated with said objective to form an image on said reading device (24), The device comprising at least one component (20) including said light source and having a zone transparent to the emission radiation (REF_2), The reading device (24), the objective (22), the tube lens (23) and said component (20) including the light source being positioned along a single optical axis (X). Figure to be published with the abstract: Figure 2

Description

Compact reflecting microscope Technical field of the invention

The present invention relates to a reflection microscope having the advantage of being particularly compact.

State of the art

• Une source lumineuse 100 chargée d'émettre les rayonnements d'excitation EXC_1,
• Un filtre d'excitation 101 mis en vis-à-vis de la source lumineuse pour filtrer les rayonnements d'excitation,
• Un objectif 105 de microscope, permettant de former une image de l’objet à l’infini,
• Un élément optique 102 tel qu'un miroir, qui peut être dichroïque, chargé de renvoyer les rayonnements lumineux d'excitation à 90° en direction de l'objet O pour l'exciter, et de laisser passer les rayonnements lumineux REF_1 d'émission émis par l'objet,
• Un filtre d'émission 103 destiné à être traversé par les rayonnements émis par l'objet. Ce filtre est en général positionné entre l’objectif et une lentille de tube, puisque dans cette partie du trajet optique les faisceaux provenant de l’objet sont collimatés, ce qui assure un meilleur fonctionnement du filtre,
• Une lentille de tube 106 permettant de focaliser l’image de l’objet sur le dispositif de détection 104,
• Un dispositif de détection 104 placé dans l'axe de l'objet pour capturer les rayonnements lumineux de détection.

Fluorescence is the property that certain bodies have of emitting light after absorbing so-called excitation light radiation. To detect the light emitted by fluorescence, forming so-called emission radiation, an optical microscope operating in reflection is often used (the excitation of the sample and the detection of the radiation are then done on the same side of the sample through the same objective). Fluorescence microscopy is thus based on the formation of an image by detecting the light emitted by the samples after their excitation using the excitation light radiation. Currently, the microscopes used are based on the principle illustrated by the and thus include:

• A light source 100 responsible for emitting the excitation radiation EXC_1,
• An excitation filter 101 placed opposite the light source to filter the excitation radiation,
• A 105 microscope objective, allowing an image of the object to be formed at infinity,
• An optical element 102 such as a mirror, which may be dichroic, responsible for returning the excitation light rays at 90° towards the object O to excite it, and for allowing the emission light rays REF_1 emitted by the object to pass through,
• An emission filter 103 intended to be crossed by the radiation emitted by the object. This filter is generally positioned between the objective and a tube lens, since in this part of the optical path the beams coming from the object are collimated, which ensures better operation of the filter,
• A tube lens 106 for focusing the image of the object on the detection device 104,
• A detection device 104 placed in the axis of the object to capture the detection light rays.

For epifluorescence operation, the combination of the optical elements formed by the excitation filter 101, the dichroic mirror 102 and the emission filter 103 makes it possible to detect at the detection device 104 only the fluorescence emission radiation, the excitation radiation having been cut off. For a reflection microscope working with other contrasts (absorption, polarization, etc.), the combination of these three elements generally makes it possible to optimize the quantity of detection signal coming from the object at the detection device 104. In this conventional embodiment, the source and the detection device are not in the same optical axis but placed at 90° relative to each other. For this type of embodiment, a first arm is used to support the source and a second arm is used to support the detection device. The other beam shaping components are then distributed over one or the other of the two arms.

The presence of two separate arms makes these earlier designs bulky, difficult to transport and quite complex.

The aim of the invention is therefore to propose a microscope operating in reflection, possibly usable for detecting fluorescence, which is compact, simple and easy to transport and deploy in the field.

• Une source lumineuse adaptée pour émettre des rayonnements lumineux, dits d'excitation, à destination de l'objet pour l'illuminer,
• Un dispositif de lecture de rayonnements, dits d'émission, en provenance de l'objet, générés après illumination,
• Un objectif et une lentille de tube associée audit objectif pour former une image sur ledit dispositif de lecture,
• Le dispositif comportant au moins un composant incluant ladite source lumineuse et présentant une zone transparente aux rayonnements d'émission,
• Le dispositif de lecture, l'objectif, la lentille de tube et ledit composant incluant la source lumineuse étant positionnés suivant un seul et même axe optique.

This goal is achieved by a reflection microscope used to observe/detect an object, said microscope comprising several components:

• A light source suitable for emitting light rays, called excitation rays, towards the object to illuminate it,
• A device for reading radiation, called emission, coming from the object, generated after illumination,
• An objective and a tube lens associated with said objective for forming an image on said reading device,
• The device comprising at least one component including said light source and having a zone transparent to emission radiation,
• The reading device, the objective, the tube lens and said component including the light source being positioned along a single optical axis.

According to a particular embodiment, the component including the light source is positioned between the objective and the tube lens.

According to another particular embodiment, the component including the light source is positioned between the objective and the object to be detected.

According to another particular embodiment, the component including the light source is positioned between the tube lens and the reading device.

According to a particular feature, the microscope comprises a first spectral filter positioned between the light source and the reading device.

According to another feature, the component including the light source comprises a layer forming a second spectral filter, completing said first spectral filter.

According to another feature, the component including the light source comprises an organic light-emitting diode.

According to another particularity, the reading device comprises a CMOS or CCD type sensor.

According to another particularity, the components of the microscope are united in a single single-block assembly in which the components are integral with each other.

According to another particularity, the microscope has a single arm for holding said assembly.

Brief description of the figures

• La montre le principe de fonctionnement d'un microscope en réflexion utilisé en détection de fluorescence, selon l'état de la technique ;
• La montre en éclaté l'architecture du microscope en réflexion conforme à l'invention ;
• La montre l'architecture du microscope en réflexion conforme à l'invention et illustre son principe de fonctionnement en détection de fluorescence ;
• La montre la structure d'une diode électroluminescente organique pouvant être utilisée dans le microscope en réflexion de l'invention ;

Other features and advantages will become apparent in the detailed description which follows, given in relation to the attached drawings in which:

• There shows the operating principle of a reflection microscope used in fluorescence detection, according to the state of the art;
• There shows an exploded view of the architecture of the reflection microscope in accordance with the invention;
• There shows the architecture of the reflection microscope in accordance with the invention and illustrates its operating principle in fluorescence detection;
• There shows the structure of an organic light-emitting diode suitable for use in the reflection microscope of the invention;

Detailed description of at least one embodiment

The invention relates to a microscope 2 operating in reflection. In a preferred architecture, it is particularly suitable for detecting fluorescence emitted by an object. In the case of fluorescence detection, subjected to excitation radiation EXC_2, the object O emits in response so-called emission radiation, referenced REF_2 in the attached figures.

In the following description, terms such as "upper", "lower", "above", "below" are to be taken into account with reference to the optical axis oriented vertically.

The optical axis (X) corresponds to the axis along which the rays pass through the microscope, between the object to be observed and the reading device or vice versa.

The object O to be observed can be of any kind. In the field of biology, it can be a sample placed on a transparent slide or in a Petri dish.

In reference to the and to the , microscope 2 has a light source, responsible for emitting the excitation radiation EXC_2 along the optical axis (X) towards the object O.

The light source is integrated into a component 20 having at least one zone transparent to the emission radiation REF_2, so as to be able to be crossed by these radiations along the optical axis (X).

Advantageously, the component 20 comprises at least one organic light-emitting diode (OLED for Organic Light Emitting Diode). This component 20 has the particularity of being able to play the role of a light source and of being transparent to REF_2 emission radiation.

• Une première électrode transparente ou semi-transparenteC1,
• Une première couche d'interface C2,
• Une couche active C3,
• Une deuxième couche d'interface C4,
• Une deuxième électrode transparente ou semi-transparente C5.

Such an organic light-emitting diode is described in patent application EP3816612A1 . With reference to the , it is produced in the form of a stack of layers which includes:

• A first transparent or semi-transparent electrodeC1,
• A first C2 interface layer,
• An active layer C3,
• A second C4 interface layer,
• A second transparent or semi-transparent electrode C5.

The deposition can be carried out on a transparent C0 substrate, for example made of glass or a transparent polymer.

• Une couche d'argent formant la première électrode métallique C1,
• La première couche d'interface C2 et la deuxième couche d'interface C4 peuvent correspondre à une couche injectrice d'électrons ou à une couche injectrice de trous,
• La couche active C3 est la couche émettrice. Elle comporte au moins un matériau organique et peut comporter un empilement ou un mélange de plusieurs matériaux organiques,
• Une couche réalisée dans un oxyde transparent conducteur (couramment appelé TCO). Il peut s'agir par exemple d'un oxyde d'Indium dopé à l'étain (ITO) formant la deuxième électrode non métallique C4.

Without limitation, the layers of the light-emitting diode may be as follows:

• A layer of silver forming the first metal electrode C1,
• The first interface layer C2 and the second interface layer C4 may correspond to an electron injecting layer or a hole injecting layer,
• The active layer C3 is the emitting layer. It comprises at least one organic material and may comprise a stack or a mixture of several organic materials,
• A layer made of a transparent conductive oxide (commonly called TCO). This can be, for example, a tin-doped indium oxide (ITO) forming the second non-metallic electrode C4.

The stack comprising the first interface layer, the active layer and the second interface layer may have a thickness of between 50nm and 400nm, preferably between 80nm and 150nm.

The stack of layers may comprise one or more upper layers C6, placed on the first electrode, and composed of a spectral filter, which may be supplemented by the spectral filter 21 positioned in the optical chain of the microscope 2 (see below) for fluorescence detection.

In the case of fluorescence detection, the radiation emitted by the light source is excitation radiation EXC_2.

The REF_2 emission radiation, emitted by the object by florescence, is caused to pass through the component 20, by transparency. It should be noted that it is particularly important that the component 20 including the light source itself has a high optical quality in transparency, so that the performances in terms of spatial resolution are well limited by the optics used (objective, tube lens) and not degraded by the passage of this component. The spatial uniformity of the lighting allowed by the OLED is advantageous.

The microscope 2 also includes an objective 22 (which may include one or more lenses) in front of which the object to be analyzed is placed. Associated with the objective 22, the microscope includes a tube lens 23.

The microscope comprises a reading device 24 used to read/detect the emission radiation REF_2 coming from the object. The reading device 24 may be a simple eyepiece (for viewing/reading with the naked eye) or may comprise a sensor 240 of the CMOS or CCD type (as in the attached figures). In the latter case, it may for example be a camera.

• L'objectif 22 est situé en vis-à-vis de l'objet ; Il est positionné à une distance proche de la distance de travail de l’objectif de l’objet O
• Dans le cas d'une détection de fluorescence, un filtre spectral 21 peut être positionné au-dessus du composant 20 incluant la source lumineuse ; Ce filtre spectral permet de bloquer/limiter les rayonnements d'excitation EXC_2 émis par la source lumineuse et laisse passer les rayonnements REF_2 de détection, émis par fluorescence en provenance de l'objet O. Le composant 20 de type OLED peut également, par construction intégrer des couches supplémentaires permettant tout ou partie du filtrage spectral.
• La lentille de tube 23 est placée au-dessus du filtre spectral lorsque celui-ci est présent ;
• Le dispositif de lecture 24, comportant par exemple le capteur 240, est placé au-dessus de la lentille de tube 23 ;
• Le composant 20 incluant la source lumineuse est placé, suivant l'axe optique, au-dessus de l'objet O, entre l'objet O et l'objectif 22 (position P2 sur la ) ou au-dessus de l'objectif 22 ; Dans ce dernier cas, il peut être placé entre l'objectif 22 et le filtre spectral 21 (si présent – position P1 sur la ) ou la lentille de tube 23 (si filtre spectral non présent). Il pourrait également être placé entre la lentille de tube 23 et le dispositif de lecture 24 (position P3 sur la ) ; Sa zone transparente, située dans l'axe optique (X) lui permet d'être traversée par les rayonnements d'émission REF_2 ;

Within the framework of the invention, going from bottom to top and following a single optical axis (X), the different components of the microscope can be arranged in the following manner:

• The objective 22 is located opposite the object; it is positioned at a distance close to the working distance of the object objective O
• In the case of fluorescence detection, a spectral filter 21 can be positioned above the component 20 including the light source; This spectral filter makes it possible to block/limit the excitation radiation EXC_2 emitted by the light source and allows the detection radiation REF_2, emitted by fluorescence from the object O, to pass. The OLED-type component 20 can also, by construction, integrate additional layers allowing all or part of the spectral filtering.
• The tube lens 23 is placed above the spectral filter when the latter is present;
• The reading device 24, comprising for example the sensor 240, is placed above the tube lens 23;
• The component 20 including the light source is placed, along the optical axis, above the object O, between the object O and the objective 22 (position P2 on the ) or above the lens 22; In the latter case, it can be placed between the lens 22 and the spectral filter 21 (if present – position P1 on the ) or the tube lens 23 (if spectral filter not present). It could also be placed between the tube lens 23 and the reading device 24 (position P3 on the ) ; Its transparent zone, located in the optical axis (X) allows it to be crossed by REF_2 emission radiation;

According to a particular aspect of the invention, these different components of the microscope 2 are assembled together so as to form a single monobloc and mono-axial assembly (which can be fixed for example on a mechanical arm). The fixing of the different components together can be carried out in different ways.

The objective 22 may have a screw thread onto which the tube lens 23, provided with a complementary screw thread, is screwed. The component 20 including the light source and the spectral filter 21 are then housed in a cavity formed between the objective 22 and the tube lens 23. Towards the top, the tube lens 23 has a second screw thread, allowing it to be screwed onto a corresponding screw thread of the reading device 24. The reading device 24 is thus in the form of a housing integrating the sensor 240 and provided with the screw thread onto which the tube lens is screwed. The spectral filter 21 and the component 20 integrating the light source are for example embedded between the objective 22 and the tube lens 23. Along the optical axis, a passage is of course created to allow the signals to pass through the components of the microscope 2.

• La source lumineuse émet les rayonnements d'excitation EXC_2 dans toutes les directions, préférentiellement en direction de l'objet O, à travers l'objectif 22. L'objet O absorbe au moins en partie ces rayonnements et, par fluorescence, émet des rayonnements de détection REF_2 dans toutes les directions, notamment en direction du dispositif de lecture 24 suivant l'axe optique (X) ;
• Les rayonnements d'émission REF_2 traversent, suivant l'axe optique (X), par transparence, le composant 20 incluant la source lumineuse ;
• Les rayonnements d'émission REF_2 traversent le filtre spectral 21 et la lentille de tube 23 pour atteindre la surface du capteur 240 du dispositif de lecture 24 ;
• Une image se forme sur la surface du capteur 240 ;
• Des moyens de traitement (non représentés) peuvent être utilisés pour traiter chaque image acquise ;

In operation, in the case of fluorescence detection, the operating principle is as follows:

• The light source emits the excitation radiation EXC_2 in all directions, preferably in the direction of the object O, through the objective 22. The object O absorbs at least part of this radiation and, by fluorescence, emits detection radiation REF_2 in all directions, in particular in the direction of the reading device 24 along the optical axis (X);
• The REF_2 emission rays pass through, along the optical axis (X), by transparency, the component 20 including the light source;
• The emission radiation REF_2 passes through the spectral filter 21 and the tube lens 23 to reach the surface of the sensor 240 of the reading device 24;
• An image is formed on the surface of the sensor 240;
• Processing means (not shown) may be used to process each acquired image;

If an eyepiece is used instead of the 240 sensor, a naked eye reading/detection would be conducted.

• Ce montage en réflexion peut être utilisé pour faire de l’imagerie par contraste d’absorption sur des échantillons opaques et/ou réfléchissants. Dans cette application, les deux filtres spectraux (en utilisant une OLED sans filtre intégré) doivent être retiré.
• Ce montage en réflexion peut également être utilisé pour faire de l’imagerie par contraste de polarisation sur des échantillons opaques (pas de possibilité d’éclairer de l’autre côté de l’objet) et/ou réfléchissants. Dans cette application, les deux filtres spectraux (en utilisant une OLED sans filtre intégré) doivent être retiré et des éléments optiques permettant de contrôler/moduler la polarisation sont alors ajoutés (pas de possibilité d’éclairer de l’autre côté de l’échantillon).

The microscope 2 described above and its reflection architecture is particularly suitable for fluorescence detection. But, it should be noted that:

• This reflection setup can be used to perform absorption contrast imaging on opaque and/or reflective samples. In this application, both spectral filters (using an OLED without an integrated filter) must be removed.
• This reflection setup can also be used to perform polarization contrast imaging on opaque (no possibility to illuminate the other side of the object) and/or reflective samples. In this application, the two spectral filters (using an OLED without an integrated filter) must be removed and optical elements to control/modulate the polarization are then added (no possibility to illuminate the other side of the sample).

As indicated above, the component 20 can be inserted in different positions along the optical axis, position P1, P2 or P3 ( ).

• Une architecture particulièrement compacte et facilement transportable, réalisée sous la forme d'un unique ensemble monobloc et suivant un unique axe optique, ne nécessitant qu'un seul bras de support.
• Une architecture qui utilise un principe d'imagerie en réflexion à l'aide d'un composant de type OLED transparent.
• Une architecture qui utilise des composants déjà existants et facilement disponibles (objectif, lentille de tube, capteur CMOS par exemple).

The solution of the invention thus presents numerous advantages, among which:

• A particularly compact and easily transportable architecture, produced in the form of a single monobloc assembly and following a single optical axis, requiring only a single support arm.
• An architecture that uses a reflective imaging principle using a transparent OLED type component.
• An architecture that uses already existing and readily available components (lens, tube lens, CMOS sensor for example).

Claims (10)

Reflecting microscope (2) used to observe/detect an object (O), said microscope comprising several components:

• A light source suitable for emitting light rays (EXC_2), called excitation rays, to the object (O) to illuminate it,
• A device (24) for reading radiation, called emission (REF_2), coming from the object (O), generated after illumination,
• An objective (22) and a tube lens (23) associated with said objective for forming an image on said reading device (24),

Characterized in that:

• The device comprises at least one component (20) including said light source and having a zone transparent to emission radiation (REF_2),
• The reading device (24), the objective (22), the tube lens (23) and said component (20) including the light source are positioned along a single optical axis (X).

Reflection microscope according to claim 1, characterized in that the component (20) including the light source is positioned (P1) between the objective (22) and the tube lens (23). Reflection microscope according to claim 1, characterized in that the component (20) including the light source is positioned (P2) between the objective (22) and the object (O) to be detected. Reflection microscope according to claim 1, characterized in that the component (20) including the light source is positioned (P3) between the tube lens (23) and the reading device (24). Reflection microscope according to one of claims 1 to 4, characterized in that it comprises a first spectral filter (21) positioned between the light source and the reading device (24). Reflection microscope according to claim 5, characterized in that the component (20) including the light source comprises a layer (C6) forming a second spectral filter, completing said first spectral filter (23). Reflection microscope according to one of claims 1 to 6, characterized in that the component (20) including the light source comprises an organic light-emitting diode. Reflection microscope according to one of claims 1 to 7, characterized in that the reading device (24) comprises a CMOS or CCD type sensor (240). Reflection microscope according to one of claims 1 to 8, characterized in that the components of the microscope (2) are combined in a single single-block assembly in which the components are integral with each other. Reflection microscope according to claim 9, characterized in that it comprises a single arm for holding said assembly.