Apparatus for observing a living cell or a set of living cells

Abstract

The present invention relates to an imaging device (100) for observing the development of a living cell or set of living cells (80), such as an embryo, comprising an illumination system (110), means for relative movement, and an imaging system (120) equipped with a wide-field camera (121) adapted to allow identification, imaging, and observation of one or more living cells or sets of living cells (80) to be observed. The present invention also relates to a method for observing embryo development by means of such a device (100).

Description

[Technical field]

[0001] The present invention relates to an imaging device for observing a living cell or a set of living cells in the context of reproduction research, in particular for in vitro fertilization (IVF).

[0002] More specifically, the present invention relates to an imaging device for observing a living cell or a set of living cells, an associated Petri dish, as well as a method and a computer program product suitable for this observation. [Background technology]

[0003] Current imaging devices for observing a living cell or set of living cells, such as an embryo, are most often microscopes equipped with lenses of different magnifications, monocular or binocular direct observation systems, and offset observation systems, typically for viewing the living cell or set of living cells on a video screen.

[0004] A living cell or a set of living cells is placed on a support and observed using different lenses to enlarge or reduce the size of the image obtained, thus observing the living cell or set of living cells according to different magnifications, i.e. in detail or globally.

[0005] These devices are not very precise in terms of handling and are not easy to use: one must start by viewing a live cell or set of live cells at a low magnification, locating it, manually moving it to center it in the microscope field, and then changing to a lens using a higher magnification to view the live cell or set of live cells in detail.

[0006] In the specific example of observing embryo development and performing in vitro fertilization (IVF), the embryo to be transferred is selected according to its cell quality, i.e. its cell number, its regularity (different sized cells or not) and its fragmentation. The selected embryos are, as a priority, those in which the time series of cell division is observed, with regular cells and no fragmentation. These parameters are indicators of a better chance of conception.

[0007] This observation is generally carried out outside an embryo culture enclosure adapted to reproduce the ideal environment in temperature and gas concentration for good embryo development. Document US2015 / 0278625A1 describes such an observation mode outside an incubator. However, it has been demonstrated that observations outside the chamber, and especially sudden changes in the environment of the embryo, play a potentially detrimental role in its development.

[0008] Embryo imaging devices have therefore been developed in the context of in vitro fertilization (IVF) techniques to allow the in vitro observation of the development of the embryo after fertilization in this type of environment ideal for its development.

[0009] This observation, which may continue for 2-5 days depending on the IVF center, helps to improve the success rate of IVF, as it allows a better selection of embryos to be transferred to the patient's reproductive tract.

[0010] There are two existing categories of observation devices: either they are directly integrated into a system adapted to recreate the ideal environment for successful embryo development and contain individual culture chambers that receive specific dishes for each device, or the observation device is placed into an existing enclosure with separate, dedicated observation dishes.

[0011] All these peculiarities make current equipment and its use very costly, limiting access for budget-constrained medical facilities or requiring increased treatment and / or costs for IVF recipients.

[0012] In any case, whether in the case of a living cell or a set of living cells or in the specific case of an embryo, the optical quality of the image, the precise identification of the living cell or set of living cells and the regular temporal tracking of its development are important parameters for the use of these methods. However, on the one hand, current systems do not allow to identify the exact location of the living cell or set of living cells, the latter being confined to a reduced space that limits the observation field in the three axes x, y and z. In the case of the embryo, its natural movement may cause it to go out of the observable zone without the option to reposition it or to appear at the edge of the field where optical aberrations are large, while the observation at a single incidence has so far made it possible to create algorithms that can assist in the interpretation.

[0013] Another important parameter is the observation time of a living cell or set of living cells forming a sample, which affects the efficiency of the analysis carried out on the sample. Current devices generally have means for mechanical movement along three axes, usually called x, y (defining the horizontal plane containing the sample to be observed) and z (perpendicular to the previous plane), in the direction of the depth of the sample in order to optimally position the sample. These mechanical movement means imply potentially time-consuming movements. A prior art solution to reduce the depth movement time is to use a liquid lens, the focal length of which can be electronically varied, as in patent applications in the dental field US2014 / 0002626A1 for an autofocus intrabuccal camera and EP2161607A1 related to a dental camera. Summary of the Invention [Problem to be solved by the invention]

[0014] There is therefore a real need for an imaging device that overcomes the deficiencies, shortcomings and obstacles of the prior art, and in particular for a device and method that allows improving the optical quality and time efficiency of the observation of a living cell or a set of living cells, while reducing the costs of manufacturing and using the device. In the particular case of IVF, better accessibility through reduced costs is required for the people involved.

[0015] In order to address one or more of the above shortcomings, the Applicant specifically provides: 1. An imaging device for observing the development of a living cell or a set of living cells (80) in the context of reproductive research, in particular for in vitro fertilization (IVF), comprising: an imaging system comprising a wide-field camera adapted for the identification and observation of one or more live cells or sets of live cells to be observed, the live cells or sets of live cells being placed in a Petri dish containing a specific compartment for each live cell or set of live cells, a support capable of receiving a Petri dish and positioned between the illumination system and the imaging system; - an illumination system with three light sources adapted to illuminate an object, which is a living cell or a set of living cells to be observed, comprising a central source, a first lateral source arranged on a first side of the central source, and a second lateral source arranged on a second side of the central source, The lighting system includes the following types of lighting: - "detection" type illumination, in which the light source (111) is configured for detecting a living cell or a set of living cells observed by an imaging system, said "detection" type illumination being generated using a central source, a first lateral source and a second lateral source, the light rays from the light source being focused on the object according to a cone with an angular aperture between 26° and 34°, - "contour" type illumination, the light source (111) being configured for counting cells present in a live cell or a set of live cells observed by an imaging system, the "contour" type illumination being generated using a first lateral source and a second lateral source, the light rays from the first lateral source and the second lateral source being separated into two symmetric collimated beams with respect to a vertical axis with respect to a plane formed by a Petri dish support or to the Petri dish itself, the first beam from the first lateral source illuminating the object according to an angle of incidence between 10° and 14° with respect to the vertical axis, and the second beam from the second lateral source illuminating the object according to an angle of incidence between 10° and 14° with respect to the vertical axis, an illumination system configured to implement each of the "relief" type illuminations, the light source (111) being configured for visualizing cellular texture and granularity present in a live cell or set of live cells observed by an imaging system, the "relief" type illumination being generated using a first lateral source, a single collimated light beam from the first lateral source propagating along an axis inclined at an angle between 8° and 16° with respect to a vertical axis; characterised in that it comprises means for the relative movement of a Petri dish with respect to an assembly formed by an illumination system and said wide-field camera so as to be able to observe living cells or sets of living cells located in different compartments of said Petri dish, The present invention proposes an imaging device, in which the wide-field camera has a field of view covering at least the entire surface area of ​​one of the compartments of a Petri dish, and the imaging device is adapted to image a live cell or a set of live cells at any position in the compartment in a Petri dish without relative movement of the Petri dish using a movement means with respect to the assembly formed by the illumination system and the wide-field camera, and the wide-field camera has a resolution adapted to observe details of the live cell or set of live cells, the details having a micrometer size.

[0016] The particular lighting system of the device according to the invention allows it to be compact and multifunctional, without requiring the removal of any parts to switch from one type of lighting to another. Handling movements are reduced to those strictly necessary, providing ideal observation conditions by facilitating its use directly in the incubator, for long periods of time and without risk of destruction of the embryos.

[0017] "Contour" type illumination allows easy counting of the number of cells in a single view, including when the cells are stacked.

[0018] The "relief" type of illumination provides information about the texture of the cell envelope. This illumination is present in two opposite directions (above / below) providing additional information that can be useful to understand the shape of the embryo, thus forming a third combination.

[0019] The field of view of the wide-field camera, covering at least the entire surface area of ​​one of the compartments of the Petri dish, thus makes it possible to directly image a live cell or set of live cells positioned at any position within the compartment of the Petri dish, thus without any means of movement.

[0020] The "wide field" camera according to the invention is particularly 2 This refers to a camera with a field of view greater than 1000 nm and a pixel resolution of less than 1 μm.

[0021] Preferably, the living cell or set of living cells observed is one or more embryos.

[0022] Preferably, in the imaging device according to the invention, the illumination system comprises one or more of the following specific types of illumination: - "detection" type illumination, the light source being configured for detecting a living cell or a set of living cells observed by an imaging system; - a "contour" type illumination, the light source being configured for counting cells present in a live cell or set of live cells observed by an imaging system; - "Texture" type illumination, the light source may be selected from at least one of those configured for visualizing the texture and granularity of cells present in a live cell or set of live cells observed by the imaging system.

[0023] In an embodiment of the device according to the invention, the light sources of the illumination system can be arranged in a plane parallel to a horizontal plane defined by a support capable of receiving a Petri dish, and a device for shaping the beam emitted by the at least one light source is arranged between the light source and the Petri dish.

[0024] According to an alternative embodiment, the moving means are capable of moving the Petri dish so that the live cell or set of live cells to be observed is placed relative to an assembly formed by the illumination system and the wide-field camera of the imaging system.

[0025] According to another alternative embodiment, a moving means is capable of moving an assembly formed by the illumination system and the wide-field camera of the imaging system relative to a Petri dish in which the live cell or set of live cells to be observed is placed.

[0026] Advantageously, the imaging system is adapted to count the number of cells present in the observed live cell or set of live cells.

[0027] Advantageously, the system is also adapted to visualise the texture and granularity of cells present in the observed live cell or set of live cells.

[0028] In one embodiment, the imaging device may further comprise a liquid lens adapted to control the focal length of the live cell or set of live cells along its depth.

[0029] The present application also relates to a Petri dish for observing the development of a living cell or a set of living cells by means of an imaging device according to the invention, said Petri dish comprising: - a receptor adapted to receive one or more living cells or sets of living cells to be observed -lid - an identification element adapted to identify the observed live cell or set of live cells - a specific compartment or well intended to place a living cell or set of living cells, said compartment or well being produced in the form of a cup adapted to receive a drop of culture medium in addition to the living cell or set of living cells in the cup.

[0030] The present application also relates to a method for observing the development of a living cell or a set of living cells by means of an imaging device according to the invention, comprising the following steps: a preparation step consisting of successively placing a live cell or a set of live cells in a Petri dish, which can be chosen from at least one Petri dish defined above and arranged on the support of the imaging device, followed by the dropping of a medium in said Petri dish, the operation being repeated the required number of times according to the desired number of live cells or sets of live cells to be observed, and then covering the whole with a liquid such as oil or water; - a step of identifying the Petri dish, using a dedicated reader to detect and visually identify an identifying element of the Petri dish containing the live cell or set of live cells to be observed; - observing a first live cell or set of live cells using an imaging system.

[0031] Advantageously, the step of identifying the Petri dish is preceded by an optional step of approximate positioning of the Petri dish relative to the assembly formed by the illumination system and the wide field camera of the imaging system using the movement means, so that the Petri dish is roughly aligned with the illumination system and the wide field camera of the imaging system.

[0032] Advantageously, the method for observing the development of a living cell or a set of living cells may, after the step of observing a first living cell or a first set of living cells, comprise a step of relative movement of the Petri dish with respect to an assembly formed by the illumination system and the wide-field camera of the imaging system in order to observe another living cell or another set of living cells located in another compartment of the Petri dish.

[0033] In an embodiment in which the imaging device according to the present invention further comprises a liquid lens, in a method for observing the development of a live cell or a set of live cells, the step of observing a first live cell or a set of live cells can be performed in a horizontal observation plane parallel to a support capable of receiving a Petri dish and perpendicular to the depth direction of the first live cell or set of live cells, which plane is determined in an additional determination step by the configuration of the liquid lens.

[0034] Advantageously, in the same embodiment in which the imaging device according to the present invention further comprises a liquid lens, the step of determining an observation plane of the first living cell or set of living cells is repeated for different planes perpendicular to the depth direction of the first living cell or set of living cells by different configurations of the liquid lens, so as to observe the first living cell or set of first living cells in the determined different observation planes.

[0035] Therefore, observation of the first living cell or set of living cells in different planes perpendicular to the depth direction, made possible by the presence of a liquid lens in the imaging device according to the present invention, makes it possible to eliminate movement of the first living cell or set of living cells in the depth direction, i.e., to save time in the procedure for analyzing and observing the living cell or set of living cells.

[0036] The present application also relates to a computer program product downloadable from a communications network and / or recorded on a computer readable medium and / or executable by a processor, characterized in that it comprises program code instructions for implementing the method for observing and developing a living cell or a set of living cells according to the invention.

[0037] Further advantages and specificities of the invention will emerge from the following description, given by way of non-limiting example with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 shows a first embodiment of an imaging device according to the invention. [Diagram 2] FIG. 2 represents a first embodiment of the "detection" type of illumination. [Diagram 3] FIG. 3 represents a first embodiment of a “contour” type illumination. [Figure 4] FIG. 4 represents a first embodiment of a “texture” type of lighting. [Diagram 5] FIG. 5 illustrates a second embodiment of an imaging device according to the invention. [Figure 6] FIG. 6 illustrates an imaging device according to another embodiment of the present invention disposed within an incubator. [Figure 7a] FIG. 7a shows a longitudinal section of an imaging device disposed within the incubator of FIG. [Figure 7b] FIG. 7b shows a top view of the imaging device disposed within the incubator of FIG. [Figure 8a] FIG. 8a depicts several two-dimensional views (top, side, cross section) of examples of Petri dishes that can be used in the present invention. [Figure 8b] FIG. 8b depicts a 3D view of the example Petri dish of FIG. 8a. [Figure 9a] FIG. 9a shows a longitudinal section of an embodiment of a Petri dish according to the invention, placed on a support. [Figure 9b] FIG. 9b shows a top view of the Petri dish of FIG. 9a placed on a support. [Figure 10] FIG. 10 depicts an embryo reservoir residing in the bottom of a Petri dish according to an embodiment of the present invention. [Figure 11a] FIG. 11a shows a longitudinal section of another embodiment of a Petri dish according to the invention. [Figure 11b] FIG. 11b shows a top view of another embodiment of a Petri dish according to the invention. [Figure 11c] FIG. 11c shows another embodiment of a Petri dish according to the invention in longitudinal section through the Petri dish. [Figure 12] FIG. 12 depicts an overview of a method for observing the development of a living cell or a set of living cells according to a first embodiment of the invention. [Figure 13] FIG. 13 shows a full-field image obtained by a first embodiment of an observation method according to the invention, using a wide-field camera of an imaging system of an imaging device according to the invention. [Figure 14] FIG. 14 depicts an overview of a method for observing the development of a living cell or a set of living cells according to a second embodiment of the invention. [Figure 15] FIG. 15 depicts an overview of a method for observing the development of a living cell or a set of living cells according to a third embodiment of the invention. [Figure 16] FIG. 16 shows two images of an embryo obtained using an imaging device and a first embodiment of the observation method according to the present invention, with two different types of illumination, respectively a "contour" type illumination on the left and a "texture" type illumination on the right. [Figure 17a] FIG. 17a represents a series of cross sections at that depth of the embryo obtained with the device according to the invention and with a third embodiment of the observation method according to the invention, using "contour" type illumination. [Figure 17b] FIG. 17b represents a series of cross sections at that depth of the embryo obtained with the device according to the invention and with a third embodiment of the observation method according to the invention, using "texture" type illumination. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] 16-17b are commented on in more detail in the following examples which illustrate the invention without limiting its scope. Other figures illustrating different embodiments of the device, the Petri dish and the observation method according to the invention and used or implemented in the examples are detailed below.

[0040] Imaging device according to the present invention An imaging device 100 according to a first embodiment of the invention as shown in FIG. 1 is embodied from the following elements: - Lighting system 110: LED (light emitting diode) type lighting an imaging system 120 comprising a wide-field camera 121 equipped with an image sensor having a resolution of the order of 5 pixels per micrometer observed; -Support 1: Glass plate - Means of relative movement such as motorized plates

[0041] These elements are shown as examples and the device according to the invention can be implemented using elements having different parameters.

[0042] The embodied imaging device 100 is intended to observe a living cell or set of living cells 80 contained in a Petri dish 10 arranged on a support 1. The term "observe" means to detect and distinguish structural details.

[0043] In the embodiment shown in Fig. 1, the device 100 is embodied in an "inverted microscope" configuration, i.e. the cell or set of live cells 80 to be observed is illuminated from above and the imaging system 120 is located below and observes the light transmitted by the live cell or set of live cells 80, which is a semi-transparent object. This configuration will be retained in the remainder of the description, but the device 100 can also be embodied in a "normal" configuration (cell or set of live cells 80 illuminated from below and observed from above, as in a standard microscope) as in the "inverted" configuration.

[0044] Image capture by the wide-field camera 121 is performed in transmission through the cell or set of living cells 80, i.e. by the so-called glass dip technique, which means that the illumination is located on one side of the object to be imaged and the camera is located on the other side of the object.

[0045]

[37] Thus, the Petri dish 10 containing the live cell or set of live cells 80 to be observed must be transparent to the light used to illuminate the live cell or set of live cells 80. As in the embodiment depicted in FIG. 1, the Petri dish 10 is placed on a support 1. This support 1 must also be transparent to the light used to illuminate the live cell or set of live cells 80, such as a glass plate for a conventional microscope, a light bulb whose light is emitted in the visible range. In the device shown here, this support 1 is a glass plate, but it can also be a sliding drawer in which one or more Petri dishes 10 can be arranged.

[0046] Advantageously, this support 1 can be provided with a contact block or rim for prepositioning the Petri dish 10 in a predefined position. In this case, thanks to the wide field of view of the wide-field camera, no preliminary lateral setting is necessary to observe the living cell or set of living cells 80, i.e. in a plane parallel to the plane defined by the support 1, but the wide-field camera 121 covering at least the entire compartment 61, also called well or cup, of the Petri dish 10, makes it possible to directly detect the living cell or set of living cells 80, regardless of their position within the compartment.

[0047] Advantageously, the illumination system 110 and the wide field camera 121 are rigidly connected and mounted in a "U" shaped configuration, with the illumination system 110 facing the wide field camera 121, as in FIG.

[0048] Advantageously, the lighting system 110 provides the following specific types of lighting: - a "detection" type of illumination, in which at least one light source 111 is configured for detecting a live cell or a set of live cells 80 observed by an imaging system 120; - a "contour" type illumination, in which at least one light source 111 is configured for counting cells present in a live cell or set of live cells 80 observed by an imaging system 120; - In "relief" type illumination, at least one light source 111 is selected from at least one of those configured to display the texture and granularity of cells present in the live cell or set of live cells 80 observed by the imaging system 120.

[0049] The "detection" type illumination is preferably used for detection and visualization by the imaging system 120, for imaging and analyzing the identification elements 50 present in the Petri dish 10, as well as for identifying the observed live cell or set of live cells 80. Therefore, the selection of at least one light source 111 and the wide-field camera 121 must be set. For example, the sensitivity of the wide-field camera 121 must be optimized according to the wavelengths emitted by the light source 111.

[0050] This “detection” type illumination can also be used to aid in the detection of a live cell or set of live cells 80 by the wide field camera 121 and imaging system 120 .

[0051] Fig. 2 represents an embodiment of illumination of the "detection" type. In this Fig. 2, the object to be identified or detected using the wide-field camera 121 and the imaging system 120, which can be an identification element 50 of a Petri dish or, as in Fig. 2, a live cell or set of live cells 80, is illuminated with uniform or diffuse type illumination. The light rays 112 from the light source 110 are concentrated or focused on the object to be identified or detected in a cone with an angular aperture α_GC of the order of 30°. The observed object (identification element 50 or a live cell or set of live cells 80) is thus illuminated according to an angle of incidence varying from -15° to +15° with respect to the normal axis P relative to the plane formed by the support 1 or to the Petri dish 10 itself.

[0052] The cone of angular aperture α_GC is of the order of 30°±4°. The incidence angle varies from -15°±2° to +15°±2°.

[0053] Fig. 3 represents an embodiment of a "contour" type of illumination. In this Fig. 3, a live cell or set of live cells 80 observed by a wide-field camera 121 can be illuminated along an axis that is preferably symmetrical with respect to a vertical axis P relative to the plane formed by the support 1 of the Petri dish or to the Petri dish 10 itself. Thus, a light beam from at least one light source 111 is split into two collimated beams 113 and 114. The first beam 113 illuminates the embryo 80 according to an angle of incidence β_PC1 with respect to the vertical axis P, and the second beam 114 illuminates the embryo 80 according to an angle of incidence β_PC2. As mentioned above, the angles β_PC1 and β_PC2 are preferably equal and opposite (i.e. β_PC1=-β_PC2). That is to say, the beams 113 and 114 are preferably symmetrical with respect to the vertical axis P. Typically, the angles β_PC1 and β_PC2 are of the order of + / -12°±2°. The total angle between the two directions formed by beams 113 and 114 is therefore typically of the order of 24°±4°. The high resolution of the wide-field camera 121 (for observing details of the living cell or set of living cells 80, said details having a micrometer size) cooperates with this "contour" type of illumination.

[0054] “Texture” type illumination is preferably used to optimize the observation by the imaging system 120 of the texture and granularity of the cells present in the observed live cell or set of live cells 80, to assess their relief and their depth.

[0055] Fig. 4 represents an embodiment of a "texture" type illumination. In this Fig. 4, a live cell or set of live cells 80, as well as its texture and granularity, observed by a wide-field camera 121, can be illuminated by a single light beam 115. This beam 115 is collimated and propagates along an axis inclined by an angle γ_PC with respect to the vertical axis P. The absolute value of this angle γ_PC typically varies between 8° and 16°. With respect to the vertical axis P or the live cell or set of live cells 80, the incidence direction is not favorable.

[0056] To facilitate these different illumination embodiments, the illumination system 110 can consist of one or more light sources 111 of the LED type. Each of these light sources 111 can be controlled individually. It is noted that LEDs have the advantage of being very compact, so that it is possible to replicate the illumination by a set of LEDs, such that the LEDs illuminate a living cell or set of living cells 80.

[0057] In the particular case where the set of living cells observed is an embryo, the wavelength of the at least one light source 111 (LED or other) used may preferably be located in the red range (about 630 nm), which is the range that is least harmful to the embryo 80. Furthermore, the light source 111 is preferably controlled in a pulsed mode so as to limit the cumulative exposure time of the embryo 80 to light to a few tens of seconds per day, so as not to cause damage.

[0058] Figure 5 shows an embodiment of the invention, where an illumination system 110 comprises several light sources 111a, 111b, 111c, in this example three, namely a central source 111b, a first lateral source 111a arranged on a first side of the central source 111b, and a second lateral source 111c arranged on a second side of the central source 111b. Plane A in Figure 5 corresponds to the orthogonal plane P in Figures 2-4. Plane A passes through the central source 111b.

[0059] In order to collimate the light emitted by the light source 111 and to emit uniform light or to emit diffuse light, the illumination system 110 may further comprise an optical system 140 for shaping the light beam emitted by the light source 111, such as a lens or a lens array, a filter, etc.

[0060] To adjust the illuminated zone, this optical shaping system 140 may also comprise a mask, such as a plate with holes, which can be moved to allow light from the light source 111 to pass through and illuminate the latter the live cell or set of live cells 80 to be observed. This may be especially true if the at least one light source 111 is extended or if the illumination system 110 comprises several light sources 111, which can optionally be controlled separately. Moreover, the use of such a mask also makes it possible to filter out parasitic light that may interfere with the observation of the live cell or set of live cells 80.

[0061] Thus, in the context of FIG. 5 , to generate a “texture” type of illumination and illuminate a live cell or set of live cells 80 observed laterally, the light source 111b located above the embryo 80 can be switched off and one of the two light sources 111a or 111c located to the side of the central light source 111b can be switched on, thus projecting oblique light onto the live cell or set of live cells 80.

[0062] 1 and 5, the imaging system 120 is located below the Petri dish 10 and thus below the live cell or set of live cells 80 to be observed (principle of an inverted microscope). It comprises a wide-field camera 121 adapted to identify the live cell or set of live cells 80 to be observed, which live cell or set of live cells 80 is placed in the adapted Petri dish 10.

[0063] For this purpose, the wide field camera 121 can be set to image the identification element 50 present on the Petri dish 10, observing the identification of the live cell or set of live cells 80 thus observed. For this purpose, the imaging system 120 can further comprise a processing unit for analysing the images captured by the wide field camera 121 to detect the position of the live cell or set of live cells 80 using the position of the identification element 50 and the characteristics of the Petri dish 10.

[0064] If the live cell or set of live cells 80 are one or more embryos, this processing unit also makes it possible, using the moving means 160, to analyze the image captured by the wide field camera 121 when it is positioned so as to image the identification elements 50 of the Petri dish 10 and to find, in the previously completed database, information regarding the contents of the Petri dish 10, i.e. the embryos 80, e.g. the couple to which the embryos 80 belong, the number of embryos 80 present in the dish, the dates of preparation and insertion of the embryos 80, etc.

[0065] Thanks to its high resolution, the wide-field camera 121 is adapted to image the illuminated living cell or set of living cells 80 and to observe its development. The wide-field camera 121 is typically equipped with an image sensor with a resolution of the order of 5 pixels per μm observed, providing a quantity of pixels capable of covering the entire surface area to be observed, supplemented by a surface area that ensures a safety margin in terms of estimating positioning uncertainties.

[0066] Furthermore, in order to be able to separate the different cell layers present in the living cell or set of living cells 80, the wide-field camera 121 must have a relatively small depth of field, of the order of 100 μm.

[0067] In order to view the different cell layers and thus observe the living cell or set of living cells 80 in its depth direction, the imaging device 100 may further comprise a liquid lens adapted to control the focal length of the living cell or set of living cells 80 in its depth direction, i.e. along an axis Z perpendicular to the plane defined by the Petri dish 10 or by the support 1. For example, a liquid lens 170 configured by electrowetting actuation, using an electroactive polymer membrane or configured by a piezoelectric actuator may be used. In the case of a liquid lens 170 configured by electrowetting actuation, a voltage is used to modify the shape of the interface separating the two different liquids, thus modifying the focal length of the liquid lens 170. In the case of a liquid lens 170 using an electroactive polymer membrane, the focal length of the liquid lens 170 is also modified by applying a voltage between the electrode and the substrate, thus modifying the curvature of the liquid lens 170. In the case of a liquid lens 170 configured by a piezoelectric actuator, the piezoelectric actuator has the effect of swelling the membrane filled with liquid. Thus, a plane corresponding to the cell layer to be observed is configured by setting the liquid lens. The advantage is therefore that it is possible to eliminate the need for a means for moving along the z-axis, the actuation of which is time consuming.

[0068] Thanks to the processing unit, the imaging system 120 can be used to count the number of cells present in the observed live cell or set of live cells 80. To this end, the processing unit of the imaging system 120 can analyze the images captured by the wide-field camera 121, and through imaging processing operations such as contour detection, it becomes possible to count the number of cells present in the observed live cell or set of live cells 80.

[0069] Counting, and especially image processing operations, are optimal when the observed live cell or set of live cells 80 is illuminated using a "contour" type of illumination.

[0070] The imaging system 120 can also be used to observe the texture and granularity of cells present in the observed embryo 80. To this end, the processing unit of the imaging system 120 analyses the images captured by the wide-field camera 121 and processes them with image processing operations that optimize the visual rendering of the texture and grain of cells present in the observed live cell or set of live cells 80.

[0071] 5, the imaging system 120, and in particular the wide field camera 121, can be advantageously offset using an optical return system 150 to make the apparatus 100 more compact. This optical return system 150 can consist of a lens 151 and a return mirror 152, for example, positioned at 45° to the optical axis A of the imaging apparatus 100, as shown by the dotted line in FIG.

[0072] 6, when the live cell or set of live cells 80 is an embryo or several embryos, the dimensions of the imaging device 100 are preferably compatible with incubators 200 found in IVF centers, so that it can be placed inside such an incubator 200. Thus, the height and depth of the device 100 may be of the order of 30 cm, and the width of the device 100 may be of the order of approximately 30 cm to 55 cm.

[0073] The imaging device 100 of FIG. 6 includes a sliding drawer onto which one or more Petri dishes 10 can be placed as described above.

[0074] Figures 7a and 7b respectively represent a longitudinal section and a top view of the imaging device 100 of Figure 6 present in an incubator 200. In this case, the support 1 covering the full width of the imaging device 100 is an assembly formed by at least an illumination system 110 and a wide field camera 121 fixed to an "elongated U" shaped structure moved by relative movement means 160 in a plane (X,Y) parallel to the plane defined by the support 1, allowing to view either another embryo 80 from the same Petri dish 10 or an embryo 80 in another Petri dish 10.

[0075] Petri dish according to the invention Secondly, a Petri dish 10 adapted for observing the development of a living cell or set of living cells 80 by means of an imaging device 100 as defined above is detailed below.

[0076] According to a first embodiment, such a Petri dish 10 comprises: a receptacle 20 adapted to receive one or more embryos 80 to be observed; A lid 30 may be provided. Figure 8a shows a two-dimensional top view and a longitudinal section as well as plane AA of an example of a Petri dish that can be used within the scope of the present invention. An example of a receptacle 20 and an example of a corresponding lid 30 are observable therein. Figure 8b shows a relief representation of the receptacle 20 and the lid 30 of Figure 8a.

[0077] A schematic diagram of this first embodiment of a Petri dish 10 is shown in FIG. 9a.

[0078] To be able to see the live cell or set of live cells 80 through the Petri dish 10, the Petri dish 10 may be made of a material transparent to the light emitted by the illumination system 110 as described above. This light is generally in the visible range (i.e. about 400 nm to 800 nm). The material used may therefore be, for example, a plastic material, glass.

[0079] Referring to FIG. 9 b , the bottom of the receptacle 20 is provided with an identification element 50 adapted to identify the observed live cell or set or live cells 80 .

[0080] According to the embodiment shown in FIG. 9b, the Petri dish 10 is rectangular in shape.

[0081] The identification element 50 can for example be a barcode or a 2D code of Data Matrix type as shown in Fig. 9b. This identification element 50 makes it possible in particular to know information about the contents of the Petri dish 10, i.e. about the living cell or set of living cells 80 contained therein. If the living cell or set of living cells 80 is one or more embryos, the information about the contents of the Petri dish 10 can be, via a previously completed database, for example the couple to which the embryo 80 belongs, and also the number of embryos 80 present, the date of preparation or insertion of the embryos 80, etc.

[0082] According to this embodiment, the living cells or sets of living cells 80 are linearly distributed over one or more rows, as in the case of the rectangular Petri dish 10 shown in FIG. 9b.

[0083] According to a particular embodiment of a Petri dish 10 shown in Fig. 10, the bottom of the receptacle 20 advantageously comprises at least one embryo reservoir 60, which comprises in its centre a cup 61, compartment or well adapted to receive a living cell or set of living cells 80, which reservoir 60 is also adapted to receive a drop of medium 62 (typically of the order of 6 μl) in addition to the living cell or set of living cells 80 in the cup 61. The diameter of the cup is typically of the order of 1 mm and its volume can be of the order of 0.75 μl. The zone in which the medium 62 is located is delimited by a protuberance of the bottom or bulge 64 of the receptacle 20.

[0084] Next, the assembly formed by the dropping of at least the living cell or set of living cells 80, medium 62, can then be covered with a liquid 63, such as oil or water. For this, the edge of the receptacle 20 must be sufficiently high, typically of the order of 5 to 10 mm.

[0085] According to another embodiment shown in Figures 11a to 11c and similar to that shown in Figures 9a and 9b, the Petri dish 10 can be firmly connected to a transparent plate 11 with gripping lugs 12 that allow easy handling of the Petri dish 10. This prevents the risk of soiling the lid 30 or the bottom of the receptacle 20, which would hinder the observation of the embryos 80 and the quality of the images captured by the device 100. Advantageously, the gripping lugs 12 are provided with a tagging area 13, on which it is possible to affix the identification element 50, rather than on the bottom of the receptacle 20.

[0086] Finally, the edge of the receptacle 20 is sufficiently high, typically on the order of 5-10 mm, to allow the placement of a volume of liquid 63, such as oil or water, covering the assembly formed by the drop of culture medium 62 and the living cell or set of living cells 80.

[0087] According to an embodiment, the identification element 50 can be created directly in the material by machining the bottom of the receptacle 20 on its inner or outer surface, or by applying a label and / or self-adhesive marker to the outer bottom of the receptacle 20.

[0088] Observation method according to the present invention Thirdly, a method for observing the development of a living cell or a set of living cells 80 by means of the imaging device 100 defined above is detailed below.

[0089] According to a first general embodiment of the method shown in FIG. 12, the method comprises three main steps: a preparatory step S300; an identification step S302; - an observation step S304.

[0090] The preparation step S300 involves successively placing live cells or sets of live cells 80 in a Petri dish 10, which can be selected from at least one Petri dish defined above and arranged on the support of the device, followed by dripping of culture medium 62 (or vice versa, dripping of culture medium 62 followed by live cells or sets of live cells 80), then repeating the operation the required number of times according to the desired number of live cells or sets of live cells 80 to be observed, and finally covering the whole with a liquid 63, such as oil or water.

[0091] A live cell or set of live cells 80 may be placed in the cup 61 and then covered with a drop of medium 62 in the live cell or set of live cells reservoir 60 .

[0092] Advantageously, and particularly where the living cell or set of living cells 80 is an embryo or multiple embryos, following the operation of preparing or placing the embryo 80, the database can be filled by the preparation technician with characteristics of the Petri dish 10, in particular with data relating to the embryo 80 placed in the Petri dish 10, such as, for example, the couple to which the embryo 80 belongs, the number of embryos 80 present in the dish, the dates of preparation and insertion of the embryo 80, etc.

[0093] The step S302 of identifying the live cell or set of live cells 80 to be observed involves using the imaging system 120 to image and analyze the identification element 50 of the Petri dish 10 containing the live cell or set of live cells 80 to be observed.

[0094] Advantageously, an optional step S301, called approximate positioning step S301, can be performed before the step called identification step S302, which involves using the movement means 160 to move the assembly formed by the illumination system 110 and the wide-field camera 121 of the imaging system 120 and the Petri dish 10 relative to each other so that the selected Petri dish 10 is roughly aligned with the assembly formed by the illumination system (110) and the wide-field camera (121) of the imaging system (120).

[0095] Therefore, when a number of Petri dishes 10 are arranged on a support, during optional step S301, it is simply necessary to position the imaging system 120 above a selected Petri dish 10 so that the selected Petri dish 10 is directly identified by identifying the identification element 50 with the wide-field camera 121 by moving the assembly formed by the illumination system 110 and the wide-field camera 121 of the imaging system 120.

[0096] The processing unit of the first imaging system 120 can then analyze the image captured by the wide field camera 121, analysing the identification element 50 and enabling it to be linked with characteristics of the Petri dish 10 observed via the previously filled database.

[0097] The observing step S304 involves imaging the first live cell or set of live cells 80 using the imaging system 120.

[0098] In configurations where the support 1 includes a contact block or rim or a notch for prepositioning the Petri dish 10 in a predefined position, the notch is prepositioned there. Since the wide field of view of the wide field camera 121 covers at least the entire compartment 61 or well of the Petri dish 10, no preliminary lateral setting, i.e. setting in a plane parallel to the plane defined by the support 1, is necessary for positioning the imaging system 120 and the wide field camera 121 with respect to the Petri dish 10. This allows, from the start of the imaging system 120, after defining the observation plane of the live cell or set of live cells 80 by setting the wide field camera 121, to directly detect the live cell or set of live cells 80 positioned in one of the compartments 61 of the Petri dish 10 in a predefined position, regardless of the position of the live cell or set of live cells 80 in one of the compartments 61 of the Petri dish 10. Thus, the illumination system 110 illuminates the first live cell or set of live cells 80 and the wide field camera 121 captures its image. 13 shows a complete image of the embryo obtained with the above observation method, which image is obtained in particular after step S304, in which it is possible to observe the embryo located to the left of its compartment 61 in the Petri dish 10 and to distinguish its constituent cells.

[0099] Advantageously, during this observation step S304, it is possible to count the number of cells present in the observed live cell or set of live cells 80 or, thanks to the imaging system 120 and also thanks to the adapted lighting, to observe the texture and grain of these live cells or set of live cells.

[0100] In the particular case where the live cell or set of live cells 80 is an embryo, images captured by wide field camera 131 may be recorded, noting the identity of the embryo 80 and the date and time of image capture, so that the developmental evolution can be observed as a whole.

[0101] According to a second general embodiment of the observation method according to the invention, it is possible to observe another live cell or set of live cells 80 located in another compartment 61 of the Petri dish 10, as shown in Fig. 14. Thus, an additional step S310 can be performed for the first embodiment of the observation method, the overview of which is shown in Fig. 12. The Petri dish 10 and the assembly formed by at least the illumination system 110 and the narrow field of view camera 131 can be moved relative to each other by means of the movement means 160. The movement is performed along a horizontal plane defined by the Petri dish 10 or along a plane parallel to the horizontal plane defined by the support 1 on which the Petri dish 10 is placed. The movement is performed such that the assembly formed by at least the illumination system 110 and the wide field of view camera 121 is positioned in the compartment 61 of the new live cell or set of live cells 80 to be observed. Fig. 14 shows the overview of this embodiment of the observation of another live cell or set of live cells 80 located in another compartment 61 of the Petri dish.

[0102] To save time when observing multiple live cells or sets of live cells 80, the processing unit of the imaging system 120 can store in memory previous positions of observed live cells or sets of live cells 80 and command the moving means to go directly to these positions. Thus, the time lapse between two image captures of the same live cell or set of live cells 80 is reduced and the evolution of the live cell or set of live cells 80 over time is observed more effectively.

[0103] If the imaging device 100 further comprises a liquid lens 170 , the observation plane of the live cell or set of live cells 80 can be determined by the setting of the liquid lens 170 .

[0104] According to a third embodiment of the observation method according to the invention, it is possible to observe a live cell or a set of live cells 80 at its depth. For this purpose, an imaging device 100 is used, which comprises a liquid lens 170. Their use makes it possible to dispense with means for mechanical movement along the z-axis defined perpendicular to the horizontal plane defined by the support 1 and thus to reduce the setting time of the imaging device for observing a live cell or a set of live cells 80. Typically, the depth range of the studied live cell or a set of live cells 80 is of the order of a few tens of micrometers (for example, 10 μm to 100 μm, typically 20 μm). The observation plane is determined by the setting of the liquid lens 170 in step S312. The setting of the liquid lens 170 should be understood as the setting of control parameters, such as voltages, for defining the focal length of the liquid lens 170. For example, a liquid lens model Cu-25H0-075 of the Varioptic brand can be used to implement the embodiment of the observation method described above.

[0105] Thus, the additional step S312 of determining the observation plane by setting the liquid lens 170 can be repeated multiple times to observe the live cell or set of live cells 80 in multiple perpendicular planes relative to the depth direction determined by different settings of the liquid lens 170. Figure 15 shows an overview of this embodiment of the observation of the live cell or set of live cells 80 at that depth.

[0106] Computer program product according to the present invention The invention also relates to a computer program product downloadable from a communication network and / or stored on a computer readable medium and / or executable by a processor, said program comprising program code instructions for implementing a method for monitoring the development of a living cell or a set of living cells 80 as defined above.

[0107] In order to optimize the observation of the live cell or set of live cells 80 and in particular to minimize the deviations between each image captured by the wide-field camera 121, the computer program can optimize the movement of the assembly formed by at least the illumination system 110 and the wide-field camera 121, thanks to knowledge of the previously recorded positions of the live cell or set of live cells 80.

[0108] Advantageously, the computer program may also make it possible to view the captured images of the living cell or set of living cells 80, either in the form of a video that follows its evolution over time, or image by image. EXAMPLES

[0109] Example 1: Observation of an embryo using an imaging device according to the invention and an observation method according to the invention with different types of illumination 16 shows two images of an embryo 80 obtained with the imaging device 100 according to the invention and with a first embodiment of the observation method according to the invention, using illumination of the "contour" type and illumination of the "texture" type, respectively. In the image with illumination of the "contour" type, the edges of the cells forming the embryo 80 and their edges, and in the image with illumination of the "texture" type, the relief and texture of these elements, respectively, are distinguished.

[0110] Example 2: Observation of an embryo at its depth using an imaging device according to the invention and by a third embodiment of the observation method according to the invention Figures 18a and 18b show different cross sections of an embryo at different depths, obtained with an imaging device including a liquid lens model of the Varioptic brand (Cu-25H0-07) with a focal length of 7.5 mm for an applied voltage of 40 V. Figure 17a shows a series of planes using "contour" type illumination, with cut planes spaced about 30 microns apart by applying a defined voltage range of the liquid lens. Figure 17b shows a series of planes using relief type illumination, with cut planes spaced 30 μm apart. In these series of images, the same properties as in Figure 16 can be observed, as well as the sharpness of each cross section.

[0111] The above-described invention has several advantages.

[0112] The imaging device is not equipped with a set of lenses with different magnifications that are manually handled like current microscopes, but with a single camera that allows direct observation without any prior setting or movement of a first cell or set of cells 80 placed at any position within compartment 61 of Petri dish 10, and the observation is high-resolution observation of a living cell or set of living cells 80.

[0113] Thus, the constraint on placing live cells or sets of live cells 80 that can move within their compartments 61 in the Petri dish 10 is relaxed in that the wide field camera 121 covers at least the entire surface area of ​​the compartments 61. This allows for no need to move the imaging device 100 in order to observe the live cells or sets of live cells 80 in their compartments 61.

[0114] The different types of illumination that can be included in the imaging device make it possible to set up image processing operations associated with different stages of the observation process, such as identifying the Petri dish 10 and thus the live cell or set of live cells 80, its detection by the imaging system 120, or counting the number of cells present in the observed live cell or set of live cells 80.

[0115] The imaging device 100 and method for observing a living cell or set of living cells described herein makes it possible to enhance the optical quality of the image, as well as the material efficiency, i.e., the use of simplified equipment with respect to devices known in the prior art, and reducing the costs of manufacturing and using the device, while enhancing the time efficiency of the reproductive research process, particularly with regard to in vitro fertilization, by limiting means of movement and eliminating movement at depth.

Claims

1. An imaging device (100) for observing the development of living cells (80) in reproductive research, including in vitro fertilization (IVF), comprising: an imaging system (120) comprising a camera (121) for the observation of one or more living cells (80) to be observed, said one or more living cells (80) being placed in a Petri dish (10) containing a specific compartment (61) for each living cell (80); a support (1) capable of receiving said Petri dish (10) and positioned between an illumination system (110) and said imaging system (120); an illumination system (110) with three light sources (111a, 111b, 111c) for illuminating an object, being one or more living cells (80) to be observed, comprising a central source (111b), a first lateral source (111a) arranged on a first side of said central source (111b), and a second lateral source (111c) arranged on a second side of said central source (111b), The lighting system (110) is adapted to provide the following specific types of lighting: - detecting said one or more living cells (80) observed by said imaging system (120); "detection" type illumination for detecting an object, said "detection" type illumination being generated using said central source (111b), said first lateral source (111a) and said second lateral source (111c), said light rays from said light sources (111a, 111b, 111c) being focused on said object according to a cone (α_GC) having an angular aperture between 26° and 34°; - "contour" type illumination for counting the cells present in the one or more living cells (80) observed by the imaging system (120), the "contour" type illumination being generated using the first lateral source (111a) and the second lateral source (111c), the light beams from the first lateral source (111a) and the second lateral source (111c) being separated into two symmetrical collimated beams (113, 114) with respect to a vertical axis (P) relative to a plane formed by the Petri dish (10) support (1) or to the Petri dish itself, the first beam (113) from the first lateral source (111a) illuminating the object according to an angle of incidence of ±12° with respect to the vertical axis (P) and the second beam (114) from the second lateral source (111c) illuminating the object according to an angle of incidence of ±12° with respect to the vertical axis (P); - an illumination system (120) configured to implement each of the "relief" type illuminations for visualizing the cellular texture and granularity present in the one or more living cells (80) observed by the imaging system (120), the "relief" type illumination being generated using the first lateral source (111a), a single collimated light beam (115) from the first lateral source (111a) propagating along an axis inclined at an angle between 8° and 16° with respect to the vertical axis (P); characterised in that it comprises means (160) for relative movement (160) of said Petri dish (10) with respect to the assembly formed by said illumination system (110) and said camera (121) so as to be able to observe one or more living cells (80) located in different compartments of said Petri dish (10); The camera (121) has a field of view that covers at least the entire surface area of ​​one of the compartments of the Petri dish (10), and the imaging device (100) images one or more living cells in the Petri dish (10) at any position in the compartment without relative movement of the Petri dish using the movement means (160) with respect to the assembly formed by the illumination system (110) and the camera (121).

2. The imaging device (100) of claim 1 , wherein the one or more living cells are one or more embryos.

3. 3. The imaging device (100) of claim 1 or 2, wherein the light source (111) of the illumination system is arranged in a plane parallel to a horizontal plane defined by the support (1) capable of receiving the Petri dish (10), and a device for shaping the beam emitted by the at least one light source (111) is arranged between the light source (111) and the Petri dish (10).

4. The imaging device (100) of claim 1 or 2, wherein the moving means (160) is capable of moving the assembly formed by the illumination system (110) and the camera (121) of the imaging system (120) relative to the Petri dish (10) in which the one or more living cells (80) to be observed are placed.

5. An imaging device (100) described in any one of claims 1 to 4, further comprising a liquid lens (170) configured to control a focal length in the depth direction of the living cell.

6. The imaging of a living cell by an imaging device (100) as defined in any one of claims 1 to 5.

1. A method for observing a subject, comprising: a preparation step (S300) consisting of successively placing one or more live cells in a Petri dish (10) which can be chosen from at least one Petri dish arranged on the support (1) of the imaging device (100), followed by the dropping of a culture medium (62) in said Petri dish (10), the operation being repeated as many times as necessary according to the desired number of one or more live cells to be observed, and then covering the entirety of said live cells and said culture medium with a liquid (63); - an identification step (S302) of the Petri dish (10) for detecting and visually identifying an identification element (50) of the Petri dish (10) containing the living cell (80) to be observed, using a dedicated reader; - observing (S304) one or more first living cells (80) using said imaging system (120).

7. The method for observing the development of living cells as described in claim 6, further comprising, prior to the identification step (S302), an approximate positioning step (S301) of using a moving means to position the selected Petri dish (10) relative to an assembly formed by the illumination system (110) and the camera (121) of the imaging system (120) so that the Petri dish (10) is aligned with the assembly formed by the illumination system (110) and the camera (121) of the imaging system (120).

8. A method for observing the development of living cells as described in claim 6 or 7, comprising, after the step of observing one or more first living cells, a step (S310) of relative movement of the Petri dish (10) with respect to an assembly formed by the illumination system (110) and the camera (121) of the imaging system (120) in order to observe another one or more living cells located in another compartment (61) of the Petri dish (10).

9. A method for observing the development of living cells as described in any one of claims 6 or 7, wherein, in the case where the imaging device (100) further comprises a liquid lens (170), the step of observing one or more first living cells (80) is performed in a horizontal observation plane parallel to a support (1) capable of receiving a Petri dish (10) and perpendicular to the depth direction of the one or more first living cells, the plane being determined in an additional determination step (S312) by the configuration of the liquid lens.

10. The method for observing the development of living cells as described in claim 9, wherein the step of determining an observation plane (S312) of the one or more first living cells (80) is repeated for different planes perpendicular to the depth direction of the one or more first living cells (80) by different configurations of the liquid lens to observe the one or more first living cells (80) at the determined different observation planes.

Images