DEVICE FOR OBSERVING A LIVING CELL OR A SET OF LIVING CELLS

Description

[Technical field]

[0001] The present invention relates to an imaging device for observing a cell or a group of living cells in the context of reproductive studies, and in particular for fertilization. In Vitro (IVF).

[0002] More specifically, the invention relates to an imaging device for the observation of a living cell or group of living cells, an associated Petri dish, and a method and product of computer program adapted for this observation. State of the art

[0003] Current imaging devices for observing a cell or a group of living cells such as embryos are mostly microscopes equipped with different magnification objectives, a direct monocular or binocular observation system and usually a remote observation system to allow visualization of the cell or group of living cells on a video screen.

[0004] The cell or group of living cells are placed on a support and then observed using different objectives allowing the size of the images obtained to be enlarged or reduced, thus allowing the cell or group of living cells to be visualized at different magnifications, and therefore in detail or globally.

[0005] These devices are not very precise in their operation, nor easy to use. One must begin by observing the cell or group of living cells at low magnification to locate and center it within the microscope's field of view by manually moving it, then change objectives using increasingly higher magnifications to observe the cell or group of living cells in detail.

[0006] In the specific example of observing embryo development and achieving fertilization In Vitro In IVF, embryos intended for implantation are selected based on their cell quality, namely: their cell number, their regularity (whether the cells are uniform in size), and their degree of fragmentation. Priority is given to embryos whose cell division sequence is respected, with well-defined, regular cells and no fragmentation. These parameters are considered indicators of the best chances of pregnancy.

[0007] This observation is generally carried out outside of embryo culture chambers designed to recreate an ideal environment, in terms of temperature and gas concentration, for proper embryo development. US document 2015 / 0278625 A1 describes such an observation method outside of an incubator. However, it has been demonstrated that observation outside of chambers, and especially the abrupt change of environment for embryos, has a potentially detrimental effect on their development.

[0008] Embryo imaging devices have therefore been developed within the framework of In Vitro Fertilization (IVF) techniques to allow in vitro observation of embryo development, after fertilization, in this type of environment ideal for their development.

[0009] This observation can continue for 2 to 5 days depending on the IVF centers and allows for better selection of the embryos that will be transferred into the patient's genital tract, thus improving IVF success rates.

[0010] Two categories of observation devices exist: either the observation device is directly integrated into a system adapted to recreate the ideal environment for the proper development of embryos, with individual culture chambers included receiving boxes specific to each device; or the observation device is individual and to be placed in pre-existing enclosures with boxes also specific for observation.

[0011] All these specificities make current devices and their use very expensive, thus limiting their access to medical structures with restricted budgets or requiring an increase in fees and / or care costs for IVF beneficiaries.

[0012] In all cases, whether dealing with living cells or groups of living cells, or specifically with embryos, the optical quality of the images, the precise identification of living cells or groups of living cells, and the regular, temporal monitoring of their development are essential parameters for the use of these methods. However, current systems do not allow for precise localization of living cells or groups of living cells, as they are confined to small spaces that limit the field of view along the three axes x, y, and z.In the case of an embryo, its natural movements can cause it to move out of the field of view of the observable area without the possibility of relocation or cause it to appear at the edge of the field, where optical aberrations are most significant; on the other hand, observation under a single incidence has not yet made it possible to create algorithms capable of providing assistance in interpretation.

[0013] Another important parameter is the observation time of the living cells or groups of living cells that constitute the samples, which impacts the efficiency of the analyses performed on them. Current devices generally have mechanical means of movement along three axes commonly referred to as x, y (defining a horizontal plane containing the samples to be observed), and z (direction perpendicular to the previous plane, in the direction of the depth of the samples) in order to optimally position the samples. These mechanical means of movement can be time-consuming.A prior art solution to reduce depth travel time is to use a liquid lens, the focal length of which can be varied electronically, as in dental patent applications US2014 / 0002626 A1, dealing with an autofocus intraoral camera, and EP 2 161 607 A1, dealing with a dental camera.

[0014] An example of an imaging device for observing living cells is disclosed in document US2016 / 187359A1. Document US 4 852 985 A describes a lighting system for a microscope.

[0015] There is therefore a real need for an imaging device that overcomes the shortcomings, drawbacks, and obstacles of the prior art, in particular a device and a method that improve the optical quality and temporal efficiency of observing living cells or groups of living cells, while reducing the manufacturing and operating costs of the device. In the specific case of IVF, the aim is to improve its accessibility to those concerned by reducing costs. Description of the invention

[0016] To resolve one or more of the aforementioned drawbacks, the applicant proposes an imaging device according to claim 1; in particular, an imaging device for observing the development of living cells or groups of living cells (80) in the context of reproductive studies and in particular for In Vitro Fertilization (IVF), characterized in that it comprises: an imaging system comprising a wide-field camera suitable for identifying and observing one or more living cells or groups of living cells, the living cells or groups of living cells being placed in a Petri dish containing a specific compartment for each living cell or group of living cells; a support suitable for receiving the Petri dish and positioned between the lighting system and the imaging system; a lighting system comprising three light sources, suitable for illuminating an object which is a living cell or group of living cells to be observed, including a central source, a first lateral source placed on one side of the central source, and a second lateral source placed on the other side of the central source, the lighting system being configured to implement each of the following particular types of lighting: a "spotting" type lighting in which said light source (111) is configured to enable the spotting of the living cell or cells or sets of living cells to be observed by the wide field camera of the imaging system, said "spotting" type lighting being achieved by implementing the central source, the first lateral source and the second lateral source, the light rays from the light sources being focused on the object according to a cone having an angular opening between 26° and 34°;contour lighting in which said light source (111) is configured to allow counting the cell or cells present in the living cell or group of living cells observed by the wide field camera of the imaging system, said contour lighting being achieved by implementing the first lateral source and the second lateral source, the rays from the first lateral source and the second lateral source being separated into two collimated beams symmetrical with respect to the axis perpendicular to the plane formed by the support of the Petri dish or the Petri dish itself, a first beam from the first lateral source illuminating the object at an angle of incidence with respect to the perpendicular axis between 10° and 14°, a second beam from the second lateral source illuminating the object at an angle of incidence with respect to the perpendicular axis between 10° and 14°;a "relief" type lighting in which said light source (111) is configured to allow visualization of the texture and granularity of the cell or cells present in the living cell or group of living cells observed by the wide field camera of the imaging system, said "relief" type lighting being achieved by implementing the first lateral source, a single collimated light beam from the first lateral source propagating along an axis inclined at an angle to the perpendicular axis of between 8° and 16°;means for relative movement of the Petri dish with respect to the assembly formed by the lighting system and said wide-field camera so as to be able to observe living cells or groups of living cells located in the different compartments of the Petri dish, said wide-field camera having a field of view covering at least the total surface of one of the compartments of the Petri dish, and said imaging device being adapted to image, without relative movement of said Petri dish with the means for movement with respect to the assembly formed by the lighting system and said wide-field camera, a living cell or a group of living cells in any position in its compartment in the Petri dish and said wide-field camera having a resolution adapted to the observation of the details of a living cell or a group of living cells, said details having a micrometric size;

[0017] The specific lighting system of the device according to the invention makes the device compact, multifunctional, and requires no disassembly of parts to switch from one type of lighting to another. Handling is reduced to the bare minimum, and its ease of use over time, directly in the incubator, without risk of disturbance to the embryo, offers ideal observation conditions.

[0018] Contour lighting makes it easy to count the number of cells in a single view, even when the cells are stacked.

[0019] Relief lighting provides information about the texture of the cell membrane. This lighting is present in two opposite directions (up / down), providing complementary information that can be useful for understanding the geometry of the embryo, thus forming the third combination.

[0020] The field of view of the wide-field camera, covering at least the total surface of one of the housings of the Petri dish, thus makes it possible to directly image, without using any means of movement, a living cell or a set of living cells positioned in any position in its housing in the Petri dish.

[0021] For the purposes of this invention, "wide field camera" means, in particular, a camera with a field of view greater than 1 mm², with a pixel resolution of less than 1µm.

[0022] Preferably, the living cells or sets of living cells to be observed are one or more embryos.

[0023] According to one variant, the means of movement may be capable of moving the assembly formed by the lighting system and the wide-field camera of the imaging system relative to the Petri dish in which the living cells or sets of living cells to be observed are deposited.

[0024] In one embodiment, the imaging device according to the invention may further include a liquid lens, adapted to control a focusing distance in the direction of the depth of the living cell or group of living cells.

[0025] Another subject matter of the present application relates to an observation method according to claim 5, in particular a method for observing the development of living cells or groups of living cells by an imaging device according to the invention, comprising the following steps: a preparation step consisting of successively depositing a living cell or a group of living cells into a Petri dish, The Petri dish includes: a container suitable for receiving one or more living cells or sets of living cells to be observed, a lid, an identification element suitable for identifying the living cell(s) or set of living cells observed, specific compartments or wells intended for the deposit of a living cell or set of living cells, said compartments or wells being made in the form of cups adapted to receive, in addition to the living cell or set of living cells in the cup, a drop of a culture medium, then a drop of a culture medium in said Petri dish, the operation being repeated as many times as necessary depending on the desired number of living cells or sets of living cells to be observed; then to cover the whole with a liquid, such as oil or water;a Petri dish identification step consisting of detecting and visualizing, using a dedicated reader, the identifying element of the Petri dish in which the living cell(s) or set of living cells to be observed are located; and an observation step of a first living cell or set of living cells using the imaging system.

[0026] Advantageously, an optional step, prior to the Petri box identification step, of approximate positioning of the Petri box relative to the assembly formed by the lighting system and the wide field camera of the imaging system relative to the Petri box using the means of movement is carried out, so that the Petri box is approximately aligned with the assembly formed by the lighting system and the wide field camera of the imaging system;

[0027] Advantageously, the method of observing the development of living cells or sets of living cells may include, after the step of observing a first living cell or a first set of living cells, a step of relative displacement of the Petri dish with respect to the assembly formed by the lighting system and the wide-field camera of the imaging system allowing the observation of another living cell or another set of living cells located in another compartment of the Petri dish.

[0028] In the embodiment where the imaging device according to the invention further comprises a liquid lens, the observation step of a first living cell or a first set of living cells in the method of observing the development of living cells or sets of living cells can be carried out in a horizontal observation plane parallel to the support suitable for receiving the Petri dish and perpendicular to the depth direction of said first living cell or said set of living cells, said plane being determined in a further determination step by a configuration of the liquid lens.

[0029] Advantageously, in the same embodiment where the imaging device according to the invention further comprises a liquid lens, the step of determining an observation plane of said first living cell or of said set of living cells is repeated for different planes perpendicular to the direction of the depth of said first living cell or of said set of living cells by different configurations of the liquid lens, so as to observe a first living cell or of a first set of living cells in the different observation planes determined.

[0030] Observation in different planes perpendicular to the depth direction of said first living cell or group of living cells, made possible by the presence of a liquid lens in the imaging device according to the invention, thus eliminates the need for movement in the depth direction of said first living cell or group of living cells, and thus saves time in the procedure for analyzing and observing the living cell(s) or group(s) of living cells.

[0031] Other advantages and features of the present invention will become apparent from the following description, given by way of non-limiting example and with reference to the accompanying figures: There figure 1 represents a first embodiment of an imaging device according to the invention; The figure 2 represents a method of implementing "location" type lighting; The figure 3 represents a method of implementing "contour" type lighting; The figure 4 represents a method of implementing "texture" type lighting; The figure 5 represents a second embodiment of an imaging device according to the invention; The figure 6 represents an imaging device according to another embodiment of the invention arranged inside an incubator; The figure 7a represents a side view of the imaging device arranged inside an incubator of the figure 6 ; There figure 7b represents a top view of the imaging device arranged inside an incubator of the figure 6 ; There figure 8a represents several two-dimensional views (top, side, cross-section) of an example of a Petri dish that can be used in the invention; The figure 8b represents a 3D view of the example Petri dish of the figure 8a ; There figure 9a represents a side view of an embodiment of a Petri dish, according to the invention, placed on a support; The figure 9b represents a top view of the Petri dish of the figure 9a placed on a support; The figure 10 represents an embryo reservoir located on the bottom of a Petri dish according to one embodiment of the invention; The figures 11a à 11c represent another embodiment of a Petri dish according to the invention, respectively the figure 11a represents a profile view, the figure 11b a top view and the figure 11c a cross-section of the Petri dish profile; The figure 12 represents the synoptic diagram of a method for observing the development of a living cell or group of living cells according to a first embodiment of the invention; The figure 13 represents a full-field image obtained by the first embodiment of the observation method according to the invention with the wide-field camera of the imaging system of the imaging device according to the invention; The figure 14 represents the synoptic diagram of a method for observing the development of a living cell or group of living cells according to a second embodiment of the invention; The figure 15 represents the synoptic diagram of a method for observing the development of a living cell or group of living cells according to a third embodiment of the invention; The figure 16 show two images of an embryo obtained with an imaging device and according to the first embodiment of the observation method according to the invention, with two different types of lighting, respectively a "contour" type lighting on the right and a "texture" type lighting on the right; The figure 17a represents a succession of cross-sections of an embryo in its depth obtained with a device according to the invention and according to the third embodiment of the observation method according to the invention, using contour lighting; The figure 17b represents a succession of cross-sections of an embryo in its depth obtained with a device according to the invention and according to the third embodiment of the observation method according to the invention, using "texture" type lighting.

[0032] THE figures 16 à 17b are discussed in more detail in the following examples, which illustrate the invention without limiting its scope. The other figures, describing 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. IMAGING DEVICE ACCORDING TO THE INVENTION

[0033] An imaging device 100 is produced according to a first embodiment of the invention as illustrated in the figure 1 based on the following elements: lighting system 110: LED (light-emitting diode) type lighting imaging system 120 comprising a wide field camera 121 equipped with an image sensor having a resolution of the order of 5 pixels per 1 micrometer observed a support 1: a pane of glass relative movement means of motorized plates.

[0034] These elements are given as examples, and the device according to the invention can be made with elements having different parameters.

[0035] The imaging device 100 produced is intended to observe a living cell or a group of living cells 80 contained in a Petri dish 10 placed on the support 1. By the term "observe", it is understood that the details of a structure can be detected and distinguished.

[0036] In the embodiment presented to the figure 1 The device 100 is configured in an "inverted microscope" position, meaning that the cell or group of living cells being observed (80) is illuminated from above, and the imaging system (120) is positioned below, observing the light transmitted by the living cell or group of living cells (80), which is a translucent object. For the remainder of this description, this configuration will be used; however, the device 100 can also be configured in a "normal" position (the cell or group of living cells (80) illuminated from below and observed from above, as with a standard microscope) or in an "inverted" position.

[0037] Image acquisition by the wide-field camera 121 is done by transmission through the cell or group of living cells 80, i.e., by the technique known as diascopy. That is to say, the illumination is on one side of the object to be imaged and the camera on the other side of the object.

[0038]

[37] Thus, the Petri dish 10 in which the living cell(s) or group of living cells 80 to be observed is located must be transparent to the light used to illuminate the living cell(s) or group of living cells 80. The Petri dish 10 is placed on the support 1, as in the embodiment shown in the figure 1 This support 1 must also be transparent to the light used to illuminate the living cell or group of living cells 80, like a glass pane for conventional microscopes whose light source is a bulb emitting in the visible spectrum. In the device presented here, this support 1 is a glass pane but could also be a sliding drawer on which one or more Petri dishes 10 can be placed.

[0039] Advantageously, this support 1 can include pads or a rim allowing the Petri dish(es) 10 to be pre-positioned in predefined positions. In this case, no preliminary lateral adjustment, i.e., in a plane parallel to the plane defined by the support 1, is necessary to observe a living cell or a group of living cells 80 thanks to the wide field of view of the wide-field camera 121 which, covering at least the entirety of a cavity 61, also called a well or cupule, of a Petri dish 10, makes it possible to directly locate a living cell or a group of living cells 80 regardless of its position in its cavity 61.

[0040] Advantageously, the lighting system 110 and the wide-field camera 121 are joined and mounted on a U-shaped structure so that the lighting system 110 is positioned in front of the wide-field camera 121, as in the Figure 1 .

[0041] Advantageously, the lighting system 110 is chosen from at least one of the following specific lighting types: "Spotting" type lighting in which at least one light source 111 is configured to allow the spotting of the living cell or living cell group 80 to be observed by the imaging system 120; "Contouring" type lighting in which at least one light source 111 is configured to allow the counting of the cell or cells present in the living cell or living cell group 80 observed by the imaging system 120; and "Relief" type lighting in which at least one light source 111 is configured to allow the visualization of the texture and granularity of the cell or cells present in the living cell or living cell group 80 observed by the imaging system 120.

[0042] "Spotting" illumination is preferably used for detection and visualization by the imaging system 120, for imaging and analyzing an identification element 50 present on the Petri dish 10, allowing the identification of the living cell(s) or group(s) of living cells 80 to be observed. Therefore, the choice of at least one light source 111 and the wide-field camera 121 must be configured. For example, the sensitivity of the wide-field camera 121 must be optimized according to the wavelength emitted by the light source 111.

[0043] This "spotting" type lighting can also be used to aid in the detection of the live cell or group of live cells 80 by the wide field camera 121 and imaging system 120.

[0044] There figure 2 represents a method of implementing "location" type lighting. In this figure 2 , the object to be identified or detected using the wide-field camera 121 and the imaging system 120, which may be the identification element 50 of the Petri dish or as in the figure 2 The living cell or group of living cells 80 is illuminated by homogeneous or diffuse lighting. The light rays 112 from the light source 110 are concentrated or focused on the object to be located or detected, within a cone having an angular opening α_GC of approximately 30°. The observed object (identification element 50 or living cell or group of living cells 80) is therefore illuminated at angles of incidence varying between -15° and +15° with respect to the axis perpendicular P to the plane formed by the support 1 of the Petri dish 10 or the Petri dish 10 itself.

[0045] The angular opening cone α_GC is on the order of 30° ± 4°. The angles of incidence vary between -15° ± 2° and + 15° ± 2°.

[0046] There figure 3 represents a method of implementing "contour" type lighting. In this figure 3 The living cell or group of living cells 80 to be observed by the wide-field camera 121 can be illuminated along two axes, preferably symmetrical with respect to the axis perpendicular P to the plane formed by the support 1 of the Petri dish 10 or the Petri dish 10 itself. Thus, the rays from at least one light source 111 are separated into two collimated beams 113 and 114. The first beam 113 illuminates the embryo 80 at an angle of incidence β_PC1 with respect to the perpendicular axis P, and the second beam 114 illuminates the embryo 80 at an angle of incidence β_PC2. As stated previously, the angles β_PC1 and β_PC2 are preferably equal and opposite (i.e. β_PC1 = -β_PC2), that is, the bundles 113 and 114 are preferably symmetric with respect to the perpendicular axis P. Typically, the angles β_PC1 and β_PC2 are on the order of + / - 12° ± 2°.The total angle between the two directions formed by beams 113 and 114 is therefore typically on the order of 24° ± 4°. The high resolution of the wide-field camera 121 (allowing observation of the details of a living cell or a group of living cells 80, said details having a micrometric size) cooperates with this "contour" type illumination.

[0047] Texture lighting is preferably used to optimize the visualization, by the imaging system 120, of the texture and granularity of the cells present in the living cell or group of living cells 80 observed so as to allow appreciation of its relief and depth.

[0048] There figure 4 represents a way of achieving "texture" type lighting. In this figure 4 The living cell or group of living cells 80 to be observed by the wide-field camera 121, and whose cell texture and granularity will be visualized, can be illuminated by a single light beam 115. This beam 115 is collimated and propagates along an axis inclined at an angle γ_PC with respect to the perpendicular axis P. The absolute value of this angle γ_PC typically varies between 8° and 16°. No direction of incidence is preferred with respect to the perpendicular axis P or the living cell or group of living cells 80.

[0049] To facilitate the implementation of these different lighting schemes, the lighting system 110 can be composed of at least one or more LED light sources 111. Each of these LED light sources 111 can be controlled individually. It should be noted that LEDs have the advantage of being very compact, making it possible to duplicate the lighting using a group of LEDs so that one LED illuminates a living cell or group of living cells 80.

[0050] In the particular case where the set of living cells to be observed is an embryo, the wavelength of at least one light source 111 used (LED or other) can preferably be in the red range (around 630 nm), which is the least harmful range for the embryo 80. In addition, it is preferable that the light source 111 be controlled in pulsed mode so as to limit the cumulative exposure time of the embryo 80 to light to a few tens of seconds per day, in order not to damage it.

[0051] There figure 5 illustrates an embodiment of the invention where the lighting system 110 comprises several light sources 111a, 111b, 111c, three in this example, namely a central source 111b, a first lateral source 111a placed on one side of the central source 111b, and a second lateral source 111c placed on the other side of the central source 111b. Plan A of the figure 5 corresponds to the orthogonal plane P of the figures 2 à 4 Plan A passes through central source 111b.

[0052] To collimate the light emitted by the light source 111 and emit homogeneous lighting or diffuse lighting, the lighting system 110 may also include an optical system for shaping the light beam emitted by the light source 111, such as a lens or a matrix of lenses, filters, etc.

[0053] To adjust the area to be illuminated, this optical shaping system 140 may also include a mask, such as a plate with a hole, that can be moved to allow light from the light source 111 to illuminate the living cell or group of living cells 80 to be observed. This may be the case, in particular, if at least one light source 111 is extended or if the lighting system 110 includes several light sources 111 that can be controlled separately or together. Furthermore, the use of such a mask also filters out stray light that could interfere with the observation of the living cell or group of living cells 80.

[0054] Thus, within the framework of the figure 5 , to achieve a "texture" type lighting and illuminate the living cell or group of living cells 80 to be observed from the side, the light source 111b located vertically above the embryo 80 could be switched off and one of the two light sources 111a or 111c located on the sides of the central light source 111b could be switched on, thus projecting an oblique light onto the living cell or group of living cells 80.

[0055] The 120 imaging system is located, in the case of the figures 1 And 5 , below the Petri dish 10 and therefore below the living cell or group of living cells 80 to be observed (principle of the inverted microscope). It includes the wide-field camera 121 adapted to allow the identification of a living cell or group of living cells 80 to be observed, the living cell or group of living cells 80 being placed in a suitable Petri dish 10.

[0056] For this purpose, the wide-field camera 121 can be adjusted to image an identification feature 50 on the Petri dish 10, thus enabling the identification of the living cell or group of living cells 80 to be observed. The imaging system 120 may further include a processing unit that analyzes the image captured by the wide-field camera 121 to detect the position of the identification feature 50 and the position of the living cell(s) or group of living cells 80 to be observed, using the characteristics of the Petri dish 10.

[0057] In the case where the living cell or group of living cells 80 is one or more embryos, this processing unit also allows the image captured by the wide field camera 121 to be analyzed when it is positioned, using the means of movement 160, so as to image the identification element 50 of the Petri dish 10, so as to find in a previously filled database the information relating to the contents of the Petri dish 10, i.e. to the embryo or embryos 80, such as the patient couple to which the embryos 80 belong, the number of embryos 80 present in the dish, the date of preparation and placement of the embryos 80, etc.

[0058] Thanks to its high resolution, the 121 wide-field camera is suitable for imaging a living cell or group of living cells under light and for observing their development. The 121 wide-field camera is typically equipped with an image sensor with a resolution of approximately 5 pixels per 1 µm observed and with a sufficient number of pixels to cover the entire surface of the observed well, plus an additional area to ensure a safety margin to account for positioning uncertainties.

[0059] Furthermore, in order to be able to separate the different layers of cells present in the living cell or group of living cells 80, the wide field camera 121 must have a relatively shallow depth of field, on the order of 100 µm.

[0060] To view the different cell layers and thus observe the living cell or group of living cells 80 in its depth, the imaging device 100 may further include a liquid lens, adapted to control the focusing distance of the living cell or group of living cells 80 in the direction of its depth, i.e., along a Z-axis 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, or 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 change the shape of the interface separating two different liquids, and thus changes 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 an electrical voltage between an electrode and a substrate, thus changing the curvature of the liquid lens 170. In the case of a liquid lens 170 configured by a piezoelectric actuator, the latter inflates a liquid-filled membrane. The plane corresponding to the cell layer to be observed is thus configured by the liquid lens configuration. The advantage is therefore the elimination of a means of movement along the z-axis, the actuation of which is time-consuming.

[0061] Thanks to the processing unit, the imaging system 120 can count the number of cells present in the observed living cell or group of living cells 80. To do this, the processing unit of the imaging system 120 analyzes the images captured by the wide-field camera 121 and, through image processing such as edge detection, counts the number of cells present in the observed living cell or group of living cells 80.

[0062] The counting and especially the image processing are optimal when the living cell or group of living cells observed is illuminated using "contour" type lighting.

[0063] The imaging system 120 can also visualize the texture and granularity of the cells present in the observed embryo 80. To do this, the processing unit of the imaging system 120 analyzes the images captured by the wide-field camera 121 and processes them with image processing techniques that optimize the visual rendering of the texture and grain of the cells present in the living cell or group of living cells 80 being observed.

[0064] As illustrated on the figure 5 The imaging system 120, and in particular the wide-field camera 121, can advantageously be relocated using an optical reflector system 150, so as to make the device 100 more compact. This optical reflector system 150 can consist, for example, of a lens 151 and a reflector mirror 152 positioned at 45° with respect to the optical axis A of the imaging device 100, shown in dashed lines on the diagram. figure 5 .

[0065] In the case where the living cell or group of living cells is an embryo or several embryos, as illustrated by the figure 6 The dimensions of the imaging device 100 are preferably compatible with the incubators 200 present in IVF centers so that it can be placed inside such incubators 200. Thus, the height and depth of the device 100 can be on the order of 30 cm and the width of the device 100 can be on the order of 30 cm to 55 cm approximately.

[0066] The 100 imaging device of the figure 6 includes a sliding drawer on which one or more Petri dishes 10 can be placed as previously indicated.

[0067] THE figures 7a et 7b represent respectively a side view and a top view of the imaging device 100 of the figure 6 present in an incubator 200. In the present case, the support 1 covering the entire width of the imaging device 100, it is the assembly formed by at least the lighting system 110 and the wide field camera 121 fixed on a structure in the shape of an "elongated U" which is moved by the relative displacement means 160 in a plane (X, Y) parallel to that defined by the support 1 so as to be able to visualize either another embryo 80 from the same Petri dish 10, or an embryo 80 in another Petri dish 10. PETRI BOWL ACCORDING TO THE INVENTION

[0068] In a second step, a Petri dish 10 adapted for the observation of the development of living cells or sets of living cells 80 by an imaging device 100 as defined previously will be detailed below.

[0069] According to a first embodiment, such a Petri dish 10 may include: a container 20 adapted to receive one or more embryos 80 to be observed; and a lid 30.

[0070] There figure 8a presents two-dimensional top and side views as well as a section AA of an example Petri dish that can be used in the context of the invention. An example of a container 20 and a corresponding example of a lid 30 are shown. figure 8b presents a relief representation of the container 20 and the lid 30 of the figure 8a .

[0071] A schematic view of this first embodiment of the Petri dish 10 is illustrated in the figure 9a .

[0072] In order to be able to see the living cells or groups of living cells 80 through the Petri dish 10, the dish can be made of a material transparent to the light emitted by the lighting system 110 as previously mentioned. This light is generally in the visible range (approximately between 400 nm and 800 nm). Thus, the material used can be glass or a plastic material, for example.

[0073] With reference to the figure 9b , the bottom of the container 20 includes an identification element 50 adapted to allow the identification of the live cell or cells or the set / sets of live cells 80 observed.

[0074] According to the embodiment presented in the figure 9b The Petri dish 10 is rectangular in shape.

[0075] The identification element 50 can, for example, be a barcode or a 2D code, such as a data-matrix code as shown on the figure 9b This identification element 50 allows, in particular, the retrieval of information relating to the contents of the Petri dish 10, that is to say, the living cells or sets of living cells 80 it contains. In the case where the living cell(s) or set(s) of living cells 80 are one or more embryos, the information relating to the contents of the Petri dish 10 may include, for example, the patient couple to whom the embryos 80 belong, the number of embryos 80 present in the dish, the date of preparation and placement of the embryos 80, etc., through a pre-populated database.

[0076] According to this embodiment, the living cells or groups of living cells 80 are arranged linearly in one or more rows, as is the case for the rectangular Petri dish 10 illustrated in the figure 9b .

[0077] According to a particular embodiment of the Petri dish 10 illustrated in the figure 10 The base of the container 20 may advantageously include at least one embryo reservoir 60 comprising in its center a well 61, or chamber, adapted to receive a living cell or group of living cells 80. The reservoir 60 is also adapted to receive, in addition to the living cell or group of living cells 80 in the well 61, a drop of culture medium 62 (typically on the order of 6 µl). The diameter of the well may typically be on the order of 1 mm and its capacity on the order of 0.75 µl. The area containing the culture medium 62 is delimited by a raised section or bump 64 on the base of the container 20.

[0078] The assembly formed by at least the living cell or group of living cells 80, the drop of culture medium 62 can then be covered with a liquid 63 such as oil or water. For this, the edges of the container 20 must be sufficiently high, typically on the order of 5 to 10 mm.

[0079] According to another embodiment, illustrated in the figures 11a à 11c and similar to that presented in the figures 9a et 9b The Petri dish 10 can be attached to a transparent plate 11 comprising a gripping lug 12 allowing easy handling of the Petri dish 10. This avoids the risk of soiling the bottom of the container 20 or the lid 30, which would disrupt the observation of the embryo 80 and the quality of the images taken by the device 100. Advantageously, the gripping lug 12 includes a marking area 13 on which the identification element 50 can be affixed rather than on the bottom of the container 20.

[0080] Finally, the edges of the container 20 are high enough to allow a quantity of liquid 63, such as oil or water, to be deposited to cover the whole formed by the drop of culture medium 62 and the living cell or group of living cells 80, typically in the order of 5 to 10 mm.

[0081] Depending on the embodiment, the identification element 50 can be made directly in the material, by machining the bottom of the container 20 on its internal or external face, or by affixing a label and / or self-adhesive markers to the external bottom of the container 20. OBSERVATION METHOD ACCORDING TO THE INVENTION

[0082] Thirdly, a method for observing the development of living cells or groups of living cells 80 by an imaging device 100 as defined previously will be detailed below.

[0083] According to a first general embodiment of the process illustrated in the figure 12 This process can include three main stages: a preparation step S300; an identification step S302; and an observation step S304.

[0084] The S300 preparation step consists of successively depositing a live cell or a set of live cells 80 and then a drop of a culture medium 62 (or conversely a drop of a culture medium 62 and then a live cell or a set of live cells 80) and then repeating the operation according to the number of live cells or sets of live cells 80 to be observed desired and finally covering everything with a liquid 63, such as oil, or water, in a Petri dish 10 as defined previously and which can be chosen from at least one Petri dish arranged on the support of the device.

[0085] The live cell or group of live cells 80 can be deposited in the cup 61 and then covered with a drop of culture medium 62 in the live cell or group of live cells reservoir 60.

[0086] Advantageously, and especially in the case where the living cell or group of living cells 80 is an embryo or several embryos, following this operation of preparing or depositing the embryos 80, a database can be filled by the preparer with the characteristics of the Petri dish 10 and especially the data related to the embryos 80 deposited in the Petri dish 10 such as: the couple of patients to whom the embryos 80 belong, the number of embryos 80 present in the dish, the date of preparation and placement of the embryos 80, etc.

[0087] The S302 identification step of the live cell or set of live cells 80 to be observed consists of imaging and analyzing, using the imaging system 120, the identification element 50 of the Petri dish 10 in which the live cell or set of live cells 80 to be observed is located.

[0088] Advantageously, an optional step S301, called the approximate positioning step S301, can be carried out prior to the step, called the identification step S302, said optional step S301 consisting of moving relative to each other the assembly formed by the lighting system 110 and the wide field camera 121 of the imaging system 120 and the Petri box 10 using the movement means 160, so that the chosen Petri box 10 is approximately aligned with the assembly formed by the lighting system (110) and the wide field camera (121) of the imaging system (120).

[0089] Thus, in the case where several Petri dishes 10 are arranged on the support, it is sufficient to position the imaging system 120, during an optional step S301, above a chosen Petri dish 10 so that it is directly identified, in particular by the identification of the identification element 50 with the wide field camera 121, by moving the assembly formed by the lighting system 110 and the wide field camera 121 imaging system 120.

[0090] Then the processing unit of the first imaging system 120 can analyze the image taken by the wide field camera 121 and allows the identification element 50 to be analyzed to trace back to the characteristics of the Petri dish 10 observed via a previously filled database.

[0091] The S304 observation step consists of imaging a first live cell or set of live cells 80 using the imaging system 120.

[0092] In a configuration where the support 1 includes lugs, ridges, or notches allowing a Petri dish 10 to be pre-positioned in predefined positions, the dish is thus pre-positioned. No preliminary lateral adjustment, i.e., in a plane parallel to the plane defined by the support 1, is necessary to position the imaging system 120 and the wide-field camera 121 relative to the Petri dish 10, because the wide field of view of the wide-field camera 121 covers at least the entirety of one well 61 of the Petri dish 10.This allows, as soon as the imaging system 120 is switched on, after defining the observation plane of the living cell or group of living cells 80 by configuring the wide-field camera 121, for the direct identification of a living cell or group of living cells 80 positioned in one of the compartments 61 of the Petri dish 10 in place, regardless of the position of the living cell or group of living cells 80 in its compartment 61. Thus, the illumination system 110 illuminates the first living cell or group of living cells 80 and the wide-field camera 121 images it. The . figure 13 shows a full image of an embryo obtained by the observation process described above, the image being obtained in particular after step S304. The embryo can be seen positioned on the left side of its housing 61 in the Petri dish 10 and the cells which constitute it can be distinguished.

[0093] Advantageously, during this S304 observation stage, it will be possible to count the number of cells present in the observed living cell or group of living cells 80 or to observe the texture and grain of these living cells or groups of living cells using the imaging system 120 and possibly also using suitable lighting.

[0094] In the particular case where the living cell or group of living cells 80 is an embryo, in order to observe the evolution of its development as a whole, the images taken by the wide field camera 131 can be recorded, taking care to identify the embryo 80 and the date and time of the shot.

[0095] According to a second general embodiment of the observation method according to the invention, and illustrated in the figure 14 It is possible to observe another living cell or group of living cells 80 located in another compartment 61 of the Petri dish 10. An additional step S310 can thus be carried out compared to the first embodiment of the observation method, a synoptic diagram of which was shown in the figure 12 The Petri dish 10 and the assembly consisting of at least the lighting system 110 and the narrow-field camera 131 can be moved relative to each other using the movement means 160. The movement is carried out along a plane parallel to the horizontal plane defined by the Petri dish 10 or to that defined by the support 1 on which the Petri dish 10 is placed. The movement is performed so that the assembly consisting of at least the lighting system 110 and the wide-field camera 121 is positioned at the level of the cavity 61 of the new living cell or set of living cells 80 to be observed. figure 14 presents the synoptic diagram of this method of observing another living cell or group of living cells 80 located in another compartment 61 of the Petri dish.

[0096] It should be noted that, to save time when observing several living cells or groups of living cells, the processing unit of the imaging system can store the previous positions of the observed living cells or groups of living cells in memory, so that the movement mechanisms can be directed to these positions. Thus, the time-lapse between two image acquisitions of the same living cell or group of living cells is reduced, and the temporal evolution of the living cell or group of living cells is observed more effectively.

[0097] In the case where the imaging device 100 further includes a liquid lens 170, the observation plane of the living cell or set of living cells 80 can be determined by a configuration of the liquid lens 170.

[0098] According to a third embodiment of the observation method according to the invention, a living cell or a group of living cells 80 can be observed in its depth. This is achieved using an imaging device 100 comprising a liquid lens 170. The use of this lens eliminates the need for a mechanical means of movement along the z-axis defined perpendicular to the horizontal plane defined by the support 1, and thus reduces the setup time of the imaging device for observing living cells or groups of living cells 80. Typically, the depth range of a living cell or group of living cells 80 explored is on the order of a few tens of micrometers (for example, from 10 µm to 100 µm, typically 20 µm). An observation plane is determined by a configuration of the liquid lens 170 in a step S312.By setting up the liquid lens 170, it must be understood that a control parameter such as an electrical voltage must be set to define the focal length of the liquid lens 170. For example, to implement the embodiment of the observation method described above, the Varioptic brand Cu-25H0-075 liquid lens model can be used.

[0099] Thus, the additional step S312 of determining an observation plane by liquid lens configuration 170 can be repeated several times to observe a living cell or group of living cells 80 in several planes perpendicular to the depth direction determined by different liquid lens configurations 170. The figure 15 presents the synoptic diagram of this method of observing a living cell or group of living cells in its depth. COMPUTER PROGRAM PRODUCT ACCORDING TO THE INVENTION

[0100] 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. This computer program includes program code instructions for implementing the method of observing the développement of a living cell or a group of living cells 80 as previously defined.

[0101] To optimize the observation of living cells or groups of living cells 80 and in particular to minimize the gap between each shot by the wide field camera 121, the computer program will be able to optimize the movement of the assembly formed by at least the lighting system 110 and the wide field camera 121 thanks to the knowledge of previously recorded positions of the living cells or groups of living cells 80.

[0102] Advantageously, this computer program will also allow viewing images taken of a living cell or a group of living cells 80 either in the form of a video retracing its evolution over time, or image by image. EXAMPLES

[0103] EXAMPLE 1: Observation of an embryo with an imaging device according to the invention and by an observation method according to the invention with different types of lighting

[0104] There figure 16 Figure 80 shows two images of an embryo obtained with an imaging device according to the invention and according to the first embodiment of the observation method according to the invention, using respectively "contour" type lighting and "texture" type lighting. In the image under "contour" type lighting, the edges of the cells constituting the embryo are visible, as well as the edges of the embryo itself, and in the image under "texture" type lighting, the relief and texture of these elements are visible.

[0105] EXAMPLE 2: Observation of an embryo in its depth with an imaging device according to the invention and according to the third embodiment of the observation method according to the invention

[0106] Figures 18a and 18b show different cross-sections of an embryo at various depths, obtained with an imaging device comprising a Varioptic brand liquid lens model (Cu-25H0-07), with a focal length of 7.5 mm and an application voltage of 40 V. figure 17a presents a series of sections with "contour" illumination, where the section planes are spaced approximately 30 microns apart by applying specific voltage ranges to the liquid lens. figure 17b presents a series of cross-sections with relief lighting, where the cross-sectional planes are spaced 30µm apart. The same properties can be observed in these image series as in the figure 16 , and also the sharpness of each cross-section.

[0107] The invention as previously described offers multiple advantages.

[0108] The imaging device is no longer equipped with a set of objectives of different magnifications to be manipulated manually as on current microscopes but it is equipped with a single camera allowing the direct observation, without preliminary adjustment or movement of a first living cell or group of living cells 80 placed in any position in a housing 61 of a Petri dish 10, the observation being a high-resolution observation of the cell or group of living cells 80.

[0109] The constraints regarding the deposition of living cells or groups of living cells 80 which can move within their housing 61 in the Petri dish 10 are therefore relaxed by the fact that the wide field camera 121 covers at least the entire surface of a housing 61. This makes it possible not to have to move the imaging device 100 to observe the living cell(s) or groups of living cells 80 in their housing 61.

[0110] The different types of lighting that the imaging device can include allow the image processing associated with the different phases of the observation process to be configured accordingly, such as the identification of the Petri dish 10 and therefore of the living cells or sets of living cells 80, their detection by the imaging system 120, or the counting of the number of cells present in the living cell or set of living cells 80 observed.

[0111] The imaging device 100 and the method for observing living cells or groups of living cells presented here improve optical and image quality, as well as material efficiency, i.e., the use of simplified equipment compared to devices known in the prior art, and temporal efficiency, thanks to the limitation of means of movement and the elimination of deep displacements, in the processes of studying reproduction, particularly fertilization. In Vitro, while reducing the manufacturing and operating costs of the device.

Claims

1. Imaging device (100) for observing the development of living cells or sets of living cells (80) in the context of studying reproduction and in particular for In Vitro Fertilisation (IVF), characterised in that it comprises: - an imaging system (120) comprising a wide-field camera (121) adapted for observing one or more living cells or sets of living cells (80) to be observed, the living cells or sets of living cells (80) being deposited in a Petri dish (10) containing a specific compartment (61) for each living cell or set of living cells (80); - a support (1) for receiving the Petri dish (10), which is positioned between the lighting system (110) and the imaging system (120); - a lighting system (110) comprising three light sources (111a, 111b, 111c), adapted for illuminating an object which is a living cell or a set of living cells (80) to be observed, including a central source (111b), a first lateral source (111a) placed on a first side of the central source (111b), and a second lateral source (111c) placed on a second side of the central source (111b), the lighting system (110) being configured to implement each of the following specific types of lighting: - a "locating" type of lighting for locating the living cell or cells or sets of living cells (80) to be observed by the wide-field camera (121) of the imaging system (120), said "locating" type of lighting being produced using the central source (111b), the first lateral source (111a) and the second lateral source (111c), the light rays (112) from the light sources (111a, 111b, 111c) being focused on the object along a cone (α_GC) having an angular aperture of between 26° and 34°; - a "contour" type of lighting for counting the cell or cells present in the living cell or set of living cells (80) observed by the wide-field camera (121) of the imaging system (120), said "contour" type of lighting being produced using the first lateral source (111a) and the second lateral source (111c), the rays from the first lateral source (111a) and the second lateral source (111c) being split into two collimated beams (113, 114) that are symmetrical with respect to the perpendicular axis (P) to the plane formed by the support (1) of the Petri dish (10) or the Petri dish itself, a first beam (113) from the first lateral source (111a) illuminating the object at an angle of incidence with respect to the perpendicular axis (P) of between 10° and 14°, a second beam (114) from the second lateral source (111c) illuminating the object at an angle of incidence with respect to the perpendicular axis (P) of between 10° and 14°; - a "relief" type of lighting for viewing the texture and granularity of the cell or cells present in the living cell or set of living cells (80) observed by the wide-field camera (121) of the imaging system (120), said "relief" type of lighting being produced 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 with respect to the perpendicular axis (P) of between 8° and 16°; - means (160) for relative movement of the Petri dish (10) with respect to the assembly formed by the lighting system (110) and said wide-field camera (121) so as to be able to observe living cells or sets of living cells (80) located in the various compartments of the Petri dish (10), said wide-field camera (121) having a field of view covering at least the total surface area of one of the compartments of the Petri dish (10), and said imaging device (100) being adapted for imaging, without relative movement of said Petri dish with respect to the assembly formed by the lighting system (110) and said wide-field camera (121) using the movement means (160), a living cell or a set of living cells in any position in its compartment in the Petri dish (10) and said wide-field camera (121) having a resolution adapted for observing the details of a living cell or a set of living cells (80), said details having a micrometric size.

2. Imaging device (100) according to claim 1, wherein the set or sets of living cells are one or more embryos.

3. Imaging device (100) according to any one of claims 1 to 2, wherein the movement means (160) are capable of moving the assembly formed by the lighting system (110) and the wide-field camera (121) of the imaging system (120) relatively with respect to the Petri dish (10) in which the living cells or sets of living cells (80) to be observed are deposited.

4. Imaging device (100) according to any one of claims 1 to 3, further comprising a liquid lens (170), adapted for controlling a focal length in the direction of the depth of the living cell or set of living cells.

5. Method for observing the development of living cells or sets of living cells by means of an imaging device (100) as defined in any one of claims 1 to 4, comprising the following steps: - a preparation step (S300) consisting in successively depositing a living cell or a set of living cells in a Petri dish (10) comprising: - a container (20) adapted for receiving one or more living cells or sets of living cells to be observed, - a cover (30), - an identification element (50) adapted for identifying the living cell or cells or sets of living cells observed, - specific compartments or wells for depositing a living cell or a set of living cells, said compartments or wells being in the form of cavities (61) adapted for receiving, in addition to the living cell or the set of living cells in the cavity, a drop of a culture medium (62), followed by a drop of a culture medium (62) in said Petri dish (10), the operation being repeated as many times as necessary depending on the desired number of living cells or sets of living cells to be observed; then covering the whole with a liquid (63), such as oil or water; - an identification step (S302) for identifying the Petri dish (10) consisting in detecting and viewing, using a dedicated reader, the identification element (50) of the Petri dish (10) in which the living cell or cells or sets of living cells (80) to be observed are located; and - an observation step (S304) for observing a first living cell or set of living cells (80) using the imaging system (120).

6. Method for observing the development of living cells or sets of living cells according to claim 5, further comprising, prior to the identification step (S302), a step (S301) of approximately positioning the selected Petri dish (10) with respect to the assembly formed by the lighting system (110) and the wide-field camera (121) of the imaging system (120) relative to a Petri dish (10) selected using the movement means, so that the Petri dish (10) is approximately aligned with the assembly formed by the lighting system (110) and the wide-field camera (121) of the imaging system (120).

7. Method for observing the development of living cells or sets of living cells according to claim 5 or 6, comprising, after the step of observing a first living cell or a first set of living cells, a step (S310) of relative movement of the Petri dish (10) with respect to the assembly formed by the lighting system (110) and the wide-field camera (121) of the imaging system (120), making it possible to observe another living cell or another set of living cells located in another compartment (61) of the Petri dish (10).

8. Method for observing the development of living cells or sets of living cells according to any one of claims 5 to 7, in the case where the imaging device (100) further comprises a liquid lens (170), wherein the step of observing a first living cell or a first set of living cells (80) is carried out in a horizontal observation plane parallel to the support (1) for receiving the Petri dish (10) and perpendicular to the direction of the depth of said first living cell or said set of living cells, said plane being determined in an additional determination step (S312) by a configuration of the liquid lens.

9. Method for observing the development of living cells or sets of living cells according to claim 8, wherein the step (S312) of determining an observation plane of said first living cell or set of living cells (80) is repeated for different planes perpendicular to the direction of the depth of said first living cell or set of living cells (80) by means of different configurations of the liquid lens, so as to observe a first living cell or a first set of living cells (80) in the different observation planes determined.