Flow-cytometry apparatus particularly for processing sperm cells

Abstract

The invention relates to a flow-cytometry apparatus, particularly for processing sperm cells and in particular for sexing animal semen, comprising an optical detection system (3) configured to differentiate, according to predetermined parameters, between a plurality of cells (26) in a jet of such cells (11) directed towards a differentiation area (17), and an imaging system (40) configured to image the jet containing the differentiated cells in the differentiation area and determine information representative of a time at which a drop containing an identified cell must be generated, to then be sorted; the optical detection system (3) is provided with a first light source (12) configured to emit a light ray (16) focused on the differentiation area (17), with at least two detection members (14) configured to each detect an amount of light re-emitted by the cells (26) that are illuminated in different re-emission directions, and at least two collection members (15) that are focused, on a first side, on the differentiation area (17) and, on a second side opposite the first side, on the respective detection members (14); the apparatus being characterized in that at least one of the two collection members (15) is formed by a lens (60, 70) having a cut edge (61, 71) and in that the imaging system (40) is provided with a camera (41) and with a second light source (42), distinct from the first light source (12), which is arranged and configured so as to generate an imaging ray (43) directed in the direction of the differentiation area (17) and camera.

Description

[0001] DESCRIPTION

[0002] TITLE: Flow cytometry installation, particularly for the processing of sperm cells

[0003] TECHNICAL FIELD OF THE INVENTION

[0004]

[0001] The invention relates generally to flow cytometry, particularly for the processing of spermatozoa-type cells, and in particular for the sexing of animal semen, for example bovine semen

[0005]

[0002] The invention relates more particularly to a flow cytometry installation comprising an optical detection system configured to differentiate animal semen cells and an imaging system configured to sort these cells.

[0006] STATE OF THE ART

[0007]

[0003] Flow cytometry facilities make it possible to identify and sort animal semen cells, in particular spermatozoa, so as to isolate cells with an X chromosome from those with a Y chromosome.

[0008]

[0004] These installations are typically provided with a feeding system configured to generate a jet of cells at the outlet of a nozzle, the cells being conveyed in a so-called sheath fluid, a droplet generation system from the jet of cells and the sheath fluid, an optical detection system which is configured to examine the jet of cells in order to differentiate the type of cell, an imaging system configured to visualize drops containing the differentiated or to be differentiated cells mixed with the sheath fluid, and a sorting system configured to apply a predetermined charge to each differentiated cell in a drop, the predetermined charge being associated with the result of the differentiation, in other words with the type of cell, as well as collection containers for the differentiated and sorted cells.

[0009]

[0005] Such installations are notably called “jet-in-air sorter” in Anglo-Saxon terminology.

[0010]

[0006] These installations are also equipped with control and command units that are connected to the power supply system, the optical detection system, the imaging system, the droplet generation system, and the sorting system, and that are configured to process certain information representative of certain parameters in order to control and command the systems to which they are connected. These parameters may, for example, correspond to the cell jet flow rate, the cell types, or the predetermined loads associated with them.

[0011]

[0007] Optical detection systems in flow cytometry installations are generally based on fluorescence technologies and are equipped with an excitation laser configured to emit a beam of light and a beam shaping device that is configured to focus the beam of light to the location where the cell jet passes.

[0012]

[0008] These optical detection systems for flow cytometry installations are also provided with collection elements and detection elements arranged both in a direction of emission of the laser beam and in a direction orthogonal to the direction of emission of the laser beam.

[0013]

[0009] In such optical detection systems for flow cytometry installations, the detection elements can be photomultiplier type sensors, while the collection elements are optical equipment of the microscopic lens type, offering for example a very high magnification, in particular on the order of 50.

[0014]

[0010] These optical detection systems for flow cytometry installations need to be adjusted to obtain the desired result, namely to differentiate cells in order to sort them.

[0015]

[0011] However, in practice, the necessary adjustments are complex because it is difficult to make the focal point of the laser coincide with the focal points of the collection organs at very high magnification, and also with the cell jet, or even with the cells inside the cell jet.

[0016]

[0012] French patent application FR 3 126 043 discloses an optical detection system for a flow cytometry installation, in particular for the processing of spermatozoon-type cells, configured to differentiate, according to predetermined parameters, a plurality of cells in a jet of such cells directed towards a differentiation zone, the optical detection system being provided with a light source configured to emit light focused on the differentiation zone, a detection organ configured to detect an amount of light re-emitted by the illuminated cells, and a collection organ which is focused, on a first side, on the differentiation zone and on a second side opposite to the first side, on the detection organ.

[0017]

[0013] The optical detection system is configured so that the collection organ has a magnification of less than 10 and the detection organ has a vision area having a surface area at least 10 times greater than a surface area of ​​the cell jet at the location of the differentiation zone.

[0018]

[0014] The collection organ therefore has a particularly low magnification and much lower than the magnifications used for example in microscopy.

[0019]

[0015] Moreover, the optical detection system has a detection element whose viewing area is much larger than the surface area of ​​the cell jet, without this impacting the actual differentiation of the cells, since what is important is to collect the light re-emitted by the illuminated cells in the differentiation zone.

[0016] In other words, it is a matter of detecting a light flux rather than capturing an image of the illuminated cells.

[0020]

[0017] This is made possible because the detection organ has the ability to detect, in the middle of a large surface forming the vision zone, particular elements formed by cells that emit more light than others.

[0021]

[0018] It is therefore only a matter of detecting that some cells emit more light than others in order to differentiate these cells.

[0022]

[0019] Note for example that light radiation can induce fluorescence on cells, so the detection organ is configured to detect an amount of light re-emitted for example by fluorescence or scattering.

[0023]

[0020] In other words, it is not necessary to zoom in heavily on the area of ​​differentiation and therefore on the cells, which are particularly small, in order to fill the surface of the vision area of ​​the sensing organ to better differentiate these cells.

[0024]

[0021] Moreover, the use of a collection organ with low magnification allows for a very large field of view and therefore a very large latitude for focusing.

[0025]

[0022] In particular, the focusing distance is very large so that it is particularly convenient to arrange the components of the optical detection system so that the differentiation zone, where in particular the focused light beam and the cell jet are located, can always be seen in the vision zone of the detection organ.

[0026]

[0023] In other words, the setup of the installation is not complicated and the positioning of the cell jet allows for a certain tolerance.

[0027]

[0024] As indicated above, the detection system makes it possible to identify the type of cells in the cell jet.

[0028]

[0025] In addition, sorting systems for flow cytometry installations are provided, for example, with a camera configured to visualize the cells identified by the detection system, a vibrating device controlled by means of the information collected by the camera and configured to generate drops in the cell jet, each drop containing, for example, an identified cell, and the sorting device is configured to apply a predetermined load to each differentiated cell in a given drop.

[0029]

[0026] Such imaging systems are therefore configured to determine information representative of the time at which a drop containing an identified cell should be generated, and subsequently sorted. This representative information is often called "drop delay" in Anglo-Saxon terminology.

[0030] DESCRIPTION OF THE INVENTION

[0027] The invention aims to provide a flow cytometry installation, in particular for the processing of spermatozoon-type cells, which is particularly convenient and efficient, both in its adjustment and in use.

[0031]

[0028] The invention thus relates, according to a first aspect, to a flow cytometry installation, in particular for the processing of spermatozoon-type cells and in particular for the sexing of animal semen, comprising an optical detection system configured to differentiate, according to predetermined parameters, a plurality of cells in a jet of such cells directed towards a differentiation zone, and an imaging system configured to visualize the jet comprising the differentiated cells at the level of the differentiation zone and to determine information representative of a moment at which the drop containing an identified cell must be generated, and then sorted;with the optical detection system which is provided with a first light radiation source configured to emit a light beam focused on the differentiation zone, at least two detection organs configured to each detect a quantity of light re-emitted by the cells which are illuminated in different directions of re-emission, and at least two collection organs which are focused, on a first side, on the differentiation zone and on a second side opposite to the first side, on the respective detection organs;the installation being characterized in that at least one of the two collection organs is formed by a lens having a cut edge and in that the imaging system is provided with a camera and a second light source, distinct from the first light source, which is arranged and configured so as to generate a viewing beam directed towards the differentiation zone and the camera.

[0032]

[0029] Having at least one of the collection elements formed of a lens with a cut edge and illuminating the differentiation zone with a viewing beam from a second light source dedicated to viewing and distinct from the first light source dedicated to collection and detection, makes it possible to propose a particularly compact and efficient arrangement of the detection system and the viewing system of the installation, while offering the possibility of using collection elements with a large numerical aperture and positioning them close to and substantially at an equal distance from the differentiation zone.This allows for a particularly small footprint and better visualization of the jet containing the drops, themselves containing the differentiated cells, at the level of the differentiation zone, so as to determine the information representative of the moment at which a drop containing an identified cell must be generated, and then sorted, namely the information representative of the drop delay.

[0033]

[0030] In other words, the arrangement of the detection system and the imaging system is carried out so as to offer large digital apertures, close to the differentiation zone, to improve collection and therefore detection, while not impacting visualization and therefore the determination of the drop delay.

[0034]

[0031] Preferred, simple, convenient and economical features of the installation according to the invention are presented below.

[0035]

[0032] The second light radiation source can be arranged and configured so as to direct the viewing ray towards the differentiation zone and at least one of the two collection members is configured to reflect scattered rays from the viewing ray coming from the differentiation zone.

[0036]

[0033] The collection element which reflects the viewing beam may include a lens which is coated with a reflective coating.

[0037]

[0034] One of the collection elements and one of the detection elements can be arranged in a substantially orthogonal arrangement with respect to the other of the collection elements and the other of the detection elements.

[0038]

[0035] One of the collection elements and one of the detection elements can be arranged substantially in a direction of emission of the light beam, and the other of the collection elements and the other of the detection elements can be arranged in a direction substantially orthogonal to the direction of emission of the light beam.

[0039]

[0036] The imaging system may be provided with a mirror disposed between the differentiation zone and the camera.

[0040]

[0037] The camera can be disposed near the collection element arranged substantially in the direction orthogonal to the direction of emission of the light ray, between this collection element and the first light source, the mirror can be disposed near the first light source between the latter and the collection element arranged substantially in the direction orthogonal to the direction of emission of the light ray, and the second light source can be disposed near the first light source between the latter and the collection element arranged substantially in the direction of emission of the light ray, opposite the mirror with the first light source interposed between the mirror and the second light source.

[0041]

[0038] The camera can be disposed near the collection element arranged substantially in the direction orthogonal to the direction of emission of the light ray, between this collection element and the first source of light radiation, the mirror can be disposed near the first source of light radiation between the latter and the collection element arranged substantially in the direction orthogonal to the direction of emission of the light ray, and the second source of light radiation can be disposed near the collection element arranged substantially in the direction of emission of the light ray between this collection element and the first source of light radiation.

[0042]

[0039] At least one of the two collecting elements may include a lens having a cut edge located on a side where another collecting element is located.

[0043]

[0040] The collection organs may include lenses having a numerical aperture of between at least 0.6 and as close as 1.

[0044]

[0041] The first source of light radiation can be a laser, and the second source of light radiation can be a stroboscopic source.

[0045]

[0042] The detection elements are formed of photomultiplier tubes, for example of the traditional PMT type or of the SiPM type for "Silicon Photomultiplier" in Anglo-Saxon terminology. In the remainder of this document, the use of the term photomultiplier includes all types of photomultiplier tubes, including PMT and SiPM.

[0046]

[0043] The installation may include a feeding device configured to generate the cell jet and direct it to the differentiation zone, which feeding device is at least partially mobile independently of the optical detection system and the imaging system.

[0047]

[0044] The feeding device may include a chamber having a first cavity provided for receiving a sheath fluid and a second cavity extending from the first cavity, a cell injection needle which is housed at least partially in the chamber and which opens into the second cavity, at the level of which the cells and the sheath fluid meet to form the cell jet.

[0048]

[0045] The installation may include a droplet generation system configured to generate drops from the cell jet based on representative information determined by the imaging system, and a sorting system configured to apply a predetermined load to each differentiated cell in a determined droplet formed by the droplet generation system.

[0049]

[0046] The installation may further include one or more control and command units which are connected to the power supply system and / or the optical detection system and / or the imaging system and / or the drop generation system and / or the sorting system, and which are configured to process certain information representative of certain parameters in order to control and command the systems to which the control and command units are connected.

[0050]

[0047] The predetermined parameters for differentiating the plurality of cells in a jet of such cells can be the X and Y chromosomes.

[0051]

[0048] The invention also relates, according to a second aspect, to a method for processing spermatozoa-type cells by flow cytometry, particularly for sexing animal semen, using an installation as described above, comprising a step of generating the cell jet, a step of illuminating the cell jet using the first light source of the detection system at the differentiation zone, of collecting the first and second re-emitted light beams using the collection elements of the detection system, and of detecting the quantities of light re-emitted by the illuminated cells, using the detection elements of the detection system, a step of visualizing the jet containing the differentiated cells, at the differentiation zone,using the imaging system and determine the representative information of the moment at which a droplet containing an identified cell should be generated, and then sorted; a step of generating drops from the cell stream using a droplet generation system and from the determined representative information, so that in each droplet to be sorted there is one or more differentiated cells, for example of the same type; and a step of applying a predetermined load to each differentiated cell in a given droplet using a sorting system so as to sort the differentiated cells.

[0052]

[0049] The step of visualizing the differentiated cells at the level of the differentiation zone using the imaging system is carried out by generating a viewing beam from the second light source, distinct from the first light source, and directed towards the differentiation zone and the camera.

[0053] BRIEF DESCRIPTION OF THE FIGURES

[0054]

[0050] The invention, according to an exemplary embodiment, will be well understood and its advantages will become more apparent upon reading the following detailed description, given by way of example and not limiting in any way, with reference to the attached drawings.

[0055]

[0051] Figure 1 schematically and partially illustrates, in perspective, a flow cytometry installation particularly for the processing of spermatozoon-type cells and in particular for the sexing of animal semen.

[0056]

[0052] Figure 2 is a block diagram illustrating steps in a process for treating spermatozoon-type cells, using the installation of Figure 1 and for the purpose of sexing animal semen.

[0057]

[0053] Figure 3 schematically and partially illustrates, in top view, the installation of Figure 1, including a particular arrangement of a detection system and an imaging system of the installation.

[0058]

[0054] Figure 4 schematically shows a detail of the detection system of Figure 3.

[0059]

[0055] Figure 5 is similar to Figure 4, showing an alternative embodiment of the detection system.

[0056] Figure 6 is similar to Figure 3, showing an alternative embodiment of the installation.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061]

[0057] Figure 1 illustrates a flow cytometry installation 1, in particular here for the sexing of animal semen, especially bovine.

[0062]

[0058] Alternatively, it could be animal semen for animals other than cattle, for example equines, or pigs, or sheep or other animals.

[0063]

[0059] By sexing of animal semen we mean the differentiation and sorting of 26 sperm-type cells, according to the types of chromosomes they contain.

[0064]

[0060] In particular, differentiation is based on predetermined parameters to differentiate a plurality of cells 26 in a jet 1 1 of such cells, and these parameters are the X and Y chromosomes that spermatozoa may contain.

[0065]

[0061] The installation 1 described below is of the "jet-in-air sorter" type in Anglo-Saxon terminology.

[0066]

[0062] The installation 1 includes a feeding system 2 configured to generate a jet 11 from a plurality of cells 26 and direct it towards a differentiation zone 17 of the installation 1.

[0067]

[0063] The supply system 2 may include a chamber 6 having a first cavity 9 intended to receive a sheath fluid via supply conduits 8 which open into this first cavity 9, as well as a second cavity 10 extending the first cavity 9 on the side of an outlet orifice of the chamber directed towards the differentiation zone 17.

[0068]

[0064] The feeding system 2 may further include an injection needle 7 intended to receive the cells 26 via an additional feeding conduit (not shown).

[0069]

[0065] The injection needle 7 is housed at least partially in the chamber 6 and opens into the second cavity 10, where the cells 26 are distributed by this needle 7.

[0070]

[0066] It is in this second cavity 10 that the cells 26 and the sheath fluid meet to form the jet 11 of cells 26.

[0071]

[0067] It will be noted that the feeding system 2 can be configured to orient or not the cells 26 in the jet 11 of cells, that is to say to ensure that the cells 26 are in a determined position, or in any position, at the level of the differentiation zone 17.

[0068] The feeding system 2 can further include a positioning member 35 configured to move together the chamber 6 and the injection needle 7 in one or more directions of movement (see below).

[0072]

[0069] For example, it may be a positioning element 35 formed of a micrometric screw and pivot mechanism.

[0073]

[0070] Installation 1 further includes an optical detection system 3 which is configured to examine the jet 11 of cells 26 in order to differentiate the cell type.

[0074]

[0071] In particular, the optical detection system 3 is configured here to differentiate, according to the chromosomal parameters X and Y, a plurality of cells 26 in the jet 11 of such cells 26 directed towards the differentiation zone 17. It should be noted that the cells 26 to be observed can re-emit light by fluorescence to allow their differentiation, in particular with a first determined wavelength.

[0075]

[0072] The optical detection system 3 can be provided with a first light radiation source 12 configured to emit a light beam 16 focused on the differentiation zone 17. It can, for example, be a laser.

[0076]

[0073] The optical detection system 3 may include a device 13 for shaping the light beam 16 from the first light radiation source 12, hereinafter referred to as the laser source 12.

[0077]

[0074] The optical detection system 3 may further include two detection organs 14 configured to detect an amount of light re-emitted, here by fluorescence, by the cells 26 which are illuminated.

[0078]

[0075] The optical detection system 3 may also include two collection elements 15 which are focused, on a first side, on the differentiation zone 17 and on a second side opposite to the first side, on the respective detection elements 14.

[0079]

[0076] One of the collection organs 15 and one of the detection organs 14 are here arranged in the direction of emission of the light beam 16, so as to collect a first re-emitted light beam 18, substantially in the main axis of the light beam 16.

[0080]

[0077] The other of the collection organs 15 and the other of the detection organs 14 are arranged in the direction orthogonal to the direction of emission of the light beam 16, so as to collect a second re-emitted light beam 19, substantially perpendicular to the main axis of the light beam 16.

[0081]

[0078] Note that the positioning member 35 of the feeding system 2 is configured here so that the feeding system 2 is at least partially mobile independently of the rest of the installation 1, and in particular with respect to the optical detection system 3.

[0079] In particular, the positioning member 35 can be configured to move the chamber 6 and the injection needle 7 according to the direction of emission of the light beam 16 from the laser source 12.

[0082]

[0080] The positioning member 35 can also be configured to move the chamber 6 and the injection needle 7 in the direction orthogonal to the direction of emission of the light beam 16 from the laser source 12.

[0083]

[0081] Such displacements make it possible in particular to position the jet 11 of cells 26 in a determined position in the differentiation zone 17.

[0084]

[0082] The installation 1 further comprises an imaging system 40 configured to visualize the jet 1 1 comprising the differentiated cells 26 here at the level of the differentiation zone 17 and to determine representative information of a time at which a drop containing an identified cell should be generated, this representative information being called "drop delay" in Anglo-Saxon terminology.

[0085]

[0083] Installation 1 further includes a drop generation system 50 configured to generate drops from the jet 11 of cells 26 from the representative information of the drop delay determined by the imaging system 40.

[0086]

[0084] The installation 1 further includes a sorting system 4 configured to apply a predetermined load to each differentiated cell in a determined drop formed by the drop generation system 50, so as to sort the differentiated cells 26.

[0087]

[0085] It should be noted that the predetermined charge is associated with the result of the differentiation, in other words with the type of cell, X or Y.

[0088]

[0086] The arrangement of the detection system 3 and the imaging system 40 will be described in more detail below, in particular with reference to Figure 3.

[0089]

[0087] The sorting system 4 may for example include conductive plates 20 configured to form electrodes which are capable of positively or negatively charging the differentiated cells 26, depending on the result of the differentiation.

[0090]

[0088] The sorting system 4 is also configured to guide the differentiated and loaded cells 26 towards collection containers 21 configured to receive the sorted cells 26.

[0091]

[0089] In the illustrated example, the installation 1 comprises three collection containers 21 configured to receive three cell streams 26 respectively, of which a first stream 23 corresponds for example to positively charged cells identified as Y, a second stream 24 corresponds for example to negatively charged cells identified as X, and a third stream 25 corresponds to uncharged and undifferentiated cells.

[0092]

[0090] The installation 1 may further include one or more control and command units 30 which are connected to the power supply system 2, the optical detection system 3, the imaging system 40, the drop generation system 50 and the sorting system 4, and which are configured to process certain information representative of certain parameters in order to control and command the systems to which the control and command units 30 are connected.

[0093]

[0091] These parameters can, for example, correspond to the drop delay, but also to at least one parameter among a flow rate of the jet 1 1 of cells 26, including a pressure value of the substance to be sorted and / or a pressure value of the sheath fluid, a laser power value, fluorescence values ​​emitted by the illuminated cells 26, and the cell types 26 and the predetermined charges associated with them.

[0094]

[0092] Figure 2 is a block diagram illustrating steps of a process 100 of treating 26 sperm-type cells, using the installation of Figure 1 and for the purpose of sexing animal semen.

[0095]

[0093] The process 100 for processing cells 26 includes the step 101 of supplying the feeding system 2, on the one hand, with a sample of animal semen in the injection needle 7 and, on the other hand, with the sheath fluid in the first cavity 9 of the chamber 6, so as to generate the jet 11 of cells 26 in the second cavity 10 at the outlet of the feeding system 2.

[0096]

[0094] The cell treatment process 100 26 includes the step 102 of illuminating the jet 11 of cells 26 using the laser source 12 of the detection system 3 at the level of the differentiation zone 17, of collecting the first and second re-emitted light beams 18, 19 using the collection elements 15 of the detection system 3, and of detecting the quantities of re-emitted light, here by fluorescence, by the illuminated cells 26, using the detection elements 14 of the detection system 3.

[0097]

[0095] The cell treatment process 100 26 includes the step 103 of visualizing the jet 1 1 comprising the differentiated cells 26, here at the level of the differentiation zone 17, using the imaging system 40 and determining the representative information of the drop delay using the control and command units 30.

[0098]

[0096] The cell treatment process 100 includes the step 104 of generating drops from the jet 11 of cells 26 using the drop generation system 50 and from the representative information of the drop delay, so as to have in each drop to be sorted one or more differentiated cells 26 for example of the same type.

[0099]

[0097] The cell treatment process 100 comprises step 105 of applying a predetermined load to each differentiated cell 26 in a determined drop using the sorting system 4 so as to sort the differentiated cells 26.

[0100]

[0098] Figure 3 shows in more detail an example of the arrangement of the detection system 3 and the imaging system 40.

[0099] The detection system 3 here comprises the two collection elements 15 which are focused, on one side, on the differentiation zone 17 and on a second side opposite the first side, on the two respective detection elements 14, with one of the collection elements 15 and one of the detection elements 14 which are arranged in the direction of emission of the light beam 16 and therefore substantially in the main axis of the light beam 16, and with the other of the collection elements 15 and the other of the detection elements 14 which are arranged in the direction orthogonal to the direction of emission of the light beam 16 and therefore substantially perpendicular to the main axis of the light beam 16, which is generated by the laser source 12 and the shaping device 13.

[0101]

[0100] The imaging system 40 here comprises a camera 41 and a second light radiation source 42 arranged to generate a viewing beam 43, here a stroboscopic beam, directed towards the differentiation zone 17.

[0102]

[0101] The viewing beam 43 can be partially scattered from the differentiation zone 17 to one of the collection organs 15 and then reflected by the latter, unlike the light re-emitted by fluorescence by the cells 26 illuminated by the light beam 16 from the first light radiation source 12, which is not reflected but collected.

[0103]

[0102] In particular here, the second light radiation source is distinct from the first light radiation source 12 and is formed by a stroboscopic source 42.

[0104]

[0103] The stroboscopic source 42 is here offset relative to the laser source 12, with the light beam 16 and the stroboscopic beam 43 therefore not coinciding.

[0105]

[0104] The imaging system 40 further includes a mirror 44 disposed in the direction of the stroboscopic ray 43 coming from the differentiation zone 17, or scattered ray.

[0106]

[0105] Thus, thanks to the stroboscopic beam 43 directed on the differentiation zone 17, the imaging system 40 is configured to visualize the jet 11 comprising the cells 26 differentiated here at the level of the differentiation zone 17 and determine the information representative of the drop delay.

[0107]

[0106] It should be noted that the stroboscopic source 42 and the mirror 44 are removable and repositionable, to adapt their respective positions to the arrangement in particular of the collection organs 15.

[0108]

[0107] As explained above, the scattered rays from the stroboscopic ray 43 towards the collection organ(s) 15 are reflected, unlike the light re-emitted by fluorescence by the cells 26 illuminated by the light ray 16 from the first light radiation source 12, which is not reflected but collected.

[0109]

[0108] In the illustrated example, the camera 41 is, for example, disposed near the collection element 15 arranged in the direction orthogonal to the direction of emission of the light beam 16, between this collection element 15 and the laser source 12; the mirror 44 is disposed near the laser source 12 between the latter and the collection element 15 arranged in the direction orthogonal to the direction of emission of the light beam 16; and the stroboscopic source 42 is disposed near the laser source 12 between the latter and the collection element 15 arranged in the direction of emission of the light beam 16, opposite the mirror 44 with the laser source 12 interposed between the mirror 44 and the stroboscopic source 42.

[0110]

[0109] Figure 4 schematically illustrates the collection organs 15 of the detection system 3 of Figure 3.

[0111]

[0110] The collection organ 15 disposed in the direction of emission of the light ray 16, on the right of the figure 4, is provided with a lens 60 having a cut edge 61 and having here a numerical aperture of about 0.83.

[0112]

[0111] The collecting organ 15 arranged in the direction orthogonal to the direction of emission of the light ray 16, at the top of the figure 4, is provided with a lens 62, with uncut edges, having here a numerical aperture of about 0.6.

[0113]

[0112] The cut edge 61 of the lens 60 is located on the side of the lens 62.

[0114]

[0113] The cut edge 61 is formed of a face inclined with respect to an external face 64 of the lens 60 which is opposite the differentiation zone 17.

[0115]

[0114] Thus, the lens 60 has a portion called oblique between its external face 64 and an internal face 66 opposite to the external face 64.

[0116]

[0115] Such an arrangement of lenses 60 and 62 not only allows for large numerical apertures while positioning the lenses close to and substantially equidistant from the differentiation zone, in a particularly small footprint.

[0117]

[0116] It should be noted that in this example of arrangement, lens 62 is smaller, i.e. less wide, than lens 60.

[0118]

[0117] In addition, the lens 60 is coated with a reflective coating 65 covering the outer face 64, the cut edge 61 and the inner face 66 opposite the outer face 64 and joining the cut edge 61.

[0119]

[0118] The reflective coating 65 is configured to reflect scattered rays from the stroboscopic ray 42 coming from the differentiation zone 17, while allowing the collection of light reflected by fluorescence by the cells 26 illuminated by the light ray 16.

[0120]

[0119] In other words, the arrangement of the detection system 3 and the imaging system 40 is designed to provide large numerical apertures near the differentiation zone to improve collection, and therefore detection, without impacting visualization and thus the determination of the drop delay.

[0120] Figure 5 shows an alternative embodiment of the collection elements 15 of the detection system 3.

[0121]

[0121] The collection organ 15 disposed in the direction of emission of the light ray 16, on the right of the figure 5, is provided as in the figure 4, with a lens 60 having a cut edge 61, with a numerical aperture of about 0.83 and a coating 65 reflecting the scattered rays of the stroboscopic ray 43 only and covering the external face 64, the cut edge 61 and the internal face 66 of the lens 60.

[0122]

[0122] The collection organ 15 arranged in the direction orthogonal to the direction of emission of the light ray 16, at the top of the figure 4, is itself provided with a lens 70 also with a cut edge 71, located opposite the cut edge 61 of the lens 60, and having here a numerical aperture of about 0.83, like the lens 60.

[0123]

[0123] Such an arrangement of lenses 60 and 70 also allows for an even larger numerical aperture on lens 70 while positioning the lenses close to and substantially equidistant from the differentiation zone, in the same particularly small footprint.

[0124]

[0124] It should be noted that in this example of arrangement, lens 70 is overall the same size as lens 60.

[0125]

[0125] In figure 5, lens 70 is also devoid of reflective coating.

[0126]

[0126] Alternatively, both lenses 60 and 70, or 60 and 62, may be without reflective coating.

[0127]

[0127] To this end, Figure 6 shows an alternative embodiment of the imaging system 40, where the detection system 3 comprises the two collection elements 15 which are focused, on one side, on the differentiation zone 17 and on a second side opposite to the first side, on the two respective detection elements 14, with one of the collection elements 15 and one of the detection elements 14 which are arranged in the direction of emission of the light beam 16 and therefore substantially in the main axis of the light beam 16, and with the other of the collection elements 15 and the other of the detection elements 14 which are arranged in the direction orthogonal to the direction of emission of the light beam 16 and therefore substantially perpendicular to the main axis of the light beam 16, which is generated by the laser source 12 and the shaping device 13.

[0128]

[0128] In addition, the imaging system 40 includes the camera 41 and the stroboscopic source 42 which is arranged here to generate the stroboscopic beam 43 directed towards the differentiation zone 17, which is visible from the camera 40 using the mirror 44.

[0129]

[0129] The stroboscopic source 42 is here offset with respect to the direction orthogonal to the direction of emission of the light ray 16.

[0130] In the illustrated example, the camera 41 is therefore disposed near the collection organ 15 arranged in the direction orthogonal to the direction of emission of the light beam 16, between this collection organ 15 and the laser source 12; the mirror 44 is disposed near the laser source 12 between the latter and the collection organ 15 arranged in the direction orthogonal to the direction of emission of the light beam 16; and the stroboscopic source 42 is disposed near the collection organ 15 arranged in the direction of emission of the light beam 16 between this collection organ 15 and the laser source 12.

[0130]

[0131] As in the arrangement of Figure 6, the two collection elements 15 of the detection system 3 may include lenses such as those described in reference to Figures 4 and 5, but both without a reflective coating, since the risk of scattering the stroboscopic ray towards the collection elements 15 is reduced or eliminated.

[0131]

[0132] Thus, such an arrangement also allows for lenses with numerical apertures of at least 0.6 while positioning the lenses close to and roughly equidistant from the differentiation zone, in a particularly small footprint.

[0132]

[0133] In addition to offering large digital openings, close to the differentiation zone to improve collection, and therefore detection, such an arrangement does not impact visualization and therefore the determination of drop delay.

[0133]

[0134] Variants not shown are described below.

[0134]

[0135] The lenses of collection organs can have a numerical aperture of at least 0.6 and more generally between 0.5 and as close as 1.

[0135]

[0136] The imaging system can be mirrorless if the camera is directly opposite the differentiation zone.

[0136]

[0137] Depending on the positioning of the stroboscopic source, one or both lenses may be provided with a reflective coating to reflect the scattered rays of the stroboscopic ray coming from the differentiation zone.

[0137]

[0138] The stroboscopic source here is a source with a determined lighting frequency allowing the visualization, in a stationary manner, of the jet containing the drops containing the droplet cells, and may for example include light-emitting diodes, or alternatively a laser.

[0138]

[0139] The stroboscopic source here has a wavelength greater than the first determined wavelength, and the coating covering one or both lenses is configured to reflect only the light from the stroboscopic source.

[0140] The detection system may include more than two collection elements, which may be arranged differently, for example at less than or more than 90° to each other and to the light beam.

[0139]

[0141] More generally, the invention is not limited to the examples described and shown.

Claims

Demands 1. Flow cytometry installation, in particular for the processing of spermatozoon-type cells and in particular for the sexing of animal semen, comprising an optical detection system (3) configured to differentiate, according to predetermined parameters, a plurality of cells (26) in a jet of such cells (11) directed towards a differentiation zone (17), and an imaging system (40) configured to visualize the jet containing the differentiated cells at the differentiation zone and to determine representative information of a time at which a drop containing an identified cell should be generated, and then sorted;with the optical detection system (3) which is provided with a first light radiation source (12) configured to emit a light beam (16) focused on the differentiation zone (17), at least two detection elements (14) configured to detect each a quantity of light re-emitted by the cells (26) which are illuminated in different directions of re-emission, and at least two collection elements (15) which are focused, on a first side, on the differentiation zone (17) and on a second side opposite to the first side, on the respective detection elements (14);the installation being characterized in that at least one of the two collection organs (15) is formed by a lens (60, 70) having a cut edge (61, 71) and in that the imaging system (40) is provided with a camera (41) and a second light radiation source (42), distinct from the first light radiation source (12), which is arranged and configured so as to generate a viewing ray (43) directed towards the differentiation zone (17) and the camera (40).

2. Installation according to claim 1, characterized in that the second light source (42) is arranged and configured so as to direct the viewing ray (43) towards the differentiation zone and at least one of the two collection members (15) is configured to reflect scattered rays of the viewing ray (43) coming from the differentiation zone (17).

3. Installation according to claim 1, characterized in that the collection element (15) which reflects the viewing ray (43) comprises a lens (60) which is coated with a reflective coating (65).

4. Installation according to any one of claims 1 to 3, characterized in that one of the collection members (15) and one of the detection members (14) are arranged in a substantially orthogonal arrangement with respect to the other of the collection members (15) and to the other of the detection members (14).

5. Installation according to any one of claims 1 to 4, characterized in that one of the collection members (15) and one of the detection members (14) are arranged substantially in a direction of emission of the light beam (16), and the other of the collection members (15) and the other of the detection members (14) are arranged substantially in a direction orthogonal to the direction of emission of the light beam (16).

6. Installation according to any one of claims 1 to 5, characterized in that the imaging system (40) is provided with a mirror (44) disposed between the differentiation zone (17) and the camera (40).

7. Installation according to claims 5 and 6, characterized in that the camera (41) is disposed near the collection element (15) disposed substantially in the direction orthogonal to the direction of emission of the light ray (16), between this collection element (15) and the first light source (12), the mirror (44) is disposed near the first light source (12) between the latter and the collection element (15) disposed substantially in the direction orthogonal to the direction of emission of the light ray (16), and the second light source (42) is disposed near the first light source (12) between the latter and the collection element (15) disposed substantially in the direction of emission of the light ray (16), opposite the mirror (44) with the first light source (12) interposed between the mirror (44) and the second light source (42).

8. Installation according to claims 5 and 6, characterized in that the camera (41) is disposed near the collecting element (15) disposed substantially in the direction orthogonal to the direction of emission of the light ray (16), between this collecting element (15) and the first light source (12), the mirror (44) is disposed near the first light source (12) between the latter and the collecting element (15) disposed substantially in the direction orthogonal to the direction of emission of the light ray (16), and the second light source (42) is disposed near the collecting element (15) disposed substantially in the direction of emission of the light ray (16) between this collecting element (15) and the first light source (12).

9. Installation according to any one of claims 1 to 8, characterized in that at least one of the two collection members (15) comprises a lens (60, 70) having a cut edge (61, 71) located on a side where another collection member (15) is located.

10. Installation according to any one of claims 1 to 9, characterized in that the collecting organs (15) comprise lenses (60, 70) having a numerical aperture of between at least 0.6 and as close as 1.

11. Installation according to any one of claims 1 to 10, characterized in that the first light radiation source (12) is a laser, and the second light radiation source (42) is a stroboscopic source.

12. Installation according to any one of claims 1 to 11, characterized in that the detection elements (14) are formed of photomultipliers.

13. Installation according to any one of claims 1 to 12, comprising a feeding device (2) configured to generate the cell jet (11) and direct it to the differentiation zone (17), which feeding device (2) is at least partially mobile independently of the optical detection system (3) and the imaging system (40).

14. Installation according to claim 13, characterized in that the feeding device (2) comprises a chamber (6) having a first cavity (9) provided for receiving a sheath fluid and a second cavity (10) extending from the first cavity (9), an injection needle (7) of the cells (26) which is housed at least partially in the chamber (6) and which opens into the second cavity (10), at the level of which the cells (26) and the sheath fluid meet to form the cell jet (11).

15. Installation according to any one of claims 1 to 14, comprising a droplet generation system (50) configured to generate droplets from the jet (11) of cells (26) from the representative information determined by the imaging system (40), and a sorting system (4) configured to apply a predetermined load to each differentiated cell in a determined droplet formed by the droplet generation system (50).

16. Installation according to any one of claims 1 to 15, characterized in that the predetermined parameters for differentiating the plurality of cells (26) in a batch of such cells (11) are the X and Y chromosomes.

17. A method for processing, in particular, sperm cells and especially for sexing animal semen, using an installation according to any one of claims 1 to 16, comprising a step (101) of generating the jet (11) of cells (26), a step (102) of illuminating the jet (11) of cells (26) using the first light source (12) of the detection system (3) at the differentiation zone (17), of collecting the first and second re-emitted light beams (18, 19) using the collection elements (15) of the detection system (3), and of detecting the quantities of light re-emitted by the illuminated cells (26) using the detection elements (14) of the detection system (3), a step (103) of visualizing the jet (11) comprising the cells (26) differentiated, at the level of the differentiation zone (17),using the imaging system (40) and determine the representative information of the time at which a droplet containing an identified cell should be generated, and then sorted, a step (104) of generating drops from the jet (11) of cells (26) using a droplet generation system (50) and from the determined representative information, so as to have in, each droplet to sort one or more differentiated cells (26), for example of the same type, and a step (105) of applying a predetermined load to each differentiated cell (26) in a given droplet using a sorting system (4) so ​​as to sort the differentiated cells (26).

18. Method according to claim 17, characterized in that the step of visualizing the differentiated cells (26) at the differentiation zone (17) using the imaging system (40) is carried out by generating a viewing beam (43) from the second light source (42), separate from the first light source (12), and directed towards the differentiation zone (17) and the camera (40).

Images