ELECTRO-OPTIC FLOW MEASURING DEVICE

Description

[0001] The invention relates to the field of electro-optical flow measurement devices for the characterization of microparticles, particularly biological cells, comprising a measurement chamber through which the flow of the fluid to be characterized circulates and which contains the cells to be characterized. This field relies on the use of analytical methods based on electrical and optical measurements to count and differentiate the cells present in a sample to be analyzed.

[0002] The present invention relates more specifically to a multiparameter electro-optical device for cell counting and characterization. The fluid to be characterized is preferably a blood sample, but it can also be a biological fluid of another nature such as cerebrospinal fluid, urine, pleural fluid, synovial fluid, a cell suspension, bone marrow, etc. The sample may also contain particles of any kind (cells, proteins, biomarkers, etc.) that will need to be differentiated and counted.

[0003] More specifically, the invention relates to devices comprising at least two light sources, a resistivity or impedance measurement device and several detectors enabling the measurement of optical parameters, typically extinction measurements, large angle diffraction measurements and fluorescence measurements.

[0004] These measurements allow for the characterization and counting of biological cells or particles present in the fluid.

[0005] Electrical impedance measurements allow for particle counting and provide information about their sizes.

[0006] Optical parameters (refraction, scattering, absorption, and reflection) allow the extraction of morphological information from cells, such as shape, volume, size, and internal structure. Light sources (such as lasers, halogen lamps, or LEDs) generate light that is focused by lenses and illuminates biological cells moving through a measurement chamber. Upon contact with the biological cell, the light interacts with it. Along the incident axis of the light beam, the light is collected by several lenses and can be spatially filtered by diaphragms for detection by a photodiode detector. This measurement, within the chosen range of angles, provides an indication of the size of the biological element combined with structural information. Furthermore, another measurement can be performed along the incident axis of the light beam.Incident light is blocked by a beam stopper, and the signals scattered by the cells are detected by a photodiode sensor to generate the forward scatter (FSC) measurement. This measurement, within the chosen angle range, provides an indication of the size of the biological element and / or can trigger the measurement for small particles. Another portion of the light is collected orthogonally and passes through another set of lenses and a set of semi-reflective mirrors to be measured by a sensor to generate the side scatter (SSC) signal. This orthogonal light measurement provides an indication of the density of the biological element, as well as its granularity (structure) or its intracellular content.

[0007] Fluorescence measurement is used to detect fluorescent dyes used as cell markers or as molecular probes specific to a biological structure or function. For example, if an antibody specific to a certain cell class is coupled to a fluorochrome, this cell class can be detected, allowing for precise characterization and cell counting. Using multiple fluorescence patterns simultaneously allows for a more detailed characterization of the biological cells of interest.

[0008] When the fluid is a blood sample, the formed elements of the blood are determined quantitatively (count) and qualitatively (differential) by the blood cell count. These elements are the red blood cell (or erythrocyte) lineages, the white blood cell (or leukocyte) lineages, and the platelet (or thrombocyte) lineages. This analysis is called a complete blood count (CBC). Abnormalities in the CBC can alert the doctor to underlying pathologies (anemia, cancer, etc.).

[0009] All these cells originate from the same stem cell located in the bone marrow, called a hemocytoblast. These stem cells then differentiate into several subpopulations.

[0010] Thus, in the case of hematopoietic cells, experts know that cell analysis, obtained by impedance, diffraction, or absorption spectroscopy, allows for the differentiation of the main cell lineages, including erythrocytes (red blood cells), thrombocytes (platelets), and leukocytes (white blood cells). The latter population is itself subdivided into several categories, such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Blood is normally composed of mature cells that no longer divide.

[0011] Cell counting and differentiation can be performed by simultaneously determining the volume and apparent extinction under white light, as described in US patent 5,138,181 filed by the Applicant. A device developed in an embodiment under quasi-monochromatic light is, for example, described in patent WO 2006 / 053960.

[0012] Other exemplary devices are described in US 2013 / 308122 and US 2018 / 348112 A1.

[0013] For each of the cell types mentioned above, the different stages of maturation are known. Thus, red blood cells, also called erythrocytes, are first produced as proerythroblasts, then as basophilic erythroblasts, then as polychromatophilic erythroblasts, which develop into acidophilic erythroblasts, and finally into reticulocytes, which are formed after the nucleus of the acidophilic erythroblast is expelled. It is these reticulocytes that, after complete loss of residual RNA, differentiate into erythrocytes in the circulating blood.

[0014] White blood cells or leukocytes originate from the bone marrow in the form of myeloblasts, which will then give rise to pro-granulocytes, which will then transform into basophilic, eosinophilic or neutrophilic granulocytes, initially unsegmented, but whose nuclei will increasingly segment as they mature.

[0015] These myeloblasts are also the origin of the monocytic lineage which will give rise to monoblasts, promonocytes and then monocytes which will pass into the peripheral blood.

[0016] The pluripotent stem cell from which the myeloblast originates also gives rise to the lymphocyte lineage through differentiation into lymphoid stem cells, part of which, the T lymphocyte lineage, will continue its maturation in the thymus and lymph nodes, and the other part will remain in the bone marrow to give rise to the B lymphocyte lineage. These B lymphocytes, once activated in the form of plasma cells, produce antibodies to fight pathogenic antigens.

[0017] Blood platelets, or thrombocytes, originate from megakaryoblasts, which themselves derive from the myeloid progenitor cell from which the myeloblast originates. Once these megakaryoblasts reach their final stage of maturation as thrombocytogenic megakaryocytes, they produce platelets through the breakdown of their cytoplasm. Young platelets, known as reticulated platelets, contain a charge of RNA, a remnant of their original cell.

[0018] The diagnosis of certain diseases requires increasingly precise enumeration and characterization of hematopoietic cells in circulating blood. In particular, it is becoming necessary to be able to identify specific populations such as reticulocytes and erythroblasts, which are the immature versions of erythrocytes. Similarly, the identification of immature cells, precursors of leukocytes, called lymphocytes, monocytes, or immature granulocytes, is of great importance. The classification and counting of activated lymphocytes or reticular platelets would also significantly improve patient diagnosis.

[0019] Specific fluorescent labeling of cells provides high specificity and enables the detection of immature or atypical cells. Different types of lymphocytes can be precisely characterized and counted using specific antibodies coupled to a fluorochrome. Other cells abnormally found in the blood, such as atypical lymphocytes or immature precursor cells, can also be detected after fluorescence labeling. Similarly, cross-linked platelets can be specifically labeled with a fluorochrome.

[0020] To better differentiate each of the particle populations contained in a sample, the Applicant proposed an electro-optical flow measurement device in patent application FR 2 971 337. This device offers numerous advantages. However, due to its configuration, this device is complicated to adjust and offers very limited access to the measuring cell.

[0021] The invention improves the situation.

[0022] To this end, the invention proposes an electro-optical flow measurement device comprising a measurement tank in which a flow of fluid to be characterized circulates, at least two light emission guns presenting disjoint spectra, a trigger gun allowing the measurement of diffraction at small angles and a receiving gun allowing to perform an extinction measurement and at least one fluorescence.

[0023] This electro-optical device is such that: The first emission gun comprises a light source having a central wavelength greater than 580nm and defining a principal optical axis perpendicular to the fluid flow; the second emission gun comprises a second light source, having a central wavelength less than 580nm and defining a secondary optical axis substantially orthogonal to the principal optical axis and to the fluid flow; the first and second emission guns are arranged on one side of the measuring tank; the receiving gun is arranged on the other side of the measuring tank along the principal optical axis; and the triggering gun is arranged on the other side of the measuring tank along the secondary optical axis; the receiving gun comprises a detection channel for measuring extinction and at least one detection channel for measuring at least one fluorescence signal.as well as a single beam collection objective for the beam resulting from the interaction of the light beam between the first emission gun and the second emission gun and the particles in the fluid flow, which collection objective is arranged so that the light beam it transmits is a beam substantially collimated along the principal optical axis towards the detection channel to measure extinction, the receiving gun forming a single mechanical block at least part of which is movable relative to the measuring tank, the receiving gun further comprising a first dichroic mirror placed downstream of the collection objective where the light beam is substantially collimated, arranged to partially transmit the light beam resulting from the interaction between the first emission gun and the particles in the fluid flow towards the detection channel to measure extinction,and to partially reflect the light beam resulting from the interaction between the second emission gun and the particles in the fluid flow towards at least one detection channel to measure at least one fluorescence signal.

[0024] This device is advantageous because it offers unobstructed access to the measuring tank, as well as easy adjustment thanks to the receiving nozzle being a single mechanical unit. Furthermore, it offers increased robustness compared to the device described in patent application FR 2 971 337.

[0025] In various versions, the invention may have one or more of the following features: The receiving gun includes a detection channel for measuring wide-angle diffraction and a second dichroic mirror placed downstream of the collecting objective and arranged to partially reflect the light beam from the interaction between the second emitting gun and the particles in the fluid flow towards at least one of the following: at least one detection channel for measuring fluorescence and the detection channel for measuring wide-angle diffraction; the detection channel for measuring extinction includes a detector arranged to measure an amount of red light; at least one detection channel for measuring fluorescence includes a detector arranged to measure an amount of green light and an amount of orange or near-infrared light; and the detection channel for measuring wide-angle diffraction includes a detector arranged to measure an amount of blue light.The detector arranged to measure a quantity of red light is a photodiode, the detector arranged to measure a quantity of green light is a photomultiplier or a silicon photomultiplier, and the photodetector arranged to measure a quantity of blue light is a photodiode. The receiving gun is a single piece and is fully movable relative to the measuring cell. The second dichroic mirror is placed downstream of the first dichroic mirror in the path of the reflected beam and is arranged to partially reflect the beam towards the detection channel to measure fluorescence and to partially transmit it towards the detection channel to measure diffraction at large angles. The detection channel is used to measure extinction.one or more detection channels for measuring one or more fluorescence signals and the detection channel for measuring wide-angle diffraction each comprise optics followed by a diaphragm upstream of their respective detector; the collection objective comprises two lenses, one of which is movable relative to the measuring cuvette, the remainder of the receiving barrel not being movable relative to the measuring cuvette, and a diaphragm disposed downstream of the two lenses; the second dichroic mirror is placed upstream of the first dichroic mirror in the path of the reflected beam and is arranged to partially reflect the beam towards the detection channel for measuring wide-angle diffraction and to partially transmit it towards the first dichroic mirror; the first emission barrel comprises a first emission source which is a red LED,The second emitting gun comprises a second emission source, which is a blue laser; the device further comprises one or more adjustment elements arranged to allow adjustment of the device by displacement of all or part of the receiving gun and measurement in the detection channel; and the device further comprises one or more mirrors arranged to offset one or more substantially collimated beams.

[0026] Other features and advantages of the invention will become clearer upon reading the following description, drawn from illustrative and non-limiting examples taken from the drawings shown: [ Fig 1 ] represents a first embodiment of a device according to the invention, [ Fig 2 ] represents a second embodiment of a device according to the invention, [ Fig 3 ] represents a third embodiment of a device according to the invention.

[0027] The drawings and description below contain, for the most part, elements of a definite nature. They can therefore not only serve to better explain the present invention, but also contribute to its definition, if necessary.

[0028] There figure 1 represents a first embodiment of a device according to the invention. Device 2 includes a first emission gun 4 and a second emission gun 6 arranged on one side of a measuring tank 8, a trigger gun 9 and a receiving gun 10 arranged on the other side of the measuring tank 8. The receiving gun 10 includes a collection lens 11, three detection channels referenced 12, 14 and 16, as well as dichroic mirrors 18 and 20 described below allowing the light beam propagating in the measuring tank 8 to be separated into several light beams having different wavelengths (in the example described here red, blue, green) and specific to each of the three detection channels 12, 14 and 16. The detection channel 12 is used to measure extinction, the detection channel 14 is used to measure fluorescence, and the detection channel 16 is used to measure 90° diffraction (also called SSC).

[0029] In the example described here, the first emission gun 4 includes a first emission source 40, a shaping optic 42 and a reticle 44 and a focusing optic 45 which serves to reduce the size of the rectangle from the reticle 44 which is projected onto the measuring tank 8.

[0030] The first emission source 40, in the example described here, is an Epitex SMB660NR-1100 red LED and has an intensity distribution close to that of a Lambertian source, allowing for the desired uniformity. The emission spectrum of LED 40 is broad and centered at 660 nm. The LED 40 chip has an active area of ​​1 x 1 mm². The optical power delivered by LED 40 at the output of the first emission gun 4, i.e., at the center of the measuring cell 8, is 45 µW. Generally speaking, this first emission source exhibits an emission spectrum with a central wavelength greater than 580 nm. Conversely, as will be seen below, the second emission source exhibits an emission spectrum with a central wavelength less than 580 nm. This dichotomy is made in order to be able to separate a measurement in the red and a measurement in the blue or green.

[0031] The shaping optics 42, as described here, includes two collecting lenses located between the LED 40 and the reticle 44. These two lenses project the aperture diaphragm, ensuring uniformity of the beam illuminating the biological cell stream propagating in the measuring cuvette 8. To account for the fact that the spectrum of the LED 40 overlaps part of the fluorescence emission spectrum of Thiazole Orange, and to limit the resulting noise on the fluorescence measurement detection channel 14, a colored or interference filter can optionally be placed between the two lenses of the shaping optics 42, where the light beams are collimated. This cuts off the green component of the LED 40.

[0032] The reticle 44 in the example described here is rectangular in shape (150 x 500 µm). Thus, in combination with the LED 40 and the shaping optics 42, the paraxial magnification of the system up to the cells is 0.172. The paraxial image of the reticle 44 on the cell stream is therefore a rectangle of 86 µm x 25.8 µm (500 µm x 0.172 and 150 µm x 0.172, respectively). On the other hand, the simulation on ZEMAX shows that a system " réel » A perfect image (limited by diffraction) should yield a 90 µm x 28.8 µm spot (size obtained with Zemax Cross X and Cross Y at the curve foot). Experimental measurements of the image dimensions are very close to those produced by a " réel » Perfect.

[0033] At the measuring tank 8, the beam from a point on LED 40 (corresponding to the image of LED 40 and without a reticle) is collimated, meaning that the aperture diaphragm is projected to infinity. This was achieved by aligning its image with the focal point of the system.

[0034] The first emission gun 4, in conjunction with the detection gun 12, measures the extinction of an LED beam around 660nm by moving biological cells. This measurement is enhanced by precise control of the beam shape and the uniformity of illumination at the cell level.

[0035] In the example described here, the detection gun 12 for measuring extinction is located downstream of the dichroic mirror 18 of the receiving channel 10, and includes a focusing lens 122, a diaphragm 124 and a detector 126.

[0036] In the example described here, the dichroic mirror 18 is a Semrock FF605-Di02 filter, which transmits to the detection channel 12 the portion of the beam collimated by the collecting lens 11 that has a wavelength greater than 605 nm, while the other portion of the beam collimated by the collecting lens 11 that has a wavelength less than 605 nm is reflected to the detection channel 14 and the detection channel 16. The dichroic mirror 18 described here transmits 98% of the red while reflecting 99% of the blue and green. According to the specifications of the dichroic mirror 18, the collecting lens 11 collimates the beam from the measuring cell 8, so that it strikes the filter 18 at a half-angle of less than 2°.

[0037] In the example described here, the bandpass filter 120 is a Semrock FF01-655 / 40 bandpass interference filter that transmits only the red wavelengths (from 635 nm to 675 nm) from LED 40. In this example, the focusing lens 122 focuses the light beam into the diaphragm 124, which is 1 mm in diameter and positioned 22 mm from the lens. The focusing lens 122 in this example is a custom-made model. This plano-convex lens has a radius of curvature of 12.42 mm and is made of N-BK7 material. However, other lenses can be used. Finally, the detector 126 in this example is a Hamamatsu S1223 photodiode located 6.75 mm from the diaphragm 124 and therefore from the focal point.The half-angle of the beam entering the detection channel 12 is less than the 7° prescribed for the bandpass filter 120, and the numerical aperture of this beam at the output of the measuring tank is limited to 0.31 by means of a diaphragm in the receiving barrel 12.

[0038] In the example described here, the second emission gun 6 includes a second emission source 60, and a shaping optic 62.

[0039] The second emission source 60 is in the example described here a blue laser source which includes a 50mW laser diode emitting at 488nm from the Osram brand, whose output is shaped by anamorphic lenses so that the beam is elliptical at the output of the laser source 60.

[0040] The shaping optics 62, as described here, include a spherical lens with a focal length of 75 mm, for example, a Thorlabs AC127-075-A doublet. Having a lens with a relatively long focal length allows for a greater depth of field at the interaction between biological cells and the light beam. The ellipse then has a size of approximately 200 µm x 30 µm (at 1 / e²) in the measuring cuvette and is vertically polarized.

[0041] The laser source 60 has the advantage of being at the focal point within the cell beam of the measuring tank 8, resulting in superior beam quality and improved reproducibility. Furthermore, this allows for high tolerances in lateral and longitudinal positioning. Lateral positioning is facilitated by the length of the laser beam within the measuring tank (flattened ellipse), while the longitudinal tolerance depends on the distance between the laser and the measuring tank.

[0042] The laser source 60 is used by the fluorescence measurement detection channel 14 and the 90° diffraction measurement detection channel 16. These two detection channels process the portion of the beam collimated by the collection lens 11 that has a wavelength below 605 nm and is reflected by the dichroic mirror 18. The dichroic mirror 20 is positioned downstream of the dichroic mirror 18 in the beam path used to split the beam in two. In the example described here, the mirror 20 is a Semrock FF506-Di03 filter that reflects wavelengths below 506 nm and transmits wavelengths above 506 nm.

[0043] Thus, the fluorescence detection channel 14 measures wavelengths in the green range emitted by the fluorescence of Thiazole Orange, which labels the nucleic acids of biological cells. The detection channel 14 is positioned along the axis of the beam reflected by the dichroic mirror 18. The detection channel 14 comprises a lens doublet 140, a diaphragm 142, and a detector 144. The lens doublet 140 focuses the beam transmitted by the dichroic mirror 20 onto the diaphragm 142, which has a diameter of 1.5 mm. The diaphragm 142 provides spatial filtering and enables a high signal-to-noise ratio. The fluorescence signal is then measured by the detector 144, which in this example is a Hamamatsu H10723 photomultiplier tube. The numerical aperture of the detection channel 14 is 0.6.

[0044] The part of the beam that is reflected by the dichroic mirror 20 is directed to the detection channel 16 for the measurement of 90° diffraction from the laser-biological cell interaction in the blue.

[0045] The detection channel 16, in the example described here, comprises a focusing lens 160, a diaphragm 162, and a detector 164. The focusing lens 160 is, in this example, a Thorlabs LA-1270-A reference lens, and the diaphragm 162 has a diameter of 1.5 mm, while the photodiode 164 is a Hamamatsu S1223 photodiode. Here again, the diaphragm 162 provides spatial filtering and enables a high signal-to-noise ratio.

[0046] As mentioned above, the collection lens 11 downstream of the measuring cell 8 is common to the detection channels 12, 14, and 16. In the example described here, the collection lens 11 has a relatively large numerical aperture of 0.6, in order to collect as much flux as possible from the measuring cell 8. The focusing distance of this lens is chosen to be sufficiently long to facilitate access to the measuring cell (the distance between the measuring cell and the doublet being 5.2 mm in the example described here). In the example described here, the collection lens 11 is a doublet of lenses, which also helps to limit chromatic aberrations given the wide range of wavelengths present (from 488 nm to 700 nm).

[0047] The adjustment of the receiving barrel 10 is carried out with a diaphragm of diameter of 0.3mm (not shown on the figure 1 ), which is introduced in place of the diaphragm 124 of the detection barrel 12. The use of a small diameter diaphragm (0.3 mm) allows for high adjustment accuracy. The optimum position of the receiving barrel 10 corresponds to the maximum intensity detected by the photodiode 126. To achieve this, the position of the measuring cell 8 relative to the receiving barrel 10 is adjusted along three axes by means of a moving device for the mechanical block that houses all the elements of the receiving channel 10. This allows the three detectors to be adjusted with a single precise adjustment. This adjustment is simplified since it uses the same detector as the extinction detector 126. This avoids the need for bulky equipment such as a camera, which is particularly advantageous for after-sales service personnel.

[0048] Optionally, dedicated bandpass filters for extinction 120 (Red), 90° diffraction (SSC) (Blue) and fluorescence (Green) with respective transmissions of 85%, 90% and 96% can be added to the respective detection channels 12, 14 and 16. Thus, taking into account the characteristics of the dichroic mirrors 18 and 20, for a reflection of 1% on the lens interfaces, the transmission is 78% in extinction, 83% in diffraction and 86% in fluorescence.

[0049] In extinction, the percentage of stray light is 2 x 10⁻⁶% in both blue and green. In 90° diffraction, this percentage is 2 x 10⁻⁶% in red and 9 x 10⁻³% in green. Since the fluorescence signal is weak, the gain of the 144 photomultiplier tube is quite high; therefore, it is important to consider the stray light in this channel, particularly the blue light associated with the laser, which is 3 x 10⁻⁴% in both blue and red.

[0050] The trigger channel 9 includes a beam blocker 90 and a detector 92. The on-axis signal detection at 488 nm is used as the trigger signal in the example described here. The trigger channel 9 includes a beam blocker to suppress the incident laser beam. In the example described here, the beam blocker is a vertical bar 0.8 mm wide, located 5.5 mm from the measuring cell 8, on the axis of the laser source 60. Its width is sufficient to block the laser beam from the source 60, whose size on the horizontal axis is quite stable and remains below 300 µm. The detector 92, in the example described here, is a Hamamatsu S1223 photodiode, which allows for small-angle diffraction (SAD) measurements.

[0051] The Applicant's clinical studies have demonstrated that this device allows for leukocyte differentiation: LMNE (lymphocyte, monocyte, neutrophil, eosinophil), Baso (basophils), IG (immature granulocytes), LYA (atypical lymphocytes), HRC (high RNA content), and ERB (erythroblasts). The device can discriminate between erythrocytes, three reticulocyte maturation indices, and platelets.

[0052] Moreover, this device, with its one-piece receiving barrel 10, is significantly easier to adjust than known devices, while offering much greater access to the measuring tank 8, which greatly facilitates its maintenance, and therefore both the maintenance of the quality of measurements over the long term and the optimization of its cost of use.

[0053] The device shown on the figure 2 represents a second embodiment in which the concept of the one-piece receiving barrel has been further developed in order to further simplify the adjustment procedure and reduce the production costs of the device.

[0054] In this embodiment, the first emission gun 4, the measuring tank 8 and the trigger gun 9 are identical to the device described figure 1 .

[0055] The second emission gun 6 differs in that the laser source 60, in the example described here, is a laser diode with a power of approximately 10 mW. The low power of 10 mW eliminates the need for Peltier-effect thermal regulation, thus reducing size and cost. However, to increase power density, the height of the light beam in the measurement cell has been reduced. The laser diode's light beam is shaped by anamorphic lenses so that the beam is elliptical at the laser output. The laser beam is focused onto the flow of biological cells in the measurement cell 8 using the same shaping optics 62.

[0056] As will be seen below, in order to limit the moving part of the device to a simple lens, the detection channels 12, 14, and 16 share a common axis that includes the dichroic filters 18 and 20. The dichroic filter 20 is positioned upstream of the dichroic filter 18 to keep the detection channel 12 aligned with the axis of the first emission source 4. Thanks to the design of the collection lens 11, the size of the dichroic filters 18 and 20 is smaller than that of the dichroic filters in the first embodiment, resulting in significant cost savings and miniaturization of the optical system, making it more compact. Furthermore, since the adjustment is made via the detection channel 12, bringing detector 144 closer to detector 124 allows for more precise positioning of the light beam on detector 144.The dichroic filter 18 remains unchanged, and the dichroic filter 20 is here a Semrock FF518-Di01 filter with a separation wavelength of 518 nm. Thus, blue radiation is sent to detection channel 16, green radiation to detection channel 14, and red radiation to detection channel 12.

[0057] Thanks to the reduction in beam size, the extinction detection channel 12 is simplified to include only the photodiode 126. Optionally, a focusing lens and / or a bandpass filter substantially centered on the emission wavelength of the emission source 40 can be provided.

[0058] Similarly, the fluorescence detection channel 14 is simplified to include only a Semrock FF01-550 / 49 interference filter (optional and not shown) at the input followed by a focusing lens 140 which focuses the beam onto the photomultiplier 144 which is here simplified to a silicon photomultiplier (SiPM).

[0059] Thanks to the reduction in beam size, the 90° diffraction detection channel 16 is simplified to include only the photodiode 164. Optionally, a focusing lens and / or a bandpass filter centered on the detection wavelength (e.g. a Semrock FF01-482 / 35 filter) can be provided.

[0060] Since the dichroic mirrors 18 and 20 and the bandpass filters are fixed to the same mechanical part (i.e., the receiving barrel 10, excluding the movable lens 112), the accuracy is increased. Indeed, the angle of the dichroic filter is better controlled and the tolerances are smaller.

[0061] As mentioned above, the main change in this embodiment lies in the collection objective 11, which is here made up of two lenses 110 and 112 downstream of the measuring tank 8, close to it and in the emission axis of the first emission channel 4, a diaphragm 114, and a lens 116 placed downstream of the diaphragm 114 so that the latter is at its focal plane.

[0062] Lens 110 has a high numerical aperture (here, 0.6) and a large diameter (25.4 mm). Its focal length is long enough to allow space between the measuring tank and lens 110. This facilitates access to the measuring tank, as the center of the first surface of lens 110 is located 4.6 mm from the measuring tank 8. In the example described, lens 110 is an aspheric lens, which helps to limit spherical aberrations.

[0063] Lens 112 then focuses the light beam onto diaphragm 114, which provides spatial filtering. The filtered beam is then reflected to infinity by lens 116, which has a diameter of 6 mm. In this example, diaphragm 114 has a diameter of 1.5 mm. Lens 112 reduces the size of the light beam, resulting in a more compact optical system and lowering the cost of the optical components.

[0064] Focusing the beam after lenses 110 and 112 allows for spatial filtering while reducing the size of the light beam. This allows for smaller filters and detectors in the rest of the device, resulting in a more compact design. The long focal length and large diameter of the objective lens allow for greater distance from the measuring cell 8, thus facilitating access, particularly for replacement.

[0065] This embodiment of the collection objective 11 is also highly advantageous because it allows for even simpler and more reliable adjustment than in the first embodiment. Indeed, unlike the first embodiment where it was necessary to move the entire receiving barrel 10 relative to the measuring tank 8, in the second embodiment, it is only necessary to move the lens 112, which has been made movable. To make the adjustment, the photodiode 126 of the detection channel 12 is removed to insert an adjustment tool comprising a focusing lens, a diaphragm with a diameter of 0.3 mm and a thickness of 1 mm positioned at the focal plane of the focusing lens, and the photodiode is then replaced. Because the diameter of the diaphragm is very small, there is only one operating point when the adjustment is made, which corresponds to the maximum intensity measured on the photodiode.To achieve this, the position of lens 112 relative to diaphragm 114 is adjusted along three axes. Furthermore, since the photodiode 126 constitutes the electronics used for this adjustment, it is reliable and simple to implement. The addition of the adjustment tool also allows for a more compact optical bench.

[0066] The device produced in this second embodiment is more compact and less expensive to manufacture than the first embodiment. It nevertheless offers the same advantages in terms of accessibility to the measuring tank, and above all, its mechanical design is significantly simplified because the lens 112 is the only moving part of the device.

[0067] The device shown on the figure 3 represents a third embodiment in which two fluorescence signals are measured. While adding a measurement, the concept of the monobloc receiving gun remains essential.

[0068] In this embodiment, the first emission gun 4, the measuring tank 8 and the triggering gun 9 are identical to those of the devices of figures 1 et 2 The emission cannon 6 can either be identical to that of the device of the figure 1 , or be identical to that of the device of the figure 2 This choice may depend on the light energy required in the measuring cuvette, for example depending on the antibodies and fluorochrome used.

[0069] In this embodiment, the objective 11 and the adjustment of the receiving barrel are identical to those of the device of the figure 2 .

[0070] The dichroic filter 18 remains unchanged; the dichroic filter 20 is here a Semrock FF500-Di01 filter with a separation wavelength of 500 nm. In this configuration, a dichroic filter is added: the Semrock FF555-Di03 filter, whose separation wavelength is centered on 555 nm. Thus, blue radiation is directed to detection channel 16, green radiation to detection channel 14, yellow radiation to detection channel 22, and red radiation to detection channel 12.

[0071] The extinction detection channel 12, the objective 11, the 90° diffraction detection channel 16, and the optical bench adjustment are identical to those of the device of the figure 2 Regarding the fluorescence detection channel 14, it is identical to that of the figure 2 except that the detector used is a photomultiplier or a silicon photomultiplier (SiPM).

[0072] The device further includes a fluorescence detection channel 22 which includes an FF01-585 / 40 interference filter (optional and not shown) at the input followed by a focusing lens 240 identical to the lens 140 which focuses the beam onto the photomultiplier or onto a silicon photomultiplier (SiPM) 244.

[0073] Configurations have been described above with respect to specific parts whose precise references have been provided. It goes without saying that the invention is not limited to these parts alone, and that other comparable elements could be used, which a person skilled in the art will be able to select and adapt as needed, with regard to laser wavelengths, filters, optics, objectives or lenses, and detectors. Furthermore, the trigger gun could also be used to perform on-axis diffraction measurements for size measurements. It should also be noted that the embodiments described above allow the use of both emission channels together, enabling all measurements to be performed simultaneously. This results in a system with a higher measurement rate. Alternatively, the emission guns could be activated sequentially.Furthermore, in the foregoing and in the claims, the beam can be subsequently shifted by one or more mirrors, particularly when it is mentioned that a beam is substantially collimated along an axis.

Claims

1. Electro-optical device for taking measurements of flow, comprising a measurement tank (8) through which a flow of fluid to be characterised flows, at least two guns (4, 6) for emitting light having separate spectra, a triggering gun (9) allowing small-angle diffraction to be measured and a receiving gun (10) allowing a measurement of attenuation and at least one fluorescence to be taken, the electro-optical device being such that: - the first emitting gun (4) comprises a light source (40) having the centre wavelength greater than 580 nm and defining a main optical axis perpendicular to the flow of fluid, - the second emitting gun (6) comprising a second light source (60), having a centre wavelength of less than 580 nm and defining a secondary optical axis substantially orthogonal to the main optical axis and to the flow of fluid, - the first emitting gun (4) and the second emitting gun (6) are disposed on one side of the measurement tank (8), the receiving gun (10) is disposed on the other side of the measurement tank (8) along the main optical axis, and the triggering gun (9) is disposed on the other side of the measurement tank (8) along the secondary optical axis, - the receiving gun (10) comprises a detection channel (12) for measuring the attenuation and at least one detection channel (14) for measuring at least one fluorescence signal, as well as a single objective (11) for collecting the beam coming from the interaction of the light beam between the first emitting gun (4) and the second emitting gun (6) and the particles in the flow of fluid, said collecting objective (11) being arranged so that the light beam that it transmits is a beam substantially collimated along the main optical axis towards the detection channel (12) for measuring the attenuation, the receiving gun (10) forming a single mechanical unit at least a part of which is able to move with respect to the measurement tank (8), the receiving gun (10) furthermore comprising a first dichroic mirror (18) placed downstream of the collecting objective (11) where the light beam is substantially collimated, arranged for partially transmitting the light beam resulting from the interaction between the first emitting gun (4) and the particles in the flow of fluid towards the detection channel (12) for measuring the attenuation, and for partially reflecting the light beam resulting from the interaction between the second emitting gun (6) and the particles in the flow of fluid towards at least one detection channel (14) for measuring at least one fluorescence signal.

2. Electro-optical device for taking measurements of flow according to claim 1, wherein the receiving gun (10) comprises a detection channel (16) for measuring the large-angle diffraction and a second dichroic mirror (20) placed downstream of the collecting objective (11) and arranged to partially reflect the light beam resulting from the interaction between the second emitting gun (6) and the particles in the flow of fluid towards at least one from the at least one detection channel (14) for measuring the fluorescence and the detection channel (16) for measuring the large-angle diffraction.

3. Device according to claim 1 or 2, wherein the detection channel (12) for measuring attenuation comprises a detector (126) arranged for measuring a quantity of red light, the at least one detection channel (14) for measuring at least one fluorescence comprising a detector (144) arranged for measuring a quantity of green light and a quantity of orange light or near infrared, and the detection channel (16) for measuring large-angle diffraction comprises a detector (164) arranged for measuring a quantity of blue light.

4. Electro-optical device for taking measurements of flow according to claim 3, wherein the detector (126) arranged for measuring a quantity of red light is a photodiode, the detector (144) arranged for measuring a quantity of green light is a photomultiplier or a silicon photomultiplier, and the photodetector (164) arranged for measuring a quantity of blue light is a photodiode.

5. Electro-optical device for taking measurements of flow according to one of claims 1 to 4, wherein the receiving gun (10) is in a single piece and is able to move integrally with respect to the measurement tank (8).

6. Electro-optical device for taking measurements of flow according to claim 5, wherein the second dichroic mirror (20) is placed downstream of the first dichroic mirror (18) in the path of the reflected beam and is arranged for partially reflecting the latter towards the detection channel (14) to measure the fluorescence and to partially transmit it to the detection channel (16) to measure the large-angle diffraction.

7. Electro-optical device for taking measurements of flow according to claim 6, wherein the detection channel (12) for measuring attenuation, one or more detection channels (14) for measuring one or more fluorescence signals and the detection channel (16) for measuring large-angle diffraction each comprise an optic (122, 140, 160) followed by a diaphragm (124, 142, 162) upstream of their respective detector (126, 144, 164).

8. Electro-optical device for taking measurements of flow according to one of claims 1 to 4, wherein the collecting objective (11) comprises two lenses (110, 112), one of which is able to move with respect to the measurement tank (8), the remainder of the receiving gun (10) not having mobility with respect to the measurement tank (8), and a diaphragm (114) disposed downstream of the two lenses (110, 112).

9. Electro-optical device for taking measurements of flow according to claim 8, wherein the second dichroic mirror (20) is placed upstream of the first dichroic mirror (18) in the path of the reflected beam and is arranged for partially reflecting the latter towards the detection channel (16) to measure the large-angle diffraction and to partially transmit it to the first dichroic mirror (18).

10. Electro-optical device for taking measurements of flow according to one of the preceding claims, wherein the first emitting gun (4) comprises a first emission source (42) that is a red LED.

11. Electro-optical device for taking measurements of flow according to one of the preceding claims, wherein the second emitting gun (6) comprises a second emission source (62) that is a blue laser.

12. Electro-optical device for taking measurements of flow according to one of the preceding claims, further comprising one or more adjustment elements arranged for allowing the adjustment of the device by moving all or part of the receiving gun (12) and measurement in the detection channel (12).

13. Electro-optical device for taking measurements of flow according to one of the preceding claims, further comprising one or more mirrors arranged for offsetting one or more substantially collimated beams.