Electro-optical device for flow measurement

Abstract

The electro-optical device for measuring flow includes a measurement vessel through which a fluid to be characterized flows, at least two light-emitting guns for emitting light having distinct spectra, a trigger gun for enabling measurement of small-angle diffraction, and a receiving gun for enabling measurement of attenuation and at least one fluorescence. The first light-emitting gun includes a light source defining a primary optical axis perpendicular to the fluid flow, and the second light-emitting gun includes a second light source defining the primary optical axis and a secondary optical axis substantially perpendicular to the fluid flow. The first light-emitting gun and the second light-emitting gun are disposed on one side of the measurement vessel, the receiving gun is disposed on the other side of the measurement vessel along the primary optical axis, and the trigger gun is disposed on the other side of the measurement vessel along the secondary optical axis. The receiving gun comprises a detection channel for measuring attenuation, at least one detection channel for measuring at least one fluorescence signal, and a single focusing lens for collecting beams resulting from interactions of the light beams between the first and second emission guns and particles in the fluid stream, the focusing lens being positioned to form a substantially collimated beam of transmitted light along a main optical axis toward the detection channel for measuring attenuation, and the receiving gun comprises a single mechanical unit, at least a portion of which is movable relative to the measurement chamber. The receiving gun further comprises a first dichroic mirror positioned downstream of the focusing lens at a position where the light beams are substantially collimated, the mirror being positioned to partially transmit the light beam resulting from interactions between the first emission gun and particles in the fluid stream toward the detection channel for measuring attenuation, and to partially reflect the light beam resulting from interactions between the second emission gun and particles in the fluid stream toward at least one detection channel for measuring at least one fluorescence signal.

Description

[Technical Field]

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

[0002] More particularly, the present invention relates to a multi-parametric electro-optical device for cell counting and characterization. The fluid to be characterized is preferably a blood sample, but may also be a biological fluid of another nature, including cerebrospinal fluid, urine, pleural fluid, synovial fluid, cell suspension, bone marrow, etc. The sample may also contain particles of any nature (cells, proteins, biomarkers, etc.) that need to be differentiated and counted.

[0003] More precisely, the invention relates to an apparatus comprising at least two light sources, a device for measuring resistivity or impedance, and several detectors for measuring optical parameters, typically attenuation, wide-angle diffraction and fluorescence. [Background technology]

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

[0005] Electrical impedance measurements allow particles to be counted and information about their size obtained.

[0006] Optical parameters (refraction, diffusion, absorption, and bending) can extract morphological information about cells, such as their shape, volume, size, and internal structure. A light source (such as a laser, halogen source, or light-emitting diode) can generate light that is focused by a lens and illuminates the biological cells passing through the measurement chamber. Upon contact with the biological cells, the light interacts with the cells. At the incident axis of the light beam, the light is collected by multiple lenses, spatially filtered by a diaphragm, and detected by a photodiode detector. This measurement indicates the size of the biological element combined with structural information over a selected angular range. Another measurement can also be performed at the incident axis of the light beam. The incident light is blocked by a stopper (beam stop), and the signal scattered by the cells is detected by a photodiode-type sensor to perform a forward scatter (FSC) measurement. This measurement can indicate the size of the biological element or trigger the measurement of small particles over a selected angular range. Another portion of the light is collected in an orthogonal direction, passed through another set of lenses and semi-reflective mirrors, and measured by a sensor to generate a side-scattered light (SSC) signal, which indicates the density, granularity (structure), or subcellular content of the biological element.

[0007] Fluorescence measurements are used to reveal fluorescent dyes that are used as cell markers or molecular probes specific to the structure or function of biological elements. For example, antibodies specific to a specific cell class can be conjugated to fluorescent dyes to reveal this cell class, i.e., to accurately characterize the cells, which can then be counted. The simultaneous use of multiple fluorescent dyes allows for better characterization of biological cells of interest.

[0008] If the fluid is a blood sample, the representative elements of blood—red blood cells, white blood cells, and platelets—are measured quantitatively (counts) and qualitatively (formulas). This analysis is called a blood count or complete blood count (BFC). Abnormalities in the BFC can alert the physician to potential pathologies (anemia, cancer, etc.).

[0009] All of these cells originate from the same stem cells, called hemoblasts, found in the bone marrow, which then differentiate into several subpopulations.

[0010] For example, in the case of hematopoietic cells, those skilled in the art know that analysis of cells obtained by impedance, diffraction, or absorption allows the differentiation of major cell lineages, including erythrocytes, platelets, and leukocytes. The latter population is itself subdivided into several categories, e.g., lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Blood is normally composed of mature cells that no longer divide.

[0011] Cell counting and differentiation can be achieved by simultaneously measuring volume and apparent white light attenuation, as described in commonly owned U.S. Patent No. 5,138,181. For example, WO 2006 / 053960 describes an apparatus developed in one embodiment with quasi-monochromatic light.

[0012] Various maturation stages are known for each of the above cell types. Red blood cells are first produced in the order of proerythroblasts, basophilic erythroblasts, and polychromatic erythroblasts, which then evolve into acidophilic erythroblasts, from which the nucleus is removed to give reticulocytes. It is these reticulocytes that differentiate into red blood cells in the circulation after complete loss of residual RNA.

[0013] White blood cells are born in the bone marrow in the form of myeloblasts, which then give rise to progranulocytes, which then transform into basophilic, eosinophilic, or neutrophilic granulocytes.They are initially undifferentiated, but as they mature, their nuclei become more and more segmented.

[0014] These myeloblasts also give rise to the monocytic lineage, which gives rise to monoblasts, promonocytes, and monocytes that pass through the peripheral blood.

[0015] The pluripotent stem cells produced by myeloblasts differentiate into lymphoid stem cells to give rise to the lymphoid lineage, some of which, the T lymphocyte lineage, continue to mature in the thymus and ganglia, while others remain in the bone marrow and give rise to the B lymphocyte lineage, which, when activated in the form of plasma cells, produce antibodies to combat pathogenic antigens.

[0016] Platelets in the blood are derived in part from megakaryoblasts, which themselves are derived from myeloid precursors produced by myeloblasts and which mature to the final stage, platelet-forming megakaryocytes, where their cytoplasm divides to produce platelets. Another type of platelet, the crosslinked platelet, contains an RNA load remnant of the original cell.

[0017] Diagnosis of some pathologies requires more detailed enumeration and characterization of circulating hematopoietic cells. This requires identifying specific populations, particularly immature red blood cell types such as reticulocytes and erythroblasts. Similarly, detecting immature cells known as immature lymphocytes, monocytes, or granulocytes, which are precursors to white blood cells, is also crucial. Additionally, classification and enumeration of activated lymphocytes or crosslinked platelets can improve patient diagnostic accuracy.

[0018] Specific fluorescent labeling of cells can provide high specificity and detection of immature or atypical cells. Thus, specific antibodies conjugated with fluorescent dyes can be used to accurately characterize and count various types of lymphocytes. Other abnormal cells present in the blood, such as atypical lymphocytes and immature progenitor cells, can also be detected after fluorescent labeling. Similarly, crosslinked platelets can be specifically labeled with fluorescent dyes.

[0019] In order to best differentiate between the populations of particles contained in the sample, the Applicant has, in French Patent Application No. 2 971 337, flow An electro-optical device for measurements has been proposed, which has many advantages. However, due to its construction, the adjustment of this device is complicated and access to the measuring chamber is very limited. Summary of the Invention [Problem to be solved by the invention]

[0020] The present invention improves this situation. [Means for solving the problem]

[0021] To this end, the present invention provides flow An electro-optical device for measurements is proposed, which comprises a measurement chamber in which a fluid to be characterized flows, at least two light emitting guns for emitting light having distinct spectra, a trigger gun enabling the measurement of small-angle diffraction, and a receiving gun enabling the measurement of attenuation and at least one fluorescence.

[0022] This electro-optical device is configured as follows. The first light emitting gun comprises a light source having a central wavelength greater than 580 nm, the light source defining a main optical axis perpendicular to the fluid flow. The second light emitting gun comprises a second light source having a center wavelength less than 580 nm, the second light source defining a major optical axis and a minor optical axis substantially orthogonal to the fluid flow. The first and second light-emitting guns are positioned on one side of the measuring chamber, the receiving gun is positioned on the other side of the measuring chamber along the main optical axis, and the trigger gun is positioned on the other side of the measuring chamber along the secondary optical axis. the receiving gun comprises a detection channel for measuring attenuation, at least one detection channel for measuring at least one fluorescence signal, and a single focusing lens for collecting beams resulting from interactions of the light beams between the first and second emission guns and particles in the fluid stream, the focusing lens being positioned to form a substantially collimated light beam along a main optical axis towards the detection channel for measuring attenuation; the receiving gun constituting a single mechanical unit, at least a portion of which is movable relative to the measurement vessel; and the receiving gun further comprises a first dichroic mirror positioned downstream of the focusing lens at a position where the light beams are substantially collimated, the first dichroic mirror being positioned to partially transmit the light beam resulting from interactions between the first emission gun and particles in the fluid stream towards the detection channel for measuring attenuation and to partially reflect the light beam resulting from interactions between the second emission gun and particles in the fluid stream towards the at least one detection channel for measuring at least one fluorescence signal.

[0023] This device has the advantage of allowing free access to the measuring chamber and being easy to adjust since the receiving gun is made as a single mechanical unit, and is more robust than the device of French patent application no. 2 971 337.

[0024] In various variations, the invention can have one or more of the following features. the receiving gun comprises a detection channel for measuring wide-angle diffraction and a second dichroic mirror arranged downstream of the collecting lens, the second dichroic mirror being arranged to partially reflect a light beam resulting from an interaction between the second emitting gun and particles in the fluid stream towards at least one of the detection channel for measuring fluorescence and the detection channel for measuring wide-angle diffraction. The detection channel for measuring attenuation comprises a detector arranged to measure the amount of red light, the at least one detection channel for measuring at least one fluorescence comprises a detector arranged to measure the amount of green light and the amount of orange light or near-infrared light, and the detection channel for measuring wide-angle diffraction comprises a detector arranged to measure the amount of blue light. The detector positioned to measure the amount of red light is a photodiode, the detector positioned to measure the amount of green light is a photomultiplier tube or a silicon photomultiplier tube, and the photodetector positioned to measure the amount of blue light is a photodiode. The receiving gun is integrally formed and can move integrally with the measuring tank. The second dichroic mirror is positioned downstream of the first dichroic mirror in the path of the reflected beam and is positioned to partially reflect the beam towards the detection channel to measure fluorescence and to partially transmit the beam to the detection channel to measure wide-angle diffraction. The detection channel for measuring attenuation, the one or more detection channels for measuring one or more fluorescence signals, and the detection channel for measuring wide-angle diffraction each in turn comprise an optical system and a diaphragm upstream of the respective detector. The focusing lens comprises two lenses and a diaphragm arranged downstream of the two lenses, one of which is movable relative to the measuring chamber, and the remaining part of the receiving gun is not movable relative to the measuring chamber. The second dichroic mirror is positioned upstream of the first dichroic mirror in the path of the reflected beam and is positioned to partially reflect the beam towards a detection channel for measuring wide-angle diffraction and to partially transmit the beam to the first dichroic mirror. The first light gun includes a first light source, which is a red LED. The second light gun includes a second light source, which is a blue laser. The device further comprises one or more adjustment elements arranged to allow adjustment of the device by moving all or part of the receiving gun and to allow measurements in the detection channel. The apparatus further comprises one or more mirrors positioned to offset the one or more substantially collimated beams. [Brief explanation of the drawings]

[0025] Further characteristics and advantages of the present invention will become more apparent from the following description, given by way of non-limiting example with reference to the accompanying drawings, in which: FIG. [Figure 1] 1 shows a first embodiment of the device according to the invention; [Figure 2] FIG. 2 shows a second embodiment of the device according to the invention. [Figure 3] FIG. 3 shows a third embodiment of the device according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The accompanying drawings and the following description essentially contain elements of a particular nature of the present invention, which not only serve to better understand the present invention but can also contribute appropriately to its definition.

[0027] FIG. 1 shows a first embodiment of the device according to the invention.

[0028] The device 2 includes a first light-emitting gun 4 and a second light-emitting gun 6, which are disposed on one side of the measurement chamber 8, and a trigger gun 9 and a receiving gun 10, which are disposed on the other side of the measurement chamber 8. The receiving gun 10 includes a focusing lens 11, three detection channels 12, 14, and 16, and dichroic mirrors 18 and 20, which will be described later. The dichroic mirrors 18 and 20 separate the light beam propagating within the measurement chamber 8 into multiple light beams having different wavelengths (here, red, blue, and green) specific to each of the three detection channels 12, 14, and 16. The detection channel 12 is used to measure attenuation, 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 this embodiment, the first light-emitting gun 4 comprises a first light source 40, a shaping optical system 42, a reticle 44, and a focusing optical system 45 that functions to reduce the size of the rectangle projected from the reticle 44 onto the measurement tank 8.

[0030] In this embodiment, the first light source 40 is an Epitex SMB660NR-1100 red LED, which has a luminous intensity distribution similar to that of a Lambertian source, thereby achieving the required uniformity. The LED 40 has a broad emission spectrum centered at 660 nm. The LED 40 chip is 1.1 mm 2 The LED 40 has an active surface area of ​​1000 Å. At the output of the first light source 4, i.e., at the center of the measurement chamber 8, the optical power supplied by the LED 40 is 45 μW. Typically, this first light source has an emission spectrum with a central wavelength greater than 580 nm. On the other hand, as will be described later, the second light source has an emission spectrum with a central wavelength less than 580 nm. This is a configuration implemented so that red measurements can be separated from blue or green measurements.

[0031] In this embodiment, the shaping optical system 42 includes two focusing lenses disposed between the LED 40 and the reticle 44. These two lenses project light onto an aperture diaphragm, ensuring uniformity of the beam irradiated onto the flow of biological cells propagating within the measurement chamber 8. To account for the fact that the spectrum of the LED 40 overlaps with part of the fluorescence spectrum of thiazole orange and to limit the resulting noise in the detection channel 14 that measures fluorescence, a color filter or interference filter can be optionally disposed between the two lenses of the shaping optical system 42 at the position where the light beam is collimated. This can filter out the green component of the LED 40.

[0032] In this embodiment, the reticle 44 has a rectangular shape (150×500 μm 2 ) used in conjunction with the LED 40 and shaping optics 42, the paraxial magnification of the system relative to the cells is 0.172. The paraxial image of the reticle 44 relative to the cell stream is therefore a rectangle measuring 86 μm × 25.8 μm (500 μm × 0.172 and 150 μm × 0.172, respectively). Simulations in Zemax also showed that a perfect "real" system (diffraction limited) would produce a spot measuring 90 μm × 28.8 μm (the dimensions obtained in Zemax at the base of the cross-X and cross-Y curves). The experimentally measured image dimensions were very close to those produced by the perfect "real" system.

[0033] In the measuring chamber 8, the beam from a point on the LED 40 (corresponding to the image of the LED 40, there is no reticle) is collimated. This means that the aperture diaphragm is projected to infinity, and it is designed to align its image with the focal point of the system.

[0034] The first emission gun 4 and detection gun 12 allow the measurement of the attenuation of an approximately 660 nm LED beam by moving biological cells, which can be improved by precisely controlling the beam morphology and illumination uniformity at the cells.

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

[0036] In this embodiment, dichroic mirror 18 is a Semrock FF605-Di02 filter, which transmits the portion of the beam collimated by condenser lens 11 having wavelengths greater than 605 nm to detection channel 12 and reflects the other portion of the beam collimated by condenser lens 11, i.e., the portion having wavelengths less than 605 nm, toward detection channels 14 and 16. Here, dichroic mirror 18 transmits 98% of red light and reflects 99% of blue and green light. The specifications of dichroic mirror 18 allow condenser lens 11 to collimate the beam from measurement chamber 8 so that it reaches filter 18 with a half angle of less than 2°.

[0037] In this example, the bandpass filter 120 is a Semrock FF01-655 / 40 bandpass interference filter, which transmits only red wavelengths (635 nm to 675 nm) from the LED 40. In this example, the focusing lens 122 has a diameter of 1 mm and focuses the light beam onto a diaphragm 124 located 22 mm from the LED 40. In this example, the focusing lens 122 is a custom-made model. This plano-convex lens is made of N-BK7 material and has a radius of curvature of 12.42 mm. However, other lenses can be used. Finally, in this example, the detector 126 is a Hamamatsu S1223 photodiode, located 6.75 mm from the diaphragm 124, i.e., from the focal position. The half angle of the beam entering the detection channel 12 is less than 7° as defined by the bandpass filter 120, and the numerical aperture of this beam from the measurement chamber is limited to 0.31 by the diaphragm of the receiving gun 12.

[0038] In this embodiment, the second light emitting gun 6 includes a second light source 60 and a shaping optical system 62 .

[0039] In this embodiment, the second light source 60 is a blue laser source from Osram, comprising a 50 mW laser diode emitting at 488 nm, the output of which is shaped by an anamorphic lens to produce an elliptical beam at the output of the laser source 60.

[0040] In this embodiment, the shaping optics 62 comprises a spherical lens with a focal length of 75 mm, e.g., a pair of Thorlabs AC127-075-A lenses. Using a lens with a relatively long focal length allows for a greater depth of field in the interaction between the biological cells and the light beam. The ellipse is approximately 200 μm × 30 μm (1 / e) in the measurement chamber. 2 at ) and is vertically polarized.

[0041] The laser source 60 has the advantage of being at the focal point of the cell stream in the measurement chamber 8. This allows for high beam quality and high reproducibility. It also allows for high tolerances for lateral and vertical positioning, since the lateral positioning is favored by the length of the laser beam in the measurement chamber (flattened ellipse), while the vertical tolerance depends on the distance between the laser and the measurement chamber.

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

[0043] Thus, the detection channel 14 for measuring fluorescence measures the green wavelength from the fluorescence of thiazole orange, which labels the nucleic acids of living cells. Thus, the detection channel 14 is positioned on the axis of the beam reflected by the dichroic mirror 18. The detection channel 14 comprises a pair of lenses 140, a diaphragm 142, and a detector 144. The pair of lenses 140 allows the beam transmitted by the dichroic mirror 20 to be focused by the diaphragm 142, which has a diameter of 1.5 mm. The diaphragm 142 performs spatial filtering, thereby achieving a high signal-to-noise ratio. The fluorescence signal is then measured by the detector 144, which in this embodiment is a Hamamatsu H10723 photomultiplier tube. The numerical aperture of the detection channel 14 is 0.6.

[0044] The portion of the beam reflected by dichroic mirror 20 is directed into detection channel 16 for measurement of 90° diffraction from the interaction between the laser and living cells in the blue.

[0045] In this example, detection channel 16 comprises a focusing lens 160, a diaphragm 162, and a detector 164. In this example, focusing lens 160 is a Thorlabs LA-1270-A reference lens, diaphragm 162 has a diameter of 1.5 mm, and photodiode 164 is a Hamamatsu S1223 photodiode. Again, diaphragm 162 provides spatial filtering, thereby achieving a high signal-to-noise ratio.

[0046] As mentioned above, the condenser lens 11 downstream of the measurement chamber 8 is common to the detection channels 12, 14, and 16. In this example, the condenser lens 11 has a very large numerical aperture of 0.6 in order to maximize collection of the flow emitted from the measurement chamber 8. The focal length of this condenser lens is chosen to be large enough to allow easy access to the measurement chamber (in this example, the distance between the measurement chamber and the lens is 5.2 mm). In this example, the condenser lens 11 is a pair of lenses, which allows the wavelengths present to be varied (488 nm to 700 nm), thereby also limiting chromatic aberrations.

[0047] The receiver gun 10 is adjusted using a 0.3 mm diameter diaphragm (not shown in FIG. 1) that replaces the diaphragm 124 of the detector gun 12. The use of a diaphragm with a small diameter (0.3 mm) allows for high adjustment precision. The optimum position of the receiver gun 10 corresponds to the maximum light intensity detected by the photodiode 126. To achieve this, the position of the measuring chamber 8 relative to the receiver gun 10 is adjusted in three axes by a device that moves a mechanical unit that receives all elements of the receiver channel 10. This allows for the adjustment of three detectors with a single precise adjustment. This adjustment is simplified because the same detector is used as the attenuation detector 126. This eliminates the need for bulky equipment, such as a camera, which is particularly advantageous for after-sales service personnel.

[0048] Optionally, dedicated bandpass filters for attenuation 120 (red), 90° diffraction (SSC) (blue), and fluorescence (green) with transmittances of 85%, 90%, and 96%, respectively, can be added to each detection channel 12, 14, and 16. Thus, taking into account the characteristics of dichroic mirrors 18 and 20, a 1% reflectance at the lens interface results in transmittances of 78% for attenuation, 83% for diffraction, and 86% for fluorescence.

[0049] In attenuation, the proportion of parasitic light is 2.10 for blue and green -6%. At 90° diffraction, this ratio is 2.10 for red -6 %, 9.10 in green -3 %. Due to the weak fluorescence signal, the gain of the photomultiplier 144 is very high. Therefore, it is important to consider the parasitic light in this channel, especially the blue light associated with the laser, where 3.10 in blue and red. -4 It is %.

[0050] The trigger channel 9 includes a beam blocker 90 and a detector 92. In this example, on-axis signal detection at 488 nm is used as the trigger signal. The trigger channel 9 includes a beam blocker to block the incident laser beam. In this example, the beam blocker is a vertical bar with a width of 0.8 mm, positioned 5.5 mm from the measurement chamber 8 in the axial direction of the laser light source 60. Its width is sufficient to block the laser beam from the light source 60, and its size in the horizontal direction is very stable, remaining less than 300 μm. In this example, the detector 92 is a Hamamatsu S1223, which allows small-angle diffraction measurements (also known as FSC).

[0051] Clinical trials conducted by the applicant have shown that the device can distinguish between the following white blood cells: LMNE (lymphocytes, monocytes, neutrophils, eosinophils), Baso (basophils), IG (immature granulocytes), LYA (atypical lymphocytes), HRC (high RNA content), and ERB (erythroblasts).The device can distinguish between red blood cells, three maturity indicators of reticulocytes, and platelets.

[0052] Furthermore, since the present device has an integrated receiving gun 10, adjustment is much easier than with conventional devices. Furthermore, since the measuring tank 8 is very easy to access, maintenance is very easy, making it possible to both maintain measurement quality over the long term and optimize usage costs.

[0053] The device shown in Figure 2 shows a second embodiment that further develops the concept of an integrated receiving gun, thereby further simplifying the adjustment procedure and reducing the manufacturing costs of the device.

[0054] In this embodiment, the first light-emitting gun 4, the measuring tank 8 and the trigger gun 9 are the same as those in the device shown in FIG.

[0055] In this embodiment, the second light-emitting gun 6 differs in that its laser light source 60 is a laser diode with an output of approximately 10 mW. The low output of 10 mW eliminates the need for temperature control using the Peltier effect, thereby saving space and reducing costs. Nevertheless, the height of the light beam within the measurement chamber is reduced to increase power density. The light beam from the laser diode is shaped by an anamorphic lens so that the beam becomes elliptical at the laser output. The light beam is then focused onto the flow of biological cells within the measurement chamber 8 by the same shaping optics 62.

[0056] As will be described later, to limit the moving parts of the device to a single lens, detection channels 12, 14, and 16 share a common axis, including dichroic filters 18 and 20. Dichroic filter 20 is positioned upstream of dichroic filter 18 to maintain detection channel 12 on the axis of first light source 4. Due to the design of focusing lens 11, the size of dichroic filters 18 and 20 is smaller than that of the dichroic filters in the first embodiment. This results in significant cost savings and allows for a more compact and compact optical system. Furthermore, because adjustments are made through detection channel 12, detector 144 can be moved closer to detector 124, allowing for more precise positioning of the light beam on detector 144. Here, dichroic filter 18 remains unchanged, and dichroic filter 20 is a Semrock FF518-Di01 filter with a separation wavelength of 518 nm. Thus, blue radiation is directed to detection channel 16, green radiation is directed to detection channel 14, and red radiation is directed to detection channel 12.

[0057] By reducing the beam size, the attenuation detection channel 12 is simplified to include only a photodiode 126. Optionally, a focusing lens and / or bandpass filter substantially centered on the emission wavelength of the light source 40 may be provided.

[0058] Similarly, the detection channel 14 for fluorescence 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. The focusing lens 140 focuses the beam onto a photomultiplier tube 144 (here simplified to a silicon photomultiplier tube (SiPM)).

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

[0060] Because the dichroic mirrors 18 and 20 and the bandpass filter are fixed to the same mechanical part (i.e., fixed to the receiving gun 10 but separate from the movable lens 112), precision is improved. In fact, the angles of the dichroic filters can be better controlled and tolerances can be tighter.

[0061] As described above, the main change in this embodiment is the condenser lens 11. Here, the condenser lens 11 is realized by a configuration including two lenses 110 and 112 on the light-emitting axis of the first light-emitting channel 4, a diaphragm 114, and a lens 116 arranged downstream of the diaphragm 114 so that the diaphragm 114 is in its focal plane, located downstream of and near the measurement tank 8.

[0062] Lens 110 has a very large numerical aperture (here 0.6) and a large diameter (25.4 mm). Its focal length is large enough to ensure space between the measurement chamber and lens 110. This allows for easy access to the measurement chamber, as the center of the first surface of lens 110 is located 4.6 mm from the measurement chamber 8. In this embodiment, lens 110 is an aspheric lens, which limits spherical aberration.

[0063] Lens 112 then focuses the light beam on diaphragm 114, which performs spatial filtering, and the filtered beam is reflected to infinity by lens 116, which has a diameter of 6 mm. In this embodiment, diaphragm 114 has a diameter of 1.5 mm. Lens 112 allows the size of the light beam to be reduced, resulting in a more compact optical system and reducing the cost of the optical components.

[0064] By focusing the beam after lenses 110 and 112, spatial filtering can be performed while reducing the size of the light beam. This allows smaller filters and detectors to be selected for other parts of the device, making the mechanism more compact. Increasing the focal length and diameter of the focusing lens allows it to be located farther away from the measurement chamber 8, making it easier to access, especially when replacing it.

[0065] This implementation of the focusing lens 11 is highly advantageous because it allows for easier and more reliable adjustment than the first embodiment. Indeed, unlike the first embodiment, which requires moving the entire receiving gun 10 relative to the measuring chamber 8, the second embodiment requires only moving the movable lens 112. Adjustment is performed by removing the photodiode 126 of the detection channel 12, placing an adjustment tool comprising a focusing lens and a 0.3 mm diameter, 1 mm thick diaphragm in the focal plane of the focusing lens, and then returning the photodiode. Because the diaphragm diameter is very small, only one operating point is required for adjustment, which corresponds to the maximum light intensity measured by the photodiode. To achieve this, the position of the lens 112 relative to the diaphragm 114 is adjusted in three axes. Furthermore, the photodiode 126 constitutes the electronics for the adjustment, making it highly reliable and easy to use. The addition of the adjustment tool also allows for a more compact optical bench.

[0066] The device implemented in this second embodiment is more compact and less expensive to manufacture than the first embodiment, yet has the same advantages of accessibility to the measurement chamber and, in particular, a much simpler mechanical design since the only moving part of the device is the lens 112.

[0067] The device shown in Figure 3 shows a third embodiment in which two fluorescent signals are measured. The concept of an integrated receiving gun with additional measurements remains fundamental.

[0068] In this embodiment, the first light-emitting gun 4, the measurement chamber 8, and the trigger gun 9 are the same as those in the apparatus of Figures 1 and 2. The light-emitting gun 6 may be the same as that in the apparatus of Figure 1, or may be the same as that in the apparatus of Figure 2. This selection may depend on the light energy required for the measurement chamber, for example, depending on the antibody and fluorescent dye used.

[0069] In this embodiment, the adjustment method of the condenser lens 11 and the receiving gun is the same as that of the device in FIG.

[0070] Here, dichroic filter 18 remains unchanged, and dichroic filter 20 is a Semrock FF500-Di01 filter with a split wavelength of 500 nm. In this configuration, an additional dichroic filter has been added. This dichroic filter is a Semrock FF555-Di03 filter with a split wavelength centered at 555 nm. Thus, blue radiation is routed 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 attenuation detection channel 12, the condenser lens 11, the 90° diffraction detection channel 16, and the method of adjusting the optical bench are the same as those in the apparatus of Figure 2. The fluorescence detection channel 14 is the same as that in Figure 2, except that the detector used is a photomultiplier tube or a silicon photomultiplier tube (SiPM).

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

[0073] The configuration of the present invention has been described above with precise reference to the above-mentioned parts. Needless to say, the present invention is not limited to these parts, and those skilled in the art can use other equivalent elements related to the laser wavelength, filters, optics, focusing lenses, or lenses and detectors that can be appropriately selected and adapted. Furthermore, a trigger gun can be used to measure diffraction along an axis for making size measurements. It should be noted that the above-described embodiment allows for the use of both emission channels, thereby allowing all measurements to be made simultaneously, resulting in a system with a high measurement rate. Alternatively, the emission guns can be operated sequentially. Furthermore, where the above and the claims specifically refer to a beam being substantially collimated along an axis, the beam can be subsequently offset by one or more mirrors.

Claims

1. An electro-optical device for measuring flow, comprising: a measuring chamber (8) through which a fluid to be characterized flows, at least two light emitting guns (4, 6) for emitting light having separate spectra, a trigger gun (9) enabling measurement of small angle diffraction, and a receiving gun (10) enabling measurement of attenuation and at least one fluorescence, the first light gun (4) comprises a light source (40) having a central wavelength greater than 580 nm, the light source (40) defining a main optical axis perpendicular to the flow of the fluid; the second light gun (6) comprises a second light source (60) having a central wavelength of less than 580 nm, the second light source (60) defining a major optical axis and a minor optical axis substantially perpendicular to the flow of the fluid; the first light-emitting gun (4) and the second light-emitting gun (6) are arranged on one side of the measuring chamber (8), the receiving gun (10) is arranged on the other side of the measuring chamber (8) along the main optical axis, and the trigger gun (9) is arranged on the other side of the measuring chamber (8) along the secondary optical axis; the receiving gun (10) comprises a detection channel (12) for measuring attenuation, at least one detection channel (14) for measuring at least one fluorescence signal, and a single focusing lens (11) for collecting beams resulting from interactions of light beams between the first and second emission guns (4, 6) and particles in the fluid flow, the focusing lens (11) being arranged to form a substantially collimated beam of the transmitted light beam along the main optical axis towards the detection channel (12) for measuring the attenuation, the receiving gun (10) constituting a single mechanical unit, at least a part of which is movable relative to the measuring chamber (8); the receiving gun (10) further comprises a first dichroic mirror (18) arranged downstream of the collecting lens (11) at a position where the light beam is substantially collimated, the first dichroic mirror (18) being arranged to partially transmit the light beam resulting from the interaction between the first light emitting gun (4) and particles in the fluid flow towards the detection channel (12) for measuring the attenuation, and to partially reflect the light beam resulting from the interaction between the second light emitting gun (6) and particles in the fluid flow towards the at least one detection channel (14) for measuring the at least one fluorescence signal; the receiving gun (10) comprises a detection channel (16) for measuring wide-angle diffraction and a second dichroic mirror (20) arranged downstream of the collecting lens (11), the second dichroic mirror (20) being arranged to partially reflect the light beam resulting from the interaction between the second emission gun (6) and particles in the fluid stream towards at least one of the at least one detection channel (14) for measuring the at least one fluorescence and the detection channel (16) for measuring the wide-angle diffraction; the detection channel (12) for measuring the attenuation comprises a photodiode (126) arranged to measure the amount of red light, the at least one detection channel (14) for measuring the at least one fluorescence comprises a detector (144) arranged to measure the amount of green light and the amount of orange light or near-infrared light, and the detection channel (16) for measuring the wide-angle diffraction comprises a photodiode (164) arranged to measure the amount of blue light; the detector (126) arranged to measure the amount of red light is a photodiode, the detector (144) arranged to measure the amount of green light is a photomultiplier tube or a silicon photomultiplier tube, and the detector (164) arranged to measure the amount of blue light is a photodiode. Electro-optical device for flow measurement.

2. 2. The electro-optical device for measuring flow according to claim 1, wherein the receiving gun (10) is integrally formed and is movable integrally with the measuring chamber (8).

3. An electro-optical device for flow measurement as described in claim 2, wherein the second dichroic mirror (20) is positioned downstream of the first dichroic mirror (18) in the path of the reflected beam and is positioned so as to partially reflect the beam towards the detection channel (14) to measure fluorescence and to partially transmit the beam to the detection channel (16) to measure wide-angle diffraction.

4. An electro-optical device for flow measurement as described in claim 3, wherein each of the detection channel (12) for measuring the attenuation, the at least one detection channel (14) for measuring the at least one fluorescence signal, and the detection channel (16) for measuring the wide-angle diffraction is sequentially provided with an optical system (122, 140, 160) and a diaphragm (124, 142, 162) upstream of the respective detector (126, 144, 164).

5. 2. The electro-optical device for flow measurement according to claim 1, wherein the focusing lens (11) comprises two lenses (110, 112) and a diaphragm (114) arranged downstream of the two lenses (110, 112), one of the lenses (110, 112) being movable relative to the measuring chamber (8), and the remaining part of the receiving gun (10) not being movable relative to the measuring chamber (8).

6. An electro-optical device for flow measurement as described in claim 5, wherein the second dichroic mirror (20) is positioned upstream of the first dichroic mirror (18) in the path of the reflected beam and is positioned so as to partially reflect the beam towards the detection channel (16) to measure wide-angle diffraction and to partially transmit the beam to the first dichroic mirror (18).

7. Electro-optical device for flow measurement according to any one of claims 1 to 6, wherein the first light emitting gun (4) comprises a first light emitting source (40) which is a red LED.

8. 8. An electro-optical device for flow measurement according to any one of claims 1 to 7, wherein the second light emitting gun (6) comprises a second light emitting source (60) which is a blue laser.

9. 9. An electro-optical device for flow measurement according to any one of claims 1 to 8, further comprising one or more adjustment elements arranged to allow adjustment of the device by moving all or part of the receiving gun (12) and to allow measurement in the detection channel (10).

10. 10. An electro-optical device for measuring flow according to any one of claims 1 to 9, further comprising one or more mirrors arranged to offset one or more of the substantially collimated beams.

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