Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1434—Optical arrangements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
  • G01N15/1427—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
  • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N2015/1486—Counting the particles

Definitions

• the present invention relates to the general field of devices and methods for measuring photoluminescence in a fluid present in a measuring vessel. Such devices and methods are in particular intended for counting microscopic objects in a fluid, for example a biological fluid. More specifically, the invention relates to devices based on the use of partially coherent light sources, such as a light emitting diode, as means for exciting molecules, for example fluorescent, present in a fluid.
• partially coherent light sources such as a light emitting diode
• Photoluminescence is a substantially isotropic radiative phenomenon induced during the return to the ground state of a molecule excitable and excited by a light energy at a wavelength of its own.
• the emission of fluorescence light is always at a frequency lower than that of excitation.
• the measurement is generally performed outside the excitation axis by the incident light and through a color filter transmitting only the spectral band of interest to the detector.
• the invention is particularly interested in the development of optical and optoelectronic means for the detection of very weak photoluminescence signals such as those derived from the labeling of biomolecules, proteins or nucleic acids, for example.
• very weak photoluminescence signals such as those derived from the labeling of biomolecules, proteins or nucleic acids, for example.
• the measurement of photoluminescence signals is particularly useful to the practitioner to develop a diagnosis and more specifically a cytological diagnosis for which the detection and counting of rare cell lines such as hematopoietic stem cells or other elements of the blood or blood. another biological fluid will be particularly useful.
• Photoluminescence measurements of biological elements are widely used in the fields of flow cytometry and automated cytology including hematology.
• the molecular probes used may be vital or supravital dyes having an intrinsic affinity for a particular type of molecule such as nucleic acid intercalating dyes. They may also be immunological probes such as conjugated products composed of an antibody on which is grafted a dye molecule, usually a fluorochrome, alone or in tandem, or sometimes a nano crystal.
• the antibody will be able to bind specifically to molecules or portions of molecules called antigens or antigenic determinants that it will be possible to count down by photoluminescence measurement.
• the mode of labeling by the implementation of immunological probes has become widely used for cytological identification, in particular using flow cytometry techniques.
• the excitation light sources must be able to deliver sufficient energy so that each labeled biological element can be detected with sufficient sensitivity as it passes in front of the excitation beam.
• Laser-type sources provide very good spatial coherence and power, but the Gaussian structure of the laser beam affects the homogeneity of the light field at the measurement point. It is necessary to implement a complex optics, thus expensive, to obtain a homogeneity of this field greater than 0.5% at the measurement point.
• laser sources therefore has the major disadvantage of the cost which can, in the case in particular of ultraviolet, dye or multiband excitation beam devices, be prohibitive and reserve their use for very specific devices in fields point of biological analysis.
• Laser diodes less expensive, have the advantage of having a high power density related to their high spatial coherence, but the choice of wavelengths is more limited compared to the choices provided by the light emitting diodes.
• FIG. 1 represents such a device generally using sources of low spatial coherence, such as arc lamps or light-emitting diodes.
• a device can be used for a photoluminescence measurement in a measurement vessel CM at the center of which is injected a fluid, for example biological, to be analyzed.
• the proposed optical system is generally called editing in epifluorescence mode.
• the device comprises a light source S for generating an excitation beam and an element, for example a photodetector PD, for capturing the light of a photoluminescence emission beam.
• beam or light or excitation radiation is intended to mean the light coming from the light source serving to illuminate the fluid to be analyzed.
• beam or light or emission radiation is meant the light resulting from the inelastic interaction of the excitation beam with microscopic objects present in the fluid to be analyzed, such as fluorescence or photoluminescence lights.
• the excitation beam generated by the light source S and the emission beam captured by the photodetector PD are coaxial along an axis called axis of the system X, the same optics allowing the emission and reception of lights.
• the device comprises a DC dichroic plate for dissociating the excitation and emission beams.
• the device further comprises two filters F1 and F2 respectively intended to filter the light of the source S emitted towards the measurement vessel CM and the fluorescence light or lights resulting from the inelastic interaction of the excitation light emitted by the source S with microscopic objects of the fluid present in the measurement vessel CM.
• Optics L1 and L3 allow the excitation and emission beams to be parallel beams as they pass through the filters F1 and F2 and the dichroic plate DC.
• a lens or an optical combination L2 with a large numerical aperture makes it possible to focus the excitation beam on a small volume centered at a measuring point M of the measurement vessel CM.
• the fluorescence light from a microscopic object present in the fluid and passing at the point M illuminated by the excitation beam is focused in a parallel beam by the lens L2, transmitted through the DC blade, filtered by the filter F2 and received by the PD photodetector after focusing by the lens L3.
• the power of the excitation beam obtained at the selected central wavelength relative to the fluorochrome used is low. This also reduces the discrimination capabilities of known devices whose fields of application are restricted. They are indeed limited to the detection of high fluorescence signals, for example corresponding to a large number of epitopes or to high fluorescence yield markers.
• the main object of the present invention is thus to overcome the drawbacks presented by the devices of the prior art by proposing a device for measuring photoluminescence and measuring absorbance and / or diffraction that is precise, sensitive and inexpensive, comprising at least two optical systems each including a low spatial coherence light source sending an excitation beam to the measurement vessel along an axis of the system and a photoluminescence emission beam capture element centered on the axis of the system , said optical systems operating simultaneously and being positioned so that their axes form between them a non-zero and distinct obtuse angle of 180 ° around the measurement vessel, said photoluminescence measurement being deduced from a coupling of data obtained from the beams emission captured simultaneously by the capture elements.
• the optical systems are positioned such that there is at least one partial overlap beam between the source excitation beam of a first optical system and the emission beam captured by the capture element of a second optical system and that the device is provided with at least one so-called extinction capture element in the vicinity of at least one of the sources for sensing light at the wavelength of the excitation in the partial recovery beam, a measurement absorbance and / or diffraction being deduced from data obtained from the light captured by the extinguishing capture element.
• the invention proposes in particular to multiply the number of optical systems, using a source of low spatial coherence, while coupling the transmitted transmission beams.
• This allows, for n optical systems, that the excitation power at the measurement point is n times greater than for a single system and allows the received photoluminescence emission power to be n 2 times greater than for a single system since it is received on n optical systems simultaneously.
• fluorescence emission beams are collected simultaneously by the two capture elements of the two. optical systems.
• the two systems are mounted epifluorescence, that is to say that emission and reception are along the same axis using the same optics, and that the two systems are arranged so as to make an angle strictly obtuse, the fluorescence emission beam received by each optical system is angularly offset from the excitation beam of the other optical system.
• the received fluorescence emission beam is then little parasitized by direct light illumination and also twice as intense since two excitation beams are used instead of a single excitation beam as this is the case in the devices of the prior art.
• the device according to the invention is therefore more precise and more sensitive.
• the two optical systems between them an obtuse angle around the measuring tank, the existence of a covering beam, insofar as its extent is reduced, ensures that the maximum power arrives in the tank while by generating a minimum of stray light.
• the invention proposes to use an extinction capture element capable of capturing the light of the covering beam which reveals changes in intensity indicative of absorption and / or diffraction by a microscopic object having passed through the beam. recovery took place.
• a suitable capture element makes it possible to quantify this absorption.
• the device comprises an odd number of optical systems positioned so that, in pairs, their axes make between them non-zero obtuse angles and distinct from 180 ° around the measurement vessel.
• the optical systems are positioned so that their axes form between them identical angles around the measurement vessel.
• the number of optical systems is equal to three.
• the device then comprises three optical systems positioned around the measuring vessel such that their axes form between them identical angles around the measuring vessel.
• This particular feature makes it possible to limit the steric hindrance around the measurement vessel while multiplying by three the intensity of each fluorescence emission beam received by each capture element from the measurement vessel CM with respect to a excitation with a single optical system.
• the 120 ° positions around the measuring chamber of the optical systems and the need for a cover beam require the use of excitation and emission beams having large numerical apertures.
• the implementation of three optical systems represents a preferred configuration in terms of angles between the optical systems, available light power, recovery, uniformity of the light field, cost and sensitivity.
• the capture elements of the emission beam are connected to the same photodetector or the same set of photodetectors.
• This implementation makes it possible to sum the fluorescence signals received simultaneously by the capture elements directly within the common photodetector. The data is then coupled immediately upon acquisition by the same photodetector. This characteristic makes it possible to perform an optical addition of the light signals.
• Photodetectors are generally sensitive to a single wavelength. The use of a single photodetector is therefore rather suitable when a single wavelength of photoluminescence is expected, which generally corresponds to a single excitation wavelength.
• the use of the same set of photodetectors for the capture elements makes it possible to detect several wavelengths of photoluminescence. This will therefore be better suited when several photoluminescence wavelengths are expected, which more generally corresponds to an excitation on several wavelengths. This will correspond for example to a configuration where the three optical systems do not necessarily emit each light of the same wavelength. In all cases, the photodetectors perform an optical addition of the light signals.
• the photodetector or photodetectors are connected to data processing means able to deduce the photoluminescence measurement from the data received from the photodetector (s).
• the extinction capture element is connected to a photodetector, itself connected to data processing means able to deduce an absorbance and / or diffraction measurement from the data received in from the photodetector.
• the capture elements of the emission and / or extinction beams are optical fibers of circular or rectangular section.
• the light sources include a light coherence electroluminescent diode coupled to an optical element intended to render the excitation beam homogeneous.
• the optical element is a light conductor, for example an optical fiber.
• the measurement vessel has a polyhedral section in the plane where the optical systems are placed, the polyhedron being such that its faces are perpendicular to the axes of the optical systems.
• the tank thus has an equilateral triangular section.
• the measuring vessel is cylindrical.
• each optical system includes aberration correction means for correcting the aberrations introduced by the geometry of the measurement vessel on the different beams.
• the fluid is a biological fluid.
• a device according to the invention can be used for the detection and counting of fluorescently labeled biological elements.
• the Applications are numerous in the field of flow cytometry in particular and more particularly for the identification and counting of biological cells in peripheral blood samples or bone marrow or any other biological fluid.
• the invention also relates to a method for measuring photoluminescence and measuring absorbance and / or diffraction in a fluid present in a measuring vessel, characterized in that the fluid in the measuring vessel simultaneously receives at least two beams of excitation from two optical systems each comprising a low spatial coherence light source sending said excitation beam to the measurement vessel along an axis of the system and a capture element for receiving a fluorescence emission beam centered on the axis of the system from the fluid, said optical systems being positioned so that their axes form between them a non-zero and distinct obtuse angle of 180 ° around the measurement vessel, said photoluminescence measurement being deduced from a coupling of data obtained from the emission beams captured simultaneously by the capture elements.
• the optical systems are positioned in such a way that there is a partial overlap beam between the excitation beam of the source of a first optical system and the emission beam captured by the capture element.
• at least one light at the wavelength of the excitation is picked up in the partial overlap beam by at least one so-called extinction capture element placed in the vicinity of at least one sources, an absorbance and / or diffraction measurement being deduced from data obtained from the light captured by the extinction capture element.
• FIG. 1 represents a photoluminescence measuring device as known from the prior art
• FIG. 2 is a block diagram of a device for measuring a photoluminescence according to the invention
• FIG. 3 represents the profile of the intensity of the light field in the horizontal direction in the measurement vessel of a device according to FIG. 2;
• FIG. 4 gives examples of spectral characteristics of filters and of dichroic plate as implemented in a device according to FIG. 2;
• FIG. 6 illustrates in three dimensions the volume analyzed by a photoluminescence measuring device according to the invention
• FIG. 7 represents in perspective a first embodiment of a device for measuring a photoluminescence according to the invention.
• FIG. 8 represents means for correcting the aberrations due to the geometry of the measurement vessel
• FIG. 9 represents the transmission coefficient of the emission beam as a function of the angle of incidence on a glass / air interface
• FIG. 10 represents in perspective a second embodiment of a device for measuring a plurality of photoluminescences according to the invention.
• FIGS. 11a, 11b and 11c show several positions for an extinction capture element in a device according to the invention
• FIGS. 12a and 12b show the overlapping bundles, respectively in a triangular tank and in perspective
• FIG. 13 schematically illustrates the principle of the absorption and diffraction measurements according to the invention
• FIG. 14 illustrates the result observed at the output of an optical fiber used as an extinction capture element
• FIG. 15 represents the absorption spectra, in solid lines, and emission, in dashed line, in fluorescence of the dye Phycoerythrin Cyanine 5;
• FIG. 16 is a diagram bearing the characteristics of a population of reticulocytes measured by means of a device according to FIG. 2. Detailed description of an embodiment
• FIG. 2 diagrammatically represents a photoluminescence measuring device in a cylindrical measurement vessel CM according to the invention.
• This device comprises three optical systems Ca, Cb, and Cc similar and each centered on an axis Xa, Xb, Xc. These axes Xa, Xb, Xc make non-zero and distinct angles of 180 ° between them.
• the optical systems Ca, Cb, and Cc being regularly distributed around the measurement vessel CM these angles are identical and equal to 120 °.
• These are optical systems called epifluorescence assemblies, in which the same optics are used for the emission and reception of light. In these assemblies, the axes of the light emitted towards the measurement vessel and the light received from the measuring vessel are merged.
• Each optical system Ci comprises a source Si, for emitting an excitation beam, schematized in solid line, to the measurement vessel CM, and a capture element CEi, for capturing fluorescence emission beams, partially schematized in FIG. dashed line, coaxial with the excitation beam along the axis Xi.
• the sources Si are electroluminescent diodes of high brightness and low spatial coherence.
• high-brightness light-emitting diodes are integrated circuits which comprise, on their surfaces, inhomogeneous zones due to the presence of electrical contacts serving to supply current to the semiconductor junction.
• the beam obtained is then not homogeneous and to project the image of the diode into a measurement volume does not allow precise discrimination of the microscopic objects analyzed.
• the electroluminescent diodes Si are therefore advantageously coupled with an optical element EOi whose function is to render the excitation light field homogeneous.
• the optical element EOi is advantageously a light conductor, for example an optical fiber, or a specific optical element such as a non-imaging optical beam transformer.
• gradient or index jump fibers of circular or rectangular section may be used.
• the emitting area of the integrated circuit comprising the diode may be placed on the input face of the light-conducting optical element EOi.
• Such coupling is economical and simple to perform.
• the temperature of the integrated circuit can reach values greater than 100 ° C., it is advisable to use materials bearing such temperatures, for example silica.
• plastic EO light conductors by inserting a specific optics, such as a glass ball lens, silica or synthetic ruby, between the light conductor EO and the integrated circuit.
• a specific optics such as a glass ball lens, silica or synthetic ruby
• the ball lens can further improve the homogeneity of the excitation light field by placing, for example, the integrated circuit in the focal plane of the ball lens.
• the light conductor EO is illuminated in parallel beam, each point of the source emitting a coupled wave in the fiber.
• the divergence of the excitation beam at the output of a light conductor E0 is given by its numerical aperture which, in the case of an optical fiber, is a function of the difference in index between the guiding portion and the sheath which the envelope.
• the light conductor EO is a silica optical fiber with a diameter of 940 ⁇ m and a numerical aperture of 0.22.
• the coupled power in each fiber is 1.5 mW making a total of 4.5 mW in the CM measuring vessel.
• the optical fiber is placed in contact with a light emitting diode OSRAM brand (Golden Dragon type) powered by a current of 2000 mA.
• the power supply of the integrated circuit can be well beyond the manufacturer data, it is advisable to provide means for cooling the junction especially when the excitation beam is used in continuous lighting mode.
• a cooling circuit composed of a radiator of low thermal resistance coupled to a Peltier effect element is for example implemented in a device according to the invention.
• cooling can be avoided when the light source is used in pulses triggered by an auxiliary means such as an optical extinction signal or an electrical impedance transducer of the type called Coulter effect.
• Extinguishing or electrical measurements are then performed upstream of the photoluminescence measuring device according to the invention in the direction of flow of the fluid flow in the measurement vessel CM.
• this direction of flow is perpendicular to the plane of the figure.
• the triggering of the excitation beams is advantageously delayed by the travel time of the detected microscopic object to go from the sensor of the detector. Impedance at the optical measurement point located at the measuring point M.
• each optical system Ci the excitation beam coming from the light conductor EOi is made parallel by a collimation optic LIi.
• This excitation beam is advantageously then filtered by a filter element FIi which is a bandpass filter defined by the absorption spectra or spectra of the fluorescent compound (s) to be detected.
• a filter element FIi which is a bandpass filter defined by the absorption spectra or spectra of the fluorescent compound (s) to be detected.
• FIG. 3 represents a normalized profile of the intensity IL of the light field centered at the point M in the horizontal direction as obtained in the measurement vessel CM. There is a good spatial homogeneity over the width of the M point which is, there, 300 microns.
• FIG. 4 describes an example of spectral characteristics, in this case gains G as a function of the wavelength, for the filters F1 and F2 and for the dichroic plate DC in an application intended for the measurement of colored microscopic objects using the orange thiazole dye or any other dye with the same spectrometric characteristics.
• a dye whose absorption properties, solid line, and fluorescence, dotted line, are shown in Figure 5, is for example used for the differential count of reticulocytes which is one of the major applications of the invention. . It is also possible to detect with the invention nucleated cells or other biological elements. As shown diagrammatically in FIG. 5, the excitation is carried out in a narrow band centered on 488 nm and the fluorescence measurement is carried out on a 30 nm band centered on 530 nm.
• the filter F1 is centered at the excitation wavelength of 470 nm with a width of 15 nm
• the filter F2 is centered at the fluorescence emission wavelength of 540 nm. with a width of 20 nm.
• the filter F2 is mono channel.
• a multi-channel filter is advantageously used.
• the DC dichroic filter has a very fast rising edge from its minimum transmission to its maximum transmission at about 10 nm. It is a high-pass filter allowing the fluorescence emission wavelengths to pass and reflecting the excitation wavelengths.
• filters are for example available from manufacturers OMEGA or SEMROCK.
• magnification Gr of the optical assembly constituted by the optical combinations L1 and L2 is then a parameter determining this power.
• the projected image in the counting chamber has a size of
• the focusing of the excitation beam produces a power density equal to P Gr 2 / (axb), that is to say that the power density is Gr 2 times greater than at the level of the exit face of the light conductor EO.
• magnification Gr it will therefore be advantageous for the magnification Gr to be as large as possible and it is therefore advantageous for the optical combinations L 2 to have large numerical apertures.
• each microscopic object of the measurement volume perceives three excitation beams, thus enjoying a triple excitation fluorochromes.
• the fluorescence lights being isotropic, fluorescence emission beams are collected by the three optical combinations L2a, L2b, L2c.
• the emission beam is then transmitted through the dichroic plate DCi and then filtered using the filter F2i.
• the emission beam is then focused using the optical combination L3i to the capture element CEi.
• the capture elements CEa, CEb, CEc are advantageously light conductors, for example optical fibers, one end of which is placed at the focal center of the lenses L3a L3b, L3c and the other end is oriented towards a sensitive surface of a PD single photodetector, which can be a photomultiplier, a single photodiode or avalanche effect.
• the photodetector PD simultaneously receives the emission beams from the three capture elements CEa, CEb and CEc and then realizes the sum of the light energies captured by the optical fibers CEa, CEb, CEc.
• the quantity of light collected by this set is therefore greater than the sum of the luminous quantities collected by each system taken separately, and this as soon as two optical systems are used according to the principles of the invention.
• the excitation beams in the device according to FIG. 2 are offset with respect to each other because the epifluorescence assemblies make a non-zero obtuse angle distinct from each other. 180 °. This configuration avoids a total overlap of the excitation and emission beams which minimizes background light, the main source of noise at the PD photodetector.
• I b the more discriminating the measuring device will be.
• I b is minimized in the devices according to the invention because the excitation beams do not overlap or overlap only partially.
• the device of FIG. 2 further comprises spatial filters D, for example simple pinholes, placed in front of the capture elements CE.
• This filtering has the effect of eliminating certain signals undesirable reflections, such as spurious reflections on the walls of the measuring vessel CM. It thus contributes to the decrease of the component I b and thus to the improvement of the signal-to-noise ratio.
• FIG. 7 represents, in perspective, a first embodiment of a device according to the invention. in which a triangular CM measuring vessel is used. The measurement vessel CM is then such that its faces are perpendicular to the axes Xa, Xb, Xc of the optical systems Ca, Cb and Cc located at 120 ° from each other.
• a DC dichroic plate is placed at 45 degrees at the intersection of the excitation and emission beams and has the spectral transmission characteristics shown in Figure 4.
• the embodiment shown in FIG. 7 is suitable for the detection and counting of a single fluorescence wavelength and uses three optical systems Ca, Cb and Cc according to the principles of the invention.
• This device can be used in particular for the detection and counting of reticulocytes in peripheral whole blood samples.
• the passage of microscopic objects in the illumination plane of the optical systems is represented by a succession of aligned balls passing through the measurement vessel CM.
• the three emission beams are captured by three capture elements CEa, CEb, CEc, which are conjugated light conductors on a single PD photodetector.
• Each emission beam is spectrally filtered by means of a DC dichroic plate and interferential filters, not shown and preferably positioned between the DC dichroic plates and the L3 optical combinations.
• the spectral filtering is provided by an interference filter positioned between the three arrivals of the capture elements CEa, CEb, CEc and the photosensitive detector PD.
• each optical system Ca, Cb, Cc it is advantageous for each optical system Ca, Cb, Cc to comprise means for correcting the optical aberrations introduced by the thick diopter constituted by each face of the measurement vessel CM.
• the optical combination L2 is advantageously corrected geometric aberrations related on the one hand to the large numerical aperture of the beam which may be greater than 0.6, and on the other hand to the crossing of thick diopters, in particular the diopter of the measuring vessel CM and the fluid thickness traversed to the measuring point M.
• FIG. 8 shows an exemplary embodiment of a correction implementing a set of lenses of curvature and refractive indices chosen, the interval between two lenses being also a sizing parameter.
• the correction is made for a measurement vessel CM of equilateral triangular section. It is an association of three flat dioptres for example composed of a 2.5 mm glass wall and 1.5 mm of silica.
• the optical combination L1 is an achromatic doublet that minimizes the chromatic aberration at 488 nm
• the optical combination L2 is composed of a four-lens train consisting of a paired doublet including the diopters of Figure 8.
• the optical combination L3 is a convex plane lens.
• aspheric optics can also be used to make corrections of similar aberrations or other kinds.
• the described optics correct the geometric and chromatic aberrations introduced by the crossing of the thick dioptres constituted by the glass wall of the tank and the thickness of water between the wall of the measuring tank CM and the passage of the microscopic objects, by example the cells, in point M.
• the excitation beam then passes through a first air / glass interface and then a second glass / water interface which reduces the amount of light by a factor equal to the Fresnel transmission at the interfaces considered.
• optical assembly consisting of the optical combinations L1 and L2 can be optimized by correcting, in addition to geometric aberrations, chromatic aberration related to the width of the spectrum of the excitation.
• optical assembly consisting of the optical combinations L2 and L3 can be optimized by correcting the chromatism linked, on the one hand, to the fact that the fluorescence lights are centered on wavelengths shifted towards the long wavelengths. and on the other hand that the detection of these lights is performed on a spectral band of finite width.
• the fluorescence emission beams are collected by the three capture elements CEa, CEb and CEc, which are light conductors gathered in a single beam coupled to a photoelectric detector PD, which can be a photomultiplier or an avalanche photodiode.
• This photodetector PD realizes the sum of the light energies collected from the three optical fibers.
• a calculation of the fluorescence is then carried out according to the knowledge of those skilled in the art, especially after a prior calibration of the device. A measurement of the fluorescence generated in the measurement volume v is thus obtained.
• FIG. 10 represents, in perspective, a second embodiment of a device according to the invention adapted to the measurements of several fluorescence wavelengths of a fluid in a measurement vessel CM.
• the microscopic objects present in the measurement vessel CM are, here again, illuminated by the three excitation beams coming from the sources Si of three systems Ca, Cb, Cc, via a filtering by means of a possible spectral filter, not shown. and a DC dichroic splitter plate.
• the dichroic plate DCi reflects light from Si to the measurement vessel CM where L2i concentrates it.
• This dichroic plate DCi transmits the higher wavelengths from the illuminated microscopic objects to the capture elements CEa, CEb and CEc which are preferentially light conductors such as optical fibers.
• the three capture elements CEa, CEb, CEc are then conjugated on a common spectrometric detection assembly consisting, for example, of a diffraction grating DG and of photodetectors PD1 to PDn.
• the n photodetectors are positioned spatially with respect to the DG network to collect and measure each a wavelength band corresponding to one of the target fluorescences emitted by the biological elements passing through the tank CM.
• These photodetectors PD1 to PDn may be detectors chosen from possibly avalanche-effect photodiodes, for example arranged in line or in strips, photo-multiplier tubes, multiple optical sensors of the CCD type, for example organized in matrix or in line. .
• Distinct fluorescence intensities are then obtained for a plurality of distinct wavelength bands.
• the presence of fluorescences at distinct wavelengths may be due to differences between the detected particles or to the plurality of wavelengths used in the emission, this plurality generating fluorescence at different wavelengths.
• the three capture elements CEa, CEb, CEc are conjugated on a detection assembly consisting, for example, of separating plates, possibly dichroic, distributing the light beam towards photodetectors PD1 to PDn.
• the emission beams Prior to the measurement, the emission beams can be filtered by means of interference filters adapted to the fluorophores used.
• At least one of the optical systems includes an extinction capture element DT placed near the source, here Sa, of the system concerned.
• This extinction capture element DT is intended to capture lights having the same properties in terms of wavelengths as the source Sa. These lights are thus reflected by the dichroic plate DC coming from the tank CM towards the Sa source.
• the extinction capture element DT is coupled to a photodetector PDT.
• FIG. 11 proposes a number of possible positions for an extinction capture element DTa near the source Sa, represented by the section of the optical element EOa ensuring the homogeneity of the beam produced by the source Sa.
• Such an extinction capture element DT makes it possible to visualize the intersections of the excitation and emission beams, also called recovery beams. Geometrically, these intersections correspond to the intersection of six cones resting on the pupil of the optics L2i and pointing to the center of the measuring chamber CM; these volumes or FC cover beams are shown in Figure 12. They exist as soon as the numerical aperture of the lens L2 is sufficiently large.
• Figure 12a shows a horizontal section of the measuring vessel CM on which are indicated the axes Xa, Xb and Xc of the three optical systems Ca, Cb and Cc.
• the overlapping beams respectively corresponding to excitation by the excitation beam of the optical system Cb and Cc received by the system Ca are denoted FCb 3 and FCc 3 . This notation is used for the other overlay beams received by the Cb and Cc systems.
• Figure 12b gives a three-dimensional representation of these same overlapping beams.
• the existence of such overlapping beams is advantageously exploited to measure the absorption and diffraction by the microscopic objects present in the measurement vessel.
• the extinction capture elements DT are intended to capture the light of these beams.
• the section of the optical element EOa is rectangular and written in a square whose upper part is used to place a plurality of extinction capture elements DTa ', to receive signals indicative of an absorption , and DTa ", to receive signals indicative of a diffraction
• the use of two rectangular extinction sensors DTa 'placed on either side of the source Sa captures the lights of the beams of recovery since the geometry of these beams is such that they are located on either side of the excitation beam, the use of the central extinction sensor DTa "makes it possible to capture the possible diffracted lights.
• the sections of the other optical elements EOb, EOc can advantageously be, for their part, square.
• FIG. 13 schematically illustrates the principle of an absorption and / or diffraction measurement in a photoluminescence measuring device according to FIG. 7.
• the measurement vessel CM is illuminated by two beams of light. excitation from the optical systems Cb and Cc.
• the opening of the excitation beams from sources Sb and Sc is such that overlap beams FCb 3 and FCc 3 exist with the transmission beam of the system Ca.
• the emission beam of the fluorescence wavelength captured by the system Ca crosses the plate DCa without being deflected while the light received from the sources Sb and Sc constituting the covering beams is deflected by the dichroic blade DCa.
• FIG. 13 only these overlapping light beams FCb 3 and FCc 3 of the same wavelength as the sources Sb and Sc are represented. They come from the measurement vessel CM and go towards the extinguishing capture element DTa via the dichroic plate DCa.
• the extinguishing capture element DTa is advantageously an optical fiber of circular section.
• FC 3 overlay beam Those that do not belong to any FC 3 overlay beam only include RD diffracted rays in the CM measuring vessel and are indicative of the diffraction in the CM measuring vessel.
• RD rays will appear in the angular sector bordered by the overlapping beams FCb 3 and FCc 3 only when a microscopic object has diffracted the excitation light into the measurement vessel CM.
• FCc 3 of the source Sc include diffracted rays from one of the sources Sb, Sc, or even Sa when the latter is active, and the beams of the beam from the source Sc and having passed through the measuring vessel CM without deviation or absorption.
• the radii of a covering beam are partly a reflection of diffraction but also of absorption since extinction due to an absorbing microscopic object will be visible in the angular sectors defined by a covering beam.
• One of the advantages of the invention is the ability to visualize and exploit the FC overlay beams and the RD diffracted beams in the angular sector bordered by the FC overlay beams.
• an optical fiber preferably of circular section is used to produce the extinguishing capture element DTa, when it is unique.
• the optical characteristics of such an optical fiber make it possible to exploit the differences in entry angles in the optical fiber of the rays belonging to and not belonging to a covering beam FCb 3 or FCc 3 . Indeed, as illustrated in FIG. 13, the radii of the covering beams FCb 3 and FCc 3 enter the fiber at an angle greater than the axis of the fiber than those RD, diffracted, located in the angular sector. bordered by overlapping bundles.
• This angular property is conserved along the fiber because the rays of the overlapping beams twist along the fiber but remain close to the index jump line or index gradient while the other diffracted rays entered with a smaller angle to the axis of the fiber are found throughout the section of the fiber.
• each element targeting a specific part of the section of the fiber DTa by imaging the output of the fiber DTa on a multi-element PDT photodetector, each element targeting a specific part of the section of the fiber DTa, it can be seen that the CNT contour of the fiber DTa is permanently illuminated and undergoes extinction at the moment of passage of a microscopic object, this extinction being due to absorption by this object. The light then observed on the CNT contour is indicative of the absorption and partially of the diffraction which has no reason to be zero in the angular sectors of the overlapping beams.
• the CTR center of the fiber DTa only becomes illuminated when a microscopic object passes through, signifying the diffraction of the light by this object.
• diffraction is isotropic, it is possible, by connecting the photodetector PDT to processing means, deduce its intensity to the light observed on the contour of the optical fiber to obtain a value of the absorption.
• Such use of the overlay beams is particularly advantageous for differentiating biological cells according to their absorption and / or diffraction characteristics.
• extinction measures can be used for the cytological study for which they are interpreted as morphological or chemical information.
• each microscopic object for example a biological object passing through the measurement vessel CM
• the three epifluorescence being conjugated in one and the same photodetector for each fluorescence wavelength considered.
• the identification and differential counting of biological elements is commonly done in flow cytometry. To do this the blood sample is incubated with antibodies specific biological elements to identify. These antibodies are conjugated to markers, most often fluorochromes. These fluorochromes are usually chosen for identify each antibody specifically and the measurement of a fluorochrome therefore corresponds to the identification of the antibody on which it is conjugated. It is thus possible to identify several different antibodies by measuring as many different wavelengths. In the device described in FIG. 10, it is possible to measure several different wavelengths. It is thus possible to measure at least as many antibodies or specific markings as wavelengths.
• FIG. 5 The spectrum of FIG. 5, already presented, is that of orange thiazole. It can also be applied to the dye Fluorescein Iso ThyoCyanate (FITC) very commonly used in conjugation to an antibody.
• FITC Fluorescein Iso ThyoCyanate
• Figure 15 depicts another dye, the tandem Phycoerythrin Cyanine 5, also commonly used in flow cytometry. These two dyes can be used to identify at least two different antibodies in the device described.
• the tank CM is arranged to measure sequentially on all the elements passing through it, their volume by the method of impedance measurement as described in patent FR 2,653,885. ⁇
• the measurements can be performed on a single wavelength or a plurality depending on the device used and the markers used.
• the previously presented steps have been carried out for the differentiation and counting of reticulocytes with the device of FIG. 7.
• Reticulocytes are young forms of erythrocytes or red blood cells. They are characterized by the intracytoplasmic presence of reticulum consisting of RNA. These traces are the remainder of the expulsion of the nucleus during its transition from the erythroblast stage to the reticulocyte stage within the bone marrow. About twenty-four hours after this expulsion, the reticulocytes pass from the bone marrow into the blood.
• the ribosomes degrade to transform the reticulocyte into a mature erythrocyte. Since the average life span of a red blood cell is 120 days, the normal regeneration rate must be 0.83%. The generally accepted average normal percentage is between 0.5 and 1.5%, with these values being higher in infants under 3 weeks of age (2-6%).
• the observation and counting of reticulocytes is therefore an indicator of the erythropoietic activity and thus a particularly useful parameter especially in the follow-up of the medullary recovery after chemotherapy, in the follow-up of treatment with recombinant erythropoietin (rHuEpo), in the balance sheet. investigation of anemia or in the search for hemolysis or compensated haemorrhage.
• the dilution step of the whole blood sample is carried out using a reagent containing Thiazole Orange, in particular as described in patent FR 2,759,166.
• the results of the fluorescence and volume measurements are reconstituted and advantageously arranged to allow the absolute and differential count of the biological elements observed. It is then possible to extract the number of erythrocytes and the number and percentage of reticulocytes based on the fluorescence of the intracellular RNA. It is also possible to calculate an index of reticulocyte immaturity (IRF) based on the distribution of elements according to their fluorescence. The most fluorescent elements being considered as the youngest ones.
• IRF index of reticulocyte immaturity
• FIG. 16 is a diagram bearing the results obtained by means of the invention: the reticulocyte population is placed on the diagram as a function of their VC cell volume in ⁇ m 3 measured by impedancemetry on the abscissa and on the ordinate, the intensity IF of the picoWatts fluorescence signal.
• the invention can be implemented with various numbers of optical systems, starting from two and shifted in pairs by a non-zero angle and distinct from 180 °.
• such a property is very useful for discriminating distinct microscopic objects.
• the wavelengths of the sources Sa, Sb, Sc it is also possible to envisage varying the wavelengths of the sources Sa, Sb, Sc. It would thus be possible to illuminate the microscopic objects passing through the measurement vessel CM with an excitation beam comprising two or several wavelength ranges or with different wavelength excitation beams and to individually measure the resulting epifluorescence.

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