WO2005001449A1 - Method for dosing a biological or chemical sample - Google Patents

Method for dosing a biological or chemical sample Download PDF

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Publication number
WO2005001449A1
WO2005001449A1 PCT/FR2004/050289 FR2004050289W WO2005001449A1 WO 2005001449 A1 WO2005001449 A1 WO 2005001449A1 FR 2004050289 W FR2004050289 W FR 2004050289W WO 2005001449 A1 WO2005001449 A1 WO 2005001449A1
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WO
WIPO (PCT)
Prior art keywords
sample
image
biological
tank
measurement
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PCT/FR2004/050289
Other languages
French (fr)
Inventor
François PERRAUT
Emmanuelle Schultz
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Commissariat A L'energie Atomique
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Application filed by Commissariat A L'energie Atomique filed Critical Commissariat A L'energie Atomique
Priority to CA002530382A priority Critical patent/CA2530382A1/en
Priority to EP04767851A priority patent/EP1639349A1/en
Priority to JP2006516356A priority patent/JP2007516411A/en
Priority to US10/560,973 priority patent/US7396650B2/en
Priority to AU2004252258A priority patent/AU2004252258A1/en
Publication of WO2005001449A1 publication Critical patent/WO2005001449A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • 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
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
    • G01N15/0227—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging using imaging, e.g. a projected image of suspension; using holography
    • G01N15/1433—

Definitions

  • the present invention relates to a method for assaying a biological or chemical sample.
  • the field of the invention is notably that of concentration measurements of fluorescent molecules called fluorochromes contained in solutions. Such molecules are used to measure the quantity of a given biological species. The quantity of molecules of this biological species is then linked to the quantity of fluorescent molecules. A measurement of the intensity emitted during the excitation of these fluorescent molecules makes it possible, by calibrating the measuring device used, to deduce the quantity or the concentration of biological molecules. Such measurements are commonly used in biology, chemistry and physics. In the following description, for reasons of simplification of description, the invention is described in this field of fluorescence measurements of samples.
  • pixels are sometimes associated in the direction orthogonal to that of the dispersion in order to carry out an operation called “binning” which makes it possible to increase the signal to noise ratio of each spectral measurement by reducing the noise of reading of the detectors in front of the photon flux .
  • Multiple detectors are also used in some multi-sample devices. The presence of several samples then requires the use of several cells and the measurement for each sample is made via an image sensor. The detection sensitivity of such devices is insufficient to measure small quantities of molecules, typically of the order of picoMolar, either to diagnose a disease or to study the purity of a solution.
  • assays are even impossible to do below a certain concentration: in the field of immunoassays (assay of antigens), the statistical detection threshold of known art, expressed in concentration of targets, the lowest obtained for a measurement in solution is of the order of a hundred picoMolar.
  • tank-based trade devices do not allow fluorescence to be measured below a concentration of lnM (nanoMolar) targets.
  • the light from a laser source can be focused in a very small volume, as is done in capillary electrophoresis. The sample then passes through a capillary a few hundred micrometers in diameter.
  • the detection limit obtained is of the order of nM, as described in the document referenced [1] at the end of the description.
  • the excitation light can be focused.
  • the increase in the power density makes it possible to increase the number of photons emitted in fluorescence.
  • a high power density is accompanied by a photo-extinction the more rapid the greater the light energy.
  • Factors limiting the detection threshold reside in the auto-fluorescence of liquids, which is an intrinsic fluorescence of these media, and in Raman scattering.
  • the level of light emitted reduces, in fact, the detection performance because the photoluminescence "offset" of the buffer used is of the same nature as the signal, called “specific”, which one wants to detect.
  • the smallest signal measurable in the statistical sense S m i n is equal to where "Offset” is a measure expressed in primary electrons (electrons resulting directly from the conversion of photons by a photocathode in the case of a photomultiplier or a semiconductor surface) and 3 is an arbitrary factor which allows ensure discriminate between 99% S m i n and Offset.
  • the solutions of the known art realize: 1) a judicious choice of liquids, 2) a choice of the marker, 3) an increase in the measurement time for accumulating photons, 4) an increase in power 25 of excitation to collect more photons.
  • the main factor limiting the detection threshold is the non-reproducibility of the measurements, which, for low signal levels, very quickly become dominant.
  • This non-reproducibility 30 essentially results from a poor mechanical repositioning of the measurement tank and of the light which is collected by the liquid meniscus in this tank and which is randomly directed into space.
  • One solution to reduce such non-reproducibility consists in injecting into the tank a larger volume of solution. However, such an injection is insufficient to obtain good sensitivity. In addition it is not always possible or desirable to work with large volumes. Mechanical positioning cannot be easily improved. Furthermore, in such a solution, variations are not treated, for example, caused by ambient lighting and the variation in the shape of the meniscus.
  • Another solution to reduce such non-reproducibility consists in using cuvettes comprising a transparent window in “black” glass, which corresponds to the zone considered for the measurement. But this solution reduces the flow of photons collected by the measurement system, and therefore raises the detection limit. In addition, it does not allow to know the variations of the "offset", which can be caused by a modification of the ambient light, by a bad surface condition of the faces of the tank.
  • the object of the invention is to overcome these drawbacks by proposing a new method for assaying a biological or chemical sample which uses a device for spatial recording of the image of the interaction between the light coming from a source and from this sample as a means for selecting the useful information.
  • the invention relates to a method for dosing a biological or chemical sample, comprising the following steps: - possible introduction of the sample into a tank, all of the faces of which are transparent, - illumination of the sample at means of a light beam from a source, characterized in that it further comprises the following steps: - production of an image comprising the image of the light scattered by the sample, - analysis of the image according to reference criteria, - extraction of information specific to the interaction of light beam / sample, - calculation of the dosage.
  • the diffusion can be Raman diffusion, fluorescence diffusion, molecular or particulate diffusion.
  • the analysis may consist of studying the spatial structure of the image and the distribution of light energy in this image.
  • the dosage can be calculated in relation to a calibration between the measurement of light energy and the concentration or quantity of sample.
  • the dosage calculation can also be to do in relation to the analysis of the kinetics of the biological or chemical reaction.
  • a first area of interest is defined around the area of excited volume, and a second area of interest arranged next to this first area, and the specific signal is measured by performing the subtraction between the sum of all the pixels in the first area and the sum of all the pixels in the second area.
  • the invention has the following advantages: - It makes it possible to reach an experimental detection limit much lower than that of conventional systems. - It makes it possible to carry out a dosage using a large volume of solution with a high flow rate, and therefore to envisage applications such as an analysis of river water, aeration systems in buildings. - It does not require focusing the light in a small volume. The photo-bleaching of fluorescent molecules is therefore very low. - It allows, because of the geometry of the cell, to simultaneously excite a large number of molecules, which makes it possible to collect numerous photons. - Neither auto-fluorescence nor Raman diffusion of the liquid medium is a limitation on the sensitivity of the invention. It therefore allows work with all commercial markers, which reduces marking constraints.
  • the non-reproducibility due to the mechanics of the tank, the cleaning or alteration of the optical faces of the tank, and the presence of artefacts (bubbles, dust) are no longer a constraint.
  • the invention makes it possible, by image analysis, to reduce or even eliminate them. A shift in the position of the tank in front of the measuring system of the invention or a translation of the excitation light from the medium can be completely corrected after measuring the position of the fluorescent trace in the recorded image.
  • dust present in the excited volume which could significantly modify the measurement, is small in front of the excited volume, and can thus be identified and removed from the measurement without losing it, which is impossible to be performed with a single detector.
  • the invention makes it possible to know the variations in the effective power and to correct the measurement.
  • the variations in ambient light caused either by the sample or by the environment are compensated for, by a measurement in the image, then a withdrawal of the possible "offsets".
  • the analysis of the information in an image can be carried out on the basis of elements determined in advance (lighting function, predetermined positions of the various useful zones), or dynamically to deal with random and / or unforeseen disturbances by applying image processing methods (maximization of entropy, neural network).
  • the assay dynamic of the invention which, for a certain type of measurement, makes it possible to experimentally achieve a biological assay dynamic of 2,200, is much higher than that of the known art, which, for the same type of measurements, is typically between 5 and 10.
  • the invention is applicable in many fields, and in particular: - in all fields where it is useful to measure a fluorescent solution, - in biology, more particularly for the assay of biological molecules or of biological interest: antigens, antibodies, peptides, DNA, cells, bacteria ..., for clinical diagnosis, - in chemistry (assay), - in pharmacy: activity assay, contamination, etc. ., in physics: search for product traces, fluidics, mixture analysis, etc.
  • FIGS. 1 and 2 illustrate an image of the tank obtained with the image recording device shown in FIG. 1.
  • Figures 4 to ⁇ illustrate a second embodiment of a device implementing the method of the invention.
  • FIG. 7 illustrates a third embodiment of a device implementing the method of the invention.
  • FIGS. 8 to 10 illustrate three exemplary embodiments of a device implementing the method of the invention.
  • the method of the invention is a method for assaying (measuring a concentration or a quantity) of a biological sample or of biological interest (antigens, antibodies, peptides, DNA, cells, bacteria, toxins) or chemical (solvent, dissolved gas, formulation, chemical activity), which can be a solid, a liquid, a gel, etc.
  • a sample 10 is illuminated by a light beam 17 from from a source 11 tuned with the fluorochro e used.
  • a source 11 tuned with the fluorochro e used.
  • an Argon 488 n laser can be used for fluorescein or a Helium-Neon laser at 633 nm for Cy5.
  • the sample 10 is placed in a tank 12, for example of rectangular section, all the faces of which are transparent.
  • the section of this tank 12 can, in fact, be rectangular, square, cylindrical or elliptical.
  • a lens system 13, equipped with a stop filter 14, is mounted in front of a device for recording the spatial structure of an image 15, for example a CCD camera or a scanning system, connected to a processing member 16.
  • the device 15 which receives the beam 18 scattered by the sample 10, allows the recording of an image from which can be extracted a specific measurement signal.
  • the method of the invention comprises the following steps: - illumination of the sample 10 by means of the light beam 17 coming from the source 11, which can be a gas laser, a solid laser, a laser diode, a light-emitting diode, an organic diode, a spectral lamp such as a halogen, mercury, xenon, deuterium lamp, - production of an image of the light beam 18 scattered by the sample 10, the origin of the scattering being possibly Raman scattering, fluorescence scattering, molecular scattering (Rayleight scattering) or particulate scattering (use of nanoparticles), - analysis of the image in relation to references, this analysis then consisting in studying the spatial structure of the image and the distribution of light energy in this image, these references being constituted, for example, by the experience of a user or by morphological criteria (shape and position of a light trace), photo metric (frequencies of spatial variations of light in the image), statistics (variation of measurement estimators, entropy in the image
  • this extraction consisting, for example, of arithmetic operations between the image and other images or constants (for example, subtractions, additions, divisions, multiplications), morphological (erosion, dilation, binarization, clipping, segmentation, offset correction) or photometric (polynomial corrections, convolutions, 10 filters, thresholds), - dosage calculation, this calculation being done in relation a calibration between the measurement of light energy and the concentration or quantity of the biological or chemical sample.
  • This calculation can also be performed by recording the kinetics of the biological or chemical reaction and analyzing this kinetics by methods known to those skilled in the art.
  • the measurement is made in an image obtained by the device 20 for recording the spatial structure of an image 15.
  • the invention does not lie in the use of such a device 15 but mainly in: - the fact of recording the beam 18 diffused by the sample 10 in the form of an image, 25 - the fact of extracting the information from this image, the adaptive side obtained by the application of a image analysis.
  • FIG. 2 illustrates the image of the tank 12 obtained with the device for recording the spatial structure of an image 15.
  • the tank may have dimensions smaller than those of the image. It can for example be replaced by one or more capillaries.
  • the light beam can be either smaller or larger than the tank.
  • an area 20 of illuminated volume which corresponds to the volume of the tank 12 excited by the beam 17, - the area 21 of entry of this beam 17 into the tank 12, - the area 22 outlet of this bundle 17 from the tank 12, - a meniscus area 23, - an artefact area 24. It is thus possible, as illustrated in FIG. 3, to define a first region of interest 25 around the area 20 of illuminated volume and a second region of interest 26 disposed next to this area 25. The measurement of the specific signal is then given by calculation: ⁇ RI ⁇ - ⁇ RI2; that is to say the subtraction between the sum of all the pixels of the first region of interest 25 and the sum of all the pixels of the second region of interest 26.
  • ⁇ RI ⁇ - ⁇ RI2 that is to say the subtraction between the sum of all the pixels of the first region of interest 25 and the sum of all the pixels of the second region of interest 26.
  • the two regions of interest 25 and 26 are the same size, which is not essential. When these regions do not have the same size, it suffices either to perform an averaging of the gray levels of each region, or a weighting of the values by the number of pixels. Analysis of the image thus represented leads to certain observations:
  • the fluorescent trace of the light beam (area 20) gives the specific signal.
  • the meniscus (zone 23), delimiting the liquid from the air, is strongly luminous. Its origin comes from said trace. The shape of this meniscus is highly random. The amplitude of the signal from this meniscus is therefore very variable.
  • An artefact (zone 24) can be, for a given assembly, a spring washer intended to ensure good mechanical positioning of the tank 12.
  • Zones 21 and 22 correspond respectively to the entry and exit points of the light beam in the tank 12. With another adjustment of the display thresholds, it is easier to highlight the weakest light levels.
  • the method of the invention makes it possible to improve the coefficient of variation CV (standard deviation / average). Indeed, the CV coefficient obtained with the method of the invention is much lower than the CV coefficient calculated by summing all the pixels of a CCD camera, which corresponds to a measurement made with a mono-detector.
  • the CV coefficient obtained with the method of the invention is moreover of the same order of magnitude as that obtained with a large volume of solution, as envisaged previously in the introduction to said application. This CV coefficient obtained with the method of the invention is lower than that obtained by measuring in each of the zones. of interest 25 and 26.
  • a single detector associated with a matrix of pixels with programmable transparency 30 such as a matrix of liquid crystals or micro-mirrors or any other equivalent system is placed in front of the tank, 12 illustrated in the figure 4. This matrix 30 is interposed between the tank 12 and the detector via an image forming system or not.
  • a first measurement is then carried out by "opening" the pixels corresponding to the first area of interest 25, as illustrated in FIG. 5, then a second measurement by opening the pixels corresponding to the second area of interest 26, as illustrated in FIG. 6.
  • the use of such a matrix 30 with variable transparency makes it possible to avoid the systematic recording of an image, by carrying out for example the following steps: - recording of the image of the beam scattered by the aperture / successive closure of all the pixels of the matrix 30 in synchronization with the measurement carried out by the mono-detector, - analysis of the image and definition of the area or areas of interest making it possible to extract the specific information, - recording of such parameters for a subsequent use, - during the analysis of a given sample, successive openings of the regions defined during the analysis step and recording of the measurement results for each of these zones, - extraction of useful information, - calculation dosage.
  • two mono-pixel detectors 35 and 36 each observe a region of interest 25 or 26.
  • Two imaging means 37 and 38 are placed respectively in front of each of these two detectors 35 and 36.
  • the measurement of the signal from the first region of interest 25 is made with the detector 35, that for the second region of interest 26 is made with the detector 36.
  • the area 39 represents the fluorescent trace with a view to face.
  • the invention makes it possible to adapt the extraction of the specific signal to the experimental conditions. For example, if the cell has moved between two series of measurements, it is possible, by automatic image analysis, to reposition the regions of interest automatically, an operation impossible to perform with a static system.
  • a light source 40 for example a laser or a light-emitting diode, excites, in a tank 41, the liquid containing fluorescent molecules through shaping optics 10 not shown, and various accessories such as a shutter 42, and a diaphragm 43.
  • a first objective 44 arranged for example perpendicular to the main direction of the light beam, collects part of this light beam 15, emitted by the fluorescent molecules in the tank 41.
  • a stop filter 45 is disposed behind the first objective 44, just in front of a second objective 46.
  • this detector 47 can be a detector of 512 x 512 pixels with a side of 10 ⁇ m.
  • the first objective 44 can have a focal length 50 mm and the second objective 46 25 a focal length 25 mm.
  • a variable transparency matrix 56 which may be a liquid crystal matrix or a micro-mirror matrix, plays the role of a field diaphragm.
  • the matrix 56 can be replaced by a movable slot actuated by a mechanical or electro-mechanical actuator, for example, an electromagnet or an electric motor.
  • a mechanical or electro-mechanical actuator for example, an electromagnet or an electric motor.
  • the configuration for exciting the interior of the tank 41 is the same as for FIG. 8 and the configuration for the collection of light is the same as for that of FIG. 9.
  • Two mono-detectors 50 and 51 make it possible to observe two different regions of the tank 41.
  • a recovery optic 52 and 53 makes it possible to form the image of the region d interest on a field diaphragm 54 and 55 which limits the region observed.
  • a stop filter can be placed before, in or behind the diaphragm.
  • Zone 56 represents the fluorescent trace.

Abstract

The invention relates to a method for dosing a biological or chemical sample, said method consisting of the following steps: the sample (10) is illuminated by means of a light beam (17) emitted from a source (11); an image containing the image of the beam (18) emitted by the sample (10) is produced; said image is analysed according to reference criteria; information relating to the interaction between the light beam and the sample is extracted; and the dosage is calculated.

Description

PROCEDE DE DOSAGE D'UN ECHANTILLON BIOLOGIQUE OU CHIMIQUE METHOD FOR DETERMINING A BIOLOGICAL OR CHEMICAL SAMPLE
DESCRIPTIONDESCRIPTION
DOMAINE TECHNIQUE La présente invention concerne un procédé de dosage d'un échantillon biologique ou chimique. Le domaine de l'invention est notamment celui des mesures de concentration de molécules fluorescentes appelées fluorochromes contenues dans des solutions. De telles molécules sont utilisées pour doser la quantité d'une espèce biologique donnée. La quantité de molécules de cette espèce biologique est alors liée à la quantité de molécules fluorescentes. Une mesure de l'intensité émise lors de l'excitation de ces molécules fluorescentes permet, par calibrage de l'appareil de mesure utilisé, de déduire la quantité ou la concentration de molécules biologiques . De telles mesures sont couramment utilisées en biologie, en chimie et en physique. Dans la suite de la description, pour des raisons de simplification d'exposé, l'invention est décrite dans ce domaine de mesures de fluorescence d' échantillons .TECHNICAL FIELD The present invention relates to a method for assaying a biological or chemical sample. The field of the invention is notably that of concentration measurements of fluorescent molecules called fluorochromes contained in solutions. Such molecules are used to measure the quantity of a given biological species. The quantity of molecules of this biological species is then linked to the quantity of fluorescent molecules. A measurement of the intensity emitted during the excitation of these fluorescent molecules makes it possible, by calibrating the measuring device used, to deduce the quantity or the concentration of biological molecules. Such measurements are commonly used in biology, chemistry and physics. In the following description, for reasons of simplification of description, the invention is described in this field of fluorescence measurements of samples.
ETAT DE LA TECHNIQUE ANTERIEURE De nombreux appareils de commerce, tels que les fluorimètres et les spectro-fluorimètres, permettent de mesurer la fluorescence d'une solution. Ces appareils permettent d'effectuer une mesure dans une cuve dont la géométrie est variable et fonction de 1' application . D'autres appareils de mesure en solution utilisent des capillaires comme cuve. Ce sont par exemple les systèmes de mesures pour appareil d' électrophorèse. Dans tous ces appareils on mesure un échantillon placé dans une cuve, auquel on associe un détecteur unique. Dans certaines applications de spectro- fluorimétrie on utilise des détecteurs multiples . Le spectre d'émission des molécules fluorescentes est alors dispersé sur un capteur d' image afin de mesurer simultanément l'énergie dans toutes les longueurs d'onde, ce qui revient à associer un détecteur unique pour chaque intervalle spectral. Plusieurs pixels sont parfois associés dans la direction orthogonale à celle de la dispersion afin de réaliser une opération appelée « binning » qui permet d'augmenter le rapport signal sur bruit de chaque mesure spectrale en réduisant le bruit de lecture des détecteurs devant le flux de photons . Des détecteurs multiples sont également utilisés dans certains appareils multi-échantillons. La présence de plusieurs échantillons impose alors l'emploi de plusieurs cuves et la mesure pour chaque échantillon est faite via un capteur d'image. La sensibilité de détection de tels appareils est insuffisante pour doser de faibles quantités de molécules, typiquement de l'ordre du picoMolaire, soit pour faire un diagnostic de maladie, soit pour étudier la pureté d'une solution. Certains types de dosages sont même impossibles à faire en dessous d'une certaine concentration : dans le domaine des immuno-analyses (dosage des antigènes), le seuil de détection statistique de l'art connu, exprimé en concentration de cibles, le plus bas obtenu pour une mesure en solution est de l'ordre de la centaine de picoMolaire. Typiquement, les appareils de commerce en cuve ne permettent pas de mesurer une fluorescence en dessous d'une concentration de cibles de lnM (nanoMolaire) . Pour diminuer la limite de détection on peut focaliser la lumière d'une source laser dans un très petit volume, comme cela est réalisé en électrophorèse capillaire. L'échantillon passe alors dans un capillaire de quelques centaines de micromètres de diamètre. La limite de détection obtenue est de l'ordre du nM, comme décrit dans le document référencé [1] en fin de description. On retrouve une telle limite de détection en marqueurs dans un système complexe comportant une optique confocale intégrée scrutant l'intérieur d'un capillaire, comme décrit dans le document référencé [2]. Les systèmes de mesure utilisant des capillaires ne permettent pas un débit d' échantillon très grand, par exemple d'une dizaine à une centaine de microlitre/minute. Une mesure sur un échantillon de grand volume est donc impossible. Le prélèvement qui s'ensuit réduit l'échantillonnage moléculaire et augmente le seuil de détection. Par exemple, s'il est possible de détecter la présence d'une molécule unique par des techniques de corrélation de fluorescence, comme décrit dans document référencé [3], le volume sondé est très faible, de l'ordre du femtolitre . Détecter une molécule dans un tel volume donne une limite de détection de l'ordre du nM. Pour augmenter la densité de puissance dans le volume excité on peut focaliser la lumière d'excitation. Comme l'émission de fluorescence est quasi proportionnelle à la quantité d'énergie reçue lors de l'excitation, l'augmentation de la densité de puissance permet d'augmenter le nombre de photons émis en fluorescence. Pour un système de mesure bien conçu, comme c'est le cas de la plupart des instruments du commerce, plus le nombre de photons mesurés est grand et plus l'incertitude relative de cette mesure est faible . Cette incertitude varie comme y r— où N est le nombre de photons transformés en électrons lors de la conversion par le détecteur. Ceci justifie le choix d'une augmentation de la densité de puissance dans le volume excité. Cependant, une forte densité de puissance s'accompagne d'une photo-extinction d'autant plus rapide que l'énergie lumineuse est grande. Pour la mesure d'une très faible concentration de molécules fluorescentes, il faut alors exposer ce volume pendant un temps supérieur au temps de photo-blanchiment, qui correspond à une propriété des molécules fluorescentes consistant à s'arrêter d'émettre de la lumière au bout d'un certain temps. Il est alors impossible de collecter suffisamment de photons pour atteindre le seuil de détection requis. B 14352.3 DBSTATE OF THE PRIOR ART Many commercial devices, such as fluorimeters and spectro-fluorimeters, make it possible to measure the fluorescence of a solution. These devices make it possible to measure in a tank whose geometry is variable and depends on the application. Other solution measurement devices use capillaries as a vessel. These are, for example, measurement systems for electrophoresis apparatus. In all these devices, a sample is placed in a tank, with which a single detector is associated. In certain spectro-fluorimetry applications, multiple detectors are used. The emission spectrum of the fluorescent molecules is then dispersed on an image sensor in order to simultaneously measure the energy in all wavelengths, which amounts to associating a single detector for each spectral interval. Several pixels are sometimes associated in the direction orthogonal to that of the dispersion in order to carry out an operation called “binning” which makes it possible to increase the signal to noise ratio of each spectral measurement by reducing the noise of reading of the detectors in front of the photon flux . Multiple detectors are also used in some multi-sample devices. The presence of several samples then requires the use of several cells and the measurement for each sample is made via an image sensor. The detection sensitivity of such devices is insufficient to measure small quantities of molecules, typically of the order of picoMolar, either to diagnose a disease or to study the purity of a solution. Some types of assays are even impossible to do below a certain concentration: in the field of immunoassays (assay of antigens), the statistical detection threshold of known art, expressed in concentration of targets, the lowest obtained for a measurement in solution is of the order of a hundred picoMolar. Typically, tank-based trade devices do not allow fluorescence to be measured below a concentration of lnM (nanoMolar) targets. To reduce the detection limit, the light from a laser source can be focused in a very small volume, as is done in capillary electrophoresis. The sample then passes through a capillary a few hundred micrometers in diameter. The detection limit obtained is of the order of nM, as described in the document referenced [1] at the end of the description. We find such a detection limit in markers in a complex system comprising an integrated confocal optics scanning the inside of a capillary, as described in the document referenced [2]. Measuring systems using capillaries do not allow a very large sample flow, for example from ten to one hundred microliter / minute. A measurement on a large volume sample is therefore impossible. The resulting sampling reduces molecular sampling and increases the detection threshold. For example, if it is possible to detect the presence of a single molecule by fluorescence correlation techniques, as described in document referenced [3], the volume probed is very low, of the order of femtoliter. Detecting a molecule in such a volume gives a detection limit of the order of nM. To increase the power density in the excited volume, the excitation light can be focused. As the fluorescence emission is almost proportional to the quantity of energy received during the excitation, the increase in the power density makes it possible to increase the number of photons emitted in fluorescence. For a well-designed measurement system, as is the case with most commercial instruments, the greater the number of photons measured and the lower the relative uncertainty of this measurement. This uncertainty varies as yr— where N is the number of photons transformed into electrons during conversion by the detector. This justifies the choice of an increase in the power density in the excited volume. However, a high power density is accompanied by a photo-extinction the more rapid the greater the light energy. To measure a very low concentration of fluorescent molecules, this volume must then be exposed for a time greater than the photo-bleaching time, which corresponds to a property of fluorescent molecules consisting in stopping to emit light at after a while. It is then impossible to collect enough photons to reach the required detection threshold. B 14352.3 DB
Des facteurs de limitation du seuil de détection résident dans l'auto-fluorescence des liquides, qui est une fluorescence intrinsèque de ces milieux, et dans la diffusion Raman. Le niveau de 5 lumière émis réduit, en effet, les performances de détection car l'« offset » de photoluminescence du tampon utilisé est de même nature que le signal, dit « spécifique », que l'on veut détecter. Si le système de mesure est seulement limité par le « bruit de 10 grenaille », dit également « bruit de photons », alors le plus petit signal mesurable au sens statistique Smin est égal à
Figure imgf000006_0001
où « Offset » est une mesure exprimée en électrons primaires (électrons résultants directement de la conversion des photons par une photo- 15 cathode dans le cas d'un photomultiplicateur ou d'une surface semi-conductrice) et 3 est un facteur arbitraire qui permet de garantir une discrimination à 99% entre Smin et Offset. Pour résoudre un tel problème les solutions de l'art connu réalisent : 20 1) un choix judicieux des liquides, 2) un choix du marqueur, 3) une augmentation du temps de mesure pour accumuler des photons, 4) une augmentation de la puissance 25 d'excitation pour collecter plus de photons. Mais le principal facteur de limitation du seuil de détection est la non-reproductibilité des mesures, qui, pour de faibles niveaux de signaux, devient très vite dominante. Cette non reproductibilité 30 provient essentiellement d'un mauvais repositionnement mécanique de la cuve de mesure et de la lumière qui est collectée par le ménisque liquide dans cette cuve et qui est dirigée dans l'espace de façon aléatoire. Une solution pour réduire de telles non - reproductibilités consiste à réaliser l'injection dans la cuve d'un plus grand volume de solution. Mais une telle injection est insuffisante pour obtenir une bonne sensibilité. De plus il n'est pas toujours possible ou souhaitable de travailler avec des grands volumes. Le positionnement mécanique n' est pas améliorable facilement . De plus dans une telle solution on ne traite pas, alors, des variations provoquées, par exemple, par l'éclairage ambiant et la variation de la forme du ménisque. Une autre solution pour réduire de telles non-reproductibilités consiste à utiliser des cuves comprenant une fenêtre transparente en verre « noir », qui corresponde à la zone considérée pour la mesure. Mais cette solution réduit le flux de photons collecté par le système de mesure, et donc élève la limite de détection. De plus, elle ne permet pas de connaître les variations de l'« offset », qui peuvent être causées par une modification de la lumière ambiante, par un mauvais état de surface des faces de la cuveFactors limiting the detection threshold reside in the auto-fluorescence of liquids, which is an intrinsic fluorescence of these media, and in Raman scattering. The level of light emitted reduces, in fact, the detection performance because the photoluminescence "offset" of the buffer used is of the same nature as the signal, called "specific", which one wants to detect. If the measurement system is only limited by the "noise of 10 shots", also called "photon noise", then the smallest signal measurable in the statistical sense S m i n is equal to
Figure imgf000006_0001
where "Offset" is a measure expressed in primary electrons (electrons resulting directly from the conversion of photons by a photocathode in the case of a photomultiplier or a semiconductor surface) and 3 is an arbitrary factor which allows ensure discriminate between 99% S m i n and Offset. To solve such a problem, the solutions of the known art realize: 1) a judicious choice of liquids, 2) a choice of the marker, 3) an increase in the measurement time for accumulating photons, 4) an increase in power 25 of excitation to collect more photons. However, the main factor limiting the detection threshold is the non-reproducibility of the measurements, which, for low signal levels, very quickly become dominant. This non-reproducibility 30 essentially results from a poor mechanical repositioning of the measurement tank and of the light which is collected by the liquid meniscus in this tank and which is randomly directed into space. One solution to reduce such non-reproducibility consists in injecting into the tank a larger volume of solution. However, such an injection is insufficient to obtain good sensitivity. In addition it is not always possible or desirable to work with large volumes. Mechanical positioning cannot be easily improved. Furthermore, in such a solution, variations are not treated, for example, caused by ambient lighting and the variation in the shape of the meniscus. Another solution to reduce such non-reproducibility consists in using cuvettes comprising a transparent window in “black” glass, which corresponds to the zone considered for the measurement. But this solution reduces the flow of photons collected by the measurement system, and therefore raises the detection limit. In addition, it does not allow to know the variations of the "offset", which can be caused by a modification of the ambient light, by a bad surface condition of the faces of the tank.
(salissures, rayures etc.), par une diffusion provenant du ménisque et des parois. Ainsi ces différentes solutions de l'art connu ne permettent ni une bonne sensibilité, ni une bonne reproductibilité des mesures . L'invention a pour objet de pallier ces inconvénients en proposant un nouveau procédé de dosage d'un échantillon biologique ou chimique qui utilise un dispositif d'enregistrement spatial de l'image de l'interaction entre la lumière issue d'une source et de cet échantillon comme moyen pour sélectionner les informations utiles .(dirt, scratches etc.), by diffusion from the meniscus and the walls. Thus, these various solutions of the known art do not allow either good sensitivity or good reproducibility of the measurements. The object of the invention is to overcome these drawbacks by proposing a new method for assaying a biological or chemical sample which uses a device for spatial recording of the image of the interaction between the light coming from a source and from this sample as a means for selecting the useful information.
EXPOSÉ DE L'INVENTION L'invention concerne un procédé de dosage d'un échantillon biologique ou chimique, comportant les étapes suivantes : - introduction éventuelle de l'échantillon dans une cuve dont toutes les faces sont transparentes, - éclairement de l'échantillon au moyen d'un faisceau lumineux issu d'une source, caractérisé en ce qu'il comporte, en outre, les étapes suivantes : - réalisation d'une image comprenant l'image de la lumière diffusée par l'échantillon, - analyse de l'image selon des critères de référence, - extraction d'une information spécifique à l'interaction faisceau lumineux/échantillon, - calcul du dosage. Dans ce procédé la diffusion peut être une diffusion Raman, une diffusion par fluorescence, une diffusion moléculaire ou particulaire. L 'analyse peut consister en l'étude de la structure spatiale de l'image et de la distribution de l'énergie lumineuse dans cette image. Le calcul du dosage peut se faire par rapport à un étalonnage entre la mesure d'énergie lumineuse et la concentration ou la quantité d'échantillon. Le calcul du dosage peut également se faire par rapport à l'analyse de la cinétique de la réaction biologique ou chimique. Dans ce procédé, avantageusement, on définit une première zone d' intérêt autour de la zone de volume excité, et une seconde zone d'intérêt disposée à côté de cette première zone, et on mesure le signal spécifique en réalisant la soustraction entre la somme de tous les pixels de la première zone et la somme de tous les pixels de la seconde zone.PRESENTATION OF THE INVENTION The invention relates to a method for dosing a biological or chemical sample, comprising the following steps: - possible introduction of the sample into a tank, all of the faces of which are transparent, - illumination of the sample at means of a light beam from a source, characterized in that it further comprises the following steps: - production of an image comprising the image of the light scattered by the sample, - analysis of the image according to reference criteria, - extraction of information specific to the interaction of light beam / sample, - calculation of the dosage. In this process, the diffusion can be Raman diffusion, fluorescence diffusion, molecular or particulate diffusion. The analysis may consist of studying the spatial structure of the image and the distribution of light energy in this image. The dosage can be calculated in relation to a calibration between the measurement of light energy and the concentration or quantity of sample. The dosage calculation can also be to do in relation to the analysis of the kinetics of the biological or chemical reaction. In this method, advantageously, a first area of interest is defined around the area of excited volume, and a second area of interest arranged next to this first area, and the specific signal is measured by performing the subtraction between the sum of all the pixels in the first area and the sum of all the pixels in the second area.
L'invention présente les avantages suivants : - Elle permet d' atteindre une limite de détection expérimentale beaucoup plus faible que celle des systèmes conventionnels . - Elle permet de réaliser un dosage en utilisant un grand volume de solution avec un grand débit, et donc d'envisager des applications comme une analyse des eaux de rivière, des systèmes d'aération dans des immeubles. - Elle n'impose pas de focaliser la lumière dans un petit volume. Le photo-blanchiment des molécules fluorescentes est donc très faible. - Elle permet, du fait de la géométrie de la cuve, d'exciter simultanément un grand nombre de molécules, ce qui permet de collecter de nombreux photons . - Ni l'auto -fluorescence, ni la diffusion Raman du milieu liquide ne sont une limitation à la sensibilité de l'invention. Elle permet donc de travailler avec tous les marqueurs du commerce, ce qui réduit les contraintes de marquage. Les non-reproductibilités dues à la mécanique de la cuve, le nettoyage ou l'altération de faces optiques de la cuve, et la présence d'artefacts (bulles, poussières) ne sont plus une contrainte. L'invention permet, par analyse d'image, de les réduire voire de les supprimer. Un décalage de la position de la cuve devant le système de mesure de l'invention ou une translation de la lumière d'excitation du milieu peut être totalement corrigé après mesure de la position de la trace fluorescente dans l'image enregistrée. De même, des poussières présentes dans le volume excité, qui pourraient modifier de façon significative la mesure, sont de petite taille devant le volume excité, et peuvent ain si être repérées et retirées de la mesure sans perdre celle-ci, ce qui est impossible à réaliser avec un mono-détecteur. En cas d'altération des faces optiques de la cuve, qui peut entraîner une modification de la quantité de lumière qui excite effectivement l'échantillon, l'invention permet de connaître les variations de la puissance effective et de corriger la mesure. Les variations de lumière ambiante causées soit par l'échantillon soit par l'environnement sont compensées, par une mesure dans l'image, puis un retrait des « offsets » possibles . - L'analyse de l'information dans une image peut être conduite à partir d' éléments déterminés à l'avance (fonction d' éclairement, positions prédéterminées des différentes zones utiles), ou de façon dynamique pour faire face à des perturbations aléatoires et/ou imprévues par application de méthodes de traitement des images (maximisation de l'entropie, réseau de neurones) . - La dynamique de dosage de l'invention, qui, pour un certain type de mesures, permet d'atteindre expérimentalement une dynamique de dosage biologique de 2 200, est très supérieure à celle de l'art connu, qui, pour un même type de mesures, est comprise typiquement entre 5 et 10. L' invention est applicable dans de nombreux domaines, et notamment : - dans tous les domaines où il est utile de faire la mesure d'une solution fluorescente, - en biologie, plus particulièrement pour le dosage de molécules biologiques ou d' intérêt biologique : antigènes, anticorps, peptides, ADN, cellules, bactéries..., pour le diagnostic clinique, - en chimie (dosage) , - en pharmacie : dosage d'activité, contamination..., en physique : recherche de traces de produits, fluidique, analyse de mélange....The invention has the following advantages: - It makes it possible to reach an experimental detection limit much lower than that of conventional systems. - It makes it possible to carry out a dosage using a large volume of solution with a high flow rate, and therefore to envisage applications such as an analysis of river water, aeration systems in buildings. - It does not require focusing the light in a small volume. The photo-bleaching of fluorescent molecules is therefore very low. - It allows, because of the geometry of the cell, to simultaneously excite a large number of molecules, which makes it possible to collect numerous photons. - Neither auto-fluorescence nor Raman diffusion of the liquid medium is a limitation on the sensitivity of the invention. It therefore allows work with all commercial markers, which reduces marking constraints. The non-reproducibility due to the mechanics of the tank, the cleaning or alteration of the optical faces of the tank, and the presence of artefacts (bubbles, dust) are no longer a constraint. The invention makes it possible, by image analysis, to reduce or even eliminate them. A shift in the position of the tank in front of the measuring system of the invention or a translation of the excitation light from the medium can be completely corrected after measuring the position of the fluorescent trace in the recorded image. Likewise, dust present in the excited volume, which could significantly modify the measurement, is small in front of the excited volume, and can thus be identified and removed from the measurement without losing it, which is impossible to be performed with a single detector. In the event of alteration of the optical faces of the tank, which can cause a modification of the quantity of light which effectively excites the sample, the invention makes it possible to know the variations in the effective power and to correct the measurement. The variations in ambient light caused either by the sample or by the environment are compensated for, by a measurement in the image, then a withdrawal of the possible "offsets". - The analysis of the information in an image can be carried out on the basis of elements determined in advance (lighting function, predetermined positions of the various useful zones), or dynamically to deal with random and / or unforeseen disturbances by applying image processing methods (maximization of entropy, neural network). - The assay dynamic of the invention, which, for a certain type of measurement, makes it possible to experimentally achieve a biological assay dynamic of 2,200, is much higher than that of the known art, which, for the same type of measurements, is typically between 5 and 10. The invention is applicable in many fields, and in particular: - in all fields where it is useful to measure a fluorescent solution, - in biology, more particularly for the assay of biological molecules or of biological interest: antigens, antibodies, peptides, DNA, cells, bacteria ..., for clinical diagnosis, - in chemistry (assay), - in pharmacy: activity assay, contamination, etc. ., in physics: search for product traces, fluidics, mixture analysis, etc.
BRÈVE DESCRIPTION DES DESSINS La figure 1 illustre une vue de dessus d'un premier mode de réalisation d'un dispositif mettant en œuvre le procédé de l'invention. Les figures 2 et 3 illustrent une image de la cuve obtenue avec le dispositif d'enregistrement d' image représenté sur la figure 1. Les figures 4 à β illustrent un second mode de réalisation d'un dispositif mettant en oeuvre le procédé de l'invention. La figure 7 illustre un troisième mode de réalisation d'un dispositif mettant en oeuvre le procédé de l'invention. Les figures 8 à 10 illustrent trois exemples de réalisation d'un dispositif mettant en oeuvre le procédé de l'invention.BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a top view of a first embodiment of a device implementing the method of the invention. FIGS. 2 and 3 illustrate an image of the tank obtained with the image recording device shown in FIG. 1. Figures 4 to β illustrate a second embodiment of a device implementing the method of the invention. FIG. 7 illustrates a third embodiment of a device implementing the method of the invention. FIGS. 8 to 10 illustrate three exemplary embodiments of a device implementing the method of the invention.
EXPOSÉ DÉTAILLÉ DE MODES DE RÉALISATION PARTICULIERSDETAILED PRESENTATION OF PARTICULAR EMBODIMENTS
Le procédé de l'invention est un procédé de dosage (mesure d'une concentration ou d'une quantité) d'un échantillon biologique ou d'intérêt biologique (antigènes, anticorps, peptides, ADN, cellules, bactéries, toxines) ou chimique (solvant, gaz dissous, formulation, activité chimique) , qui peut être un solide, un liquide-, un gel... Dans un premier mode de réalisation illustré sur la figure 1, un échantillon 10 est éclairé par un faisceau lumineux 17 issu d'une source 11 accordée avec le fluorochro e employé. On peut utiliser par exemple un laser Argon 488 n pour de la fluorescéine ou un laser Hélium-Néon à 633 nm pour du Cy5. L'échantillon 10 est placé dans une cuve 12, par exemple de section rectangulaire dont toutes les faces sont transparentes. La section de cette cuve 12 peut, en effet, être rectangulaire, carrée, cylindrique ou elliptique. Un système d'objectif 13, équipé d'un filtre d'arrêt 14, est monté devant un dispositif d' enregistrement de la structure spatiale d' une image 15, par exemple une caméra CCD ou un système à balayage, relié à un organe de traitement 16. Le dispositif 15 qui reçoit le faisceau 18 diffusé par l'échantillon 10, permet l'enregistrement d'une image de laquelle peut être extraite un signal spécifique de mesure . Le procédé de l'invention comprend les étapes suivantes : - éclairement de l'échantillon 10 au moyen du faisceau lumineux 17 issu de la source 11, qui peut être un laser à gaz, un laser solide, une diode laser, une diode électroluminescente, une diode organique, une lampe spectrale comme une lampe halogène, à mercure, à xénon, à deutérium, - réalisation d'une image du faisceau lumineux 18 diffusé par l'échantillon 10, l'origine de la diffusion pouvant être une diffusion Raman, une diffusion par fluorescence, une diffusion moléculaire (diffusion Rayleight) ou particulaire (utilisation de nano-particules) , - analyse de l'image par rapport à des références, cette analyse consistant alors en l'étude de la structure spatiale de l'image et de la distribution de l'énergie lumineuse dans cette image, ces références étant constituées, par exemple, par l'expérience d'un utilisateur ou par des critères morphologiques (forme et position d'une trace lumineuse) , photométrique (fréquences des variations spatiales de la lumière dans l'image), statistiques (variation d'estimateurs de mesures, d'entropie dans l'image) , B 14352.3 DB 13The method of the invention is a method for assaying (measuring a concentration or a quantity) of a biological sample or of biological interest (antigens, antibodies, peptides, DNA, cells, bacteria, toxins) or chemical (solvent, dissolved gas, formulation, chemical activity), which can be a solid, a liquid, a gel, etc. In a first embodiment illustrated in FIG. 1, a sample 10 is illuminated by a light beam 17 from from a source 11 tuned with the fluorochro e used. For example, an Argon 488 n laser can be used for fluorescein or a Helium-Neon laser at 633 nm for Cy5. The sample 10 is placed in a tank 12, for example of rectangular section, all the faces of which are transparent. The section of this tank 12 can, in fact, be rectangular, square, cylindrical or elliptical. A lens system 13, equipped with a stop filter 14, is mounted in front of a device for recording the spatial structure of an image 15, for example a CCD camera or a scanning system, connected to a processing member 16. The device 15 which receives the beam 18 scattered by the sample 10, allows the recording of an image from which can be extracted a specific measurement signal. The method of the invention comprises the following steps: - illumination of the sample 10 by means of the light beam 17 coming from the source 11, which can be a gas laser, a solid laser, a laser diode, a light-emitting diode, an organic diode, a spectral lamp such as a halogen, mercury, xenon, deuterium lamp, - production of an image of the light beam 18 scattered by the sample 10, the origin of the scattering being possibly Raman scattering, fluorescence scattering, molecular scattering (Rayleight scattering) or particulate scattering (use of nanoparticles), - analysis of the image in relation to references, this analysis then consisting in studying the spatial structure of the image and the distribution of light energy in this image, these references being constituted, for example, by the experience of a user or by morphological criteria (shape and position of a light trace), photo metric (frequencies of spatial variations of light in the image), statistics (variation of measurement estimators, entropy in the image), B 14352.3 DB 13
- extraction de l'information spécifique à l'interaction entre le faisceau 17 et l'échantillon 10, cette extraction consistant, par exemple, en des opérations arithmétiques entre l'image et d'autres 5 images ou des constantes (par exemple, des soustractions, additions, divisions, multiplications) , morphologiques (érosion, dilatation, binarisation, détourâge, segmentations, correction de décalage) ou photométriques (corrections polynomiales, convolutions, 10 filtres, seuillages) , - calcul du dosage, ce calcul se faisant par rapport à un étalonnage entre la mesure d'énergie lumineuse et la concentration ou la quantité de l'échantillon biologique ou chimique. Ce calcul peut 15 aussi être effectué en enregistrant la cinétique de la réaction biologique ou chimique et en analysant cette cinétique par des méthodes connues de l'homme de l'art. Dans le procédé de l'invention la mesure est faite dans une image obtenue par le dispositif 20 d'enregistrement de la structure spatiale d'une image 15. L'invention ne réside pas dans l'emploi d'un tel dispositif 15 mais principalement dans : - le fait d'enregistrer le faisceau 18 diffusé par l'échantillon 10 sous la forme d'une image, 25 - le fait d'extraire l'information de cette image, le côté adaptatif obtenu par l'application d'une analyse de l'image. La figure 2 illustre l'image de la cuve 12 30 obtenue avec le dispositif d' enregistrement de la structure spatiale d'une image 15. La cuve peut avoir des dimensions plus petites que celles de l'image. Elle peut par exemple être remplacée par un ou plusieurs capillaires. De plus, le faisceau lumineux peut être indifféremment plus petit ou plus grand que la cuve. Dans cette image on peut distinguer plusieurs zones : une zone 20 de volume éclairé, qui correspond au volume de la cuve 12 excité par le faisceau 17, - la zone 21 d'entrée de ce faisceau 17 dans la cuve 12, - la zone 22 de sortie de ce faisceau 17 de la cuve 12, - une zone 23 de ménisque, - une zone 24 d'artefact. On peut ainsi, comme illustré sur la figure 3, définir une première région d'intérêt 25 autour de la zone 20 de volume éclairé et une deuxième région d'intérêt 26 disposée à côté de cette zone 25. La mesure du signal spécifique est alors donné par le calcul : ∑RIι-∑RI2 ; c'est-à-dire la soustraction entre la somme de tous les pixels de la première région d'intérêt 25 et la somme de tous les pixels de la seconde région d'intérêt 26. Sur la figure 3 les deux régions d'intérêt 25 et 26 ont la même taille, ce qui n'est pas indispensable. Lorsque ces régions n'ont pas la même taille, il suffit soit de réaliser un moyennage des niveaux de gris de chaque région, soit une pondération des valeurs par le nombre de pixels . L'analyse de l'image ainsi représentée conduit à certaines observations : La trace fluorescente du faisceau lumineux (zone 20) donne le signal spécifique. - Le ménisque (zone 23) , délimitant le liquide de l'air, est fortement lumineux. Son origine provient de ladite trace. La forme de ce ménisque est fortement aléatoire. L'amplitude du signal provenant de ce ménisque est donc très variable. - Un artefact (zone 24) peut être, pour un montage donné, une rondelle ressort destinée à assurer un bon positionnement mécanique de la cuve 12. Les zones 21 et 22 correspondent respectivement aux points d'entrée et de sortie du f isceau lumineux dans la cuve 12. Avec un autre réglage des seuils d'affichage, on peut mieux mettre en évidence les niveaux de lumière les plus f ibles . Le procédé de l'invention permet d'améliorer le coefficient de variation CV (écart type/moyenne) . En effet le coefficient CV obtenu avec le procédé de l'invention est bien inférieur au coefficient CV calculé en faisant la somme de tous les pixels d'une caméra CCD, qui correspond à une mesure faite avec un mono-détecteur . Le coefficient CV obtenu avec le procédé de l'invention est d'ailleurs du même ordre de grandeur que celui obtenu avec un grand volume de solution, comme envisagé précédemment dans l'introduction de ladite demande. Ce coefficient CV obtenu avec le procédé de l' invention est plus faible que celui obtenu par la mesure dans chacune des zones d'intérêt 25 et 26. La soustraction des mesures effectuées dans ces deux zones d' intérêt permet de corriger des variations d' éclairage qui affectent toutes les régions. L'invention permet d'effectuer ainsi un filtrage spatial dans le plan qui permet d'extraire le signal contenant l'information spécifique du phénomène de fluorescence. De plus l'invention permet d' extraire les régions d' intérêt qui sont vraiment pertinentes dans une image ou une pseudo- image, alors qu'un système de mesure utilisant un seul détecteur mono-pixel ne peut pas réaliser une telle fonction. Dans un second mode de réalisation, un mono-détecteur associé à une matrice de pixels à transparence programmable 30 telle qu'une matrice de cristaux liquides ou de micro-miroirs ou tout autre système équivalent est disposé devant la cuve, 12 illustrée sur la figure 4. Cette matrice 30 est intercalée entre la cuve 12 et le détecteur via un système de formation des images ou non. On réalise alors une première mesure en « ouvrant » les pixels correspondant à la première zone d'intérêt 25, comme illustré sur la figure 5, puis une deuxième mesure en ouvrant les pixels correspondant à la seconde zone d'intérêt 26, comme illustré sur la figure 6. L'emploi d'une telle matrice 30 à transparence variable permet d'éviter l'enregistrement systématique d'une image, en réalisant par exemple les étapes suivantes : - enregistrement de l'image du faisceau diffusé par l' ouverture/fermeture successives de tous les pixels de la matrice 30 en synchronisation avec la mesure effectuée par le mono-détecteur, - analyse de l'image et définition de la ou des zones d'intérêt permettant d'extraire l'information spécifique, - enregistrement de tels paramètres pour une utilisation ultérieure, - lors de l'analyse d'un échantillon donné, ouvertures successives des régions définies lors de l'étape d'analyse et enregistrement des résultats de mesures pour chacune de ces zones, - extraction de l'information utile, - calcul du dosage. Dans un troisième mode de réalisation illustré sur la figure 7, deux détecteurs mono-pixel 35 et 36 observent chacun une région d'intérêt 25 ou 26. Deux moyens de formation d'image 37 et 38 sont placés respectivement devant chacun de ces deux détecteurs 35 et 36. La mesure du signal provenant de la première région d'intérêt 25 est faite avec le détecteur 35, celle pour la seconde région d'intérêt 26 est faite avec le détecteur 36. La zone 39 représente la trace fluorescente en vue de face. L'invention permet d'adapter l'extraction du signal spécifique aux conditions d'expériences. Par exemple, si la cuve a bougé entre deux séries de mesures, il est possible, par analyse automatique de l'image, de repositionner les régions d'intérêt de manière automatique, opération impossible à effectuer avec un système statique. B 14352 .3 DB 18extracting information specific to the interaction between the beam 17 and the sample 10, this extraction consisting, for example, of arithmetic operations between the image and other images or constants (for example, subtractions, additions, divisions, multiplications), morphological (erosion, dilation, binarization, clipping, segmentation, offset correction) or photometric (polynomial corrections, convolutions, 10 filters, thresholds), - dosage calculation, this calculation being done in relation a calibration between the measurement of light energy and the concentration or quantity of the biological or chemical sample. This calculation can also be performed by recording the kinetics of the biological or chemical reaction and analyzing this kinetics by methods known to those skilled in the art. In the method of the invention, the measurement is made in an image obtained by the device 20 for recording the spatial structure of an image 15. The invention does not lie in the use of such a device 15 but mainly in: - the fact of recording the beam 18 diffused by the sample 10 in the form of an image, 25 - the fact of extracting the information from this image, the adaptive side obtained by the application of a image analysis. FIG. 2 illustrates the image of the tank 12 obtained with the device for recording the spatial structure of an image 15. The tank may have dimensions smaller than those of the image. It can for example be replaced by one or more capillaries. In addition, the light beam can be either smaller or larger than the tank. In this image we can distinguish several zones: an area 20 of illuminated volume, which corresponds to the volume of the tank 12 excited by the beam 17, - the area 21 of entry of this beam 17 into the tank 12, - the area 22 outlet of this bundle 17 from the tank 12, - a meniscus area 23, - an artefact area 24. It is thus possible, as illustrated in FIG. 3, to define a first region of interest 25 around the area 20 of illuminated volume and a second region of interest 26 disposed next to this area 25. The measurement of the specific signal is then given by calculation: ∑RIι-∑RI2; that is to say the subtraction between the sum of all the pixels of the first region of interest 25 and the sum of all the pixels of the second region of interest 26. In FIG. 3 the two regions of interest 25 and 26 are the same size, which is not essential. When these regions do not have the same size, it suffices either to perform an averaging of the gray levels of each region, or a weighting of the values by the number of pixels. Analysis of the image thus represented leads to certain observations: The fluorescent trace of the light beam (area 20) gives the specific signal. - The meniscus (zone 23), delimiting the liquid from the air, is strongly luminous. Its origin comes from said trace. The shape of this meniscus is highly random. The amplitude of the signal from this meniscus is therefore very variable. - An artefact (zone 24) can be, for a given assembly, a spring washer intended to ensure good mechanical positioning of the tank 12. Zones 21 and 22 correspond respectively to the entry and exit points of the light beam in the tank 12. With another adjustment of the display thresholds, it is easier to highlight the weakest light levels. The method of the invention makes it possible to improve the coefficient of variation CV (standard deviation / average). Indeed, the CV coefficient obtained with the method of the invention is much lower than the CV coefficient calculated by summing all the pixels of a CCD camera, which corresponds to a measurement made with a mono-detector. The CV coefficient obtained with the method of the invention is moreover of the same order of magnitude as that obtained with a large volume of solution, as envisaged previously in the introduction to said application. This CV coefficient obtained with the method of the invention is lower than that obtained by measuring in each of the zones. of interest 25 and 26. Subtracting the measurements made in these two areas of interest makes it possible to correct variations in lighting which affect all the regions. The invention thus makes it possible to perform spatial filtering in the plane which makes it possible to extract the signal containing the specific information of the phenomenon of fluorescence. In addition, the invention makes it possible to extract the regions of interest which are really relevant in an image or a pseudo-image, whereas a measurement system using a single mono-pixel detector cannot perform such a function. In a second embodiment, a single detector associated with a matrix of pixels with programmable transparency 30 such as a matrix of liquid crystals or micro-mirrors or any other equivalent system is placed in front of the tank, 12 illustrated in the figure 4. This matrix 30 is interposed between the tank 12 and the detector via an image forming system or not. A first measurement is then carried out by "opening" the pixels corresponding to the first area of interest 25, as illustrated in FIG. 5, then a second measurement by opening the pixels corresponding to the second area of interest 26, as illustrated in FIG. 6. The use of such a matrix 30 with variable transparency makes it possible to avoid the systematic recording of an image, by carrying out for example the following steps: - recording of the image of the beam scattered by the aperture / successive closure of all the pixels of the matrix 30 in synchronization with the measurement carried out by the mono-detector, - analysis of the image and definition of the area or areas of interest making it possible to extract the specific information, - recording of such parameters for a subsequent use, - during the analysis of a given sample, successive openings of the regions defined during the analysis step and recording of the measurement results for each of these zones, - extraction of useful information, - calculation dosage. In a third embodiment illustrated in FIG. 7, two mono-pixel detectors 35 and 36 each observe a region of interest 25 or 26. Two imaging means 37 and 38 are placed respectively in front of each of these two detectors 35 and 36. The measurement of the signal from the first region of interest 25 is made with the detector 35, that for the second region of interest 26 is made with the detector 36. The area 39 represents the fluorescent trace with a view to face. The invention makes it possible to adapt the extraction of the specific signal to the experimental conditions. For example, if the cell has moved between two series of measurements, it is possible, by automatic image analysis, to reposition the regions of interest automatically, an operation impossible to perform with a static system. B 14352 .3 DB 18
Exemples de réalisation de l'invention Les trois exemples de réalisation décrits ci-dessous correspondent respectivement aux trois modes de réalisation définis précédemment. 5 Dans un premier exemple de réalisation illustré sur la figure 8, une source lumineuse 40, par exemple un laser ou une diode électroluminescente, excite, dans une cuve 41, le liquide contenant des molécules fluorescentes au travers d'optiques de mise 10 en forme non représentées, et de divers accessoires tels qu'un obturateur 42, et un diaphragme 43. Un premier objectif 44, disposé par exemple perpendiculairement à la direction principale du faisceau lumineux, collecte une partie de ce faisceau 15 lumineux, émis par les molécules fluorescentes dans la cuve 41. Un filtre d'arrêt 45 est disposé derrière le premier objectif 44, juste devant un second objectif 46. L'association des objectifs 44 et 46 permet de former une image de la cuve 41 sur le détecteur d'image 20 47 qui est relié à un système de commande et de contrôle 49. Pour une cuve 41 d'une largeur intérieure de 1 cm, ce détecteur 47 peut être un détecteur de 512 x 512 pixels de 10 μm de côté. Le premier objectif 44 peut avoir une focale 50 mm et le second objectif 46 25 une focale 25 mm. Dans un second exemple de réalisation, illustré sur la figure 9, dont la configuration est la même que celle de la figure 8 pour l'excitation, on n'utilise qu'un seul détecteur. Une matrice à 30 transparence variable 56, qui peut être une matrice à cristaux liquide ou une matrice de micro-miroirs, joue le rôle d'un diaphragme de champ. La matrice 56 peut être remplacée par une fente mobile actionnée par un actuateur mécanique ou électro-mécanique, par exemple, un électro-aimant ou un moteur électrique. Dans un troisième exemple de réalisation illustré sur la figure 10, la configuration pour exciter l'intérieur de la cuve 41 est la même que pour la figure 8 et la configuration pour la collection de lumière est la même que pour celle de la figure 9. Deux mono-détecteurs 50 et 51, par exemple des photomultiplicateurs décalés, permettent d'observer deux régions différents de la cuve 41. Devant chaque détecteur 50 et 51, une optique de reprise 52 et 53 permet de former l'image de la région d'intérêt sur un diaphragme de champ 54 et 55 qui limite la région observée. Un filtre d'arrêt peut être disposé avant, dans ou derrière le diaphragme. La zone 56 représente la trace fluorescente. Examples of embodiments of the invention The three embodiments described below correspond respectively to the three embodiments defined above. 5 In a first embodiment illustrated in FIG. 8, a light source 40, for example a laser or a light-emitting diode, excites, in a tank 41, the liquid containing fluorescent molecules through shaping optics 10 not shown, and various accessories such as a shutter 42, and a diaphragm 43. A first objective 44, arranged for example perpendicular to the main direction of the light beam, collects part of this light beam 15, emitted by the fluorescent molecules in the tank 41. A stop filter 45 is disposed behind the first objective 44, just in front of a second objective 46. The association of the objectives 44 and 46 makes it possible to form an image of the tank 41 on the image detector 20 47 which is connected to a command and control system 49. For a tank 41 with an internal width of 1 cm, this detector 47 can be a detector of 512 x 512 pixels with a side of 10 μm. The first objective 44 can have a focal length 50 mm and the second objective 46 25 a focal length 25 mm. In a second exemplary embodiment, illustrated in FIG. 9, the configuration of which is the same as that of FIG. 8 for excitation, only one detector is used. A variable transparency matrix 56, which may be a liquid crystal matrix or a micro-mirror matrix, plays the role of a field diaphragm. The matrix 56 can be replaced by a movable slot actuated by a mechanical or electro-mechanical actuator, for example, an electromagnet or an electric motor. In a third exemplary embodiment illustrated in FIG. 10, the configuration for exciting the interior of the tank 41 is the same as for FIG. 8 and the configuration for the collection of light is the same as for that of FIG. 9. Two mono-detectors 50 and 51, for example offset photomultipliers, make it possible to observe two different regions of the tank 41. In front of each detector 50 and 51, a recovery optic 52 and 53 makes it possible to form the image of the region d interest on a field diaphragm 54 and 55 which limits the region observed. A stop filter can be placed before, in or behind the diaphragm. Zone 56 represents the fluorescent trace.
REFERENCESREFERENCES
[1] "So e applications of near-ultraviolet laser- induced fluorescence détection in nanomolar- and subnanomolar-range high-performance liquid chromatography or micro-high performance liquid chromatography" de N. Siméon, R. Myers, C. Bayle, M. Nertz, J.K. Ste art, F. Couderc (2001, Journal of Chromatography A, Vol 913, I 1-2, pages 253-259) .[1] "So e applications of near-ultraviolet laser- induced fluorescence detection in nanomolar- and subnanomolar-range high-performance liquid chromatography or micro-high performance liquid chromatography" by N. Siméon, R. Myers, C. Bayle, M Nertz, JK Ste art, F. Couderc (2001, Journal of Chromatography A, Vol 913, I 1-2, pages 253-259).
[2] "Performance of an integrated microoptical System for fluorescence détection in microfluidic Systems" de J.C. Roulet, R. Volkel, H. P. Herzig, E. Verpoorte, N.F. Rooij , R. Dandliker (2002, Anal ytical Che istry, Vol. 74 (14), pages 3400-3407).[2] "Performance of an integrated microoptical System for fluorescence detection in microfluidic Systems" by JC Roulet, R. Volkel, HP Herzig, E. Verpoorte, NF Rooij, R. Dandliker (2002, Anal ytical Che istry, Vol. 74 ( 14), pages 3400-3407).
[3] "Single molécule détection of spécifie nucleic acid séquences in unamplified genomic DNA" de A. Castro, et J.G. Williams (1997, Analytical chemistry, Vol. 69 (19) , pages 3915-3920) . [3] "Single molecule detection of specifies nucleic acid sequences in unamplified genomic DNA" by A. Castro, and J.G. Williams (1997, Analytical chemistry, Vol. 69 (19), pages 3915-3920).

Claims

B 14352.3 DB 21REVENDICATIONS B 14352.3 DB 21 CLAIMS
1. Procédé de dosage d'un échantillon biologique ou chimique, comportant une étape 5 d' éclairement de l'échantillon (10) au moyen d'un faisceau lumineux (17) issu d'une source (11), caractérisé en ce qu'il comporte, en outre, les étapes suivantes : réalisation d'une image comprenant 10 l'image du faisceau (18) diffusé par l'échantillon (10), - analyse de l'image selon des critères de référence, - extraction d'une information spécifique à 15 l'interaction faisceau lumineux/échantillon, - calcul du dosage, et en ce que l'analyse consiste en l'étude de la structure spatiale de l'image et de la distribution de l'énergie lumineuse dans cette image. 20 2. Procédé selon la revendication 1, qui comporte une étape préalable d' introduction de l'échantillon (10) dans une cuve (12) dont toutes les faces sont transparentes . 25 3. Procédé selon la revendication 1, dans lequel la diffusion est une diffusion Raman, une diffusion par fluorescence, une diffusion moléculaire ou particulaire. 30 1. Method for dosing a biological or chemical sample, comprising a step 5 of illuminating the sample (10) by means of a light beam (17) coming from a source (11), characterized in that it further comprises the following steps: production of an image comprising the image of the beam (18) scattered by the sample (10), - analysis of the image according to reference criteria, - extraction of information specific to the interaction of light beam / sample, - calculation of the assay, and in that the analysis consists in the study of the spatial structure of the image and of the distribution of light energy in this picture. 2. Method according to claim 1, which comprises a preliminary step of introducing the sample (10) into a tank (12), all the faces of which are transparent. 3. The method of claim 1, wherein the scattering is Raman scattering, fluorescent scattering, molecular or particulate scattering. 30
B 14352. B 14352.
3 DB 223 DB 22
4. Procédé selon la revendication 1, dans lequel le calcul du dosage se fait par rapport à un étalonnage entre la mesure d' énergie lumineuse et la concentration ou la quantité d' échantillon .4. Method according to claim 1, in which the calculation of the dosage is made with respect to a calibration between the measurement of light energy and the concentration or the quantity of sample.
5. Procédé selon la revendication 1, dans lequel le calcul du dosage se fait par rapport à l'analyse de la cinétique de la réaction biologique ou chimique. 10 6. Procédé selon la revendication 1, dans lequel on définit une première zone d'intérêt (25) autour de la zone de volume excité, et une seconde zone d'intérêt (26) disposée à côté de cette première zone, 15 et on mesure le signal spécifique en réalisant la soustraction entre la somme de tous les pixels de la première zone (25) et la somme de tous les pixels de la seconde zone (26) .5. Method according to claim 1, in which the calculation of the dosage is made with respect to the analysis of the kinetics of the biological or chemical reaction. 6. The method as claimed in claim 1, in which a first zone of interest (25) is defined around the zone of excited volume, and a second zone of interest (26) disposed next to this first zone, and the specific signal is measured by performing the subtraction between the sum of all the pixels in the first area (25) and the sum of all the pixels in the second area (26).
20 7. Application du procédé selon l'une quelconque des revendications précédentes à la fluorescence.7. Application of the method according to any one of the preceding claims to fluorescence.
25 25
PCT/FR2004/050289 2003-06-27 2004-06-24 Method for dosing a biological or chemical sample WO2005001449A1 (en)

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JP2006516356A JP2007516411A (en) 2003-06-27 2004-06-24 Method for quantitative analysis of biological or chemical samples
US10/560,973 US7396650B2 (en) 2003-06-27 2004-06-24 Method for dosing a biological or chemical sample
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