WO2004055502A2 - Method of measuring a quantity of photons which is proportional to the quantity of photons received by the object and associated device - Google Patents

Method of measuring a quantity of photons which is proportional to the quantity of photons received by the object and associated device Download PDF

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Publication number
WO2004055502A2
WO2004055502A2 PCT/FR2003/050160 FR0350160W WO2004055502A2 WO 2004055502 A2 WO2004055502 A2 WO 2004055502A2 FR 0350160 W FR0350160 W FR 0350160W WO 2004055502 A2 WO2004055502 A2 WO 2004055502A2
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sample
photon
fluorescence
photons
measurement
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PCT/FR2003/050160
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French (fr)
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WO2004055502A3 (en
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Philippe Rizo
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Commissariat A L'energie Atomique
Apibio
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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
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the aim of the method and the device according to the invention is to increase the precision of the fluorescence measurement systems, in particular in the field of reading fluorescence by microscopy.
  • the method and the device described below relate as well to measurements obtained by epi-illumination as to measurements coming from an excitation by evanescent waves, or to measurements of fluorescence obtained by means of a guide. wave.
  • the detection of the fluorescence originating from a solution or from a surface makes it possible to obtain information on said solution or said surface which will be useful for the analysis, the characterization or imaging of said sample.
  • the quantity of fluorescence light emitted by the fluorescent sample will depend, on the one hand, on the vicinity of the sample, and on the other hand, on the excitation photon flux, as well as on the spectrum of said photon flux and of the quantity of photons received by the sample. To ensure good reproducibility of the measurements, it is therefore necessary to ensure that these different parameters can be controlled.
  • the excitation spectrum obtained after filtering does not fluctuate or is negligible. If we consider a sample in a given environment and within a small range of variation in the illumination flux (of the order of some 10%), the amount of fluorescent light re-emitted by the sample will be directly proportional to the amount of excitation photons which have reached said sample.
  • the illumination level is measured by acquisition on a reference sample at a given time, which makes it possible to standardize the measurements.
  • part of the photons emitted by the source are taken at a given time.
  • this measurement serves as a reference measurement. It is considered that this luminous flux does not vary during the measurement of the fluorescence compared to the time when the reference measurement was taken. However, this hypothesis is only valid for measurements where
  • the repeatability is not critical, but is largely insufficient when one seeks a repeatability of the order of 1%, in particular in the field of imaging when one seeks to correct images.
  • control of the luminous flux is done either by measurement of time, or by acquisition on a reference sample.
  • the proposed device and method make it possible to overcome measurement errors linked to variations in intensity of the lighting device.
  • the solution is to take part of the incident beam continuously during the measurement, measure this quantity and use it as a photon counter in order to know exactly how much photons are received by the sample.
  • the object of the invention is to provide a precise and repeatable estimate of the quantity of photons sent to the sample for a given measurement.
  • a fluorescence measurement device comprising a photon emitting source intended to emit a beam of photons towards a sample to be analyzed, the device also comprising means for detecting a fluorescence light emitted by the sample in response to the beam photons received by it, characterized in that it also comprises suitable sampling means taking, during the fluorescence measurement, a determined fraction of the photon beam emitted by the photon source, means for measuring said fraction of the sampled photon beam, and integration means making it possible to integrate the fraction determined of the photon beam.
  • said fluorescence measurement device will also include a microscope objective situated between the sample to be analyzed and the means for detecting the fluorescence light emitted by the sample and on the path of the fluorescence light.
  • the sampling means are a semi-transparent blade.
  • the beam emitted by the source is, in general, not perfectly geometrically stable over time. These small fluctuations must be averaged and to take account of this problem, it is advantageous to take a number of photons directly proportional to the total number of photons included in the beam.
  • One way of carrying out this operation consists in using a semi-transparent plate which reflects a fixed percentage of the whole beam on an optic which focuses all of these photons taken from the means for measuring this fraction of photons taken.
  • the means for detecting the fluorescence light emitted by the sample and the means for measuring the fraction of the photon beam taken are chosen from the group consisting of a CCD sensor, a photomultiplier and a photodiode .
  • the integration means comprise a unitary sensor which integrates all of the determined fraction of the photon beam.
  • the integration means comprise a set of unitary sensors capable of providing an image of the spatial distribution of the determined fraction of the photon beam along a section of said beam.
  • the fluorescence measuring device can comprise cut-off means which control the cut-off of the beam of photons emitted at a given integration time.
  • the fluorescence measurement device further comprises means for cutting off the beam of photons emitted to a sufficient number of photons of the sampled beam detected.
  • said cutting means will act on interrupting means placed on the optical path of the light emitted by the source between the photon emitting source and the sampling means. In a second case, said cutting means will act on means for interrupting the supply of the photon emitting source.
  • said cutting means will act on interrupting means placed on the optical path of the light coming from the sample between said sample and the detection means.
  • said cutting means will act on means for interrupting the operation of the detection means.
  • these cut-off means will make it possible to interrupt the measurement of fluorescence by the detection means.
  • the means for interrupting the emission of the photon source or the fluorescence measurement will be a shutter.
  • the means for cutting off the emitted photon beam will furthermore comprise a system for comparing the quantity of light integrated by the means for integrating the sampled light and a level of clearly taken as a reference. When the two values are identical, the cutoff means will act on at least one. means of interruption.
  • the terms: "measurement of the monitor”, “measurement of the reference”, “measurement of the monitor at the reference” are constants which do not vary from one measurement to another .
  • the excitation beam or the light entering the fluorescence measurement means is cut off.
  • two measurements made on two identical samples with approximately identical lighting levels give two identical measured fluorescence values.
  • the variation in light was thus automatically compensated for by a variation in the time necessary to reach the required quantity of light.
  • these integration means will include a capacitor. Indeed, it will then suffice to use the current supplied by the means for measuring the light sampled to charge a capacitor and to compare the charge of the capacitor with a fixed threshold to know when the sample will have received the right quantity of photons.
  • Another object of the invention consists of a method capable of providing an accurate and repeatable estimate of the quantity of photons sent to the sample for a given measurement which uses the device according to the invention and which is characterized in that it comprises at least one step of comparison between the value obtained by the means for measuring the fraction of the photon beam sampled and a reference value, this comparison giving a value called normalized fluorescence value.
  • this reference value will be a predetermined value. In a second case, this reference value will be the result of a measurement made beforehand by the measuring means.
  • the fluorescence level of the sample can be calculated.
  • a value of the type can be used as the normalized fluorescence value:
  • - reference monitor measurement value supplied by the device for measuring the light sampled during the examination of the reference object
  • the device according to the invention has many advantages. First, it improves the accuracy of the fluorescence measurement of a sample.
  • the signal sensor or fluorescence sensor (CCD detector for example) has an accuracy of 1%
  • the sampling of the direct beam and its measurement by the monitor are carried out with an accuracy of 1%
  • the comparator on the number of integrated photons has a repeatability of 1%.
  • the photometric accuracy of the assembly will then be of the order of
  • V3x (0.0l) 2 + (0.03) 2 0.035 that is to say a repeatability of the order of 3.5% which does not allow a simple comparison with fluorescence measurements carried out on a device of the same type.
  • the precision of the fluorescence measurement can be further refined by using an imaging sensor (CCD sensor for example) as a device for measuring the light taken instead of a photodiode or a photomultiplier.
  • CCD sensor CCD sensor for example
  • the use of a signal sensor making it possible to produce an image of the sampled beam should make it possible to take account in the long term in the correction of the inhomogeneity of the beam.
  • the device also makes it possible to take into account the intensity fluctuations of the light source in fluorescence microscopy. There is no need to wait for the photon source flux (laser or arc lamp for example) to be completely stabilized before making measurements (although it is still preferable to wait).
  • Another advantage of the invention is that with a system for measuring the intensity delivered on the sample, it is easy to calibrate the ratio between the signal taken and the signal sent to the sample. Then, by replacing the sample with a calibrated albedo object, we can calibrate the ratio between the light emitted by the sample and the light measured by the fluorescence sensor. With these two calibrations, if the proposed invention is used, the same experiment carried out on two different systems will give the same result.
  • FIG. 2 is a diagram illustrating the implementation of the device according to the invention in the case of lighting by evanescent waves.
  • This light sampling device 3 divides the light beam into two: part of the beam, the reflected beam, will illuminate a device 4 for measuring the light collected; the other part of the beam, the transmitted beam, will reflect in a dichroic cube 7.
  • the device for measuring the sampled light 4 which receives the reflected beam can be, for example, a photodiode, a photomultiplier or a CCD sensor.
  • the measuring device 4 converts light into a signal which will be integrated by a system 5.
  • This integration can also be carried out directly on the light signal, for example using a CCD camera or a photographic plate. Then, possibly, the beam can arrive in a device 6 whose function is to cut the beam at a given integration time or at a sufficient number of detected photons. For this, this cut-off device is connected and controls the operation of two shutters 2 and 10 placed respectively in front of the light source and in front of the fluorescence sensor. It should be noted that the two shutters are not necessary: only one is sufficient.
  • the part of the beam which arrives on the dichroic cube 7 will be separated into two beams. Indeed, the dichroic cube has the effect of separating the excitation light from the fluorescence light.
  • the excitation light is reflected by the dichroic cube, passes through a microscope objective 8 and arrives at the sample 9, which can be, for example, a DNA chip having fluorescent markers.
  • This sample will in turn emit a beam which will pass through the microscope objective 8, and which, as it is a beam of fluorescent light, will pass through the dichroic cube 7 and the shutter 10 of the measurement sensor fluorescence.
  • This transmitted beam finally arrives on the signal sensor or fluorescence 11, which can in particular be a CCD sensor, a photodiode or a photomultiplier.
  • a light beam coming from a light source 21 passes through a shutter 22 of the illumination source and arrives on a light sampling system 23, which shares the light beam in two: part of the beam will be received by a system 24 for measuring the light sampled to go into a system 25 integrating the signal sampled in the light beam, then optionally in a system 26 for cutting the beam to a given integration time or at a sufficient number of detected photons, the other part of the beam will propagate in the sample 29.
  • the shutters 2, 22, 10, 30 can be replaced by interrupting means placed directly either on the photon source, or on the detection means.
  • One of the simplest possibilities may consist in cutting the electrical supply from the source or from the detection means.

Abstract

The invention relates to a fluorescence-measuring device comprising a photon-emitting source (1) which is intended to emit a photon beam in the direction of a sample to be analysed. The inventive device also comprises means (11) of detecting a fluorescence light which is emitted by the sample in response to the photons of the beam received by said sample. In addition, the device comprises: sampling means (3) which, during the fluorescence measuring process, can sample a determined fraction of the photon beam emitted by the photon source; means (4) of measuring the sampled fraction of the photon beam; and integration means (5) which can be used to integrate the determined fraction of the photon beam. The invention also relates to a method of obtaining a precise and repeatable estimation of the quantity of photons sent to the sample for a given measurement, using the inventive device. Said method comprises at least one comparison step whereby the value obtained by the measuring means (4) is compared with a reference value, said comparison producing a value known as the normalised fluorescence value.

Description

PROCEDE DE MESURE D'UNE QUANTITE DE PHOTONS METHOD FOR MEASURING A QUANTITY OF PHOTONS
PROPORTIONNELLE A LA QUANTITE DE PHOTONS REÇUS PARPROPORTIONAL TO THE QUANTITY OF PHOTONS RECEIVED BY
L'OBJET ET DISPOSITIF ASSOCIETHE OBJECT AND ASSOCIATED DEVICE
DESCRIPTIONDESCRIPTION
DOMAINE TECHNIQUETECHNICAL AREA
Le procédé et le dispositif selon l'invention ont pour but d'augmenter la précision des systèmes de mesure de la fluorescence, en particulier dans le domaine de la lecture de la fluorescence par microscopie.The aim of the method and the device according to the invention is to increase the precision of the fluorescence measurement systems, in particular in the field of reading fluorescence by microscopy.
Le procédé et le dispositif décrits ci- dessous se rapportent aussi bien à des mesures obtenues par épi-illumination qu'à des mesures provenant d'une excitation par ondes évanescentes, ou à des mesures de fluorescence obtenues par l'intermédiaire d'un guide d' onde .The method and the device described below relate as well to measurements obtained by epi-illumination as to measurements coming from an excitation by evanescent waves, or to measurements of fluorescence obtained by means of a guide. wave.
La détection de la fluorescence provenant d'une solution ou d'une surface, que l'on appellera de manière générale « échantillon », permet d'obtenir des informations sur ladite solution ou ladite surface qui vont être utiles pour l'analyse, la caracterisation ou l'imagerie dudit échantillon. La quantité de lumière de fluorescence rëémise par l'échantillon fluorescent dépendra, d'une part, du voisinage de l'échantillon, et d'autre part, du flux de photons d'excitation, ainsi que du spectre dudit flux de photons et de la quantité de photons reçue par l'échantillon. Il convient donc, pour assurer une bonne reproductibilité des mesures, de s'assurer qu'on peut contrôler ces différents paramètres .The detection of the fluorescence originating from a solution or from a surface, which will generally be called “sample”, makes it possible to obtain information on said solution or said surface which will be useful for the analysis, the characterization or imaging of said sample. The quantity of fluorescence light emitted by the fluorescent sample will depend, on the one hand, on the vicinity of the sample, and on the other hand, on the excitation photon flux, as well as on the spectrum of said photon flux and of the quantity of photons received by the sample. To ensure good reproducibility of the measurements, it is therefore necessary to ensure that these different parameters can be controlled.
Dans la plupart des systèmes de mesures de fluorescence, le spectre d'excitation obtenu après filtrage ne fluctue pas ou bien de manière négligeable. Si on considère un échantillon dans un environnement donné et dans une petite plage de variation du flux d'éclairement (de l'ordre de quelques 10%), la quantité de lumière de fluorescence réémise par l'échantillon sera directement proportionnelle à la quantité de photons d'excitation qui aura atteint ledit échantillon.In most fluorescence measurement systems, the excitation spectrum obtained after filtering does not fluctuate or is negligible. If we consider a sample in a given environment and within a small range of variation in the illumination flux (of the order of some 10%), the amount of fluorescent light re-emitted by the sample will be directly proportional to the amount of excitation photons which have reached said sample.
Pour avoir une bonne estimation du niveau de fluorescence de l'échantillon observé, il faut donc pouvoir relier la quantité de photons arrivant sur le détecteur de fluorescence et la quantité de photons envoyés sur l'échantillon. Or la quantité de photons reçue par l'échantillon varie et les mesures obtenues vont donc être entachées d'erreur. Il faut donc pouvoir contrôler à chaque instant l'exposition de 1' échantillon.To have a good estimate of the fluorescence level of the observed sample, it is therefore necessary to be able to relate the quantity of photons arriving on the fluorescence detector and the quantity of photons sent to the sample. However, the quantity of photons received by the sample varies and the measurements obtained will therefore be vitiated by error. It is therefore necessary to be able to control the exposure of the sample at all times.
ETAT DE LA TECHNIQUE ANTERIEURESTATE OF THE PRIOR ART
En mesure de fluorescence et en particulier en microscopie de fluorescence, que l'on travaille en excitation par épi-illumination ou par transparence à travers l'échantillon, le contrôle de l'exposition de l'échantillon quand il est pris en compte est fait par une mesure de temps . En général, on définit un temps d' intégration et on considère que le niveau d'éclairement pendant cette durée est constant.In fluorescence measurement and in particular in fluorescence microscopy, whether one works in excitation by epi-illumination or by transparency through the sample, the control of the exposure of the sample when it is taken into account is made by a time measurement. In general, an integration time is defined and it is considered that the level of illumination during this time is constant.
Dans les systèmes un peu régulés, on mesure 5 le niveau d'éclairement par une acquisition sur un échantillon de référence à un temps donné, ce qui permet de normaliser les mesures.In slightly regulated systems, the illumination level is measured by acquisition on a reference sample at a given time, which makes it possible to standardize the measurements.
Mais ce type de correction est très limité si la source, c'est-à-dire la quantité de photons émis, 10 varie pendant le temps d'intégration car alors cette variation ne peut être prise en compte.However, this type of correction is very limited if the source, that is to say the quantity of photons emitted, varies during the integration time because then this variation cannot be taken into account.
Dans d'autres systèmes, on prélève une partie des photons émis par la source à un temps donnéIn other systems, part of the photons emitted by the source are taken at a given time.
15 et on mesure ce flux lumineux : cette mesure sert de mesure de référence. On considère que ce flux lumineux ne varie pas pendant la mesure de la fluorescence par rapport au moment où on a pris la mesure de référence. Or cette hypothèse n'est valide que pour des mesures où15 and this luminous flux is measured: this measurement serves as a reference measurement. It is considered that this luminous flux does not vary during the measurement of the fluorescence compared to the time when the reference measurement was taken. However, this hypothesis is only valid for measurements where
20 la répétabilité n'est pas critique, mais est largement insuffisante quand on recherche une répétabilité de l'ordre de 1%, notamment dans le domaine de l'imagerie quand on cherche à corriger des images .20 the repeatability is not critical, but is largely insufficient when one seeks a repeatability of the order of 1%, in particular in the field of imaging when one seeks to correct images.
Il ne semble pas qu'on n'ait jamais mesuré,It doesn't seem like we've ever measured,
25 directement par prélèvement et en continu, une valeur proportionnelle au nombre de photons qui atteignent l'échantillon. Aucun des constructeurs actuels de microscopes travaillant en fluorescence ne fournit une mesure proportionnelle au nombre de photons ayant25 directly by sampling and continuously, a value proportional to the number of photons reaching the sample. None of the current manufacturers of fluorescence microscopes provide a measure proportional to the number of photons having
30. atteint l'échantillon afin de pouvoir corriger les mesures. De même, aucun des scanners de lecture de biopuce actuel (qu'il soit confocal ou non) n'assure ce type de contrôle .30. reaches the sample in order to correct the measurements. Likewise, none of the scanning scanners Current biochip (whether confocal or not) does not provide this type of control.
En résumé, dans l'art antérieur, le contrôle du flux lumineux se fait soit par mesure du temps, soit par acquisition sur un échantillon de référence.In summary, in the prior art, the control of the luminous flux is done either by measurement of time, or by acquisition on a reference sample.
Tous les procédés actuellement utilisés ne permettent pas de suivre les fluctuations en temps réel. On peut toujours faire l'hypothèse que l'intensité de la source ne varie pas pendant la mesure effective, mais cette hypothèse n'est pas acceptable quand on veut obtenir des mesures avec une grande précision.Not all of the procedures currently in use allow fluctuations to be followed in real time. We can always assume that the intensity of the source does not vary during the actual measurement, but this assumption is not acceptable when we want to obtain measurements with high precision.
Le dispositif et le procédé proposés permettent de s'affranchir des erreurs de mesures liées aux variations d'intensité du dispositif d'éclairage.The proposed device and method make it possible to overcome measurement errors linked to variations in intensity of the lighting device.
EXPOSÉ DE L'INVENTIONSTATEMENT OF THE INVENTION
La solution consiste à prélever une partie du faisceau incident en continu au cours de la mesure, de mesurer cette quantité et de l'utiliser comme compteur de photons afin de savoir exactement quelle quantité de photons est reçue par l'échantillon.The solution is to take part of the incident beam continuously during the measurement, measure this quantity and use it as a photon counter in order to know exactly how much photons are received by the sample.
Le but de l'invention est de fournir une estimation précise et répétable de la quantité de photons envoyés sur l'échantillon pour une mesure donnée .The object of the invention is to provide a precise and repeatable estimate of the quantity of photons sent to the sample for a given measurement.
Ce but et d'autres encore sont atteints, conformément à l'invention par un dispositif de mesure de fluorescence comportant une source émettrice de photons destinée à émettre un faisceau de photons vers un échantillon à analyser, le dispositif comprenant également des moyens de détection d'une lumière de fluorescence émise par l'échantillon en réponse aux photons du faisceau reçus par celui-ci, caractérisé en ce qu'il comporte en outre des moyens de prélèvement aptes à prélever, au cours de la mesure de fluorescence, une fraction déterminée du faisceau de photons émis par la source de photons, des moyens de mesure de ladite fraction du faisceau de photons prélevée, et des moyens d'intégration permettant d' intégrer la fraction déterminée du faisceau de photons .This object and others are achieved, in accordance with the invention by a fluorescence measurement device comprising a photon emitting source intended to emit a beam of photons towards a sample to be analyzed, the device also comprising means for detecting a fluorescence light emitted by the sample in response to the beam photons received by it, characterized in that it also comprises suitable sampling means taking, during the fluorescence measurement, a determined fraction of the photon beam emitted by the photon source, means for measuring said fraction of the sampled photon beam, and integration means making it possible to integrate the fraction determined of the photon beam.
Avantageusement, ledit dispositif de mesure de fluorescence comportera également un objectif de microscope situé entre l'échantillon à analyser et les moyens de détection de la lumière de fluorescence émise par l'échantillon et sur le trajet de la lumière de fluorescence.Advantageously, said fluorescence measurement device will also include a microscope objective situated between the sample to be analyzed and the means for detecting the fluorescence light emitted by the sample and on the path of the fluorescence light.
Avantageusement, les moyens de prélèvement sont une lame semi-transparente .Advantageously, the sampling means are a semi-transparent blade.
En effet, le faisceau émis par la source n'est, en général, pas parfaitement stable géométriquement dans le temps. Ces petites fluctuations doivent être moyennées et pour tenir compte de ce problème, il est avantageux de prélever un nombre de photons directement proportionnel au nombre total de photons compris dans le faisceau. Une manière de réaliser cette opération consiste à utiliser une lame semi-transparente qui réfléchit un pourcentage fixe de l'ensemble du faisceau sur une optique qui focalise l'ensemble de ces photons prélevés sur les moyens de mesure de cette fraction de photons prélevée.In fact, the beam emitted by the source is, in general, not perfectly geometrically stable over time. These small fluctuations must be averaged and to take account of this problem, it is advantageous to take a number of photons directly proportional to the total number of photons included in the beam. One way of carrying out this operation consists in using a semi-transparent plate which reflects a fixed percentage of the whole beam on an optic which focuses all of these photons taken from the means for measuring this fraction of photons taken.
Avantageusement, les moyens de détection de la lumière de fluorescence émise par l'échantillon et les moyens de mesure de la fraction du faisceau de photons prélevée sont choisis parmi le groupe constitué d'un capteur CCD, d'un photomultiplicateur et d'une photodiode .Advantageously, the means for detecting the fluorescence light emitted by the sample and the means for measuring the fraction of the photon beam taken are chosen from the group consisting of a CCD sensor, a photomultiplier and a photodiode .
Selon un premier mode de réalisation, les moyens d'intégration comportent un capteur unitaire qui intègre l'ensemble de la fraction déterminée du faisceau de photons.According to a first embodiment, the integration means comprise a unitary sensor which integrates all of the determined fraction of the photon beam.
Selon un deuxième mode de réalisation, les moyens d' intégration comportent un ensemble de capteurs unitaires apte à fournir une image de la répartition spatiale de la fraction déterminée du faisceau de photons selon une section dudit faisceau.According to a second embodiment, the integration means comprise a set of unitary sensors capable of providing an image of the spatial distribution of the determined fraction of the photon beam along a section of said beam.
Selon un autre mode de réalisation, le dispositif de mesure de fluorescence peut comporter des moyens de coupure qui commandent la coupure du faisceau de photons émis à un temps d'intégration donné.According to another embodiment, the fluorescence measuring device can comprise cut-off means which control the cut-off of the beam of photons emitted at a given integration time.
Selon un autre mode de réalisation, le dispositif de mesure de fluorescence comporte en outre des moyens de coupure du faisceau de photons émis à un nombre de photons du faisceau prélevé détectés suffisant .According to another embodiment, the fluorescence measurement device further comprises means for cutting off the beam of photons emitted to a sufficient number of photons of the sampled beam detected.
Selon un premier cas, lesdits moyens de coupure agiront sur des moyens d' interruption placés sur le trajet optique de la lumière émise par la source entre la source emettrice de photons et les moyens de prélèvement . Selon un deuxième cas, lesdits moyens de coupure agiront sur des moyens d'interruption de l'alimentation de la source emettrice de photons.In a first case, said cutting means will act on interrupting means placed on the optical path of the light emitted by the source between the photon emitting source and the sampling means. In a second case, said cutting means will act on means for interrupting the supply of the photon emitting source.
Dans les deux cas, ces moyens de coupure permettront d'interrompre l'émission de la source de photons .In both cases, these cut-off means will make it possible to interrupt the emission of the photon source.
Selon un autre cas, lesdits moyens de coupure agiront sur des moyens d'interruption placés sur le trajet optique de la lumière provenant de l'échantillon entre ledit échantillon et les moyens de détection.In another case, said cutting means will act on interrupting means placed on the optical path of the light coming from the sample between said sample and the detection means.
Selon un deuxième autre cas, lesdits moyens de coupure agiront sur des moyens d'interruption du fonctionnement des moyens de détection. Dans ces deux cas, ces moyens de coupure permettront d'interrompre la mesure de fluorescence par les moyens de détection.In a second other case, said cutting means will act on means for interrupting the operation of the detection means. In these two cases, these cut-off means will make it possible to interrupt the measurement of fluorescence by the detection means.
Avantageusement, les moyens d'interruption de l'émission de la source de photons ou de la mesure de fluorescence seront un obturateur.Advantageously, the means for interrupting the emission of the photon source or the fluorescence measurement will be a shutter.
Comme la plupart des fluorophores sont sensibles au phénomène de photo-extinction, si l'on désire s'affranchir des fluctuations de mesures dues à ce phénomène et ceci afin d'améliorer la précision de l'estimation de la fluorescence de l'échantillon, il convient d'effectuer les mesures à un niveau d'éclairement à peu près constant, et surtout de travailler à quantité de photons intégrés constante. Pour cela, on a le choix entre travailler à un temps d'intégration fixé ou bien à une quantité de photons fixée . Si l'on travaille à un temps d'intégration fixé, les moyens qui commandent la coupure du faisceau ou de la mesure de la fluorescence par le biais des moyens d'interruption agiront lorsque ledit temps sera atteint. Dans ce cas, on utilisera la mesure obtenue par les moyens d'intégration comme correction pour obtenir une valeur normalisée (on utilisera une des formules présentées plus loin) .As most fluorophores are sensitive to the phenomenon of photo-extinction, if one wishes to overcome the fluctuations of measurements due to this phenomenon and this in order to improve the precision of the estimation of the fluorescence of the sample, measurements should be made at an almost constant level of illumination, and above all to work with a constant quantity of integrated photons. For this, we have the choice between working at a fixed integration time or a fixed quantity of photons. If one works at a fixed integration time, the means which control the cutting of the beam or the measurement of the fluorescence by means of the interruption means will act when said time is reached. In this case, the measurement obtained by the integration means will be used as a correction to obtain a normalized value (one of the formulas presented below will be used).
Si l'on veut travailler à quantité de photons intégrés constante, les moyens de coupure du faisceau de photons émis comprendront en outre un système de comparaison entre la quantité de lumière intégrée par les moyens d'intégration de la lumière prélevée et un niveau d'éclairement pris comme référence. Quand les deux valeurs seront identiques, les moyens de coupure agiront sur au moins un. des moyens d'interruption.If it is desired to work with a constant quantity of integrated photons, the means for cutting off the emitted photon beam will furthermore comprise a system for comparing the quantity of light integrated by the means for integrating the sampled light and a level of clearly taken as a reference. When the two values are identical, the cutoff means will act on at least one. means of interruption.
Dans ce cas, pour les formules présentées plus loin, les termes : « mesure du moniteur », « mesure de la référence », « mesure du moniteur à la référence » sont des constantes qui ne varient pas d'une mesure à l'autre. Dès qu'on a obtenu un ratio donné, on coupe le faisceau d'excitation ou la lumière entrant dans les moyens de mesure de la fluorescence. Avec un tel dispositif, deux mesures faites sur deux échantillons identiques avec des niveaux d'éclairement à peu près identiques donnent deux valeurs de fluorescence mesurées identiques. On a ainsi automatiquement compensé la variation de lumière par une variation de temps nécessaire pour atteindre la quantité de lumière prélevée requise. On remarquera qu'il est plus facile, pour assurer la précision du dispositif, d'intégrer une quantité de lumière donnée et de couper le faisceau lumineux dès que cette quantité est intégrée. Avantageusement, ces moyens d'intégration comprendront un condensateur. En effet, il suffira alors d'utiliser le courant fourni par les moyens de mesure de la lumière prélevée pour charger une capacité et de comparer la charge du condensateur à un seuil fixé pour savoir quand l'échantillon aura reçu la bonne quantité de photons.In this case, for the formulas presented below, the terms: "measurement of the monitor", "measurement of the reference", "measurement of the monitor at the reference" are constants which do not vary from one measurement to another . As soon as a given ratio has been obtained, the excitation beam or the light entering the fluorescence measurement means is cut off. With such a device, two measurements made on two identical samples with approximately identical lighting levels give two identical measured fluorescence values. The variation in light was thus automatically compensated for by a variation in the time necessary to reach the required quantity of light. It will be noted that it is easier, to ensure the accuracy of the device, to integrate a given quantity of light and to cut the light beam as soon as this quantity is integrated. Advantageously, these integration means will include a capacitor. Indeed, it will then suffice to use the current supplied by the means for measuring the light sampled to charge a capacitor and to compare the charge of the capacitor with a fixed threshold to know when the sample will have received the right quantity of photons.
Avec ce type de dispositif, on peut ainsi obtenir une répétabilité de mesure meilleure que le 1%. Ce qui permettra de comparer, en relatif mais avec une très bonne précision, des mesures faites sur le même appareil .With this type of device, it is thus possible to obtain a repeatability of measurement better than 1%. This will allow you to compare, in relative but with very good precision, measurements made on the same device.
Un autre objet de l'invention consiste en un procédé capable de fournir une estimation précise et répétable de la quantité de photons envoyés sur l'échantillon pour une mesure donnée qui utilise le dispositif selon l'invention et qui est caractérisé en ce qu'il comprend au moins une étape de comparaison entre la valeur obtenue par les moyens de mesure de la fraction du faisceau de photons prélevée et une valeur de référence, cette comparaison donnant une valeur appelée valeur de fluorescence normalisée.Another object of the invention consists of a method capable of providing an accurate and repeatable estimate of the quantity of photons sent to the sample for a given measurement which uses the device according to the invention and which is characterized in that it comprises at least one step of comparison between the value obtained by the means for measuring the fraction of the photon beam sampled and a reference value, this comparison giving a value called normalized fluorescence value.
Selon un premier cas, cette valeur de référence sera une valeur prédéterminée . Selon un deuxième cas, cette valeur de référence sera le résultat d'une mesure faite au préalable par les moyens de mesure.In a first case, this reference value will be a predetermined value. In a second case, this reference value will be the result of a measurement made beforehand by the measuring means.
Ainsi, si la mesure de la quantité de photons envoyés sur l'échantillon est connue et si la mesure de la quantité correspondante de photons reçue par le capteur de fluorescence est connue, on peut calculer le niveau de fluorescence de l'échantillon. On peut par exemple retenir comme valeur de fluorescence normalisée une valeur du type :Thus, if the measurement of the quantity of photons sent to the sample is known and if the measurement of the corresponding quantity of photons received by the fluorescence sensor is known, the fluorescence level of the sample can be calculated. For example, a value of the type can be used as the normalized fluorescence value:
mesure de fluorescence ou mesure du moniteurfluorescence measurement or monitor measurement
mesure de fluorescence Xmesure du moniteur de la référence mesure du moniteur x mesure de la référencefluorescence measurement xmeasurement of reference monitor measurement of monitor x measurement of reference
Avec :With:
- mesure de fluorescence = valeur fournie par le capteur de fluorescence lors de l'examen de l'échantillon,- fluorescence measurement = value supplied by the fluorescence sensor during the examination of the sample,
- mesure du moniteur = valeur fournie par le dispositif de mesure de la lumière prélevée lors de l'examen de 1 ' échantillon,- monitor measurement = value supplied by the device for measuring the light taken during the examination of the sample,
- mesure du moniteur de la référence = valeur fournie par le dispositif de mesure de la lumière prélevée lors de l'examen de l'objet de référence,- reference monitor measurement = value supplied by the device for measuring the light sampled during the examination of the reference object,
- mesure de la référence = valeur fournie par le capteur de fluorescence lors de l'examen de l'objet de référence .- reference measurement = value supplied by the fluorescence sensor when examining the reference object.
Le dispositif selon l'invention possède de nombreux avantages . Tout d'abord, il permet d'améliorer la précision de la mesure de fluorescence d'un échantillon.The device according to the invention has many advantages. First, it improves the accuracy of the fluorescence measurement of a sample.
Supposons que le capteur de signal ou capteur de fluorescence (détecteur CCD par exemple) ait une précision de 1%, que le prélèvement du faisceau direct et sa mesure par le moniteur soient effectués avec une précision de 1% et que le comparateur sur le nombre de photons intégrés ait une répétabilité de 1%. La précision photométrique de l'ensemble sera alors de 1 ' ordre deSuppose that the signal sensor or fluorescence sensor (CCD detector for example) has an accuracy of 1%, that the sampling of the direct beam and its measurement by the monitor are carried out with an accuracy of 1% and that the comparator on the number of integrated photons has a repeatability of 1%. The photometric accuracy of the assembly will then be of the order of
Figure imgf000013_0001
Figure imgf000013_0001
soit une répétabilité de l'ordre de 1,7%, répétabilité qui peut être ramenée à 2% si les mesures de fluorescence ont été faites avec des appareils différents mais qui ont été étalonnés par rapport à la même référence .or a repeatability of the order of 1.7%, repeatability which can be reduced to 2% if the fluorescence measurements were made with different devices but which have been calibrated against the same reference.
Si l'on n'utilise pas ce type de contrôle de la source et qu'on se contente d'un prélèvement du flux avant la mesure ou d'une mesure de référence effectuée avant la mesure, on conserve une valeur de 1% pour la précision de la mesure par le capteur de signal, une valeur de 1% sur la mesure de la référence, mais par contre, on a au moins 3% d'incertitude sur la stabilité de la source et 1% sur le temps d'intégration. Cela conduit à une précision au mieux de l' ordre deIf one does not use this type of source control and is satisfied with a sampling of the flux before the measurement or with a reference measurement carried out before the measurement, one retains a value of 1% for the accuracy of the measurement by the signal sensor, a value of 1% on the measurement of the reference, but on the other hand, there is at least 3% of uncertainty on the stability of the source and 1% on the time of integration. This leads to an accuracy at best of the order of
V3x(0,0l)2+(0,03)2 = 0,035 soit une répétabilité de l'ordre de 3,5% qui ne permet pas une comparaison simple avec des mesures de fluorescence effectuées sur un appareil du même type.V3x (0.0l) 2 + (0.03) 2 = 0.035 that is to say a repeatability of the order of 3.5% which does not allow a simple comparison with fluorescence measurements carried out on a device of the same type.
On remarquera qu'on peut encore affiner la précision de la mesure de fluorescence en utilisant un capteur d'imagerie (capteur CCD par exemple) comme dispositif de mesure de la lumière prélevée au lieu d'une photodiode ou d'un photomultiplicateur. En effet, on peut dans ce cas effectuer une mesure du flux intégré pour chaque pixel du capteur de signal. L'utilisation d'un capteur de signal permettant de réaliser une image du faisceau prélevé devrait permettre de prendre en compte à terme dans la correction l' inhomogénéité du faisceau. Le dispositif permet aussi de prendre en compte les fluctuations d'intensité de la source d'éclairement en microscopie de fluorescence. On n'a pas besoin d'attendre que le flux de la source de photons (laser ou lampe à arc par exemple) soit complètement stabilisé pour faire des mesures (bien qu'il soit quand même préférable d'attendre) .It will be noted that the precision of the fluorescence measurement can be further refined by using an imaging sensor (CCD sensor for example) as a device for measuring the light taken instead of a photodiode or a photomultiplier. Indeed, it is possible in this case to carry out a measurement of the integrated flux for each pixel of the signal sensor. The use of a signal sensor making it possible to produce an image of the sampled beam should make it possible to take account in the long term in the correction of the inhomogeneity of the beam. The device also makes it possible to take into account the intensity fluctuations of the light source in fluorescence microscopy. There is no need to wait for the photon source flux (laser or arc lamp for example) to be completely stabilized before making measurements (although it is still preferable to wait).
Un autre avantage de l'invention est qu'avec un système de mesure de l'intensité délivrée sur l'échantillon, on peut facilement calibrer le ratio entre le signal prélevé et le signal envoyé sur l'échantillon. Ensuite, en remplaçant l'échantillon par un objet d'albedo calibrée, on peut calibrer le ratio entre la lumière émise par l'échantillon et la lumière mesurée par le capteur de fluorescence. Avec ces deux calibrations, si on utilise l'invention proposée, la même expérience réalisée sur deux systèmes différents donnera le même résultat.Another advantage of the invention is that with a system for measuring the intensity delivered on the sample, it is easy to calibrate the ratio between the signal taken and the signal sent to the sample. Then, by replacing the sample with a calibrated albedo object, we can calibrate the ratio between the light emitted by the sample and the light measured by the fluorescence sensor. With these two calibrations, if the proposed invention is used, the same experiment carried out on two different systems will give the same result.
BREVE DESCRIPTION DES DESSINS L'invention sera mieux comprise et d'autres avantages et particularités apparaîtront à la lecture de la description qui va suivre, donnée à titre d'exemple non limitatif, accompagnée des dessins annexés parmi lesquels : - la figure 1 est un schéma illustrant la mise en œuvre du dispositif selon l'invention dans le cadre d'une mesure en épifluorescence,BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood and other advantages and particularities will appear on reading the description which follows, given by way of nonlimiting example, accompanied by the appended drawings among which: - Figure 1 is a diagram illustrating the implementation of the device according to the invention in the context of an epifluorescence measurement,
- la figure 2 est un schéma illustrant la mise en œuvre du dispositif selon l'invention dans le cas d'un éclairage par ondes évanescentes .- Figure 2 is a diagram illustrating the implementation of the device according to the invention in the case of lighting by evanescent waves.
EXPOSÉ DÉTAILLÉ DE MODES DE RÉALISATION PARTICULIERSDETAILED PRESENTATION OF PARTICULAR EMBODIMENTS
Dans la figure 1, un faisceau de lumière provenant d'une source de lumière 1, qui peut être un laser ou éventuellement une lampe à arc, passe à travers un obturateur 2 de la source d'éclairement et arrive sur un système de prélèvement de la lumière 3 , qui peut être constitué d'une lame semi-réfléchissante. Ce dispositif de prélèvement de la lumière 3 partage le faisceau lumineux en deux : une partie du faisceau, le faisceau réfléchi, va illuminer un dispositif de mesure 4 de la lumière prélevée; l'autre partie du faisceau, le faisceau transmis, va aller se réfléchir dans un cube dichroique 7. Le dispositif de mesure de la lumière prélevée 4 qui reçoit le faisceau réfléchi peut être, par exemple, une photodiode, un photomultiplicateur ou un capteur CCD. Le dispositif de mesure 4 convertit la lumière en un signal qui va être intégré par un système 5. Cette intégration peut aussi être réalisée directement sur le signal lumineux, par exemple à l'aide d'une caméra CCD ou d'une plaque photographique. Puis, éventuellement, le faisceau pourra arriver dans un dispositif 6 dont la fonction est de couper le faisceau à un temps d'intégration donné ou à un nombre de photons détectés suffisant. Pour cela, ce dispositif de coupure est relié et commande le fonctionnement de deux obturateurs 2 et 10 placés respectivement devant la source lumineuse et devant le capteur de fluorescence. Il est à noter que les deux obturateurs ne sont pas nécessaires : un seul est suffisant.In FIG. 1, a beam of light coming from a light source 1, which can be a laser or possibly an arc lamp, passes through a shutter 2 of the lighting source and arrives on a system for taking off light 3, which can consist of a semi-reflecting plate. This light sampling device 3 divides the light beam into two: part of the beam, the reflected beam, will illuminate a device 4 for measuring the light collected; the other part of the beam, the transmitted beam, will reflect in a dichroic cube 7. The device for measuring the sampled light 4 which receives the reflected beam can be, for example, a photodiode, a photomultiplier or a CCD sensor. The measuring device 4 converts light into a signal which will be integrated by a system 5. This integration can also be carried out directly on the light signal, for example using a CCD camera or a photographic plate. Then, possibly, the beam can arrive in a device 6 whose function is to cut the beam at a given integration time or at a sufficient number of detected photons. For this, this cut-off device is connected and controls the operation of two shutters 2 and 10 placed respectively in front of the light source and in front of the fluorescence sensor. It should be noted that the two shutters are not necessary: only one is sufficient.
La partie du faisceau qui arrive sur le cube dichroïque 7 va être séparée en deux faisceaux. En effet, le cube dichroïque a pour effet de séparer la lumière d'excitation de la lumière de fluorescence. La lumière d'excitation est réfléchie par le cube dichroïque, traverse un objectif du microscope 8 et arrive sur l'échantillon 9, qui peut être, par exemple, une puce ADN ayant des marqueurs fluorescents. Cet échantillon va émettre à son tour un faisceau qui va traverser l'objectif du microscope 8, et qui, comme il s'agit d'un faisceau de lumière fluorescente, va traverser le cube dichroïque 7 et l'obturateur 10 du capteur de mesure de fluorescence. Ce faisceau transmis arrive enfin sur le capteur de signal ou capteur de fluorescence 11, qui peut notamment être un capteur CCD, une photodiode ou un photomultiplicateur.The part of the beam which arrives on the dichroic cube 7 will be separated into two beams. Indeed, the dichroic cube has the effect of separating the excitation light from the fluorescence light. The excitation light is reflected by the dichroic cube, passes through a microscope objective 8 and arrives at the sample 9, which can be, for example, a DNA chip having fluorescent markers. This sample will in turn emit a beam which will pass through the microscope objective 8, and which, as it is a beam of fluorescent light, will pass through the dichroic cube 7 and the shutter 10 of the measurement sensor fluorescence. This transmitted beam finally arrives on the signal sensor or fluorescence 11, which can in particular be a CCD sensor, a photodiode or a photomultiplier.
Dans la figure 2, on a presque le même montage : un faisceau de lumière provenant d'une source de lumière 21 passe à travers un obturateur 22 de la source d'éclairement et arrive sur un système de prélèvement de la lumière 23, qui partage le faisceau lumineux en deux : une partie du faisceau va être reçue par un système de mesure 24 de la lumière prélevée pour aller dans un système 25 intégrant le signal prélevé dans le faisceau de lumière, puis éventuellement dans un système 26 de coupure du faisceau à un temps d' intégration donné ou à un nombre de photons détectés suffisant, l'autre partie du faisceau va aller se propager dans l'échantillon 29. On va observer les ondes évanescentes provenant de cet échantillon qui traversent l'objectif du microscope 28, l'obturateur 30 du capteur de mesure de fluorescence, et arrive sur le capteur de signal 31.In FIG. 2, we have almost the same arrangement: a light beam coming from a light source 21 passes through a shutter 22 of the illumination source and arrives on a light sampling system 23, which shares the light beam in two: part of the beam will be received by a system 24 for measuring the light sampled to go into a system 25 integrating the signal sampled in the light beam, then optionally in a system 26 for cutting the beam to a given integration time or at a sufficient number of detected photons, the other part of the beam will propagate in the sample 29. We will observe the evanescent waves coming from this sample which pass through the objective of the microscope 28, the shutter 30 of the fluorescence measurement sensor, and arrives at the signal sensor 31.
Dans ces deux figures, les obturateurs 2, 22, 10, 30 peuvent être remplacés par des moyens d'interruption placés directement soit sur la source de photons, soit sur les moyens de détection. Une des possibilités la plus simple peut consister à couper l'alimentation électrique de la source ou des moyens de détection.In these two figures, the shutters 2, 22, 10, 30 can be replaced by interrupting means placed directly either on the photon source, or on the detection means. One of the simplest possibilities may consist in cutting the electrical supply from the source or from the detection means.
Une des applications industrielles prometteuses de ce dispositif et de son procédé associé selon l'invention est le développement de lecteurs de puces ADN en épi-illumination (cas de la figure 1) . Dans ce système, on lit des séries de puces ADN fluorescentes et les niveaux de fluorescence des différentes puces doivent pouvoir être reliés entre eux. One of the promising industrial applications of this device and its associated process according to the invention is the development of DNA chip readers in epi-illumination (case of Figure 1). In this system, we read series of fluorescent DNA chips and the fluorescence levels of the different chips must be able to be linked together.

Claims

REVENDICATIONS
1. Dispositif de mesure de fluorescence comportant une source emettrice de photons (1, 21) destinée à émettre un faisceau de photons vers un échantillon (9) à analyser, le dispositif comprenant également des moyens de détection (11, 31) d'une lumière de fluorescence émise par l'échantillon en réponse aux photons du faisceau reçus par celui-ci, caractérisé en ce qu'il comporte en outre des moyens de prélèvement (3, 23) aptes à prélever, au cours de la mesure de fluorescence, une fraction déterminée du faisceau de photons émis par la source de photons, des moyens de mesure (4, 24) de ladite fraction du faisceau de photons prélevée, et des moyens d'intégration (5, 25) permettant d'intégrer la fraction déterminée du faisceau de photons.1. A fluorescence measurement device comprising a photon emitting source (1, 21) intended to emit a beam of photons towards a sample (9) to be analyzed, the device also comprising detection means (11, 31) of a fluorescence light emitted by the sample in response to the beam photons received by the latter, characterized in that it also comprises sampling means (3, 23) capable of taking, during the fluorescence measurement, a determined fraction of the photon beam emitted by the photon source, measuring means (4, 24) of said fraction of the sampled photon beam, and integration means (5, 25) making it possible to integrate the determined fraction of the photon beam.
2. Dispositif selon la revendication 1 caractérisé en ce qu'il comporte en outre un objectif de microscope (8, 28) situé entre l'échantillon à analyser et les moyens de détection (11, 31) sur le trajet de la lumière de fluorescence émise par l'échantillon.2. Device according to claim 1 characterized in that it further comprises a microscope objective (8, 28) located between the sample to be analyzed and the detection means (11, 31) on the path of the fluorescence light emitted by the sample.
3. Dispositif selon la revendication 1 caractérisé en ce que lesdits moyens de prélèvement (3, 23) sont une lame semi-transparente . 3. Device according to claim 1 characterized in that said sampling means (3, 23) are a semi-transparent blade.
4. Dispositif selon la revendication 1 caractérisé en ce que lesdits moyens de détection (11, 31) et lesdits moyens de mesure (4, 24) sont choisis parmi le groupe constitué d'un capteur CCD, d'un photomultiplicateur et d'une photodiode.4. Device according to claim 1 characterized in that said detection means (11, 31) and said measurement means (4, 24) are chosen from the group consisting of a CCD sensor, a photomultiplier and a photodiode.
5. Dispositif selon la revendication 1 caractérisé en ce que les moyens d'intégration (5, 25) comportent un capteur unitaire qui intègre l'ensemble de la fraction déterminée du faisceau de photons.5. Device according to claim 1 characterized in that the integration means (5, 25) comprise a unitary sensor which integrates all of the determined fraction of the photon beam.
6. Dispositif selon la revendication 1 caractérisé en ce que les moyens d'intégration (5, 25) comportent un ensemble de capteurs unitaires apte à fournir une image de la répartition spatiale de la fraction déterminée du faisceau de photons selon une section dudit faisceau.6. Device according to claim 1 characterized in that the integration means (5, 25) comprise a set of unitary sensors capable of providing an image of the spatial distribution of the determined fraction of the photon beam along a section of said beam.
7. Dispositif selon la revendication 1 caractérisé en ce qu'il comporte en outre des moyens de coupure (6, 26) du faisceau de photons émis à un temps d'intégration donné.7. Device according to claim 1 characterized in that it further comprises cutting means (6, 26) of the photon beam emitted at a given integration time.
8. Dispositif selon la revendication 1 caractérisé en ce qu'il comporte en outre des moyens de coupure (6, 26) du faisceau de photons émis à un nombre de photons du faisceau prélevé détectés suffisant.8. Device according to claim 1 characterized in that it further comprises cutting means (6, 26) of the beam of photons emitted to a sufficient number of photons of the sampled beam detected.
9. Dispositif selon la revendication 7 ou 8 caractérisé en ce que lesdits moyens de coupure (6, 26) agissent sur des moyens d'interruption (2, 22) placés sur le trajet optique de la lumière émise par la source entre la source emettrice de photons (1, 21) et les moyens de prélèvement (3, 23) et permettant d'interrompre l'émission de la source de photons.9. Device according to claim 7 or 8 characterized in that said cutting means (6, 26) act on interrupting means (2, 22) placed on the optical path of the light emitted by the source between the photon emitting source (1, 21) and the sampling means (3, 23) and making it possible to interrupt the emission of the photon source.
10. Dispositif selon la revendication 7 ou 8 caractérisé en ce que lesdits moyens de coupure (6, 26) agissent sur des moyens d'interruption de l'alimentation de la source emettrice de photons.10. Device according to claim 7 or 8 characterized in that said cutting means (6, 26) act on means for interrupting the supply of the photon emitting source.
11. Dispositif selon la revendication 7 ou 8 caractérisé en ce que lesdits moyens de coupure (6, 26) agissent sur des moyens d'interruption (10, 30) placés sur le trajet optique de la lumière provenant de l'échantillon (9, 29) entre ledit échantillon (9, 29) et les moyens de détection (11, 31) et permettant d' interrompre la mesure de fluorescence par les moyens de détection (11, 31) .11. Device according to claim 7 or 8 characterized in that said cutting means (6, 26) act on interrupting means (10, 30) placed on the optical path of the light coming from the sample (9, 29) between said sample (9, 29) and the detection means (11, 31) and making it possible to interrupt the fluorescence measurement by the detection means (11, 31).
12. Dispositif selon la revendication 7 ou12. Device according to claim 7 or
8 caractérisé en ce que lesdits moyens de coupure agissent sur des moyens d' interruption du fonctionnement des moyens de détection (11, 31) .8 characterized in that said cutting means act on means for interrupting the operation of the detection means (11, 31).
13. Dispositif selon la revendication 9 ou13. Device according to claim 9 or
11 caractérisé en ce que lesdits moyens d'interruption (2, 22 ; 10, 30) sont un obturateur.11 characterized in that said interruption means (2, 22; 10, 30) are a shutter.
14. Dispositif selon la revendication 8, caractérisé en ce que lesdits moyens de coupure (6, 26) du faisceau de photons émis comprennent en outre un système de comparaison entre la quantité de lumière intégrée par les moyens d'intégration (5, 25) et un niveau d'éclairement pris comme référence.14. Device according to claim 8, characterized in that said cut-off means (6, 26) of the emitted photon beam further comprises a comparison system between the quantity of light integrated by the integration means (5, 25) and a level of illumination taken as a reference.
15. Procédé fournissant une estimation précise et répétable de la quantité de photons envoyés sur l'échantillon pour une mesure donnée qui utilise le dispositif selon l'une des revendications précédentes, caractérisé en ce qu'il comprend au moins une étape de comparaison entre la valeur obtenue par les moyens de mesure (4, 24) et une valeur de référence, cette comparaison donnant une valeur appelée valeur de fluorescence normalisée.15. A method providing an accurate and repeatable estimate of the quantity of photons sent to the sample for a given measurement which uses the device according to one of the preceding claims, characterized in that it comprises at least one step of comparison between the value obtained by the measurement means (4, 24) and a reference value, this comparison giving a value called normalized fluorescence value.
16. Procédé selon la revendication 15, caractérisé en ce que la valeur de référence est une valeur prédéterminée .16. Method according to claim 15, characterized in that the reference value is a predetermined value.
17. Procédé selon la revendication 15, caractérisé en ce que la valeur de référence est une valeur obtenue au préalable par les moyens de mesure (4, 24) . 17. Method according to claim 15, characterized in that the reference value is a value obtained beforehand by the measuring means (4, 24).
PCT/FR2003/050160 2002-12-12 2003-12-11 Method of measuring a quantity of photons which is proportional to the quantity of photons received by the object and associated device WO2004055502A2 (en)

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WO2020234617A1 (en) 2019-05-22 2020-11-26 Biomerieux Methods and systems for manufacturing a production assay reactor

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