WO2003029882A2 - Method and device for sampling part of a light beam, particularly for a fluorescence analysis apparatus - Google Patents

Method and device for sampling part of a light beam, particularly for a fluorescence analysis apparatus Download PDF

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Publication number
WO2003029882A2
WO2003029882A2 PCT/FR2002/003337 FR0203337W WO03029882A2 WO 2003029882 A2 WO2003029882 A2 WO 2003029882A2 FR 0203337 W FR0203337 W FR 0203337W WO 03029882 A2 WO03029882 A2 WO 03029882A2
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WIPO (PCT)
Prior art keywords
acousto
optical
order
light
laser
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PCT/FR2002/003337
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French (fr)
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WO2003029882A3 (en
Inventor
Philippe Peltie
Olivier Peyssonneaux
Raymond Campagnolo
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Commissariat A L'energie Atomique
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Publication of WO2003029882A2 publication Critical patent/WO2003029882A2/en
Publication of WO2003029882A3 publication Critical patent/WO2003029882A3/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
    • 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
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G02—OPTICS
    • G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
    • G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
    • G02F1/29—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics for the control of the position or the direction of light beams, i.e. deflection
    • G02F1/33—Acousto-optical deflection devices

Definitions

  • the present invention relates to a method and a device for removing a light beam, in particular a laser beam.
  • the invention is particularly applicable to fluorescence analysis by microscopy and, more particularly, to the analysis of fluorescence images from biochips ("biochips”), or micro-titration plates ("microtiter plates” ), for example for the analysis of DNA, RNA, proteins and cells.
  • biochips biochips
  • micro-titration plates micro-titration plates
  • Particular applications of the invention are thus cellular analysis by fluorescence, the reading of biochips, for example of the genus MICAM (registered trademark) with 128 or 8100 electrodes, and the fluorimetry devices using lasers.
  • MICAM registered trademark
  • MICAM registered trademark
  • lasers are preferably used as light sources, for reasons of stability and lifetime of these sources.
  • the present invention aims to solve the above problems.
  • the invention uses one or a plurality of acousto-optical modulators, depending on whether one or a plurality of light sources are used.
  • an acousto-optical modulator when an acousto-optical modulator receives a light beam, it is capable of providing a diffracted beam of order +1 (or of order - 1), but only when it is activated. This acousto-optical modulator can therefore serve as a shutter vis-à-vis the optical device for which the light beam is intended.
  • the beam of order 0, which is constantly supplied by the acousto-optical modulator, can be used as a reference beam for regulating the light intensity of the source which emits the beam received by the modulator and for monitoring the operating status of this source, that is to say whether the latter is on or off or whether or not the optical device receives the diffracted beam of order +1 (or order -1).
  • the switching time of the acousto-optical modulator is much shorter than that of a mechanical shutter - it is of the order of a few microseconds - which is particularly advantageous when two or more light sources are used.
  • the subject of the present invention is a method of sampling a part of at least one beam of light supplied by a light source, this beam being intended to be used in an optical device, in which: places at least one acousto-optical modulator on the path of the light beam, this modulator thus providing a beam of order 0,
  • this acousto-optical modulator is activated so that it also provides a beam of order +1 (that is to say of order +1 or of order -1), and
  • the light source can be a laser, the light beam then being a laser beam.
  • the present invention also relates to a device for removing a part of at least one beam of light provided by a light source, this beam being intended to be used in an optical device, this device being characterized in that it comprises :
  • At least one acousto-optical modulator intended to receive this beam of light, this acousto-optical modulator thus providing a beam of order 0, and means of activation of this acousto-optical modulator so that it also provides a beam +1 diffracted, the optical device being intended to receive this +1 diffracted beam and therefore not receiving the latter when the acousto-optical modulator is not activated, this acousto-optical modulator thus forming a beam shutter vis-à-vis the optical device, the device further comprising at least one photodetector adapted to receive the order beam
  • the latter forming a reference beam, and to provide a signal representative of this reference beam, this signal being used to correct measurements which can be carried out by means of the optical device.
  • the optical device is an apparatus for analyzing biological samples by fluorescence and the signal is used to correct the measurements made using this analysis device.
  • the device which is the subject of the invention may comprise a plurality of acousto-optical modulators designed to receive respectively a plurality of light beams intended for use in the optical apparatus, these acousto-optical modulators thus forming a plurality of beam shutters vis-à-vis the optical device.
  • the device which is the subject of the invention comprises a plurality of acousto-optical modulators, provided for receiving respectively a plurality of light beams, and a plurality of photodetectors respectively associated with these acousto-optical modulators and provided for receiving the light beams. order 0 respectively provided by these acousto-optical modulators.
  • Each light source used with the device which is the subject of the invention can be a laser, each light beam then being a laser beam.
  • FIG. 1 is a schematic view of a particular embodiment of the device which is the subject of the invention
  • FIG. 3 is a schematic view of a device according to the invention, which is used with several lasers,
  • FIG. 4 is a schematic view of a synchronization circuit which can be used in the invention.
  • FIG. 5 is a timing diagram relating to the synchronization of the images obtained with a line transfer or frame transfer camera in a particular embodiment of the invention.
  • FIG. 6 is a timing diagram relating to the synchronization of the acquisitions of signals supplied by a photomultiplier, an avalanche photodiode or a silicon photodiode, in a particular embodiment of the invention.
  • FIG. 1 schematically illustrates a device according to the invention, which is used with an optical apparatus A. It is an apparatus for analyzing biological samples by microscopy at This device is intended for the observation of samples 2 placed in the wells 4 of a plate micro-titration 6 which we see the bottom 8 and the walls 10 defining the wells.
  • the samples are excited by radiation 12 from a laser 14 and provide fluorescence radiation 16 as a result of this excitation.
  • the bottom of the micro-titration plate is transparent to light from the laser as well as to this fluorescence radiation.
  • the light from the laser is shaped using an appropriate optic 18 then filtered using an appropriate filter 20 after passing through the device according to the invention D, to which we will return later, then was successively reflected by two mirrors 21a and 21b.
  • the light thus filtered is reflected, thanks to a dichroic mirror 22, in the direction of the objective 24 of the microscope. It crosses this objective 24 which focuses it on the sample studied. It is specified that the samples are studied one after the other, the micrititration plate being for this purpose arranged on suitable displacement means, not shown.
  • FIG. 1 also shows a camera 26, for example a CCD camera, which is designed to capture the image of the sample studied, thanks to the fluorescence radiation 16 emitted by this sample.
  • a camera 26 for example a CCD camera, which is designed to capture the image of the sample studied, thanks to the fluorescence radiation 16 emitted by this sample.
  • This fluorescence radiation 16 also passes through the objective 24 of the microscope then the dichroic mirror 22 (the latter being able to reflect the radiation 12 and transmit the radiation 16) and reaches camera 26 after having passed through another filter 28 designed to eliminate the light from the laser capable of also reaching this camera.
  • the device D which is schematically represented in FIG. 1, comprises an acousto-optical modulator 30 which is placed on the path of the beam coming from the laser 14, following the optics 18.
  • This acousto-optical modulator comprises an acousto-optical cell 32 provided with a transducer 34
  • the device D also includes a control circuit (“driver") 36 which is connected to the transducer 34.
  • driver control circuit
  • this acousto-optical modulator diffracts the laser beam and thus provides, in the example shown , a diffracted beam 38 of order +1 in addition to the beam 40 of order 0.
  • ⁇ the angle of incidence of the laser beam, this angle ⁇ being equal to 0.3 ° in the example.
  • the beam in order +1 is deviated from the angle 2 ⁇ with respect to this incident beam, while the beam of order 0 makes a zero angle with this incident beam.
  • the diffraction efficiency represents the percentage of light energy that is deflected. This percentage is around 80%. That means it about 20% of light energy remains in the beam of order 0.
  • the control circuit 36 is simply controlled by a TTL signal from 0 to 5V ( Figure 2).
  • this TTL signal is at 0V, the entire laser beam is in the 0 order and, when it is at 5V 7 about 80% of this laser beam is deflected in the +1 order.
  • the acousto-optical modulator 30 plays the role of a beam shutter, whereas it is traditionally used as a light modulator.
  • the beam can be used in order 0 as a reference beam, for example to monitor the operating state of the laser 14 or to correct this operation, in particular to correct the intensity of the emitted beam. by this laser 14.
  • FIG. 1 also shows electronic processing means 44 provided for processing the images supplied by the CCD camera 26 in the form of electrical signals, these electronic processing means being provided with display means 46 making it possible to display the images supplied. by the camera.
  • the electronic processing means 44 are provided to supply the TTL signal mentioned above to the control circuit 36.
  • the assembly that can be seen in FIG. 1 can be extended to two or more than two lasers, provided that an acousto-optical modulator and a laser photodetector are available.
  • FIG. 3 This is schematically illustrated by FIG. 3 in which we see another particular embodiment of the device which is the subject of the invention.
  • the device which is schematically represented in this FIG. 3 comprises three lasers 48, 50 and 52 provided for emitting excitation radiation from samples, these lasers having different wavelengths (for example 488 nm,
  • Each of these lasers 48, 50 or 52 is successively followed by an optical assembly 54, 56 or 58 for shaping the beam supplied by this laser, a mirror 60, 62 or 64 and an acousto-optical modulator 66, 68 or 70 , each mirror 60, 62 or 64 being designed to send the laser beam, which it receives via the optical assembly 54, 56 or 58, onto the corresponding acousto-optical modulator, at an angle of incidence appropriate.
  • the acousto-optical modulators 66, 68 and 70 are respectively controlled by control circuits ("drivers") 72, 74 and 76.
  • FIG. 3 also shows a fluorescence analysis device A intended to be illuminated by any one of the +1 diffracted beams from acousto-optical modulators 66, 68 and 70.
  • This apparatus A includes a CCD camera (not shown) which provides images in the form of electrical signals. These are processed by the electronic processing means 78 which are provided with display means (not shown) enabling these images to be viewed.
  • these electronic processing means are provided to supply, to the control circuits 72, 74 and 76, TTL signals of the type which have been mentioned above.
  • the beams 80, 82 and 84 of order 0 corresponding respectively to the acousto-optical modulators 66, 68 and 70, the diffracted beams 86, 88 and 90 of order +1, corresponding respectively to these modulators 66, 68 and 70, as well as the photodetectors 92, 94 and 96 respectively designed to receive the beams 80, 82 and 84 of order 0.
  • the electrical signals supplied by these photodetectors 92, 94 and 96 are sent to an acquisition card (not shown) of a computer.
  • the timing diagram in FIG. 2 is valid for each of these photodetectors.
  • the acousto-optical modulators 66, 68 and 70 also form beam shutters vis-à-vis the optical device A.
  • the device of FIG. 3 also includes mirrors 98, 100 and 102 which are associated respectively with modulators 66, 68 and 70 and are designed to send the beam, via another mirror 104, +1 diffracted from the acousto-optic modulator that was activated.
  • the devices according to the invention of Figures 1 and 3 have various advantages mentioned below.
  • Each of these devices makes it possible to control each of the laser beams: with each photodetector, it is possible to know whether the corresponding laser is stopped or switched on and whether the corresponding order 1 diffracted beam is emitted or not.
  • the device in FIG. 3 allows rapid switching from one laser beam to the other by controlling the modulator which is in operation: if for example the laser 48 is on and the modulator 66 is activated, the photodetector 92 supplies the voltage VI and if the photodetector 94 supplies the voltage V2, the laser beam 50 is not sent to the optical device A.
  • the switching time is very short: it takes just about 1 microsecond to 10 microseconds to pass from the beam that is sent to the optical device to another beam.
  • the devices of FIGS. 1 and 3 guarantee the safety of the users with respect to the lasers. Yes for example a user intervenes on the optical device A, no acousto-optical modulator is then activated so that no laser beam is sent to the optical device. If an electronic failure occurs, the control circuits receive no signal, so there is no deflection of the laser beam and therefore no laser beam is sent to the optical device. In addition, if a laser is broken, for example the laser 48, the corresponding photodetector 92 supplies a zero voltage, thus indicating that there is no laser beam.
  • FIGS. 1 and 3 make it possible to control the laser power as explained below.
  • No source is stable at less than a few percent (typically 5%).
  • the signal supplied by each photodetector strictly follows the fluctuations of the corresponding laser.
  • the voltages VI and V2 therefore follow the fluctuations of this laser.
  • Knowledge of the voltage VI therefore makes it possible to correct the fluctuations of the fluorescence image in real time, by monitoring, for each image acquisition, the laser power received by the photodetector during the image acquisition phase, which requires a synchronization circuit common to the actual image acquisition and to the acquisition of a light signal by the photodetector, as shown schematically in Figure 4.
  • FIG. 4 which relates to the photodetector 42 of FIG. 1, but which also applies to each photodetector of FIG. 3, there is seen an amplifier 106 intended to amplify the electrical signal supplied by this photodetector and a synchronization circuit 108 which receives the signal thus amplified and which is intended to follow the power fluctuations of the laser: it can be seen that the control circuit (“driver") 36 receives the pulses S which are addressing pulses for addressing the laser and are supplied by the computer (not shown). The output of the synchronization circuit 108 is connected to the computer.
  • the speed of switching of a laser beam by an acousto-optic modulator makes it possible to use, for image capture, line transfer or frame transfer cameras so that, during the phase of reading the charges and digitizing the signal, it is possible to start a phase of accumulation of images or signals as shown in FIG. 5.
  • the laser signal is used to drive a PING / PONG system (FIG. 6), for the integration of the signal (this signal being provided by each photodetector capable of being used in 1 invention and consisting for example of the output current of a photomultiplier or an avalanche photodiode or a silicon photodiode) or a system formed by two counters for counting photons.
  • a PING / PONG system FIG. 6
  • FIG. 5 is a timing diagram relating to the synchronization of the images which are obtained with a CCD camera for line transfer or for frame transfer.
  • Lines I to V of FIG. 5 respectively show the pulses corresponding to the cumulative image (line I), the pulses corresponding to the active state of the laser given by the reference photodiode (line II), the synchronizations of transfer of line or frame (line III), the pulses relating to the reading of the charges in the CCD camera (line IV) and the signal relating to the digitization and storage of the images ' (line V), the period T that the we see on line V being the period between images.
  • FIG. 6 is, for its part, a timing diagram relating to the synchronization of the acquisitions of signals supplied by a photodetector such as a photomultiplier or an avalanche photodiode or a silicon photodiode.
  • a photodetector such as a photomultiplier or an avalanche photodiode or a silicon photodiode.
  • Lines I to V of FIG. 6 respectively show the pulses corresponding to the active state of the laser by means of which the images are obtained (line I), the pulses corresponding to the signals supplied by the photodetectors considered above (line II) , the integrated PING signal (line III), the integrated PONG signal (line IV), and the pulses corresponding to the alternating PING / PONG digitization.
  • the diffracted beam of order -1 instead of the diffracted beam of order +1, to send this beam to an optical device A.

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Abstract

The invention relates to a method and device for sampling part of a light beam, particularly for a fluorescence analysis apparatus. According to the invention, at least one acousto-optic modulator (30) is positioned on the path of the beam and said modulator is activated in order to produce a diffracted beam (38) of the order of +1 or 1. The latter diffracted beam is subsequently sent to an optical apparatus (A) which only receives said beam when the modulator is activated. In this way, the modulator forms a shutter with respect to the apparatus. The aforementioned apparatus is intended to take measurements and the beam of the order of 0 is used as the reference beam in order to correct said measurements.

Description

PROCEDE ET DISPOSITIF DE PRELEVEMENT D'UNE PARTIE D'UN FAISCEAU LUMINEUX, NOTAMMENT POUR APPAREIL D'ANALYSE DE METHOD AND DEVICE FOR TAKING PART OF A LIGHT BEAM, IN PARTICULAR FOR AN ANALYSIS APPARATUS
FLUORESCENCEFLUORESCENCE
DESCRIPTIONDESCRIPTION
DOMAINE TECHNIQUETECHNICAL AREA
La présente invention concerne un procédé et un dispositif de prélèvement d'un faisceau lumineux, en particulier d'un faisceau laser.The present invention relates to a method and a device for removing a light beam, in particular a laser beam.
L'invention s'applique notamment à l'analyse de fluorescence par microscopie et, plus particulièrement, à l'analyse d'images de fluorescence provenant de biopuces ("biochips"), ou de plaques de micro-titration ("microtiter plates"), par exemple en vue de l'analyse d'ADN, d'ARN, de protéines et de cellules.The invention is particularly applicable to fluorescence analysis by microscopy and, more particularly, to the analysis of fluorescence images from biochips ("biochips"), or micro-titration plates ("microtiter plates" ), for example for the analysis of DNA, RNA, proteins and cells.
Des applications particulières de l'invention sont ainsi l'analyse cellulaire par fluorescence, la lecture de biopuces, par exemple du genre MICAM (marque déposée) à 128 ou 8100 électrodes, et les appareils de fluorimétrie utilisant des lasers.Particular applications of the invention are thus cellular analysis by fluorescence, the reading of biochips, for example of the genus MICAM (registered trademark) with 128 or 8100 electrodes, and the fluorimetry devices using lasers.
ETAT DE LA TECHNIQUE ANTERIEURESTATE OF THE PRIOR ART
Avec un grand nombre d'appareils optiques, en particulier avec les appareils de microscopie à fluorescence, on utilise de préférence des lasers en tant que sources lumineuses, pour des raisons de stabilité et de durée de vie de ces sources.With a large number of optical devices, in particular with fluorescence microscopy devices, lasers are preferably used as light sources, for reasons of stability and lifetime of these sources.
La plupart du temps, on utilise des obturateurs ("shutters") mécaniques pour couper les faisceaux lumineux émis par ces sources. Cependant, la commutation de tels obturateurs n'est pas très rapide, ce qui est particulièrement gênant lorsqu'on utilise deux ou plus de deux sources lumineuses . D'autres problèmes se posent dans ces appareils optiques, à savoir ceux de la régulation de l'intensité lumineuse des sources utilisées et de la surveillanceMost of the time, we use mechanical shutters to cut the light beams emitted by these sources. However, the switching of such shutters is not very fast, which is particularly troublesome when two or more of two light sources are used. Other problems arise in these optical devices, namely those of regulating the light intensity of the sources used and of monitoring
( "monitoring") de l'état de fonctionnement de ces sources, tout particulièrement lorsqu'il s'agit de lasers, ainsi que les problèmes de sécurité occulaire inhérents à l'utilisation de lasers.("monitoring") of the operating status of these sources, especially when it comes to lasers, as well as the ocular safety problems inherent in the use of lasers.
EXPOSÉ DE L'INVENTIONSTATEMENT OF THE INVENTION
La présente invention a pour but de résoudre les problèmes précédents.The present invention aims to solve the above problems.
Pour ce faire, l'invention utilise un ou une pluralité de modulateurs acousto-optiques, selon qu'une ou une pluralité de sources lumineuses sont utilisées.To do this, the invention uses one or a plurality of acousto-optical modulators, depending on whether one or a plurality of light sources are used.
En effet, lorsqu'un modulateur acousto-optique reçoit un faisceau lumineux, il est capable de fournir un faisceau diffracté d'ordre +1 (ou d'ordre - 1), mais seulement lorsqu'il est activé. Ce modulateur acousto-optique peut donc servir d'obturateur vis-à-vis de l'appareil optique auquel est destiné le faisceau lumineux.In fact, when an acousto-optical modulator receives a light beam, it is capable of providing a diffracted beam of order +1 (or of order - 1), but only when it is activated. This acousto-optical modulator can therefore serve as a shutter vis-à-vis the optical device for which the light beam is intended.
De plus, le faisceau d'ordre 0, qui est constamment fourni par le modulateur acousto-optique, peut être utilisé en tant que faisceau de référence pour la régulation de l'intensité lumineuse de la source qui émet le faisceau reçu par le modulateur et pour la surveillance de l'état de fonctionnement de cette source, c'est à dire pour savoir si cette dernière est allumée ou éteinte ou si oui ou non l'appareil optique reçoit le faisceau diffracté d'ordre +1 (ou d'ordre -1). En outre, le temps de commutation du modulateur acousto-optique est beaucoup plus court que celui d'un obturateur mécanique - il est de l'ordre de quelques microsecondes - ce qui est particulièrement intéressant lorsqu'on utilise deux sources lumineuses ou plus. De façon précise, la présente invention a pour objet un procédé de prélèvement d'une partie d'au moins un faisceau de lumière fourni par une source de lumière, ce faisceau étant destiné à être utilisé dans un appareil optique, dans lequel : - on place au moins un modulateur acousto- optique sur le trajet du faisceau de lumière, ce modulateur fournissant ainsi un faisceau d'ordre 0,In addition, the beam of order 0, which is constantly supplied by the acousto-optical modulator, can be used as a reference beam for regulating the light intensity of the source which emits the beam received by the modulator and for monitoring the operating status of this source, that is to say whether the latter is on or off or whether or not the optical device receives the diffracted beam of order +1 (or order -1). In addition, the switching time of the acousto-optical modulator is much shorter than that of a mechanical shutter - it is of the order of a few microseconds - which is particularly advantageous when two or more light sources are used. Specifically, the subject of the present invention is a method of sampling a part of at least one beam of light supplied by a light source, this beam being intended to be used in an optical device, in which: places at least one acousto-optical modulator on the path of the light beam, this modulator thus providing a beam of order 0,
- on active ce modulateur acousto-optique pour qu'il fournisse aussi un faisceau d'ordre +1 (c'est-à-dire d'ordre +1 ou d'ordre -1), etthis acousto-optical modulator is activated so that it also provides a beam of order +1 (that is to say of order +1 or of order -1), and
- on envoie ce faisceau diffracté d'ordre +1 dans l'appareil optique, ce dernier ne recevant donc pas ce faisceau diffracté d'ordre +1 lorsque le modulateur acousto-optique n'est pas activé, ce modulateur acousto-optique formant ainsi un obturateur de faisceau vis-à-vis de l'appareil optique, caractérisé en ce que l'appareil optique est destiné à effectuer des mesures et l'on utilise le faisceau d'ordre 0 en tant que faisceau de référence pour corriger ces mesures. La source de lumière peut être un laser, le faisceau de lumière étant alors un faisceau laser.- this diffracted beam of order +1 is sent into the optical device, the latter therefore not receiving this diffracted beam of order +1 when the acousto-optical modulator is not activated, this acousto-optical modulator thus forming a beam shutter vis-à-vis the optical device, characterized in that the optical device is intended to carry out measurements and the beam of order 0 is used as reference beam to correct these measurements . The light source can be a laser, the light beam then being a laser beam.
La présente invention concerne aussi un dispositif de prélèvement d'une partie d'au moins un faisceau de lumière founi par une source de lumière, ce faisceau étant destiné à être utilisé dans un appareil optique, ce dispositif étant caractérisé en ce qu'il comprend :The present invention also relates to a device for removing a part of at least one beam of light provided by a light source, this beam being intended to be used in an optical device, this device being characterized in that it comprises :
- au moins un modulateur acousto-optique prévu pour recevoir ce faisceau de lumière, ce modulateur acousto-optique fournissant ainsi un faisceau d'ordre 0, et des moyens d'activation de ce modulateur acousto-optique pour qu'il fournisse aussi un faisceau diffracté d'ordre +1, l'appareil optique étant destiné à recevoir ce faisceau diffracté d'ordre +1 et ne recevant donc pas ce dernier lorsque le modulateur acousto-optique n'est pas activé, ce modulateur acousto-optique formant ainsi un obturateur de faisceau vis-à-vis de l'appareil optique, le dispositif comprenant en outre au moins un photodêtecteur prévu pour recevoir le faisceau d'ordre- At least one acousto-optical modulator intended to receive this beam of light, this acousto-optical modulator thus providing a beam of order 0, and means of activation of this acousto-optical modulator so that it also provides a beam +1 diffracted, the optical device being intended to receive this +1 diffracted beam and therefore not receiving the latter when the acousto-optical modulator is not activated, this acousto-optical modulator thus forming a beam shutter vis-à-vis the optical device, the device further comprising at least one photodetector adapted to receive the order beam
0, ce dernier formant un faisceau de référence, et pour fournir un signal représentatif de ce faisceau de référence, ce signal étant utilisé pour corriger des mesures susceptibles d'être effectuées au moyen de l'appareil optique.0, the latter forming a reference beam, and to provide a signal representative of this reference beam, this signal being used to correct measurements which can be carried out by means of the optical device.
Selon un mode de mise en œuvre particulier du dispositif objet de l'invention, l'appareil optique est un appareil d'analyse d'échantillons biologiques par fluorescence et le signal est utilisé pour corriger les mesures effectuées au moyen de cet appareil d'analyse.According to a particular embodiment of the device which is the subject of the invention, the optical device is an apparatus for analyzing biological samples by fluorescence and the signal is used to correct the measurements made using this analysis device.
Le dispositif objet de l'invention peut comprendre une pluralité de modulateurs acousto- optiques prévus pour recevoir respectivement une pluralité de faisceaux de lumière destinés à être utilisés dans l'appareil optique, ces modulateurs acousto-optiques formant ainsi une pluralité d'obturateurs de faisceau vis-à-vis de l'appareil optique.The device which is the subject of the invention may comprise a plurality of acousto-optical modulators designed to receive respectively a plurality of light beams intended for use in the optical apparatus, these acousto-optical modulators thus forming a plurality of beam shutters vis-à-vis the optical device.
De préférence, le dispositif objet de l'invention comprend une pluralité de modulateurs acousto-optiques, prévus pour recevoir respectivement une pluralité de faisceaux de lumière, et une pluralité de photodétecteurs respectivement associés à ces modulateurs acousto-optiques et prévus pour recevoir les faisceaux d'ordre 0 respectivement fournis par ces modulateurs acousto-optiques.Preferably, the device which is the subject of the invention comprises a plurality of acousto-optical modulators, provided for receiving respectively a plurality of light beams, and a plurality of photodetectors respectively associated with these acousto-optical modulators and provided for receiving the light beams. order 0 respectively provided by these acousto-optical modulators.
Chaque source de lumière utilisée avec le dispositif objet de l'invention peut être un laser, chaque faisceau de lumière étant alors un faisceau laser.Each light source used with the device which is the subject of the invention can be a laser, each light beam then being a laser beam.
BRÈVE DESCRIPTION DES DESSINS La présente invention sera mieux comprise à la lecture de la description d'exemples de réalisation donnés ci-après, à titre purement indicatif et nullement limitatif, en faisant référence aux dessins annexés sur lesquels : - la figure 1 est une vue schématique d'un mode de réalisation particulier du dispositif objet de 1 ' invention,BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be better understood on reading the description of exemplary embodiments given below, by way of purely indicative and in no way limitative, with reference to the appended drawings in which: FIG. 1 is a schematic view of a particular embodiment of the device which is the subject of the invention,
- la figure 2 illustre schématiquement les diverses tensions que l'on peut obtenir à la sortie du photodétecteur que comporte le dispositif de la figure- Figure 2 schematically illustrates the various voltages that can be obtained at the output of the photodetector that includes the device of Figure
1,1
- la figure 3 est une vue schématique d'un dispositif conforme à l'invention, que l'on utilise avec plusieurs lasers,FIG. 3 is a schematic view of a device according to the invention, which is used with several lasers,
- la figure 4 est une vue schématique d'un circuit de synchronisation qui est utilisable dans 1 ' invention,FIG. 4 is a schematic view of a synchronization circuit which can be used in the invention,
- la figure 5 est un chronogramme relatif à la synchronisation des images obtenues avec une caméra à transfert de lignes ou à transfert de trames dans un mode de réalisation particulier de l'invention, etFIG. 5 is a timing diagram relating to the synchronization of the images obtained with a line transfer or frame transfer camera in a particular embodiment of the invention, and
- la figure 6 est un chronogramme relatif à la synchronisation des acquisitions de signaux fournis par un photomultiplicateur, une photodiode à avalanche ou une photodiode en silicium, dans un mode de réalisation particulier de l'invention.FIG. 6 is a timing diagram relating to the synchronization of the acquisitions of signals supplied by a photomultiplier, an avalanche photodiode or a silicon photodiode, in a particular embodiment of the invention.
EXPOSE DETAILLE DE MODES DE REALISATION PARTICULIERS La figure "1 illustre schématiquement un dispositif conforme à l'invention, que l'on utilise avec un appareil optique A. Il s'agit d'un appareil d'analyse d'échantillons biologiques par microscopie à fluorescence. Cet appareil est destiné à l'observation d'échantillons 2 disposés dans les puits 4 d'une plaque de micro-titration 6 dont on voit le fond 8 ainsi que les parois 10 délimitant les puits.DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS FIG. 1 schematically illustrates a device according to the invention, which is used with an optical apparatus A. It is an apparatus for analyzing biological samples by microscopy at This device is intended for the observation of samples 2 placed in the wells 4 of a plate micro-titration 6 which we see the bottom 8 and the walls 10 defining the wells.
Les échantillons sont excités par un rayonnement 12 issu d'un laser 14 et fournissent un rayonnement de fluorescence 16 à la suite de cette excitation.The samples are excited by radiation 12 from a laser 14 and provide fluorescence radiation 16 as a result of this excitation.
Le fond de la plaque de micro-titration est transparent à la lumière issue du laser ainsi qu'à ce rayonnement de fluorescence. La lumière issue du laser est mise en forme grâce à une optique appropriée 18 puis filtrée grâce à un filtre approprié 20 après avoir traversé le dispositif conforme à l'invention D, sur lequel on reviendra par la suite, puis été réfléchie successivement par deux miroirs 21a et 21b.The bottom of the micro-titration plate is transparent to light from the laser as well as to this fluorescence radiation. The light from the laser is shaped using an appropriate optic 18 then filtered using an appropriate filter 20 after passing through the device according to the invention D, to which we will return later, then was successively reflected by two mirrors 21a and 21b.
La lumière ainsi filtrée est réfléchie, grâce à un miroir dichroique 22, en direction de l'objectif 24 du microscope. Elle traverse cet objectif 24 qui la focalise sur l'échantillon étudié. On précise que les échantillons sont étudiés les uns à la suite des autres, la plaque de micro- titration étant pour ce faire disposée sur des moyens de déplacement appropriés, non représentés.The light thus filtered is reflected, thanks to a dichroic mirror 22, in the direction of the objective 24 of the microscope. It crosses this objective 24 which focuses it on the sample studied. It is specified that the samples are studied one after the other, the micrititration plate being for this purpose arranged on suitable displacement means, not shown.
On voit aussi sur la figure 1 une caméra 26, par exemple une caméra CCD, qui est prévue pour capter l'image de l'échantillon étudié, grâce au rayonnement de fluorescence 16 émis par cet échantillon.FIG. 1 also shows a camera 26, for example a CCD camera, which is designed to capture the image of the sample studied, thanks to the fluorescence radiation 16 emitted by this sample.
Ce rayonnement de fluorescence 16 traverse également l'objectif 24 du microscope puis le miroir dichroique 22 (ce dernier étant apte à réfléchir le rayonnement 12 et transmettre le rayonnement 16) et parvient à la caméra 26 après avoir traversé un autre filtre 28 prévu pour éliminer la lumière du laser susceptible de parvenir également à cette caméra.This fluorescence radiation 16 also passes through the objective 24 of the microscope then the dichroic mirror 22 (the latter being able to reflect the radiation 12 and transmit the radiation 16) and reaches camera 26 after having passed through another filter 28 designed to eliminate the light from the laser capable of also reaching this camera.
Le dispositif D conforme à l'invention, qui est schématiquement représenté sur la figure 1, comprend un modulateur acousto-optique 30 qui est placé sur le trajet du faisceau issu du laser 14, à la suite de l'optique 18.The device D according to the invention, which is schematically represented in FIG. 1, comprises an acousto-optical modulator 30 which is placed on the path of the beam coming from the laser 14, following the optics 18.
Ce modulateur acousto-optique comprend une cellule acousto-optique 32 munie d'un transducteur 34This acousto-optical modulator comprises an acousto-optical cell 32 provided with a transducer 34
Le dispositif D comprend aussi un circuit de commande ("driver") 36 qui est relié au transducteur 34.The device D also includes a control circuit ("driver") 36 which is connected to the transducer 34.
A titre purement indicatif et nullement limitatif, on utilise le modulateur acousto-optique commercialisé par la société Automate et Automatisme sous la référence A A.M P.110/A1.5-vis .As a purely indicative and in no way limitative, use is made of the acousto-optical modulator marketed by the company Automate et Automatisme under the reference A A.M P.110 / A1.5-vis.
Lorsqu'on applique au modulateur acousto- optique un signal de tension et de fréquence appropriées (typiquement une fréquence de 20 MHz et une tension de 24 V) , ce modulateur acousto-optique diffracté le faisceau laser et fournit ainsi, dans l'exemple représenté, un faisceau diffracté 38 d'ordre +1 en plus du faisceau 40 d'ordre 0.When a signal of appropriate voltage and frequency (typically a frequency of 20 MHz and a voltage of 24 V) is applied to the acousto-optical modulator, this acousto-optical modulator diffracts the laser beam and thus provides, in the example shown , a diffracted beam 38 of order +1 in addition to the beam 40 of order 0.
On note θ l'angle d'incidence du faisceau laser, cet angle θ étant égal à 0,3° dans l'exemple. Le faisceau dans l'ordre +1 est dévié de l'angle 2θ par rapport à ce faisceau incident, tandis que le faisceau d'ordre 0 fait un angle nul avec ce faisceau incident.We note θ the angle of incidence of the laser beam, this angle θ being equal to 0.3 ° in the example. The beam in order +1 is deviated from the angle 2θ with respect to this incident beam, while the beam of order 0 makes a zero angle with this incident beam.
L'efficacité de diffraction représente le pourcentage d'énergie lumineuse qui est dévié. Ce pourcentage est de l'ordre de 80%. Cela signifie qu'il reste environ 20% d'énergie lumineuse dans le faisceau d' ordre 0.The diffraction efficiency represents the percentage of light energy that is deflected. This percentage is around 80%. That means it about 20% of light energy remains in the beam of order 0.
Lorsque le circuit de commande n'est pas activé, seul existe le faisceau d'ordre 0 (faisceau qui n'est pas dévié par rapport au faisceau laser incident) et, dans ce cas, la quasi-totalité de l'énergie lumineuse du faisceau laser incident se trouve dans ce faisceau d'ordre 0.When the control circuit is not activated, only the order 0 beam exists (beam which is not deflected with respect to the incident laser beam) and, in this case, almost all of the light energy of the incident laser beam is in this order 0 beam.
Typiquement, pour un faisceau laser dont le diamètre est de l'ordre de 1 mm à 2 mm et la puissance de l'ordre de 10 m , environ 8 m se retrouvent dans le faisceau diffracté d'ordre +1 et environ 2 mW subsistent dans le faisceau d'ordre 0.Typically, for a laser beam whose diameter is of the order of 1 mm to 2 mm and the power of the order of 10 m, approximately 8 m are found in the diffracted beam of order +1 and approximately 2 mW remain in the beam of order 0.
Comme on l'a vu, lorsque le circuit de commande ne fournit aucun signal au modulateur acousto-optique, quasiment toute 1 ' énergie lumineuse se trouve dans le faisceau d'ordre 0.As we have seen, when the control circuit does not supply any signal to the acousto-optic modulator, almost all of the light energy is in the beam of order 0.
Le circuit de commande 36 est simplement commandé par un signal TTL de 0 à 5V (figure 2) . Lorsque ce signal TTL est à 0V, tout le faisceau laser se trouve dans l'ordre 0 et, lorsqu'il est à 5V7 environ 80% de ce faisceau laser est dévié dans l'ordre +1.The control circuit 36 is simply controlled by a TTL signal from 0 to 5V (Figure 2). When this TTL signal is at 0V, the entire laser beam is in the 0 order and, when it is at 5V 7 about 80% of this laser beam is deflected in the +1 order.
Pour l'appareil optique A, le modulateur acousto-optique 30 joue le rôle d'un obturateur de faisceau, alors qu'il est traditionnellement utilisé en tant que modulateur de lumière.For the optical device A, the acousto-optical modulator 30 plays the role of a beam shutter, whereas it is traditionally used as a light modulator.
On voit aussi sur la figure 1 un photodêtecteur qui est par exemple constitué par une photodiode 42 et destiné à recevoir le faisceau dans l'ordre 0. Cette photodiode est donc susceptible de fournir trois tensions de sortie différentes (figure 2) :We also see in Figure 1 a photodetector which is for example constituted by a photodiode 42 and intended to receive the beam in order 0. This photodiode is therefore likely to provide three different output voltages (Figure 2):
1) 0V lorsqu'il n'y a pas de faisceau laser1) 0V when there is no laser beam
(laser arrêté) , 2) une tension VI lorsque le laser et le modulateur acousto-optique fonctionnent, environ 80% de l'énergie lumineuse se trouvant alors dans le faisceau diffracté d'ordre +1,(laser stopped), 2) a voltage VI when the laser and the acousto-optic modulator are operating, approximately 80% of the light energy then being in the diffracted beam of order +1,
3) une tension V2 supérieure à VI lorsque le laser fonctionne mais le modulateur acousto-optique est arrêté , quasiment toute l'énergie lumineuse se trouvant alors dans le faisceau d'ordre 0.3) a voltage V2 greater than VI when the laser is operating but the acousto-optical modulator is stopped, almost all the light energy then being in the beam of order 0.
Grâce au photodétecteur 42, on peut utiliser le faisceau dans l'ordre 0 en tant que faisceau de référence, par exemple pour surveiller l'état de fonctionnement du laser 14 ou pour corriger ce fonctionnement, en particulier pour corriger 1 ' intensité du faisceau émis par ce laser 14.Thanks to the photodetector 42, the beam can be used in order 0 as a reference beam, for example to monitor the operating state of the laser 14 or to correct this operation, in particular to correct the intensity of the emitted beam. by this laser 14.
On peut aussi utiliser ce faisceau de référence pour corriger les mesures effectuées au moyen de l'appareil d'analyse A, comme on le verra mieux par la suite.It is also possible to use this reference beam to correct the measurements made by means of the analysis apparatus A, as will be seen more clearly below.
On voit aussi sur la figure 1 des moyens électroniques de traitement 44 prévus pour traiter les images fournies par la caméra CCD 26 sous la forme de signaux électriques, ces moyens électroniques de traitement étant pourvus de moyens d'affichage 46 permettant de visualiser les images fournies par la caméra . En outre, les moyens électroniques de traitement 44 sont prévus pour fournir le signal TTL mentionné précédemment au circuit de commande 36.FIG. 1 also shows electronic processing means 44 provided for processing the images supplied by the CCD camera 26 in the form of electrical signals, these electronic processing means being provided with display means 46 making it possible to display the images supplied. by the camera. In addition, the electronic processing means 44 are provided to supply the TTL signal mentioned above to the control circuit 36.
On précise que la tension de sortie du photodétecteur 42 est envoyée à une carte d'acquisitionIt is specified that the output voltage of the photodetector 42 is sent to an acquisition card
(non représentée) d'un calculateur et que sa valeur est proportionnelle à l'intensité du faisceau laser incident .(not shown) of a computer and that its value is proportional to the intensity of the incident laser beam.
Le montage que l'on voit sur la figure 1 est extensible à deux ou plus de deux lasers, à condition de disposer d'un modulateur acousto-optique et d'un photodétecteur par laser.The assembly that can be seen in FIG. 1 can be extended to two or more than two lasers, provided that an acousto-optical modulator and a laser photodetector are available.
Ceci est schématiquement illustré par la figure 3 sur laquelle on voit un autre mode de réalisation particulier du dispositif objet de l'invention.This is schematically illustrated by FIG. 3 in which we see another particular embodiment of the device which is the subject of the invention.
Le dispositif qui est schématiquement représenté sur cette figure 3 comprend trois lasers 48, 50 et 52 prévus pour émettre des rayonnements d'excitation d'échantillons, ces lasers ayant des longueurs d'ondes différentes (par exemple 488 nm,The device which is schematically represented in this FIG. 3 comprises three lasers 48, 50 and 52 provided for emitting excitation radiation from samples, these lasers having different wavelengths (for example 488 nm,
532 nm et 633 nm) .532 nm and 633 nm).
Chacun de ces lasers 48, 50 ou 52 est successivement suivi par un ensemble optique 54, 56 ou 58 de mise en forme du faisceau fourni par ce laser, un miroir 60 , 62 ou 64 et un modulateur acousto-optique 66 , 68 ou 70, chaque miroir 60, 62 ou 64 étant prévu pour envoyer le faisceau- laser, qu'il reçoit par l'intermédiaire de l'ensemble optique 54, 56 ou 58, sur le modulateur acousto-optique correspondant, suivant un angle d'incidence approprié. Les modulateurs acousto-optiques 66, 68 et 70 sont respectivement commandés par des circuits de commande ( « drivers ») 72, 74 et 76.Each of these lasers 48, 50 or 52 is successively followed by an optical assembly 54, 56 or 58 for shaping the beam supplied by this laser, a mirror 60, 62 or 64 and an acousto-optical modulator 66, 68 or 70 , each mirror 60, 62 or 64 being designed to send the laser beam, which it receives via the optical assembly 54, 56 or 58, onto the corresponding acousto-optical modulator, at an angle of incidence appropriate. The acousto-optical modulators 66, 68 and 70 are respectively controlled by control circuits ("drivers") 72, 74 and 76.
On voit aussi sur la figure 3 un appareil d'analyse de fluorescence A destiné à être éclairé par l'un quelconque des faisceaux diffractés d'ordre +1 issus de modulateurs acousto-optiques 66, 68 et 70.FIG. 3 also shows a fluorescence analysis device A intended to be illuminated by any one of the +1 diffracted beams from acousto-optical modulators 66, 68 and 70.
Cet appareil A comporte une caméra CCD (non représentée) qui fournit des images sous la forme de signaux électriques. Ceux-ci sont traités par les moyens électroniques de traitement 78 qui sont pourvus de moyens d'affichage (non représentés) permettant de visualiser ces images.This apparatus A includes a CCD camera (not shown) which provides images in the form of electrical signals. These are processed by the electronic processing means 78 which are provided with display means (not shown) enabling these images to be viewed.
En outre, ces moyens électroniques de traitement sont prévus pour fournir, aux circuits de commande 72, 74 et 76, des signaux TTL du genre de ceux qui ont été mentionnés plus haut.In addition, these electronic processing means are provided to supply, to the control circuits 72, 74 and 76, TTL signals of the type which have been mentioned above.
On voit aussi sur cette figure 3 les faisceaux 80, 82 et 84 d'ordre 0, correspondant respectivement aux modulateurs acousto-optiques 66, 68 et 70, les faisceaux diffractés 86, 88 et 90 d'ordre +1, correspondant respectivement à ces modulateurs 66, 68 et 70, ainsi que les photodétecteurs 92, 94 et 96 respectivement prévus pour recevoir les faisceaux 80, 82 et 84 d'ordre 0.We also see on this figure 3 the beams 80, 82 and 84 of order 0, corresponding respectively to the acousto-optical modulators 66, 68 and 70, the diffracted beams 86, 88 and 90 of order +1, corresponding respectively to these modulators 66, 68 and 70, as well as the photodetectors 92, 94 and 96 respectively designed to receive the beams 80, 82 and 84 of order 0.
Les signaux électriques fournis par ces photodétecteurs 92, 94 et 96 sont envoyés à une carte d'acquisition (non représentée) d'un calculateur.The electrical signals supplied by these photodetectors 92, 94 and 96 are sent to an acquisition card (not shown) of a computer.
Il convient de noter que le chronogramme de la figure 2 est valable pour chacun de ces photodétecteurs . Les modulateurs acousto-optiques 66, 68 et 70 forment encore des obturateurs de faisceau vis-à vis de l'appareil optique A.It should be noted that the timing diagram in FIG. 2 is valid for each of these photodetectors. The acousto-optical modulators 66, 68 and 70 also form beam shutters vis-à-vis the optical device A.
Le dispositif de la figure 3 comprend aussi des miroirs 98, 100 et 102 qui sont associés respectivement aux modulateurs 66,68 et 70 et sont prévus pour envoyer à l'appareil A, par l'intermédiaire d'un autre miroir 104, le faisceau diffracté d'ordre +1 issu du modulateur acousto-optique que l'on a activé. Les dispositifs conformes à l'invention des figures 1 et 3 présentent divers avantages mentionnés ci-après .The device of FIG. 3 also includes mirrors 98, 100 and 102 which are associated respectively with modulators 66, 68 and 70 and are designed to send the beam, via another mirror 104, +1 diffracted from the acousto-optic modulator that was activated. The devices according to the invention of Figures 1 and 3 have various advantages mentioned below.
Chacun de ces dispositifs permet de contrôler chacun des faisceaux laser : avec chaque photodétecteur, on peut savoir si le laser correspondant est arrêté ou allumé et si le faisceau diffracté d'ordre 1 correspondant est émis ou non.Each of these devices makes it possible to control each of the laser beams: with each photodetector, it is possible to know whether the corresponding laser is stopped or switched on and whether the corresponding order 1 diffracted beam is emitted or not.
Le dispositif de la figure 3 permet une commutation rapide d'un faisceau laser à l'autre en contrôlant le modulateur qui est en fonctionnement : si par exemple le laser 48 est allumé et le modulateur 66 est activé, le photodétecteur 92 fournit la tension VI et si le photodétecteur 94 fournit la tension V2 c'est que le faisceau du laser 50 n'est pas envoyé à l'appareil optique A.The device in FIG. 3 allows rapid switching from one laser beam to the other by controlling the modulator which is in operation: if for example the laser 48 is on and the modulator 66 is activated, the photodetector 92 supplies the voltage VI and if the photodetector 94 supplies the voltage V2, the laser beam 50 is not sent to the optical device A.
De plus, le temps de commutation est très court : il suffit d'un temps d'environ 1 microseconde à 10 microsecondes pour passer du faisceau que l'on envoie à l'appareil optique à un autre faisceau. Les dispositifs des figures 1 et 3 garantissent la sécurité des utilisateurs vis-à-vis des lasers. Si par exemple un utilisateur intervient sur l'appareil optique A, on n'active alors aucun modulateur acousto- optique de sorte qu'aucun faisceau laser n'est envoyé à l'appareil optique. S'il se produit une panne électronique, les circuits de commande ne reçoivent aucun signal, il n'y a donc aucune déflexion de faisceau laser et aucun faisceau laser n'est donc envoyé vers l'appareil optique. De plus, si un laser est en panne, par exemple le laser 48, le photodétecteur correspondant 92 fournit une tension nulle, indiquant ainsi qu'il n'y a pas de faisceau laser.In addition, the switching time is very short: it takes just about 1 microsecond to 10 microseconds to pass from the beam that is sent to the optical device to another beam. The devices of FIGS. 1 and 3 guarantee the safety of the users with respect to the lasers. Yes for example a user intervenes on the optical device A, no acousto-optical modulator is then activated so that no laser beam is sent to the optical device. If an electronic failure occurs, the control circuits receive no signal, so there is no deflection of the laser beam and therefore no laser beam is sent to the optical device. In addition, if a laser is broken, for example the laser 48, the corresponding photodetector 92 supplies a zero voltage, thus indicating that there is no laser beam.
Les dispositifs des figures 1 et 3 permettent de contrôler la puissance laser comme on l'explique ci- après .The devices of FIGS. 1 and 3 make it possible to control the laser power as explained below.
Aucune source n'est stable à moins de quelque pour cent (typiquement 5%) .No source is stable at less than a few percent (typically 5%).
Il est délicat et coûteux de stabiliser une source de lumière, en particulier une source laser. La fluorescence de l'objet éclairé par un laser fluctue donc au même rythme que l'excitation laser c'est-à-dire 5%.It is delicate and expensive to stabilize a light source, in particular a laser source. The fluorescence of the object lit by a laser therefore fluctuates at the same rate as the laser excitation, that is to say 5%.
L'énergie lumineuse diffractée étant une fraction (environ 80%) de l'énergie lumineuse incidente, le signal fourni par chaque photodêtecteur suit strictement les fluctuations du laser correspondant. Les tensions VI et V2 suivent donc les fluctuations de ce laser. La connaissance de la tension VI permet donc de corriger les fluctuations de 1 ' image de fluorescence en temps réel, en surveillant, pour chaque acquisition d'image, la puissance laser reçue par le photodétecteur pendant la phase d'acquisition de l'image, ce qui nécessite un circuit de synchronisation commun à l'acquisition réelle de l'image et à l'acquisition d'un signal lumineux par le photodétecteur, comme le montre schématiquement la figure 4.Since the diffracted light energy is a fraction (approximately 80%) of the incident light energy, the signal supplied by each photodetector strictly follows the fluctuations of the corresponding laser. The voltages VI and V2 therefore follow the fluctuations of this laser. Knowledge of the voltage VI therefore makes it possible to correct the fluctuations of the fluorescence image in real time, by monitoring, for each image acquisition, the laser power received by the photodetector during the image acquisition phase, which requires a synchronization circuit common to the actual image acquisition and to the acquisition of a light signal by the photodetector, as shown schematically in Figure 4.
Sur cette figure 4, qui est relative au photodétecteur 42 de la figure 1, mais qui s'applique aussi à chaque photodétecteur de la figure 3, on voit un amplificateur 106 destiné à amplifier le signal électrique fourni par ce photodétecteur et un circuit de synchronisation 108 qui reçoit le signal ainsi amplifié et qui est prévu pour suivre les fluctuations de puissance du laser : on voit que le circuit de commande ("driver") 36 reçoit les impulsions S qui sont des impulsions de commande d'adressage du laser et sont fournies par le calculateur (non représenté) . La sortie du circuit de synchronisation 108 est reliée au calculateur.In this FIG. 4, which relates to the photodetector 42 of FIG. 1, but which also applies to each photodetector of FIG. 3, there is seen an amplifier 106 intended to amplify the electrical signal supplied by this photodetector and a synchronization circuit 108 which receives the signal thus amplified and which is intended to follow the power fluctuations of the laser: it can be seen that the control circuit ("driver") 36 receives the pulses S which are addressing pulses for addressing the laser and are supplied by the computer (not shown). The output of the synchronization circuit 108 is connected to the computer.
De plus, la rapidité de la commutation d'un faisceau laser par un modulateur acousto-optique permet d'utiliser, pour la prise d'image, des caméras à transfert de lignes ou à transfert de trames de telle sorte que, pendant la phase de lecture des charges et de numérisation du signal, on peut commencer une phase de cumul d'images ou de signaux comme le montre la figure 5.In addition, the speed of switching of a laser beam by an acousto-optic modulator makes it possible to use, for image capture, line transfer or frame transfer cameras so that, during the phase of reading the charges and digitizing the signal, it is possible to start a phase of accumulation of images or signals as shown in FIG. 5.
Le signal II indiquant que le laser est actif (adressé) permet de générer la synchronisation de la caméra (signal III) : la période T est ainsi la période entre image .Signal II indicating that the laser is active (addressed) generates the synchronization of the camera (signal III): the period T is thus the period between images.
Si l'on utilise un détecteur du type photodiode ou photomultiplicateur, on utilise le signal laser pour piloter un système PING/PONG (figure 6) , pour l'intégration du signal (ce signal étant fourni par chaque photodétecteur susceptible d'être utilisé dans 1 ' invention et consistant par exemple en le courant de sortie d'un photomultiplicateur ou d'une photodiode à avalanche ou d'une photodiode en silicium) ou un système formé par deux compteurs pour un comptage de photons .If a detector of the photodiode or photomultiplier type is used, the laser signal is used to drive a PING / PONG system (FIG. 6), for the integration of the signal (this signal being provided by each photodetector capable of being used in 1 invention and consisting for example of the output current of a photomultiplier or an avalanche photodiode or a silicon photodiode) or a system formed by two counters for counting photons.
Revenons aux figures 5 et 6.Let's go back to Figures 5 and 6.
La figure 5 est un chronogramme relatif à la synchronisation des images que l'on obtient avec une caméra CCD à transfert de lignes ou à transfert de trames .FIG. 5 is a timing diagram relating to the synchronization of the images which are obtained with a CCD camera for line transfer or for frame transfer.
Les lignes I à V de la figure 5 montrent respectivement les impulsions correspondant au cumul d'image (ligne I), les impulsions correspondant à l'état actif du laser données par la photodiode de référence (ligne II) , les synchronisations de transfert de ligne ou de trame (ligne III) , les impulsions relatives à la lecture des charges dans la caméra CCD (ligne IV) et le signal relatif à la numérisation et à la mémorisation des images ' (ligne V) , la période T que l'on voit sur la ligne V étant la période entre images.Lines I to V of FIG. 5 respectively show the pulses corresponding to the cumulative image (line I), the pulses corresponding to the active state of the laser given by the reference photodiode (line II), the synchronizations of transfer of line or frame (line III), the pulses relating to the reading of the charges in the CCD camera (line IV) and the signal relating to the digitization and storage of the images ' (line V), the period T that the we see on line V being the period between images.
La figure 6 est, quant à elle, un chronogramme relatif à la synchronisation des acquisitions de signaux founis par un photodétecteur tel qu'un photomultiplicateur ou une photodiode à avalanche ou une photodiode en silicium.FIG. 6 is, for its part, a timing diagram relating to the synchronization of the acquisitions of signals supplied by a photodetector such as a photomultiplier or an avalanche photodiode or a silicon photodiode.
Les lignes I à V de la figure 6 montrent respectivement les impulsions correspondant à l'état actif du laser au moyen duquel on obtient les images (ligne I) , les impulsions correspondant aux signaux fournis par le photodétecteurs considéré ci-dessus (ligne II) , le signal intégré PING (ligne III) , le signal intégré PONG (ligne IV) , et les impulsions correspondant à la numérisation alternée PING/PONG.Lines I to V of FIG. 6 respectively show the pulses corresponding to the active state of the laser by means of which the images are obtained (line I), the pulses corresponding to the signals supplied by the photodetectors considered above (line II) , the integrated PING signal (line III), the integrated PONG signal (line IV), and the pulses corresponding to the alternating PING / PONG digitization.
Bien entendu, il est possible d'utiliser, dans un dispositif conforme à l'invention, le faisceau diffracté d'ordre -1, au lieu du faisceau diffracté d'ordre +1, pour envoyer ce faisceau à un appareil optique A. Of course, it is possible to use, in a device according to the invention, the diffracted beam of order -1, instead of the diffracted beam of order +1, to send this beam to an optical device A.

Claims

REVENDICATIONS
1. Procédé de prélèvement d'une partie d'au moins un faisceau de lumière fourni par une source de lumière (14; 48, 50, 51), ce faisceau étant destiné à être utilisé dans un appareil optique (A) , dans lequel : on place au moins un modulateur acousto- optique (32; 54, 56, 58) sur le trajet du faisceau de lumière, ce modulateur fournissant ainsi un faisceau d'ordre 0 (40; 80, 82, 84),1. Method for sampling part of at least one beam of light supplied by a light source (14; 48, 50, 51), this beam being intended for use in an optical device (A), in which : at least one acousto-optical modulator (32; 54, 56, 58) is placed on the path of the light beam, this modulator thus providing a beam of order 0 (40; 80, 82, 84),
- on active ce modulateur acousto-optique pour qu'il fournisse aussi un faisceau d'ordre +1 (38; 86, 88, 90), et - on envoie ce faisceau d'ordre +1 dans l'appareil optique, ce dernier ne recevant donc pas ce faisceau diffracté d'ordre _+l lorsque le modulateur acousto-optique n'est pas activé, ce modulateur acousto-optique formant ainsi un obturateur de faisceau vis-à-vis de 1 ' appareil -optique, caractérisé en ce que l'appareil optique (A) est destiné à effectuer des mesures et l'on utilise le faisceau d'ordre 0 (40; 80, 82, 84) en tant que faisceau de référence pour corriger ces mesures. - this acousto-optical modulator is activated so that it also provides a +1 order beam (38; 86, 88, 90), and - this +1 order beam is sent to the optical device, the latter therefore not receiving this diffracted beam of order _ + 1 when the acousto-optical modulator is not activated, this acousto-optical modulator thus forming a beam shutter vis-à-vis the optical device, characterized in that the optical device (A) is intended to make measurements and the beam of order 0 (40; 80, 82, 84) is used as the reference beam to correct these measurements.
2. Procédé selon la revendication 1, dans lequel la source de lumière est un laser (14; 48, 50, 52) , le faisceau de lumière étant alors un faisceau laser.2. Method according to claim 1, in which the light source is a laser (14; 48, 50, 52), the light beam then being a laser beam.
3. Dispositif de prélèvement d'une partie d'au moins un faisceau de lumière fourni par une source de lumière (14; 48, 50, 52), ce faisceau étant destiné à être utilisé dans un appareil optique (A) , ce dispositif étant caractérisé en ce qu'il comprend :3. Device for sampling a part of at least one light beam supplied by a light source (14; 48, 50, 52), this beam being intended to be used in an optical device (A), this device being characterized in that it comprises:
- au moins un modulateur acousto-optique (32; 54, 56, 58) prévu pour recevoir ce faisceau de lumière, ce modulateur acousto-optique fournissant ainsi un faisceau d'ordre 0 (40; 80, 82, 84), et- at least one acousto-optical modulator (32; 54, 56, 58) provided to receive this beam of light, this acousto-optical modulator thus providing a beam of order 0 (40; 80, 82, 84), and
- des moyens (36; 72, 74, 76) d'activation de ce modulateur acousto-optique pour qu'il fournisse aussi un faisceau diffracté d'ordre +1 (38; 86, 88, 90), l'appareil optique étant destiné à recevoir ce faisceau diffracté d'ordre +1 et ne recevant donc pas ce dernier lorsque le modulateur acousto-optique n'est pas activé, ce modulateur acousto-optique formant ainsi un obturateur de faisceau vis-à-vis de l'appareil optique, le dispositif comprenant en outre au moins un photodétecteur (42; 92, 94, 96) prévu pour recevoir le faisceau d'ordre 0, ce dernier formant un faisceau de référence, et pour fournir un signal représentatif de ce faisceau de référence, ce signal étant utilisé pour corriger l'intensité du faisceau de lumière, ou pour corriger des mesures susceptibles d'être effectuées au moyen de l'appareil optique.- Means (36; 72, 74, 76) for activating this acousto-optical modulator so that it also provides a +1 diffracted beam (38; 86, 88, 90), the optical device being intended to receive this diffracted beam of order +1 and therefore not receiving the latter when the acousto-optical modulator is not activated, this acousto-optical modulator thus forming a beam shutter with respect to the apparatus optical, the device further comprising at least one photodetector (42; 92, 94, 96) designed to receive the beam of order 0, the latter forming a reference beam, and to supply a signal representative of this reference beam, this signal being used to correct the intensity of the light beam, or to correct measurements capable of being carried out by means of the optical device.
4. Dispositif selon la revendication 3, dans lequel l'appareil optique est un appareil d'analyse d'échantillons biologiques par fluorescence (A) et le signal est utilisé pour corriger les mesures effectuées au moyen de cet appareil d'analyse.4. Device according to claim 3, in which the optical apparatus is an apparatus for analyzing biological samples by fluorescence (A) and the signal is used to correct the measurements carried out by means of this analysis apparatus.
5. Dispositif selon l'une quelconque des revendications 3 et 4, comprenant une pluralité de modulateurs acousto-optiques (66, 68, 70) prévus pour recevoir respectivement une pluralité de faisceaux de lumière destinés à être utilisés dans l'appareil optique, ces modulateurs acousto-optique formant ainsi une pluralité d'obturateurs de faisceau vis-à-vis de l'appareil optique.5. Device according to any one of claims 3 and 4, comprising a plurality of acousto-optical modulators (66, 68, 70) provided for respectively receiving a plurality of light beams intended for use in the optical device, these acousto-optical modulators thus forming a plurality of beam shutters vis-à-vis the optical device.
6. Dispositif selon l'une quelconque des revendications 3 et 4 , comprenant une pluralité de modulateurs acousto-optiques (66, 68, 70), prévus pour recevoir respectivement une pluralité de faisceaux de lumière, et une pluralité de photodétecteurs (92, 94, 96) respectivement associés à ces modulateurs acousto- optiques et prévus pour recevoir les faisceaux d'ordre 0 (80, 82, 84) respectivement fournis par ces modulateurs acousto-optiques. 6. Device according to any one of claims 3 and 4, comprising a plurality of acousto-optical modulators (66, 68, 70), adapted to receive respectively a plurality of light beams, and a plurality of photodetectors (92, 94 , 96) respectively associated with these acousto-optical modulators and designed to receive the beams of order 0 (80, 82, 84) respectively supplied by these acousto-optical modulators.
7. Dispositif selon l'une quelconque des revendications 3 à 6, dans lequel chaque source de lumière est un laser (14; 48, 50, 52), chaque faisceau de lumière étant alors un faisceau laser. 7. Device according to any one of claims 3 to 6, wherein each light source is a laser (14; 48, 50, 52), each light beam then being a laser beam.
PCT/FR2002/003337 2001-10-04 2002-10-01 Method and device for sampling part of a light beam, particularly for a fluorescence analysis apparatus WO2003029882A2 (en)

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