WO2007009983A1

WO2007009983A1 - Method of calibrating a particle flux sorting device

Info

Publication number: WO2007009983A1
Application number: PCT/EP2006/064357
Authority: WO
Inventors: Vincent Haguet; Nathalie Picollet-D'hahan; François Chatelain; Philippe Rizo
Original assignee: Commissariat A L'energie Atomique
Priority date: 2005-07-19
Filing date: 2006-07-18
Publication date: 2007-01-25
Also published as: FR2888932A1

Abstract

The invention relates primarily to a method of calibrating a particle flux sorting device comprising an inlet channel, at least two outlet channels, a particle-sorting means and at least one particle-detection means which is disposed in an outlet channel. The inventive method comprises a step consisting in setting at least one of the main operating parameters of the sorting means, from among the deflecting force to be applied to the particle by the sorting means and/or the length of time for the which the force is to be applied. The invention also relates to a method of correcting the operating parameters of a particle flux sorting device during the operation thereof, consisting in checking the effectiveness of the sorting operation and modifying at least one operating parameter until effective sorting is obtained.

Description

METHOD FOR CALIBRATING A PARTICLE FLOW SORTING DEVICE

DESCRIPTION

TECHNICAL FIELD AND PRIOR ART

The invention relates to the sorting of suspended particles with a view to separating them into several populations and extracting the particles of interest. More specifically, the invention relates to a method for efficiently calibrating a device for sorting particles or particles suspended in a medium, in particular microparticles such as cells moving in a microfluidic channel, and autocalibration of this device in order to adapt in real time the operating parameters of the sorting means. Documents US Pat. No. 3,827,555 and US Pat. No. 3,984,307 disclose a microparticle flow sorting system comprising an input channel and two output channels, a sorting means close upstream of the branch line between the channel and the channel. input and output channels, close to it, and photoelectric or electrical particle detectors in each of the channels. The sorting means performs particle separation individually by directing each particle to the appropriate exit route. The detection means makes it possible to check whether the particles flowing in each output channel are those expected. However, these posterior controls are not used to modify the operating parameters of the device sorting in operation. Thus in case of error detection, it is necessary to stop the device, to calibrate it again before the sorting operation can be continued.

This document describes the adjustment of a parameter called "delay" for the actuation of the sorting means formed, for example, by a piezoelectric transducer, in case of variation thereof.

The downstream detector can also be used to calculate the particle velocity and deduce the exact moment to trigger the sorting means.

The development of the classifiers into cell streams has revealed the need to parameterize the sorting means effectively. For example, in the case of a fluorescently labeled cell sorter (Fluorescence-Activated CeIl Sorter, FACS), the time of fall of the cell between the laser beam used as a detector in the input channel upstream of the light source. sorting and metal plates used as sorting means, must be corrected in real time during the separation operation, to compensate for fluctuations in the speed of the cell between the detector and the sorting means resulting from temperature variations in the piece or the liquid. In the FACS, the correction parameter is the vibration amplitude of the piezoelectric ceramic integrated in the nozzle. This amplitude of vibration makes it possible to fix the point of splitting of the jet, thus the travel time of the cell between the laser beam and the metal plates. Two phases can be distinguished for FACS calibration:

before the sorting phase, the initial vibration amplitude of the nozzle is adjusted by deflecting the trajectory of balls of calibrated diameter,

during the sorting, the drift of the splitting point of the jet is corrected in real time by modulating the vibration amplitude of the nozzle. This continual adjustment can be done manually by the operator or on the basis of automatic image recognition.

In the FACS, the sorting means is automatically triggered after a certain time interval following a detection. This technique imposes to keep constant the falling time of the particle.

The calibration methods of the known sorting devices are not completely reliable, in fact the operating parameters on which the calibration is carried out do not allow an effective action in sorting the particles.

In addition, these methods do not make it possible to modify the operation of the sorting device in the event of error or variation of the conditions of use.

(change of the nature of the particles and / or their speed and / or the viscosity of the medium), adjusting in real time the operating parameters.

It is therefore an object of the present invention to establish the effective operating parameters of the sorting means and to provide a method of calibrating a sorting device based on these parameters. It is also an object of the present invention to provide an autocalibration method which makes it possible, during the detection of a sorting error, to adjust the operating parameters in order to obtain reliable sorting.

STATEMENT OF THE INVENTION

The previously stated goals are achieved by calibrating the deflection force exerted by the sorting means to orient the particle to a determined channel depending on the characteristics of the particle and / or by a calibration of the duration during which this force deviation is applied. This calibration is performed before the actual sorting step, using detection means placed in at least one of the output channels. The detection means then make it possible to modify the operating parameters if a particle is in a channel to which it was not intended.

Advantageously, the correction of the operating parameters continues throughout the operation of the sorting device. The parameters mentioned above can be adjusted during the entire sorting phase when it is detected that a particle has been mis-oriented, i.e., it is in the wrong channel. The operation of the particle sorting device is more flexible because it adapts to a change in the speed of the particles, the nature of the particles, the viscosity of the fluid, etc.

In other words, the present invention relates to a method for: - identify a posteriori the exit route taken by each particle after the branch, and

- In return, correct the operating parameters of the sorting means if the particles are not directed towards the correct exit route.

The characterization resulting from this detection means is used to correct, if necessary, the operating parameters of the sorting means. In the case of active sorting of the particles, that is to say in the case of an activation of the sorting means only when detecting a particle to be deflected, the activation of the sorting means may be triggered by an additional means of detection and analysis located upstream of the branch.

Applied to cell sorting, the present invention makes it possible to increase the reliability of the separation of the different cell populations. It is particularly suitable for operations of sorting large cell populations and high flow rate. The invention can also be implemented in parallel with a cell flow cytometry performed upstream of the sorting means and / or downstream.

The method according to the present invention can be applied during two periods of use:

- during the settings preceding the separation operation,

during the separation operation, the possible drift of the sorting means or the drifting of the conditions in which it operates can be quickly identified, which results in an immediate recalibration of the sorting means with the method described.

The autocalibration method according to the invention allows the sorting of particles suspended in a microfluidic or nanofluidic device. It makes it possible to control the triggering of an active sorting means during the passage of an individual particle, to control the operating parameters of the active sorting means or the operating parameters of a passive sorting means.

An active sorting means means a device initially at rest and which triggers when the particle passes in order to direct it to the appropriate exit route. Passive sorting means is a non-triggering device that continuously creates a disturbance in the particle path and separates them individually based on their intrinsic characteristics. The invention relates to a method for characterizing the position of particles between at least two output channels and to calibrate the operating parameters, which have been determined to be particularly effective for the sorting means (duration of one actuation of the sorting means and force of deviation of the sorting means). A detector placed downstream of the sorting means on at least one of the output channels makes it possible to identify the path taken by the particle after the separation operation. This characterization is used to determine the success or failure of the sort operation for each particle. The downstream analysis of the sorting means can be used at two levels of operation:

- when looking for the optimal parameters to carry out the deflection of the particles towards the appropriate exit routes,

during the sorting operation, to check the individual trajectory of each particle after the sorting means and, if necessary, to correct the operating parameters of the sorting means by the method described.

The self-calibration of the particle sorting means can be carried out manually by the operator, but it is also conceivable to automate this approach by means of an electronic circuit or software in order to have a very fast reaction of the device as soon as a single error appears.

Since the particles pass successively in the separating device, counts can be implemented during the settings of the apparatus and / or during the separation operation: enumeration of the total number of particles passing through the device, and / or enumeration of the number of successful sorts and / or enumeration of the number of failed sorts. It is also possible to use the downstream detectors to count the particles passing through each output channel.

The invention thus relates to the calibration and continuous regulation of an automated particle flow sorting means. The circulating fluid can be a liquid or a solution, or a suspension, or a medium containing biological cells, and / or components and / or cellular products, especially cell lines and / or globules, and / or liposomes, and / or cell nuclei, and / or chromosomes, and / or strands of DNA or RNA, and / or nucleotides, and / or ribosomes, and / or enzymes, and / or proteins, and / or proteins, and / or parasites and / or bacteria, and / or viruses, and / or pollens and / or polymers. The particles that are manipulated can also be solid insoluble particles in the liquid such as dielectric particles or conductive particles, or magnetic particles, or pigments, or dyes, or protein crystals, or powders, or polymers, or insoluble pharmaceutical substances, or small clusters formed by agglomeration of colloids. The particles handled can also be liquid droplets and / or gas bubbles confined and separated by another immiscible fluid with the droplets and / or the bubbles transported.

The autocalibration method according to the invention is compatible with all of the sorting means and detectors described in the literature.

The proposed solution therefore makes it possible to highlight the errors as soon as they occur and to correct the sorting means in real time accordingly. For an active sort, the sorting means correction parameters are of three types: delay before the trigger, duration of actuation and force of deviation. For passive sorting, the operating parameter is the deflection force.

The method of autocalibration of a particle sorting means is particularly interesting for manufacturers of instrumentation for biology, mainly to separate two cell populations and possibly in combination with flow cytometry.

The main subject of the present invention is therefore a method for calibrating a particle flow sorting device, said device comprising an inlet channel and at least two outlet channels, a particle sorting means and at least one means particle detection device arranged in an output channel, said method comprising the step of adjusting at least one of the main operating parameters of the sorting means among the deflection force adapted to be applied by the sorting means to said particle and / or the duration of application of said force.

The setting of the main parameter can include:

the choice of a parameter value,

- measuring the effectiveness of this value by checking the success of a sorting,

- changing the value of the parameter until at least one successful sort.

The calibration method according to the present invention advantageously comprises the additional step of determining a range of effective values for the main parameter. This determination of an effective value range may include, for example, the determination of the bounds of the interval from an effective value of the main parameter. For that :

- the rms value is changed,

the effectiveness of this modified value is tested,

the value is increased or reduced according to the result of the efficiency test.

The calibration method according to the present invention also applies to a device capable of sorting at least a first and a second population of particles of interest, the method according to a first variant may then also comprise the steps:

determining a first range of effective values of the main parameter for the first particle population,

determining a second range of effective values of the main parameter for the second particle population,

selecting primary parameter values for the first and second populations in the first and second intervals respectively.

In the case of a passive sorting device, the choice of a single value of the main parameter for the different populations can be made in a recovery interval between the first and the second intervals. The calibration method according to a second variant, for calibrating a device capable of sorting at least a first and a second population of particles of interest, can comprise the steps of: determining a first range of effective values of the main parameter for the first particle population,

determining an effective value of the main parameter for the second population in the first interval,

determining a second range of effective values for the second population in the first interval,

selecting the values of the main parameter for the first and second populations in the first and second intervals respectively.

In the case of a passive sorting device, a single value of the main parameter can be determined for the different populations in the second interval.

Advantageously, other operating parameters of the device can be set, such as the duration before trigger corresponding to the time elapsed between the detection of a particle in the input channel upstream of the sorting means and the passage of said particle at the level of the sorting means.

The duration before tripping can be obtained from the determination of the speed of displacement of a particle of interest in the sorting device. In this case, the sorting means may or may not be activated when determining the speed of the particle.

The velocity of the particle can be obtained from a detection signal provided by an upstream detection means, or a means for detecting an output channel and the distance between these two detectors.

The velocity of the particle can also be calculated from the duration of a detection peak provided by a detector in the input channel and / or by a detector in an output channel and the distance traveled by the particle between a beginning and an end of this peak. The determination of the duration before triggering can then comprise:

- the choice of a duration value before triggering, the values of the deflection force and the duration of activation being kept constant, - the test of the efficiency of this value,

- the modification of this value until a successful sorting.

The method according to the present invention advantageously comprises the step of determining an interval of effective values for the duration before tripping, in which from an effective tripping duration value, effective sorting tests are performed with values duration increased and / or decreased compared to the appropriate value. The present invention also relates to a method for correcting the operating parameters of an operating particle flow sorting device, comprising the steps of: - verifying the efficiency of the sorting,

modifying at least one operating parameter until at least one efficient sorting is achieved.

Verification of the efficiency of sorting can be done continuously or at predetermined time intervals.

Advantageously, a first parameter is modified until an efficient sort is obtained.

It may be provided to modify an additional parameter if effective sorting is not obtained.

The operating parameters that can be corrected by the method according to the present invention are at least the deflection force applied by the sorting means, the duration of application of this force and / or the time between the detection of the particle at input of the device and its passage at the level of the sorting means.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with the aid of the description which follows and the attached drawings, in which:

FIGS. 1a-Ib, 2a to 2h, 3a-3b, 4a-4b are diagrammatic representations of various particle flow sorting devices, FIGS. 5a to 5d, 6a to 6f, 7a to 7d represent different particle flow sorting devices with their associated chronograph showing the detection of a particle; FIGS. 8a to 8c, 9a to 9d and 10a to

1Od are chronographs showing the different steps in the search for the duration before tripping according to the present invention,

FIGS. 11a to 11f are chronographs showing the various steps in setting an interval of values of the actuation time for which the particle is correctly deflected,

FIGS. 12a to 12c are chronographs showing the various steps in setting an interval of values of the deflection force for which the particle is correctly deflected,

FIG. 13 is a flowchart of a calibration method according to the present invention,

FIGS. 14a to 14d, 15a to 15f and 16a to 16d show different particle flow sorting devices with their associated chronograph showing the detection of a particle for adjusting the deflection force,

FIGS. 17a to 17c and 18a to 18c are chronographs showing the different steps in the search for the deflection force according to the present invention; FIG. 19 is a flowchart of a calibration method according to the present invention; FIGS. 20 and 21 are flowcharts of the method according to the present invention in the case where several populations are of interest,

FIG. 22 is a schematic representation of a cell dispenser capable of being calibrated by a method according to the present invention,

FIG. 23 is a flowchart of a self-calibration process according to the present invention,

FIG. 24 is a flowchart for a particular step of the autocalibration process of FIG. 23.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

In FIGS. 1a and 1b, it is possible to see a known type of particle flow sorting device comprising an inlet channel 2 in which the charged flow of the particles to be sorted flows, two channels (FIG. '(three output channels in Fig. 1b) receiving each of the determined particles. The device also comprises a detector 6, 6 'of particles in each outlet channel 4, 4'. We will see later that the device may have only one output channel provided with a particle detector.

In the example shown, the input channel 2 also comprises a particle detector

8, which can be used to analyze them individually.

A sorting means 10 is also provided in the inlet channel upstream of the outlet channels 4,4 'in the flow direction indicated by the arrow A The number of output channels is not limited to three, it can be four or more.

There is a sort of so-called active particles; a detector 8 located upstream of the sorting means makes it possible to detect and analyze the particles one by one and to control the sorting means. These means are configured or programmed to detect certain particles or cells. The sorting means is thus triggered as a result of the upstream detection, possibly according to the result of a flow cytometry operation.

During their journey, the particles are detected in flux a first time by the detector upstream of the sorting means, and a second time by the detector downstream of the sorting means in one of the output channels if this path is equipped with a detector. The distance between the upstream detector and the downstream detector being precisely known, the average speed of the particles in the stream can be calculated. The time required for a particle to travel between the upstream detector and the sorting means can then be deduced.

In one configuration, the detection of a particle upstream of the sorting means can trigger a clock on the electronic circuit driving the detectors downstream to characterize the passage of the particle in a predefined time interval. In another configuration, the downstream detectors are still operational, and the downstream detection signal is associated by the electronic circuit with the most recent and / or most probable upstream detection signal. There is also a sort of so-called passive particles. The sorting means continuously exerts a deflection force to passively deflect the particles to the appropriate exit route. The particles all undergo the same deflection force and are separated individually on the basis of their intrinsic properties or their associated properties by labeling. The calibration of the sorting means is entirely controlled by the downstream detection of the particles. The identification of each particle by a downstream detector makes it possible to validate the success or failure of the sorting operation.

The method described in this invention is independent of the number of output channels. In the case of active sorting, the operating parameters of the sorting means may be different for each output channel and are therefore established independently for each of them by applying the method described. On the contrary, for active sorting, the operating parameters of the sorting means are common to all the output channels and are therefore established simultaneously by applying the method described.

A given detector is continuously excited by a source whose nature is related to the type of detector used. The source is for example a light source for optical detection, or an electrode for impedance detection of the particles. The passage of a particle between the source and the detector causes a temporary change in the measured signal. The shape of the detection signal may possibly give information on the particle.

The detectors can be of different types, for example they can be optical detectors: the optical detection of the particles uses one or more photodetectors to measure the optical characteristics indicating the passage of an individual particle, such as diffusion or diffraction or the degree of extinction of the light beam, or the light emitted by the particle by fluorescence, autofluorescence (intrinsic fluorescence) or chemiluminescence.

The optical components, generally assembled or constructed in the substrate for supporting the fluidic channels, may be optical fibers, monolithic waveguides or planar photodetectors such as photomultipliers, avalanche detectors, CMOS sensors ( complementary metal oxide semi-conductor), CCD sensors (charge-coupled device). In one configuration, the trajectory of the particles is determined by at least one optical sensor positioned near the input channel and / or at least one output channel.

In another configuration, a planar photodetector is placed near the fluidic channels in such a way that it allows simultaneous observation of the particles in at least two output channels and possibly in the input channel. The spatial resolution of the photo-detector allows to detect and possibly characterize the particles in the fluidic channels. The same planar photo-detector makes it possible to analyze several output channels, and possibly the input channel, by defining different zones on the planar photo detector, each of these zones being dedicated to a particular channel. The pixels receiving an optical signal coming from the output or input channels make it possible to determine the presence or absence of the particles in the channels concerned and thus to implement the calibration method described in this patent. The pixels of the planar photodetector placed near the exit channels are used to verify that the path taken by the particle is the appropriate one. The pixels of the planar photodetector placed near the inlet channel make it possible to characterize the particles upstream of the sorting means, for example with a view to active sorting of the particles.

It can also be an electrical detector, the detection and characterization by impedancemetry of particles or unmarked living cells is based on the variation of the impedance measured between two electrodes during the passage of the particle. The electrical disturbance may be due to the difference in conductivity between the particle and the carrier fluid or intrinsic electrical properties of the particle.

Sensors with thermal, mechanical, magnetic, electrochemical, etc. operation may also be suitable. We will now explain the operation of such a device.

A charged stream of particles to be sorted is injected into the inlet channel 2. The detector 8 identifies one or more determined particles, which must be sent into the outlet channel 4.

The information is sent to a control unit (not shown) which generates an activation command by means of sorting. As previously described, a delay or duration before triggering is generally provided before the activation of the sorting means corresponding to the travel time of the particle or particles.

The sorting means then activates at the moment of the passage of the determined particle or particles and causes a deviation of the trajectory of the particle or particles to direct them towards the channel 4.

Sorting means have been developed for the flow sorting of individual cells or particles in a microfluidic chip.

The sorting of the microparticles can be passive type, that is to say according to physical criteria inherent or associated with the microparticle.

It can also be of the active type, and implemented after observation of the microparticles through a characterization system upstream of the sorting means.

Various sorting means implementing active or passive sorting are recalled here, for example: means using physical parameters, such as mass (deviation from trajectory by gravitation), the size (deflection by filtration) or the density (deviation by centrifugal force) used to separate a heterogeneous suspension of microparticles, - means using a separation of cells according to their size thanks to the parabolic profile of the velocities of a fluid moving between two plates, means described in US 5,039,426; - sorting means using the immunomagnetic flow separation described in WO 03/045565. This separation is based on the labeling of the cells of interest with an antibody coupled to a superparamagnetic nanoparticle. In the presence of a magnetic field, the marked cells are driven to a specific output channel.

means using dielectrophoresis, as described by S. Fiedler et al. (Anal Chem, 70, 1909-1915, 1998), a high frequency electric field is applied to direct the selected particles to one of the output paths.

- Dielectric particles of different size and refractive index can be separated into streams by optical means by distributing the optical field in a suitable geometry (M. P. MacDOnald et al., Nature, 426, 421-424, 2003).

The trajectory of the suspended particle in a laminar flow can also be controlled with optical tweezers (eg, Wang et al., Nat Biotechnol., 23 (1), 83-87,2005). - N. Sundararajan et al. (Chip Lab, 5, 350-354, 2005) have presented a pneumatic pressure microparticle sorting means based on microchannels made of a deformable plastic material. A transient change in the direction of the fluid carrying the microparticles is produced by controlling the diameter of the different output channels.

The use of a pressure wave to deflect the trajectory of a particle towards the appropriate exit route is particularly suitable for particles suspended in a liquid. The pressure wave can be generated by various means which comprise a piezoelectric, acoustic, electromechanical, pneumatic or thermal actuation, by means of a piezoelectric material, an electroacoustic transducer, a mechanical actuator, a piston or a solenoid valve, or a heating resistor. This list is not exhaustive, and any other appropriate means may be appropriate.

Figures 2a to 2h show the case where one or more output channels 4,4 'do not have detector 6,6' of particles downstream. These configurations can be used in two situations:

when the deviation of the particle towards the output path without a detector is deduced from the absence of a signal coming from the detectors situated on the other output channels, when only the output channels equipped with detectors constitute the channels of interest for the characterization of the sorting operation. It is therefore not considered necessary to control the presence of the particles directed towards the output channels without a detector.

In the devices of Figures 2a and 2b, the input channel 2 comprises a detector 8 and only one of the two channels 4,4 'has a detector 6,6'. The devices of FIGS. 2c to 2e comprise a detector 8 in the input channel 2 and three output channels 4, 4 ', 4' '. Two of the three channels comprise a detector 6, 6 ', 6' '.

The devices of FIGS. 2f to 2h also comprise three channels 4, 4 ', 4' ', a detector in the input channel 2 and a single output channel 4, 4', 4 '' provided with a detector 6, 6 ', 6' '.

Figures 3a and 3b show the case where the input channel does not have a particle detector and all the output channels comprise a detector. Only two cases are shown in Figure 3, but the number of output channels may be greater.

FIGS. 4a and 4b show the case where the input channel 2 does not have a detector and where one of the output channels 4, 4 ', 4''does not have a detector. Only two cases are shown in Figure 4. Here again, the number of output channels may be greater. The configurations represented by FIGS. 1 and 2 (presence of an upstream detector 8) can be applied indifferently to an active or passive sorting means, while the configurations shown in Figures 3 and 4 (absence of an upstream detector) are implemented only with a passive sorting means.

This sorting device works satisfactorily, however it is desirable to further increase its reliability.

The inventors have determined two operating parameters of the sorting means, the adjustment of which is particularly effective for improving the reliability of the sorting, that is to say for causing the separations of the particles of interest from the other particles contained in the stream. Indeed, the inventors have found that the intensity of the force with which the particle or particles were deviated and the duration of application of this force allowed to substantially increase the reliability of the sorting device. The calibration method according to the present invention therefore comprises the step of adjusting the intensity of the deflection force applied by the sorting means and / or the pulse duration during which this force is applied. We will now expose the calibration method of the deflection force according to the present invention.

For calibration, it is possible to circulate a flow of standard particles in the device or particles to be sorted thereafter. The separation device shown in FIG. 14b has no detector 8 upstream of the sorting means. In this case, the downstream detectors 6, 6 'are still operational (passive operation). As shown in Figure 14d, a detector

8 may optionally be present upstream of the sorting means, but this detector 8 is not used to trigger the sorting means 10. In one configuration, the upstream detector 8 can trigger a clock on the electronic circuit controlling the detectors 6, 6 'downstream to characterize the passage of the particle in a predefined time interval. In another configuration, the detectors 6, 6 'downstream are still operational, and the downstream detection signal is associated by the electronic circuit with the most recent and / or most probable upstream detection signal.

In the remainder of the description, the shape of the signal of detection of a particle corresponding to a disturbance of the signal measured due to the passage of a particle between the flux source and the detector is represented on the diagrammatic diagrams schematically by a signal in niche.

Figures 14a and 14c show typical timing charts for devices 14b, 14d respectively, in which:

mt represents the force applied on the sorting means 10,

dl is the signal measured by the detector 6 downstream in the output channel 4, d2 is the signal measured by the detector 6 'downstream in the output channel 4',

dO represents the signal measured by the detector 8 upstream, if it is present in the separation device.

The detection peaks pθ, pi, p2 on the diagrams d0, d1 or d2 respectively, of FIGS. 14a and 14c represent the passage of a particle in front of the detectors 8, 6, 6 'in FIGS. 14b and 14d. When the particle is deflected by the sorting means 10 towards a certain output channel 4, 4 ', the detector 6, 6' of this output channel 4, 4 'only produces a detection peak pi, p2. On the timing diagrams, the amplitude of the detection peak p0, pi, p2 is arbitrary, while the width of the detection peak indicates the time of presence of the particle in front of the detector. The shape of the detection peaks is simplified by a square-wave signal, but the actual shape of the detection signal may possibly provide information on the particle according to the type of detector employed.

The base of the signal mt, that is to say the force applied when the sorting means is at rest, may correspond to a null or non-zero deflection force, according to the sorting means used. The base of the signals d0, d1 and d2 may correspond to a zero or non-zero measured signal, depending on the detectors employed and the environment in which they are used.

The operation of the sorting means is characterized by the amplitude F of the deflection force of the sorting means 10. On the chronograms representing the signal mt, the dotted lower line indicates the base of the signal mt, that is to say the deflection force applied when the sorting means 10 is at rest. The upper line in solid line indicates the force applied on the sorting means 10 during its use.

The number of output channels 4, 4 'is not limited: there may be as many signals as possible output channels 4, 4' for the particles. Subsequently, we describe the case where the sorting device is equipped with two output channels 4, 4 ', but the method applies to a greater number of output channels with similar reasoning.

FIGS. 15a to 15f show how the reading of the signals of the downstream detectors informs about the success or failure of the sorting of the particle. In this example, it is desired that the particle be directed by the sorting means to the output channel 4 provided with the detector 6. The device of FIGS. 15b, 15d, 15f comprise two output channels 4, 4 ', both of which are provided with a detector 6, 6 'and the input channel 2 also comprises a detector 8.

Three cases are represented: the timing diagram of FIG. 15a, associated with the device of FIG. 15b, represents the case where the deflection force of the sorting means 10 is insufficient. The detection peak p2 on the diagram d2 shows that the particle has taken the wrong output channel 4 ', - the timing diagram of FIG. 15c, associated with the device of FIG. 15d represents the case where the Deviation force is appropriate. The sorting of the particle is successful, because the detection peak p1 on the diagram d1 shows that the particle has taken the right exit channel 4, - the chronogram of FIG. 15e, associated with the device of FIG. 15f represents the case where the deviation force is too important. The detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit channel 4 '. Figures 16a to 16d show the operation of a device whose output channel 4 'has no detector. The direction taken by the particle is deduced by the presence or absence of a detection peak pi in the output path 4 provided with a detector 6.

Two cases are represented:

on the timing diagram of FIG. 16a associated with the device of FIG. 16b, the detection peak pi on the diagram d1 shows that the particle has taken the exit route 4,

on the timing diagram of FIG. 16c, associated with the device of FIG. 16d, the absence of a detection peak pi on the diagram d1 shows that the particle has taken the output channel 4 '. It may be desirable for the particle to be directed by the sorting means towards an output channel provided with a detector or, on the contrary, to take a non-detector output path. When the device has an upstream detector and at least one detectorless output channel, the sorting of the particle is considered successful or failed. deducing its trajectory from the observations of the output channels equipped with detector.

Preferably, a detector 8 is present upstream of the sorting means 10 when at least one of the output channels 4,4 'is not provided with a detector. If the particle is directed towards the exit channel without detector, the upstream detector makes it possible to highlight the passage of the particle in the device, for example for counting purposes. In another configuration, the sorting device does not have an upstream detector. In this case, a particle passing in front of the sorting means 10 and then directed towards a detector-free output channel produces no signal during its passage in the sorting device. This configuration can be used when only the particles oriented towards the detector-equipped output channels are of interest for the sorting operation.

Figs. 16a to 16d show simplified sorting with two output channels, but it can also be applied to a larger number of output channels. If at least two output channels do not have a detector, these output channels can not be distinguished by the method described here. We shall describe later the case where the sorting device is equipped only with two output channels each provided with a detector, but the calibration method applies to devices provided with a greater number of output channels, provided with or not a detector. FIGS. 17a to 17c show a method of calibrating or detecting a deflection force allowing sorting of a population of particles towards a given output channel. In this example, it is desired that the particles of interest are directed by the sorting means to the output channel 4 provided with the detector 6. The particles that must be directed to another output channel are not affected by this operation of calibration. At the beginning of the adjustment, the sorting means is actuated with an arbitrarily low arbitrary deflection force F, as shown in FIG. 17a. It is estimated that the situation is that described in Figures 15a and 15b, that is to say that the deflection force is insufficient. The detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit channel 4 '. Then, the amplitude of the deflection force is increased by an arbitrary value. The progression of the force F depends on the sorting means used. Generally, the amplitude added to F is between 10% and 30% of the initial value of F. After each progression, we observe the exit path taken by the particle.

Figure 17b shows the case where the particle is again oriented towards the wrong exit way and where it is therefore necessary to further increase the force F.

Figure 17c shows the case where the deflection force F is adequate. The peak of detection pi on the diagram dl shows that the particle has borrowed the right exit channel 4 and therefore that the force F is suitable for sorting towards this exit route.

The method can also be applied starting from an arbitrarily strong deflection force F. It is estimated that the situation is that described in Figures 15e and 15f, that is, the deflection force is too great. The progression used to search for a deflection force F consists of removing an arbitrary fraction of its amplitude at F, for example between 10% and 30% of its initial value. After each progression, we observe the exit route taken by the particle. This operation stops when the detection peak pi on the diagram d1 shows that the particle has borrowed the right output channel 4 and therefore that the force F is suitable for sorting towards this output channel.

At the beginning of the adjustment, the sorting means 10 is actuated with an arbitrarily selected deflection force. This force F can be weak or strong depending on the direction one wishes to perform for progression. If the sorting of the particle is successful from the beginning with this arbitrary force, ie if the peak of detection pi on the diagram d1 shows that the particle has borrowed the right way of exit 4 with the force F chosen initially, it is not necessary to apply the progression.

The direction of the progression is chosen arbitrarily. However, it may be necessary to reverse the direction of progression after a predetermined number of progressions if a force F allowing the sorting of the particles was not found by reducing the step of further progression.

It can also be expected to determine a range of values of F, shown in Figures 18a to 18c, for which the particle is diverted to the correct output path. This search will be called fine tuning. Particles that need to be directed to another output path are not affected by this calibration operation. At the beginning of the adjustment (FIG. 18a), the detection peak pi on the diagram d1 shows that the particle has taken the right exit channel 4. The situation is that described in FIGS. 15c and 15d, that is to say that the deflection force of the sorting means is appropriate. Then, the deflection force of the sorting means is decreased by an arbitrary value. The magnitude of the reduction of the force F depends on the sorting means employed. Generally, the amplitude removed at F is between 3% and 10% of the initial value of F. After each reduction of the deflection force, the exit path taken by the particle is observed. Figure 18b shows the case where the particle is always oriented towards the correct output path. The force F can therefore be further reduced. Figure 18c shows the case where the force F is no longer adequate because the detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit path.

The method can also be applied by increasing the deflection force F of the sorting means, that is to say by adding to the force F an arbitrary fraction of its amplitude, for example between 3% and 10% of the initial value of the force F. After each progression, we observe the exit path taken by the particle. This operation stops when the detection peak p2 on the d2 diagram shows that the particle has taken the wrong exit path.

Preferably, the progression operation of F is performed successively in both directions of progression. In order to determine the two ends of the range of the possible forces F, a progression is applied in one direction until the end of the force gap is known, then a progression is applied in the other direction until the other end of the interval is known. This interval corresponds to the deflection forces that can be exerted to direct a given type of particles to the proper exit route.

The flowchart of FIG. 19 represents the different steps of the method of calibrating a passive sorting means when the fluid contains only a single particle population.

This method makes it possible to determine the deflection force of the sorting means for directing the single population of particles to the appropriate output pathway among several possible output paths. At each stage of the calibration, the decisions can be made by the operator, and / or by an electronic circuit and / or software, in particular the choice of the deflection force F in the identified force interval. In an automatic embodiment, the sorting means, the upstream and downstream detectors are connected an electronic circuit which receives the information from the detectors and modifies the operating parameters of the sorting means on the basis of this information. The sorting means is operated continuously. In step 100, the success or failure of the deflection of a particle is detected by the downstream detector.

If the particle has taken the right exit route, the calibration remains unchanged.

On the other hand, if the particle has taken an erroneous output path, a new calibration of the deflection force F, represented by FIG. 17, is implemented (step 102). This can be done as soon as a single failure is observed or after a predetermined number of incorrect sorts. If a fine adjustment of the deflection force is decided in step 104, this is done in step 108. The accuracy to be applied in the adjustment of the deflection force is defined by the operator, and / or the electronic circuit and / or the software controlling the sorting means. The number of fine adjustment loops can be adjusted during settings. It may also depend on the sorting means used for the separation operation.

If a fine adjustment is not desired, the calibration is complete (step 106).

The method according to the present invention also provides for modifying the settings of the operating parameters of the sorting device, when it is noted a drift of the sorting means or conditions in which it operates (for example, a variation in the speed of the particles or a modification of the nature of the particles). The detectors placed in the output channels can detect such a drift that is to say the presence of particles in the wrong way. This will be described further in the description.

In step 110, it is checked whether the fine adjustment of the deflection force has been successful. If so, we return to step 104. Otherwise, we return to step 102 to search for the deflection force. The flow charts of FIGS. 20 and 21 represent the various steps of the method of calibrating a passive sorting means in the case where the fluid contains several populations of particles of interest. The deflection force of the sorting means is then calibrated for each particle population so that each particle type is directed to the appropriate exit route. In a passive sorting device, the force F applied by the sorting means is identical for all the particles, a deflection force common to all the force intervals is then selected.

In an active sorting device, it is possible to modify the deflection force as a function of the particles detected at the inlet of the channel 2. The method represented by FIG. 20 identifies a force interval for each population of particles. This search is carried out successively for each population. In case of drift, a new search for the deflection force F is carried out for the population concerned, and if necessary for all the particles. Once force intervals have been identified for all particle populations, a deflection force F is selected from the intersection of the different force intervals. The sorting means is operated continuously. In step 200, the success or failure of the deviation of a particle is detected by the downstream detector

If the particle has taken the right exit route, the calibration remains unchanged. The verification of the success of the deviation is continued.

On the other hand, if the particle has borrowed an erroneous exit route, a new calibration of the deflection force F is implemented (step 202). This can be done as soon as a single failure is observed or after a predetermined number of incorrect sorts.

In step 204, when this deflection force has been found, a value range of F is sought which is capable of correctly deflecting the first particle population

In step 206, if the setting has not been successful, return to step 202.

If the determination of the interval is a success, we go to step 208, during which we search for a deflection force capable of correctly deflecting the particles of the second population. An index i takes the value 2.

In step 210, a value range of F is sought which is capable of correctly deflecting the second particle population Then, in step 212, if this determination fails, return to step 208, otherwise proceed to step 214.

In step 214, it is checked whether there is a third population of particles of interest.

The number of populations of interest is generally known before sorting by the operator, since the operator wishes to extract circulating fluid from certain predetermined types of particles. In the case where the number of populations is not known, it can be determined by a flow cytometry operation using an external device, or with the upstream detector 8, or with the downstream detectors 6 , 6 '. If there is another population, the index i is incremented by 1 and returns to step 208.

If there is no other population, a value of the common deviation force at the two intervals previously determined in steps 204, 210 is chosen at step 216.

In step 218, the calibration ends.

As previously, the method according to the present invention can also provide for modifying the settings of the operating parameters of the sorting device, when it is noted a drift of the sorting means or conditions in which it operates

(for example, a change in particle velocity or a change in the nature of the particles). The detectors placed in the output channels make it possible to detect such a drift, that is, the presence of particles in the wrong way. This will be described further in the description.

Thus the method described in relation to the flow chart 20 can be applied continuously during the whole sorting, and not only before sorting.

In the case of an active sorting device, it is not necessary to determine a common value for the deflection force (step 216), but a particular deflection force value can be determined for each of the populations. The setting of the sorting means is then adapted to each particle.

The process steps shown in the flowchart of FIG. 21 also make it possible to determine a common deflection force for several particle populations. For this, this method provides for narrowing the force interval by successively analyzing the trajectory of each particle population. First, a force interval is identified for a first population of particles, the other populations being ignored during this calibration operation. Then, the trajectory followed by the second particle population is also observed. The deflection force in the first force interval is varied from one of its ends until the force F also makes it possible to direct the second population of particles to the corresponding output path. Then, the force interval for the second population is sought by modifying the force F and identified within the limits of the first force interval. Alone the common part at both intervals of force is preserved. This operation is carried out for each population of particles, which makes it possible to reduce each time the width of the force interval. Finally, a deflection force F is chosen in the final force range.

The first phase 300.1 grouping the steps 300, 302, 304 and 306 is identical to the first step 200.1 of the method of FIG. 20. When the force interval I for the first population has been determined, we go on to step 308, an index i takes the value 2. A force is then sought to successfully deflect the particles of the second population within the interval I. When this force has been found in step 308 a range of values II for the second population within the interval I is determined in step 310.

In step 310, it is checked whether this interval II has been found.

If the interval II has not been found, it returns to step 308. If the determination of the interval II fails a predetermined number of times, it returns to the step 302, during which a force is determined. deviation for the first population.

If the determination of the interval II has been successful, proceed to step 314, in which it is checked whether there is another population of interest. If so, i is incremented by 1 and returns to step 308. Otherwise we go to step 316, in which we choose a value of F in the final interval, which corresponds when the flow comprises two populations, in the interval II. Then the calibration ends at the stage

320.

As for the processes represented by the flow charts 19 and 20, it is possible to provide continuous self-calibration of the sorting device, by continuously applying, for the duration of the sorting, the steps 300 to 320 previously described, so as to correct the drifts of the device .

The calibration method according to the invention can also be used to adjust the duration of application of the deflection force.

The adjustment is carried out in the same way as for the deflection force, so that the processes represented by the flowcharts of FIGS. 19, 20 and 21 apply in a similar manner. The calibration method according to the present invention may also comprise the step of adjusting at least one other operating parameter such as the time elapsed between the moment of detection by the detector upstream of the sorting means of the one or more particles of interest and the moment or the particle or particles reach the sorting means, called the duration before triggering.

FIG. 5a shows a typical chronogram, in which: the signal d0 represents the signal measured by the detector 8 upstream, mt represents the force applied on the sorting means 10,

d1 is the signal measured by the downstream detector 6 in the output channel 4, d2 is the signal measured by the downstream detector 6 'in the output channel 4'.

The detection peaks pθ, pi, p2 on the diagrams d0, d1 or d2 represent the passage of a particle in front of the detectors 8, 6, 6 'of the device of FIG. 5b respectively.

When the particle is diverted by the sorting means to a certain output channel, it produces a detection peak in front of the detector of that output channel only. In the timing diagrams, the amplitude of the detection peak is arbitrary, while the width of the detection peak indicates the time of presence of the particle in front of the detector. The shape of the detection peaks is simplified by a square-wave signal, but the actual shape of the detection signal may possibly provide information on the particle according to the type of detector employed.

The base of the signal mt, that is to say the force applied when the sorting means 10 is at rest, may correspond to a null or non-zero deflection force, according to the sorting means employed. The base of the signals d0, d1 and d2 may correspond to a zero or non-zero measured signal, depending on the detectors employed and the environment in which they are used.

The operation of the sorting means is characterized by three parameters: the duration of time tA represents the duration before the triggering of the sorting means. This duration is taken arbitrarily from the beginning of the detection peak pθ of the signal dO until the start of the triggering of the sorting means 10,

the duration of time tB represents the duration of the actuation or pulse duration of the sorting means 10,

the height F represents the amplitude of the deflection force of the sorting means 10.

The duration before tripping of the sorting means and the duration of the actuation can be identical for all the particles. The sorting towards the different output channels is then obtained thanks to a modulation of the deflection force. However, the duration before triggering tA and / or the duration of the actuation tB may also be specific, for example in the case where several sub-populations of particles are concerned. In this case, a specific duration tA and / or duration tB are sought and applied for each output channel. The flow analysis of each particle from the detection peak on the dO diagram makes it possible to apply for each particle the appropriate parameters for tA and / or tB and / or F.

The number of output channels is not limited, as shown in the timing diagram of FIG. 5c associated with the device of FIG. 5d: there are as many signals as possible output paths for the particles. Subsequently, we will describe the case where the sorting device is equipped with two output channels, but the method according to the present invention applies to devices having a greater number of output channels.

The devices of FIGS. 6b, 6d, 6f comprise a detector 8 in the input channel 2, and a detector 6, 6 'in each output channel 4, 4'. FIGS. 6a to 6f show how the reading of the signals of the detectors downstream informs about the success or failure of the sorting of the particle. In this example, it is desired for the particle to be directed by the sorting means towards the exit channel 4 equipped with the detector 6.

Three cases are represented:

the timing diagram of FIG. 6a, associated with the device of FIG. 6b, represents the case where the sorting means 10 is triggered too early. The detection peak p2 on the diagram d2 shows that the particle has taken the wrong output channel 4 ',

the timing diagram of FIG. 6c, associated with the device of FIG. 6d, represents the case where the sorting means is triggered at an appropriate moment. The sorting of the particle is successful, because the detection peak p1 on the diagram d1 shows that the particle has borrowed the right exit channel 4, - the chronogram of FIG. 6e, associated with the device of FIG. 6f represents the case where the sorting means is triggered too late, so the deflection force has been applied too late to change the trajectory of the particle of interest. The detection peak p2 on the d2 diagram shows that the particle has borrowed the wrong way out 4 '.

Figures 7a to 7d show the operation of a device whose output channel 4 'does not have a detector. The direction taken by the particle is deduced by the presence or absence of a detection peak in the detector-equipped output channels.

Two cases are represented: on the timing diagram of FIG. 7a, associated with the device of FIG. 7b, the detection peak pi on the diagram d1 shows that the particle has taken the exit route 4,

on the timing diagram of FIG. 7c, associated with the device of FIG. 7d, the absence of a detection peak on the diagram d1 shows that the particle has taken the output channel 4 '.

It may be desirable for the particle to be directed by the sorting means towards an output channel provided with a detector or, on the contrary, to take a non-detector output path. When the device has at least one output channel without a detector, the sorting of the particle is considered successful or unsuccessful by deducing its trajectory from the observations of the output channels provided with detector.

Figures 7a-7d show simplified sorting with two output channels, but it can also be applied to a larger number of output channels. If at least two output channels do not have a detector, these output channels can not be distinguished by the method described herein, although they may be differentiated in practice if the operating parameters of the sorting means differ for these output channels. We will describe later the case where the sorting device is equipped only with two output channels each provided with a detector, but the method according to the present invention applies similarly to devices with a higher number of sensors. exit routes.

The flowchart of FIG. 13 represents the steps of a calibration method according to the present invention of an active sorting means. It makes it possible to determine, for a given output channel and for a given type of particles, the operating parameters of the sorting means. In a normal calibration process, the protocol of Figure 13 is applied for each output channel and / or for each type of suspended particles. At each step, the decisions can be made by the operator, and / or by the electronic circuit and / or the software, such as the rule for the choice of the duration of actuation tB and / or the deflection force F and / or the time before tripping tA in the identified time and / or force intervals.

The sorting means is triggered during the passage of a particle. The success or failure of the deviation is detected by the downstream detector in step 400. If the particle has taken the right exit route, the calibration remains unchanged. The calibration ends, or the verification of the success of the deviation is continued. On the other hand, if the particle has borrowed an erroneous output path, a new calibration can be implemented, we proceed to a step 402. The calibration can be performed as soon as a single failure is observed or after a predetermined number of sorts. incorrect, a counter can then be provided.

In step 402, a duration before triggering tA is determined. This can be obtained by calculating the speed of one or more particles, represented by FIG. 8, and / or by the progression method of tA, represented by FIG. 9.

In order to determine the time tA, the speed of movement of the particle of interest can be determined. For this purpose, the time elapsed between the detection of the particle by the detector 8 upstream and the detection by one of the downstream detectors 6, 6 'is measured. Knowing the distance between the detectors upstream and downstream, we deduce V. From V, we then determine the time that the particle will put to reach the sorting means 10.

FIGS. 8a to 8c show three methods for obtaining an estimate of the average speed of the particles in front of the sorting means 10. This calculation of the particle speed makes it possible to give a first evaluation of the duration before tripping of the sorting means. In the method presented in FIG. 8a, the sorting means 10 is stopped and blocked. One or more particles are detected by the detector 8 and then by one of the detectors 6, 6 'downstream. The time separating the two detections of the particle makes it possible to calculate its velocity V and to deduce therefrom an approximate value of the duration before triggering tA.

The velocity V of the particles is calibrated at the beginning of the use of the device, but it can also be measured for each particle during the sorting operation so as to advantageously perform an autotune and a correction of the time tA if a drift occurred. The calculation of the speed of each particle makes it possible in particular to control the absence of drift of the particle velocity during the use of the device.

In the method represented by FIG. 8b, which is applied during the normal separation operation, the sorting means 10 is actuated when the particle passes. Regardless of the success or failure of the sorting of the particle, its speed is measured and compared to the expected speed. In the event of a significant difference or in case of drift of the speed for a predetermined number of particles, it may be decided to carry out a new setting of the duration before tripping tA.

The third calculation method, represented by FIG. 8c, uses the width of the detection peak. The sorting means 10 may be actuated or not during the passage of the particle. The measurement of the speed is carried out with the peak of detection on the detector 8 and / or with the detection peak on a detector 6, 6 'downstream. The calculation of the velocity of the particle consists in dividing the distance traveled by the particle between the beginning and the end of the peak, by the duration of the peak used. If the distance traveled by the particle before the trigger is known precisely, the ratio of this distance and the duration of the peak can estimate the speed V and evaluate the duration before tripping tA. In order for it to be able to give significant values, this method may require a high resolution in the measurement of the width of the detection peak and in that of the distance traveled by the particle in front of the detector. If the velocity V of the particle is constant between the upstream detector 8 and the detector 6,6 'downstream, the estimate of the duration before triggering tA is obtained in a simple manner by a cross product: the duration necessary for the Particle to make the path between the upstream detector and the sorting means is deduced by transposing the velocity V of the particle to the distance between the upstream detector and the position of the sorting means.

On the other hand, if the velocity V of the particle is not constant between the upstream detector and the downstream detector, a more complex calculation is used to estimate the velocity of the particle between the upstream detector and the sorting means. In particular, this correction is necessary if the flow rate of the fluid transporting the particle is different in the entry way and in the exit way. An alternative method for obtaining a duration before triggering tA is represented by FIGS. 9a to 9d. In this example, it is desired for the particles of interest to be directed by the sorting means towards the output channel 4 provided with the detector 6. The particles that must be directed to another output channel are not concerned by this operation of calibration.

The sorting means 10 is actuated with an arbitrarily selected deflection force F and pulse duration tB. The amplitude of the force F and the pulse duration tB are predetermined values by the operator and / or the electronic circuit and / or the software, or values resulting from a previous calibration, or determined values of the previously described (flow charts 19, 20, 21). The force F and the duration tB remain constant during the adjustment of tA.

At the beginning of the adjustment, the sorting means 10 is actuated with an arbitrarily short time before triggering tA, as shown in FIG. 9a. It is estimated that the situation is described in FIGS. 6a and 6b, that is to say that the sorting means 10 is triggered too early. The detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit channel 4 '.

Then, the triggering time of the sorting means is moved by adding a part of the duration of the actuation tB to the duration before triggering tA. The progression of tA is arbitrarily chosen and depends on the type of sorting means 10 employed. By For example, tA can be shifted by a duration of 0.5 * tB. Generally, the time added at tA is between 0.3 * tB and 0.7 * tB. After each progression, we observe the exit route taken by the particle. Figures 9b and 9c show the case where the particle is again oriented towards the wrong output channel 4 'and where it is necessary to further increase the duration tA.

FIG. 9d shows the case where the duration before triggering tA is adequate for the duration of actuation tB and the deflection force F applied. The detection peak p1 on the diagram d1 shows that the particle has borrowed the right output channel 4 and therefore that the duration tA is suitable for sorting towards this output channel 4.

The calibration method can also be applied starting from an arbitrarily long time before triggering tA. It is estimated that the situation is that described in Figures 6e and 6f, that is to say that the sorting means is triggered too late. The progression used to search for a duration before tripping tA provides to remove at tA an arbitrary fraction of the duration tB, for example a duration between 0.3 * tB and 0.7 * tB. After each progression, we observe the exit route taken by the particle. This operation stops when the detection peak pi on the diagram d1 shows that the particle has taken the right output channel 4, so that the duration tA is suitable for sorting towards this output channel 4. At the beginning of the setting, the sorting means is actuated with an arbitrary pre-trigger duration tA. This duration tA can be short or long depending on the direction one wishes to perform for the progression. If the sorting of the particle is successful from the beginning with this arbitrary duration, that is to say if the peak of detection pi on the diagram dl shows that the particle has borrowed the right way of exit 4 with the duration tA chosen initially, it is not necessary to apply the progression.

The search for tA by the progression operation is applied as an alternative method or as a complementary method to the calculation of tA by measuring the particle velocity. The choice of an initial time tA can be made after a first estimate of tA obtained from the calculation of the average speed of the particles. The value of tA obtained by calculation can be the starting point for the progression operation. The direction of the progression is chosen arbitrarily, but a reversal of the direction of the progression may be necessary after a predetermined number of progressions if a duration tA allowing the sorting of the particles has not been found. Another possibility is to arbitrarily shift the value of tA resulting from the calculation of the speed to obtain the initial duration tA for the progression operation. The shift can be made optionally to a duration tA shorter or longer than the duration tA resulting from the calculation of the speed. In step 404, it is checked whether tA has been determined.

In case of failure in the search for a duration before tripping tA, the progression of the duration tA is implemented over a larger time interval (Step 404.1). If this solution is unsatisfactory, the actuation time tB and / or the deflection force F are modified (Step 404.2). The duration tA is then the subject of a new search.

If successful in the search for a duration before tripping tA, we go to step 406 in which a fine adjustment of the duration tA can be performed. The precision that must be made in the settings of the parameters of the sorting means is defined by the operator, and / or the electronic circuit and / or the software controlling the sorting means. The number of fine adjustment loops can be adjusted during the adjustments. It may also depend on the sorting means used for the separation operation.

The method for determining the fine adjustment of the duration before tripping tA is explained using FIGS. 10a to 10d. This fine adjustment is used to determine the range of values of tA for which the particles are directed correctly to the output channel 4 provided with the detector 6, then to choose a duration tA in this interval. The force F and the duration tB remain constant during the adjustment of tA. The particles that must be directed to the other output channel 4 'are not affected by this calibration operation. At the beginning of the adjustment (FIG. 10a), the detection peak pi on the diagram d1 shows that the particle has taken the right exit channel 4. The situation is that described in FIGS. 6c and 6d, that is to say that the sorting means is actuated with a duration before triggering appropriate tA.

Then, the triggering time of the sorting means is moved by adding a part of the duration of the actuation tB to the duration before triggering tA. The progression of tA is chosen arbitrarily and depends on the type of sorting means used. For example, tA can be shifted by a duration of 0.2 * tB. Generally, the time added tA is between 0.1 * tB and 0.3 * tB. After each progression, we observe the exit route taken by the particle.

FIGS. 10b and 10c show the case where the particle is always oriented towards the correct output channel 4. The duration tA can therefore be further increased. FIG. 10d shows the case where the duration before tripping tA is no longer adequate, since the detection peak p2 on the diagram d2 shows that the particle has borrowed the wrong output channel 4 '. The possible interval of times tA is then known, for the duration of actuation tB and the deflection force F applied.

The full interval of the possible tri-trip times tA is known because the other end of the interval (minimum tA) was determined in step 402. If necessary, the low value of the interval is determined with a better accuracy by applying the direction of progression to decreasing tA values.

A duration tA within this range can then be chosen arbitrarily, for example the central value of this interval. The choice of a time tA near the center of the possible time interval can be motivated by the existence of a variability in the speed of the particles. The time tA at the center of the time interval appears to be the most appropriate for sorting towards an output path, since the width of the time interval corresponds to the time during which the presence of the particle in front of the sort is certain.

The method can also be applied by moving the timing of tripping of the sorting means in the opposite direction, ie by removing at an arbitrary fraction of the duration tB, for example a duration of between 0.1 * tB and 0.3 * tB. After each progression, we observe the exit route taken by the particle. This operation stops when the peak of detection on the diagram d2 shows that the particle has borrowed the wrong way out. It is also possible to apply a progression in one direction until the end of the gap is known, then to apply a progression in the other direction until the other end of the interval is known. Once the time interval has been identified, a duration tA within this range can be chosen arbitrarily. In step 410, it is checked whether a fine adjustment of tA has been found. If this determination has failed, for example in case of drift of the sorting means or conditions under which it operates (drift due, for example, to a change in particle velocity or a change in the nature of the particles) during the calibration of the sorting means, it is possible that the fine adjustments of the operating parameters of the sorting means can not succeed. We then return to step 402 and a new search of the duration before tripping tA is then performed.

If a fine adjustment of tA has been found, proceed to step 412, in which a fine adjustment of tB is chosen or not. If the answer is no, go to step 418. If the answer is yes, go to step 414 to determine this setting.

The method of fine adjustment of the actuating time tB, explained by FIGS. 11a to 11f, is used to determine the range of values of tB for which the particles are correctly directed towards the output path 4 provided with the detector 6, then to choose a duration tB in this interval. The force F remains constant during the adjustment of tB. Particles that need to be directed to another output path are not affected by this calibration operation.

At the beginning of the adjustment (FIG. 11a), the detection peak pi on the diagram d1 shows that the particle has taken the right exit channel 4. The situation is that described in FIGS. 6c and 6d, that is to say that the sorting means 10 is actuated with an appropriate actuation time tB. Then, the duration The operation of the sorting means 10 is reduced by an arbitrary value which depends on the type of sorting means 10 employed. For example, the end of the duration tB can be reduced by a period of time of 0.2 * tB. Generally, the time removed at tB is 0.1 to 0.3 times the initial value of tB. After each reduction of the duration tB, we observe the exit path taken by the particle.

FIG. 11b shows the case where the particle is always oriented towards the good output channel 4. The duration tB can therefore be further reduced.

FIG. 11c represents the case where the duration of the actuation tB is no longer adequate, since the detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit channel 4 '.

In this way, a lower part of the time interval tB is determined.

It may be unnecessary to search for the upper part of the time interval tB, since it is preferable to have a low time tB rather than a high time tB in a particle sorting application. Indeed, it is preferable that the actuation of the sorting means 10 be completed before the arrival of the next particle. However, it may be advantageous to look for and choose a duration tB longer than its initial value if a too small value of tB results in an unacceptable number of failed sorts. In this case, the duration tB is incrementally increased by an arbitrary value. For example, the end of the duration tB can be increased by a period of time between 0.1 and 0.3 times the initial value of tB. After each increase in the duration tB, we observe the exit path taken by the particle. If it is still oriented towards the correct output channel 4, the duration tB can be further increased, or the increase of tB can be increased. This decision can be made by the operator, and / or by the electronic circuit, and / or the software controlling the sorting means. On the other hand, if the particle has taken the wrong path 4 ', it is estimated that the higher value of tB has been exceeded.

The possible interval of times tB is then known, for the deflection force F applied. A duration tB within this range can then be chosen arbitrarily. In particular, the duration tB may be sufficiently wide so that, despite any variation in the speed of the particles, the sorting means is always actuated during the passage of a particle.

The fine adjustment of the duration tB can be done in different ways.

Figures 11a to 11c show the case where the end of the pulse duration tB is reduced. In another configuration, the end of the actuating duration tB is unchanged, while the beginning of the operating duration tB is reduced.

Figures Hd to Hf show another possibility for reducing the duration tB. In this configuration, the beginning and the end of the pulse duration tB are reduced simultaneously. This reduction of tB may be symmetrical or asymmetrical around the central value of tB. FIG. 1e represents the case where the particle is always oriented towards the right exit channel 4 while FIG. Hf represents the case where the duration of the actuation tB is no longer adequate. Since the possible interval of times tB is then known, for the applied deflection force F, the duration tB included in this interval is chosen arbitrarily.

If a sorting of the particles of interest can not be obtained by reducing the duration tB or if the sorting of the particles is unstable, the duration of operation of the sorting means can be increased in a similar manner, ie say by increasing the beginning and / or end of the pulse duration tB. One possibility for the actuation duration tB is to make it equal to the width of the time interval previously defined for the duration tA. The presence of the particle in front of the sorting means is certain if the duration tA is divided by 2 while the duration tB remains constant, that is to say if the duration tB becomes very large compared to the duration tA.

At step 416, it is checked whether this setting was successful. If so, go to step 418.

If it's a failure, we go back to step 402.

If one chooses in step 418 to make a fine adjustment of the deflection force, proceed to step 420 of adjusting this force.

The method of fine adjustment of the deflection force F, explained by FIGS. 12a to 12c, is used to determine the range of values of the deflection force for which the particles are directed correctly to the exit path 4 provided with the detector 6, then to choose a force F in this interval. The duration before triggering tA and the duration of actuation tB remain constant during the adjustment of F. The particles which must be directed towards another way of exit are not concerned by this calibration operation.

At the beginning of the adjustment, the detection peak pi on the diagram d1 shows that the particle has taken the right exit path 4 (Figure 12a). The situation is that described in FIGS. 6c and 6d, that is to say that the sorting means is actuated with a suitable force F. Then, the deflection force of the sorting means is reduced by an arbitrary value. The magnitude of the reduction of the force F depends on the type of sorting means employed. For example, the magnitude of the force F can be reduced by 20%. Generally, the amplitude removed at F is from 10% to 30% of the initial value of F. After each reduction of the deflection force, the exit path taken by the particle is observed. FIG. 12b shows the case where the particle is always oriented towards the right exit channel 4. The force F can therefore be further reduced. FIG. 12c shows the case where the force F is no longer adequate, since the detection peak p2 on the diagram d2 shows that the particle has taken the wrong exit channel 4 '. A force F included in this portion of the range of values of the deflection force can then be chosen arbitrarily The method can also be applied by increasing the deflection force of the sorting means, for example by an amplitude of between 10% and 30% of the initial value. After each increase of the force F, we observe the exit path taken by the particle. This operation stops when the detection peak p2 on the diagram d2 shows that the particle has taken the wrong output channel 4 '. A force F included in this portion of the range of values of the deflection force can then be chosen arbitrarily.

In order to obtain the entire range of values of the deflection force adapted to correctly deflect the particle, it is possible to group the two intervals determined previously.

It is also possible to increase the force F until the end of the gap is known, then to decrease the force F until the other end of the gap is known. The full range of possible forces is then known for the duration before tripping tA and the duration of actuation tB applied. A deflection force F in this range can then be chosen arbitrarily. In step 422, it is checked whether the setting has been successful. If yes, go to step 424.

If it's a failure, we go back to step 402.

If in step 418, one chooses not to make the fine adjustment of the deflection force, proceed to step 424. In step 424, it is asked if one wants to make an adjustment of one of the parameters tA, tB or deflection force.

If such adjustment is desired, return to step 406; otherwise we go to step 426 which completes the calibration.

The method shown in Figure 13 can be used to determine the deflection force or actuation time, and then perform or not fine-tune the other parameters.

The different stages of setting the operating parameters can be combined.

The present invention also provides to be able to modify the operating parameters of the sorting device during its operation.

Indeed, during the operation of the sorting device, a drift of the sorting means 10 may occur, or the conditions under which the sorting means operates may change (such as, for example, a variation in the speed of the particles or a modification of the the nature of the particles). The deflection force and / or the pulse duration tB and / or the trip duration tA may no longer be adapted to correctly modify the trajectory of the particle or particles of interest. Sorting can then become ineffective. In known devices, in order to continue the sorting, a stop of the device is necessary to perform a new calibration, which causes a loss of time and possibly a loss of particles in the case of fragile cells. The present invention then makes it possible to carry out the corrections of the operating parameters if they have suffered a drift or to adapt the device to the changes in the operating conditions. Corrections of the operating parameters can be made without suspending the particle flow. The correction phase may result in an error rate in sorting the particles that may be acceptable to the user. It is also possible that the output channels are connected to specific collection tanks during the steps of correcting the operating parameters of the sorting means. After the corrections, the particles can then be reinjected at the input of the separation device to be sorted.

FIG. 23 represents a flowchart of a correction or self-calibration process according to the present invention.

During step 500, at least one detector placed in one of the output channels 4, 4 'controls the success or failure of the deviation.

If successful, the control is continued.

Continuous control, that is, each particle detection, or control at specified time intervals, may be provided.

If unsuccessful, proceed to step 502, during which it is expected to change one or more operating parameters. At step 504, the success or failure of the sort is checked with the new parameter by identifying the exit routes taken by the particles of interest.

If the sorting is successful, return to step 500 for monitoring the operation of the device.

If the sorting has failed, return to step 502.

The step 502 of modifying one or more parameters can be carried out according to the flowchart shown in FIG. 24. It is possible to provide for a modification of the parameters simultaneously or successively for several particle populations, since the correction method or the Autocalibration according to the present invention can be applied to each particle population independently.

In step 502.1, the order of the operating parameters is defined in the list of parameters that can be modified. The value 1 is assigned to the first parameter, which has previously been determined as the paramount parameter. The value 2 is assigned to the second parameter and so on.

An index i, corresponding to the number of the parameter to be modified, is set equal to 1. The parameter given by the index i is called "main parameter".

At step 502.2, a new value range is searched for the main parameter. In step 502.3 it is ascertained that the value of the main parameter is modified in a range of limit values previously defined by the operator, and / or by the electronic circuit, and / or by the software. If the range of values proposed for the main parameter and the range of limit values overlap, then step 502.4. If the two intervals do not overlap, the main parameter is not changed, and return to step 502.2 by incrementing the index i by 1 so that the main parameter becomes the next parameter on the list of operating parameters. .

If the proposed ranges of values for all operating parameters are outside the limit value ranges, then it is necessary to redefine the limits of the limit values.

If the proposed ranges of values for one or more operating parameters cover all or part of the limit value ranges but the modified operating parameters do not make it possible to sort the particles of interest, it is necessary to redefine the intervals of limit values.

In step 502.4, a value for the main parameter is selected in the common interval between the proposed value range and the limit value range, and then step 504 in which success is checked. or the failure of sorting with this new parameter value. Various examples of applications will now be given: Example 1: Particle flow sorter, in particular cells

The microdevice is fed continuously from a reservoir containing a homogeneous or heterogeneous suspension of particles, in particular microparticles such as cells. The input and output pathways are capillaries, microfluidic channels or nanofluidic channels. The flow of the liquid carrying the particles in the fluidic channels is ensured by a difference in pressure, by the application of a flow rate or by an electric field generating an electrophoretic flow or an electro-osmotic flow. Particles flowing in the inlet channel are placed in a single stream by hydrodynamic focusing and / or narrowing of the stream through a mantle stream and / or narrowing of the channel section to dimensions close to those of the particles. The method presented for the self - calibration of a particle flow sorting means represents a generic method applicable to all microfluidic and nanofluidic separation systems. It is compatible with a wide range of technical means of detection and sorting.

The particle sorter can be triggered in combination with flow cytometry. In this case, the sorting of the particles is determined by the upstream detection of physical characteristics of the particle or characteristics that have been previously added to it by means of a marking. Of preferentially, the decision criteria are based on a measurement of the fluorescence, and / or the particle size, and / or the small angle scattering of a light beam and / or the electrical impedance. The deviation of the particle by the sorting means is conditioned on the detection of the selected criterion or criteria. For example, the cell sorting means may be triggered depending on the measurement of the volume of the cell and / or depending on the electrical characteristics of the cell such as the cytoplasmic conductivity and / or the capacity of the membrane.

During the initial settings of the separation apparatus, particles of calibrated size or having physical properties close to those which will be sorted can be used. A bin is provisionally placed behind all outlets to collect the particles used during the initial calibration of the unit.

The sorting means is triggered during the passage of the particles. The calibration operation and / or the operating correction operation of the sorting means can be performed by deflecting all the particles that arrive in front of the detector, or by diverting the particles of interest only towards a specific output channel. The success or failure of the deviation is deduced by observing the presence or absence of detection peaks on the different detectors downstream of the sorting means.

Signal processing operations from the detectors are performed simultaneously with the separation operation by means of a circuit electronics and / or software, in order to count the particles and / or measure their speed in real time. The particle count can be implemented in the input channel and / or in each of the output channels. Example 2: instrument for dispensing cells:

The cell dispenser is a droplet ejection head that incorporates an inlet and microfluidic channels for injecting and transporting a cell suspension, sensors for detecting cells in the ejection head, and an ejector for generating droplets containing cells (Figure 22). The microfluidic input channel 2 splits into two output channels 4, 4 '. The output channel 4 is provided with a detector 6. The other output channel 4 ', which has no detector, opens onto another orifice.

The autocalibration method described in this invention makes it possible to parameterize the microdispenser and to control its operation in real time. The ejector is used as sorting means for directing the cells of interest to the orifice. The ejector is for example a miniaturized solenoid valve or a piezoelectric ceramic positioned near the branch of the output channels. Initially, the ejector is at rest; consequently, the liquid takes the exit path 4 provided with the detector 6. Preferably, the detectors use optical and / or electrical measuring means to detect the cells in flow and possibly identify and characterize the cells. In case of heterogeneous cell population, the sorting means can be triggered only for the cells of interest. The other cell types are carried by the flow in the outlet channel 4 equipped with the detector 6. In FIG. 22, a schematic representation of such a microdispenser can be seen. A cell C of interest in circulation in the input microchannel 2 is first detected by the upstream detector 8. Then, when the cell arrives in front of the junction 12, the ejector 10 is triggered in order to deflect the 14. Simultaneously, the triggering of the ejector 10 deflects the trajectory of the cell of interest and generates a sufficient hydraulic pressure to create a droplet at the outlet of the orifice 14. The droplet containing the cell of interest is propelled through the orifice 14 and is deposited on a substrate 16.

The occurrence or absence of a detection peak on the detector 6 in the output channel 4 indicates whether the operation parameters of the ejector are correct or inappropriate, depending on the cell population concerned.

The calibration of the ejector and the correction of the ejector in operation apply for the sorting of the cells of interest. In this case, the ejector is considered as an active sorting means. There are two possibilities for characterizing sorting:

a detection peak on the detector 8, which is followed by a detection peak on the detector 6, shows that the sorting of the cell of interest has failed, that is, the cell has not been diverted to port 14;

a detection peak on the detector 8, which is not followed by a detection peak on the detector 6, shows that the sorting of the cell of interest was successful, that is to say that the particle has been oriented towards the orifice 14.

The calibration of the ejector and the correction of the ejector in operation also concern the sorting of the other particles. In this case, the ejector is considered a passive sorting means because it is kept at rest during the passage of the particle. When the microparticle is not a cell of interest, it must not be deposited on the substrate 16. Two possibilities exist to characterize the sorting:

a detection peak on the detector 8 followed by a detection peak on the detector 6 shows that the sorting of this particle has been successful, that is to say that the particle has not been diverted towards the orifice 14

a detection peak on the detector 8 which is not followed by a detection peak on the detector 6 shows that the sorting of this particle has failed, that is to say that the particle has been oriented towards the detector; orifice 14.

The deposition of the cells of interest in a volume of calibrated liquid may constitute another mode of use of the cell dispenser. The duration of actuation and the deflection force can be optimized during the calibration of the sorting means so that that the dispensing volume corresponds to the desired volume.

The present method can be applied for the calibration and / or correction in operation of the microdispenser described in the unpublished document FR 04 00433, but it is also applicable to any fluidic system comprising a sorting means. The method described here can be applied to the vast majority of microfluidic systems presented in the literature, in order to automate the search for the operating conditions of the sorter and correct drift in real time.

On-chip sorting, especially for isolating and enriching cell populations, is currently an important area of technological research. The separation of particles of interest is thus a basic technological brick which could be integrated on a chip with stages of chemical and / or biological analysis, characterization of populations, stimulation (biochemical or other), matrixing, storage, cell culture, ....

Claims

1. A method of calibrating a particle flow sorting device, said device comprising an inlet channel (2) and at least two outlet channels (4,4 '), a particle sorting means

(10) and at least one particle detection means

(6,6 ') disposed in an outlet channel (4,4'), said method comprising the step of adjusting at least one of the main operating parameters of the sorting means among the deflection force (F) suitable to be applied by the sorting means (10) to said particle and / or the duration of application of said force (F).

Method according to the preceding claim, wherein the setting of the main parameter comprises:

the choice of a value of the parameter,

- changing the value of the parameter until at least one successful sort.

The method of claim 1 or 2, including the further step of determining a range of rms values for the main parameter.

4. Method according to the preceding claim, wherein the determination of a range of effective values comprises the determining the bounds of the interval from an effective value of the main parameter.

5. Method according to the preceding claim, wherein:

- the rms value is changed,

the effectiveness of this modified value is tested,

6. Calibration method according to any one of claims 3 to 5, said device being able to sort at least a first and a second population of particles of interest, comprising the steps:

determining a first range of effective values of the main parameter for the first population of particles; determining a second range of effective values of the main parameter for the second population of particles;

7. Calibration method according to the preceding claim, wherein the choice of a single value of the main parameter for the different populations, is carried out in an interval. overlap between the first and second intervals.

8. Calibration method according to any one of claims 3 to 5, said device being able to sort at least a first and a second populations of particles of interest, comprising the steps:

determining an effective value of the main parameter for the second population in the first interval; determining a second range of effective values for the second population in the first interval;

9. Calibration method according to the preceding claim, wherein a single value of the main parameter for the different populations is chosen in the second interval.

Calibration method according to one of the preceding claims, wherein other operating parameters of the device can be set, such as the duration before triggering corresponding to the time elapsed between the detection of a particle in the input channel upstream of the sorting means and the passage of said particle at the level of the sorting means.

11. Method according to the preceding claim, wherein the duration before tripping is obtained from the determination of the speed of movement of a particle of interest in the sorting device.

12. Method according to the preceding claim, wherein the sorting means is not activated when determining the speed of the particle.

The method of claim 11, wherein the sorting means is activated when determining the particle velocity.

The method according to one of claims 11 to 13, wherein the velocity of the particle is obtained from the detection signals provided by an upstream detection means (8), a detection means (6,6 ') of an output channel (4,4 ') and the distance between these two detectors (8,6,6').

The method of claim 11, wherein the velocity of the particle is calculated from the duration of a detection peak provided by a detector (8) in the input channel (2) and / or by a detector (6, 6 ') in an output channel (4,4') and the distance traveled by the particle between a beginning and an end of this peak.

The method of any one of claims 10 to 15, wherein the determination of the time to trip includes:

the choice of a duration value before triggering, the values of the deflection force and the duration of application being kept constant,

- the test of the effectiveness of this value,

- the modification of this value until a successful sorting.

17. Method according to the preceding claim, comprising the step of determining a range of effective values for the duration before triggering.

18. The method according to the preceding claim, wherein from an effective trigger duration value, effective sorting tests are performed with increased and / or decreased duration values with respect to the adapted value.

19. A method for correcting the operating parameters of an operating particle flow sorting device comprising the steps of:

- verification of the efficiency of sorting, modifying at least one operating parameter until at least one efficient sorting is achieved.

20. Correction method according to the preceding claim, wherein the verification of the efficiency of sorting is carried out continuously.

21. Correction method according to claim 19, wherein the verification of the efficiency of sorting takes place at predetermined time intervals.

22. Correction method according to one of claims 19 to 21, wherein a first parameter is modified until an effective sort.

23. Correction method according to the preceding claim, wherein an additional parameter is modified if an effective sorting is not obtained.

24. Correction method according to any one of claims 19 to 23, wherein the operating parameters are at least the deflection force applied by the sorting means, the duration of application of this force and / or the time between the detection of the particle at the input of the device and its passage at the level of the sorting means.