• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    According to one aspect, the invention relates to a device (100) for the optical detection in transmission of moving nanoparticles in a fluid sample comprising: a light source (10) for the emission of an incident beam through the sample ; an imaging optical system (30) comprising a microscope objective (31); a two-dimensional optical detector (40) comprising a detection plane (41) conjugated to a focal plane object of the microscope objective by said imaging optical system (30), and allowing the acquisition of a sequence of images a volume of analysis of the sample, each image resulting from the optical interferences between the incident beam emitted by the light source and the beams scattered by each of the nanoparticles present in the analysis volume for a predetermined duration of less than one millisecond ; image processing means (50) making it possible to average a sequence of said images and to subtract the said average from each image.
    公开号:FR3027107A1
    申请号:FR1459690
    申请日:2014-10-09
    公开日:2016-04-15
    发明作者:Albert Claude Boccara；Martine Boccara
    申请人:ESPCI Innov SAS；
    IPC主号:
    专利说明:

    [0001] TECHNICAL FIELD OF THE INVENTION The present invention relates to a method and a device for the optical detection of nanoparticles in a fluid sample, for example in a liquid sample, or in air. The method is particularly applicable to the detection of free viruses present in the aquatic environment, in particular for the counting and characterization of viruses in seawater or in river water. STATE OF THE ART Viruses are nano-objects, the dimensions of which are typically between 30 nm and 200 nm. They are generally specific to a given host cell and therefore characteristic of a species, or even a variety or strain of this species. It was only from 1989, thanks to the work of a Norwegian team (see KJ Borsheim et al., "Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy", Appl., Environ Microbiol. (1990) 56 352-356), that there is an awareness of the abundance of viruses in a variety of aquatic environments, high concentrations have been measured in lakes, rivers, ice or sediments, from oceanic clouds, suggesting that they play an important role in the functioning of the biosphere through various mechanisms, such as the destruction of a dominant species for the benefit of rarer species or the transfer of viral genes to the host , viruses maintain the biodiversity of aquatic ecosystems and facilitate genetic mixing, so it is essential to characterize viruses in different aquatic ecosystems and to estimate their distribution. tion to better understand the relationship between viruses and the host. Depending on the aquatic ecosystems, the season or the depth of the samples, the concentrations of free viruses generally vary between 106 and 109 particles per milliliter. Many methods are known for the characterization and counting of viruses in aqueous media. For example transmission electron microscopy (or TEM, abbreviation of the English expression "Transmission Electron Microscopy") is known, which makes it possible to count the viruses and to characterize their morphology with very great precision. However, this technique, destructive, requires bulky and expensive equipment.
    [0002] Among the optical techniques for characterizing viruses in an aqueous medium, epifluorescence microscopy is known which makes it possible, after staining the nucleic acids with fluorescent markers, to count the free viruses (see, for example, Bettarel et al., "A Comparison of Methods for Counting Viruses in Aquatic Systems", Appl. Environ Microbiol., 66: 2283-2249 (2000)). This technique, however, requires a marker binding step, which may be troublesome for later stages of molecular and biochemical analysis. Since the viruses behave as dielectric nanometric particles whose refractive index, close to 1.5 in the visible spectrum, differs substantially from that of water (1.33), it is also known to detect their presence and potentially to characterize them by determining the disturbance that these nanoparticles induce to an incident electromagnetic field. Thus, methods based on light scattering by suspensions of viral particles have been described (see for example WM Balch et al., "Light scattering by 15 Viral Suspensions", Limnol Oceanogr, 45: 492-498 (2000). ). However, these methods are limited to analyzes of homogeneous virus solutions due to the low sensitivity of the detection and only make it possible to determine the virus concentration for a given size and shape; they are therefore not adapted to the identification of diversified viruses, which is generally the case in the natural environment.
    [0003] In order to gain in sensitivity, interferometric methods have been implemented for the detection of viruses in a liquid environment. Thus, in the article by Mitra et al. ("Real-time Optical Detection of Single Human and Bacterial Viruses Based on Dark-field Interferometry", Biosens Bioelectron.
    [0004] 2012 January 15; 31 (1): 499-504) is described an interferometric detection method for observing nanoparticles moving one by one in a nano-fluid conduit. The low light intensity scattered by a nanoparticle illuminated by means of an incident laser beam is amplified by a reference beam of high intensity. In addition, a structured illumination makes it possible to overcome the noise resulting from spurious reflections on the interfaces of the conduit (detection on a black background). However, this technique requires, besides the use of a coherent source (laser), a complex nano-fluidic assembly. The present invention presents an interferometric technique for the detection of nanoparticles in motion in a fluid, for example in an aqueous medium, which avoids the use of a laser and does not require specific assembly for the fluid under examination. The technique described in the present description, however, has a very good sensitivity, and can detect nanoparticles of a few tens of nanometers.
    [0005] SUMMARY OF THE INVENTION According to a first aspect, the invention relates to a device for the optical detection in transmission of moving nanoparticles in a fluid sample comprising: a light source for the emission of an incident beam through the sample; an imaging optical system comprising a microscope objective; a two-dimensional optical detector comprising a detection plane conjugated with a focal plane that is the object of the microscope objective by said imaging optical system, and allowing the acquisition of an image sequence of a scanning volume of 10 the sample, each image resulting from the optical interferences between the incident beam emitted by the light source and the beams scattered by each of the nanoparticles present in the analysis volume for a predetermined duration of less than one millisecond; image processing means making it possible to average a sequence of said images and to subtract the said average from each image. The device thus described, very simple to implement and not requiring particular shaping of the sample, allows the detection of nanoparticles with diameters of less than a few tens of nanometers, because of the amplification of the scattering signal obtained by interference between the signal emitted by the source and the signal diffused by each of the nanoparticles during very short periods during which the nanoparticles are "frozen". According to a variant, the light source is impulse, allowing the sequential emission of flashes of said predetermined duration; the device further comprises means for synchronizing the two-dimensional optical detector and the pulsed light source for acquiring said image sequence. The two-dimensional detector used can then be a standard camera operating at a hundred Hz. Alternatively, it will be possible to work with a continuous source and a fast camera, typically having a frequency greater than a few thousand images per second. Advantageously, the source is a spatially incoherent light source, for example an LED, making it possible to avoid any speckle effect which could generate noise at the detection level.
    [0006] Advantageously, the microscope objective used has a numerical aperture greater than or equal to 1 in order to increase the intensity of the light signal diffused by each of the nanoparticles and to allow the detection of nanoparticles of smaller diameter. According to a second aspect, the invention relates to a method for detecting the transmission of moving nanoparticles in a fluid sample comprising: - emitting an incident light beam through the sample; the formation, on a detection plane of a two-dimensional optical detector, and by means of an imaging optical system comprising a microscope objective, of images of an analysis volume of the sample located in the vicinity of a focal plane object of the microscope objective; the acquisition, by means of the two-dimensional detector, of a sequence of images of the analysis volume of the sample, each image resulting from the optical interferences between the incident beam emitted and the beam scattered by each of the nanoparticles present in the analysis volume for a predetermined duration of less than one millisecond; - Processing the images to average on a sequence of said images and subtract each image said average. According to a variant, the emission of the light beam is a sequential emission of flashes of said predetermined duration, the acquisition of the images being synchronized with the emission of the light flashes. According to a variant, the method further comprises, from the sequence of images thus processed, the determination of the trajectories of the nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS Other advantages and characteristics of the invention will appear on reading the description, illustrated by the following figures: FIG. 1, a diagram illustrating an example of a device for detecting nanoparticles in a fluid sample, according to FIG. this description; Figure 2 is a diagram showing in more detail an example of a liquid sample holder in a device of the type of Figure 1; FIG. 3 is a diagram illustrating the waves originating from nanoparticles situated before and after the object focal plane of the microscope objective, in an example of a device of the type of that of FIG. 1; FIGS. 4A and 4B, diagrams illustrating the principle of interference between the transmitted wave and the wave propagated by a nanoparticle, respectively in the case of a particle located after the object focal plane of the microscope objective and before the focal plane object of the microscope objective; FIG. 5A, a diagram illustrating the principle of interference between the transmitted wave and the wave scattered by each of several nanoparticles located before or after the focal plane object of the microscope objective and FIG. 5B, FIG. interference resulting in the detection plane of the detector; 6A to 6C, figures respectively showing an image obtained for a liquid sample after treatment by removal of the average, a zoom on an interference pattern of said image associated with a particle corresponding to a virus of the "phage X" type And the profile of the light intensity measured at said particle as a function of the number of pixels in the detecting plane of the detector. FIG. 7, a curve illustrating the trajectories of different nanoparticles (Phage type virus T4), the trajectories being marked by sequences of jumps made between two consecutive images.
    [0007] For the sake of consistency, the identical elements are identified by the same references in the different figures. DETAILED DESCRIPTION FIG. 1 schematically shows an example of a device for detecting moving nanoparticles in a fluid sample, according to the present description, and FIG. 2 shows a particular example of arrangement of the sample in a device of the type of Figure 1. The detection device 100 shown in Figure 1 comprises a light source 10 adapted for the emission of an incident beam through a liquid or gaseous sample 20. The source is advantageously a spatially incoherent source, for example a 30 thermal source or an LED (abbreviation of the English expression "Light Emitting Diode"). The light source 10 illuminates the field of a microscope objective 31 of large numerical aperture, typically greater than 1. The device 100 shown in FIG. 1 further comprises an optic 32, commonly referred to as a tube lens, which together with the microscope objective 31 forms an imaging optical system 30 adapted to form an image of the object focal plane of the microscope objective on a detection plane of a two-dimensional optical detector 40. The objective is for example a lens standard oil immersion and the two-dimensional detector is for example a CCD or CMOS type camera typically operating with a minimum rate of the order of one hundred Hz and a large sink capacity, for example of the order of a hundred thousand electrons at a minimum, the capacity of wells setting the signal-to-noise ratio and therefore the smallest size of measurable virus. In the example of FIG. 1, the detection device 100 further comprises processing means 50 connected to the detector 40 and to a screen 60, as well as, in this example, to a control unit 11 for controlling the light source 10 for ensure synchronization between the source when operating in pulsed mode and detection. As shown in more detail in FIG. 2, the sample 20 is for example a liquid sample whose volume is of the order of one microliter; the volume is formed by a circular hole (with a radius of the order of a millimeter for example), in a plastic film 23 of thickness approximately equal to a hundred micrometers, placed between 2 microscope slats 22, all forming a sample holder 21. No particular preparation is necessary for the analysis of the liquid sample, if it is not sometimes a preliminary filtering for separating the too large particles and keeping for example only the particles of diameter less than a few hundred nanometers, advantageously less than a hundred nanometers; however, in the case of particularly "clean" samples (low virus concentration), it may be necessary to "concentrate" the sample in virus using known methods.
    [0008] Although described in the case of a liquid sample, the method for detecting nanoparticles according to the present description could equally well be applied to nanoparticles moving in a gas, for example in air; in this case, the device 100 can be directly installed in the environment whose air is to be analyzed. Optionally, prior filtering may also be performed to limit the detection to particles of diameter less than a few hundred nanometers. The principle of the invention is illustrated by means of FIG. 3 for a volume corresponding to a "pixel" of the object field or "voxel"; FIG. 3 schematically represents the waves coming from two nanoparticles situated respectively in the object field, before and after the object focal plane of the microscope objective.
    [0009] A pixel of the object field or "voxel" is an elementary volume V defined in the object space of the microscope objective 31 for a pixel of the image field, the image field being defined by the effective detection surface of the image field. detector 40. A voxel V in the object field can be likened to a cylindrical volume of length L defined by the depth of field of the microscope objective 31 and section S defined by the diffraction task of the objective microscope. The depth of field L and the diameter of the section S are given by: Xn L = 1 22 NA (1) 2 (I) = 1 22NA (2) Where NA is the numerical aperture of the microscope objective , n the index of the medium in which the object space is immersed (for example a medium of index n 1.5 in the case of an oil immersion objective) and X the working wavelength of the light wave emitted by the source 10. It is thus possible to define a volume of analysis Va of the sample by all the Voxels V; the analysis volume Va represents the volume within which particles moving in the fluid can be detected. The analysis volume has a lateral dimension defined by the size of the object field, ie the dimension of the detection surface multiplied by the inverse of the magnification of the imaging system 30, and an axial dimension defined by the depth of field L.
    [0010] As shown in FIG. 3, the wave originating from a point F, from the focal plane F of the microscope objective 31 is a spherical wave Wo of center F, which the microscope objective transforms into a plane wave W ' o. The plane wave W'o, hereinafter referred to as the "reference wave", meets the microscope tube lens (not shown in FIG. 3). In the detection plane of the detector 40, this plane wave forms a diffraction task whose diameter is a function of the numerical aperture of the microscope objective and the magnification of the imaging system 30. Inside the voxel V nanoparticles Pi and P2 subwavelength, that is to say of dimension less than the working wavelength, located in the vicinity of the point F, but at the limits of the depth of field, each emit when they are illuminated by the light wave coming from the source 10, a scattered wave which the microscope objective 31 transforms into a quasi-planar wave respectively denoted W'1, W'2 in FIG. detection according to the present description is based on the acquisition, by means of the two-dimensional detector 40, of a sequence of images of the analysis volume of the sample, each image resulting from the optical interferences between the incident beam emitted during a predetermined duration is sufficiently short in relation to the travel time of a nanoparticle, typically less than one millisecond, and the beam scattered by each of the nanoparticles present in the analysis volume formed from the incident beam. Thus, in the example of FIG. 3, each of the waves W'i, W'2 interferes with the reference wave W'o. It can be shown (see below) that depending on whether the nanoparticle is downstream of the focal plane object of the microscope objective (for example the particle Pi) or upstream (for example the particle P2), the interference will be constructive or destructive. This difference is due to the "Gouy phase" which is at the origin of the 180 ° phase shift between a spherical wave coming from a point before or after the focus. FIGS. 4A and 4B thus illustrate the constructive interference mechanism, respectively destructive for nanoparticles positioned downstream, respectively upstream of the object focal plane of the microscope objective, but always in the depth of field. In these figures, only the source 10 and the microscope objective 31 are represented.
    [0011] The example of FIG. 4A illustrates the case of a nanoparticle Pi situated downstream from the focal plane that is the object of the microscope objective 31. The nanoparticle Pi is located in the analysis volume defined by the detector field (no represented in FIG. 4A) and the depth of field L of the microscope objective 31. The nanoparticle is illuminated by the source 10, advantageously a spatially and temporally incoherent source, for example an LED, in order to avoid the formation of speckle that could interfere with the interpretation of interference signals. Here Wo is called the reference wave coming from the focal point F, and intercepted by the opening of the microscope objective and W the wave scattered by the nanoparticle P1, also intercepted by the opening of the microscope objective. . In the case of FIG. 4A, the reference wave and the scattered wave are in phase. Constructive interference occurs between the waves which result in the opening of the microscope objective by a clear interference pattern. Since the shift between the position of the nanoparticle and the focus is less than the depth of field, the W and Wo waves are in phase for all the angles of the rays diffused in the opening of the microscope objective, 30 ie ie all angles formed between the optical axis of the microscope objective and the maximum aperture of the objective, typically 54 ° for an oil immersion objective. As a result, no rings are seen on the interference field of FIG. 4A. On the contrary, the example of FIG. 4B illustrates the case of a nanoparticle P2 situated downstream from the object focal plane of the microscope objective 31, but still in the analysis volume whose width is defined by the depth of the microscope. 31. In this example, the reference wave Wo and the scattered wave W are in phase opposition. Destructive interferences occur between the waves which are reflected at the opening of the microscope objective by a dark I interference pattern. As before, since the offset between the position of the nanoparticle and the focus is less than the depth of field, the waves W and Wo are in phase opposition for all angles and no rings are seen. In a phenomenon of interference between a very weak signal, such as that scattered by each of the nanoparticles and the strong signal from the source, as described by means of FIGS. 4A and 4B, an interference amplification is observed which allows the detection of very small diffusion signals and thus the identification of nanoparticles with diameters of less than a few tens of nanometers. Thus, if we call Ns the number of photoelectrons induced directly by the photons of the source and ND those produced by the diffusing nanoparticle, we obtain by interference between these two waves a number of photoelectrons N such that: N = Ns + ND + 21INsND cos (13) (3) Where is (I) is the phase shift between the source beam and the scattered beam. Here, the number ND of photoelectrons produced by the diffusing nanoparticle is very small in front of the number Ns of photoelectrons emitted by the source (ratio 1/106 typically).
    [0012] On the other hand, in our case, because of the position of the particles in the depth of field of the microscope objective, the phase shift (I) is close to zero or 180 ° depending on the relative position of the scattering particle and the home; hence cos (a)) is then + 1 or -1. Thus, if one averages over a large number of images, in view of the movement of the particles, in general a Brownian motion due to the very small size of the particles, the average performed on all the images represents the background (ND) because the signals associated with the particles are reduced at the noise level. To obtain images containing only the signals associated with the nanoparticles, one can then subtract from each acquired image the average. We obtain then the term of interference 2.INsND, which constitutes the signal after withdrawal of the bottom and which is very superior to ND. From the measurement of the interference term, it is possible to obtain ND, NS being known, and to deduce from it information such as, for example, the size of the particle, the amplitude of the scattered signal varying as the cube of the size of the particle. particle. A calculation of the signal-to-noise ratio with a detector capable of storing 160,000 electrons per pixel shows that, after treatment by subtraction of the average, the residual measurement noise corresponds to the signal which would be created by particles of 20 nanometers of diameter. Furthermore, the use of a NA lens of large numerical aperture, typically NA greater than or equal to 1, will also increase the intensity of the signal scattered by each nanoparticle and thereby reduce the minimum diameter. observable nanoparticles. Indeed, the intensity scattered by a nanoparticle varies in a / S where a is the scattering cross section of the nanoparticle and S is the surface of the diffraction spot; thus, according to equation (2) above, the intensity scattered by a nanoparticle varies with the square of the numerical aperture NA.
    [0013] FIG. 5B schematically represents an image obtained at a given instant for observing a plurality of moving nanoparticles in a fluid medium, as shown in FIG. 5A. FIG. 5A shows four nanoparticles referenced P1 to P4, the nanoparticles P1, P3, P4 being located downstream from the focal plane of the microscope objective and the particle P2 lying upstream of the focal plane of the objective of microscope. All particles are in the analysis volume Va defined by the detector field and the depth of field L of the microscope objective. The nanoparticles are moving in the fluid medium. It may be, for example, nanoparticles of a few tens of nanometers to a few hundred nanometers, for example viruses in an aqueous medium. During the implementation of the detection method according to the present description, a series of image acquisition is carried out, each image resulting from the optical interferences between the incident beam emitted and the beams scattered by each of the nanoparticles during a period of time. fixed time sufficiently short to "freeze" the movement of particles in the analysis volume.
    [0014] In a known manner, the diffusivity of a spherical nanoparticle of radius r animated by the Brownian motion is given by the formula: D = kBT / 67rrir (4) where kB is the Boltzmann constant and r1 the viscosity of the fluid in which is immersed the nanoparticle at the temperature T. For a time interval t, the jump / of the particle as it is imaged in 2 dimensions on the camera is given by: / = i4Dt where D is the diffusivity given by the equation (4). In practice, therefore, it is sought to form images for sufficiently short times t that the nanoparticle has not been able to travel a distance greater than a fraction of the diffraction spot. Typically, it is shown that the images must be formed for periods of time not exceeding one millisecond. According to a first variant, the motion can be frozen by detection, using a camera having a very high acquisition rate, typically greater than a few thousand images per second. Alternatively, it is possible to use a pulse source of duration less than one millisecond synchronized with the acquisition of each of the images on the detector. In this case, the detector may be a camera with a standard acquisition rate, for example a hundred Hz. The "jump" made by a nanoparticle of a few tens of 10 nanometers radius, for example about 40 nm, and measured between 2 consecutive images is greater than 1 micrometer which is easily measurable given the resolution of the microscope objectives used. As previously explained by means of FIGS. 4A and 4B, the nanoparticles located downstream from the focal plane of the microscope objective will give rise to constructive interferences resulting in clear diffraction tasks (P'1, P'3, P 4) on the detection plane 41 shown in FIG. 5B. On the contrary, the nanoparticles located upstream of the focal plane of the microscope objective will give rise to destructive interferences resulting in dark diffraction spots on the detection plane (P'2). In practice, as explained above, there is observed on the detection plane 41 a large background on which diffraction tasks are lighter or darker than the background, depending on whether the interferences are constructive or destructive. Advantageously, according to the detection method according to the present description, it is proceeded to the recording of a sequence of images, for example a few hundred, which is averaged. In order to obtain the images containing only the signals associated with the nanoparticles, the average can be subtracted from each image acquired. Figures 6 and 7 show first experimental results obtained with liquid samples analyzed using the detection method according to the present description for detecting and identifying potentially present viruses. Here, respectively, there is a sample comprising phage type X viruses and a sample comprising phage type T4 viruses, a sample very representative of what is found on the Brittany coast. Figure 6A is an image obtained from a liquid sample after averaging treatment. The device used to obtain this image is a device of the type shown in FIG. 1 with an Imperial Thorlabs® blue LED, an oil immersion objective Olympus® 100X, a 300 mm tube lens. focal length for oversampling the 3027107 12 diffraction spot and a CMOS Photon Focus® PHF-MV-D1024E-160-CL-12 camera. FIG. 6A shows a set of light or dark spots each corresponding to a nanoparticle located upstream or downstream of the focal plane object of the microscope objective. FIG. 6B shows a zoom on an interference figure of the image of FIG. 6A associated with an isolated nanoparticle and FIG. 6C illustrates the profile of the light intensity (in arbitrary units) measured at the level of said particle in FIG. function of the number of pixels in the detection plane of the detector. It is possible from the images obtained not only to confirm the presence of viruses but also to identify them, in particular according to their size; in this case, the measurement of the scattered light intensity makes it possible to conclude that a nanoparticle 60 nm in diameter, corresponding to the "phage X" virus. Figure 7 illustrates the trajectories of a number of particles measured over a period of the order of a tenth of a second, with a device similar to that used for forming the images shown in Figure 6A. Each trajectory is formed in this example of a series of jumps made between two successive images by about fifteen nanoparticles, each identified in FIG. 7 by a symbol shown in the legend. From these experimental measurements, it is possible as previously, to determine the diffused light intensity (value noted in arbitrary units, in the legend, vis-à-vis each symbol). Here, the scattered intensity is substantially identical for all the nanoparticles and it can be concluded that there is a homogeneous population of "phage T4" viruses of 90 nanometer diameter. The analysis of the trajectories makes it possible to provide additional information to the measured values of the diffused intensity. In fact, the analysis of the Brownian motion also makes it possible to deduce information specific to the nanoparticles, such as, for example, their dimensions, the presence of a tail which disturbs the Brownian movement, etc. Although described through a number of exemplary embodiments, the optical method for detecting nanoparticles in a fluid environment according to the invention and the device for implementing said method comprise various variants, modifications and improvements which will appear It is obvious to those skilled in the art that these various variations, modifications and improvements are within the scope of the invention as defined by the following claims.
    权利要求:
    Claims (7)
    [0001]
    REVENDICATIONS1. Apparatus (100) for optical transmission detection of moving nanoparticles in a fluid sample comprising: - a light source (10) for emitting an incident beam through the sample; an imaging optical system (30) comprising a microscope objective (31); a two-dimensional optical detector (40) comprising a detection plane (41) conjugated to a focal plane object of the microscope objective by said imaging optical system (30), and allowing the acquisition of a sequence of images a volume of analysis of the sample, each image resulting from the optical interferences between the incident beam emitted by the light source and the beams scattered by each of the nanoparticles present in the analysis volume for a predetermined duration of less than one millisecond ; image processing means (50) making it possible to average a sequence of said images and to subtract the said average from each image.
    [0002]
    2. Device according to claim 1, wherein the light source is impulse, allowing the sequential emission of flashes of said predetermined duration, and the device further comprises means for synchronizing the two-dimensional optical detector and the pulsed light source for the acquisition of said sequence of images.
    [0003]
    3. Device according to any one of the preceding claims, wherein the light source is spatially incoherent.
    [0004]
    4. Device according to any one of the preceding claims, wherein the microscope objective has a numerical aperture greater than or equal to 1.
    [0005]
    A method for optical transmission detection of moving nanoparticles in a fluid sample (20) comprising: - emitting an incident light beam through the sample (20); The formation, on a detection plane (41) of a two-dimensional optical detector (40), and by means of an imaging optical system (30) comprising a microscope objective (31), images of a volume of analysis of the sample located in the vicinity of a focal plane object of the microscope objective; The acquisition, by means of the two-dimensional detector, of a sequence of images of the analysis volume of the sample, each image resulting from the optical interferences between the incident beam emitted and the beam scattered by each of the nanoparticles present in the the analysis volume for a predetermined duration of less than one millisecond; Processing the images to average a sequence of said images and subtract each said average image.
    [0006]
    6. Method according to claim 5, wherein the emission of the light beam is a sequential emission of flashes of said predetermined duration, the acquisition of the images being synchronized with the emission of the light flashes. 15
    [0007]
    7. Method according to any one of claims 5 to 6, further comprising from the sequence of images thus processed the determination of the trajectories of the nanoparticles.
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    FR3027107B1|2014-10-09|2019-09-13|Espci Paristech|METHOD AND DEVICE FOR OPTICALLY DETECTING NANOPARTICLES IN A FLUID SAMPLE|FR3027107B1|2014-10-09|2019-09-13|Espci Paristech|METHOD AND DEVICE FOR OPTICALLY DETECTING NANOPARTICLES IN A FLUID SAMPLE|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1459690A|FR3027107B1|2014-10-09|2014-10-09|METHOD AND DEVICE FOR OPTICALLY DETECTING NANOPARTICLES IN A FLUID SAMPLE|
    FR1459690|2014-10-09|FR1459690A| FR3027107B1|2014-10-09|2014-10-09|METHOD AND DEVICE FOR OPTICALLY DETECTING NANOPARTICLES IN A FLUID SAMPLE|
    EP15783969.7A| EP3204755A1|2014-10-09|2015-09-29|Method and device for optically detecting nanoparticles in a fluid sample|
    JP2017538289A| JP6596498B2|2014-10-09|2015-09-29|Method and apparatus for optically detecting nanoparticles in a fluid sample|
    AU2015330241A| AU2015330241B2|2014-10-09|2015-09-29|Method and device for optically detecting nanoparticles in a fluid sample|
    US15/517,723| US10222319B2|2014-10-09|2015-09-29|Method and device for optically detecting nanoparticles in a fluid sample|
    KR1020177012524A| KR102316439B1|2014-10-09|2015-09-29|Method and device for optically detecting nanoparticles in a fluid sample|
    PCT/EP2015/072425| WO2016055306A1|2014-10-09|2015-09-29|Method and device for optically detecting nanoparticles in a fluid sample|
    CN201580066889.6A| CN107003228B|2014-10-09|2015-09-29|Method and apparatus for optical detection of nanoparticles in a fluid sample|
    CA2964079A| CA2964079A1|2014-10-09|2015-09-29|Methode et dispositif de detection optique de nanoparticules dans un echantillon fluide|
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