Optical device for analysing a sample comprising nanoparticles suspended in a solution with determination of a three-dimensional distribution of a scattered field

Abstract

The invention relates to an optical analysis device (100) comprising: - a light source (10); - an optical detector (30); - an optical device comprising a microscope objective (40), arranged to collect outgoing light comprising scattered light and unscattered light, the scattered light and the unscattered light following a single optical path and interfering with one another; - a computing device for image acquisition and processing, configured to: acquire a first temporal series (S1) of images determining an image (3) representative of the intensity of the unscattered light over a given period of time; subtract the representative image (3) from each image (2) of the first temporal series (S1) to obtain a second series (S2) of differential images (4) and obtain a normalised differential image from the normalised differential image (6); determine, by calculating an electromagnetic propagation, a three-dimensional distribution (D1) of a scattered field, from the three-dimensional distribution (D1) of a scattered field; identify nanoparticles as sources of the scattered intensity and determine their three-dimensional positions in the sample (1).

Description

Optical device for analyzing a sample comprising nanoparticles suspended in a solution, with determination of a three-dimensional distribution of a scattered field

[0001] The invention relates to the field of analysis of solutions containing suspended nanoparticles.

[0002] In particular, the invention relates to the field of detection and characterization of nanoparticles by optical microscopy. The invention can notably be used for the detection of nanoparticles with a largest dimension between 10 and 300 nm. Such objects include metallic nanoparticles, nanometric pollutants, and other objects of biological and pharmaceutical interest, such as viruses, phages, nanovesicles, or lipid nanoparticles. Technological background

[0003] Microscopy techniques, and in particular dark-field microscopy, make it possible to detect the position of biological nanoparticles with a diameter greater than 60 nm suspended in a solution, to count them and to follow their trajectory, which gives access to their hydrodynamic diameter.

[0004] For sizes smaller than 60 nm, the signal is too weak because the amplitude of the scattered field is proportional to the volume of the nanoparticle (i.e., to the cube of the diameter). Thus, a tenfold reduction in the diameter of a nanosphere leads to a tenfold reduction in the signal strength. 3 of the diffuse amplitude and by a factor of 10 6 of the power diffused.

[0005] The "image" of a nanoparticle by these dark-field microscopy techniques is a diffraction spot on the camera which contains two pieces of information: the position of the nanoparticle in an image acquisition plane, given by the center of the diffraction spot, and the scattering cross section of the nanoparticle which is given by the light intensity measured at the center of the diffraction spot.

[0006] A technique widely used today to characterize biological nanoparticles larger than about 60 nm is called Nanoparticle Tracking Analysis and uses a dark-field microscope.

[0007] One way to detect nanoparticles with a characteristic size of 100 nm or less is to label them, for example, with fluorescent labeling. However, this method quickly reaches its limits because fluorophores degrade rapidly over time. The effect of fluorescent labeling is therefore limited, restricting the duration of observations. The quality of detection is also affected, as the temporal resolution of the images is limited. Finally, this technique requires advance preparation for the fluorescent labeling.

[0008] Various optical microscopy techniques allow the detection of nanoparticles by elastic scattering without the need for prior labeling, notably dark-field and interferometric techniques. The latter produce a signal that decays with the volume, rather than the square of the volume, thus enabling the detection of smaller objects. Among these, the most common are iSCAT (interferential scattering) and COBRi (coherent bright-field microscopy). They can detect nanoparticles of 10 nm or even smaller (for example, 60 kDa proteins) that are adsorbed onto a microscope slide.

[0009] iSCAT-type techniques have been used to measure the mass of nanoparticles. It has been shown that the mass of a biological nanoparticle is proportional to the intensity of its scattering. This measurement can be used to distinguish between viruses, and more generally between nanoparticles used as vectors, and between empty and filled nanoparticles. The challenge here is to perform a measurement with a relative accuracy better than 10% in order to distinguish nanoparticles whose loading exceeds 10% of the vector mass.

[0010] Another technique called iNTA is described, for example, in document WO 2023 / 083433. It uses a bright-field microscope that detects nanoparticles in solution and measures both the scattered power, providing access to their scattering cross sections, and their trajectories, from which the hydrodynamic diameter can be deduced. It is then possible to determine the nanoparticle's refractive index. The smallest nanoparticles detected are polystyrene nanoparticles with a diameter of 40 nm and gold nanoparticles with a diameter of 10 nm. This corresponds to biological nanoparticles with a diameter of 60 nm.

[0011] A central challenge in interferometric and dark-field techniques is the signal-to-noise ratio. Their objective is to detect a signal of interest, which is light scattered elastically by a nanoparticle. However, various types of noise affect the detection of this signal.

[0012] The difficulty in detecting biological nanoparticles with a characteristic size of less than 60 nm lies in extracting the light scattered by the nanoparticle from the background noise of the acquired image.

[0013] Indeed, there is considerable technical noise related to fluctuations in the measurement system. Furthermore, the incident light source used in a microscope is subject to intensity fluctuations called photon noise, which are intrinsic to the physical process of light emission. These intensity fluctuations vary as the square root of the light source's intensity. If we measure N photons on average, the standard deviation of the measurement around N is N 1 / 2 and the signal-to-noise ratio is N -1 / 2 In order to collect a large number of photons, it is necessary to illuminate with a very bright source.

[0014] In practice, the light source used is a laser. This is the source of the second source of noise. Since the heterogeneities of the surrounding medium (scratches, roughness, and nanoparticles on the microscope's various optics) are much more intense scattering sources than the nanoparticles being imaged, a pattern of random interference, called speckle, appears on the camera. This pattern exhibits spatial fluctuations and obscures the desired signal. Thus, the signal is lost in both this background, called speckle noise, and the photon noise.

[0015] In iSCAT techniques, the near-field intensity illuminating a nanoparticle adsorbed onto an interface exhibits spatial fluctuations due to surface roughness and is therefore stationary. This is a source of error that limits the accuracy of mass measurements. Detecting nanoparticles in free diffusion within the solution avoids this problem and allows for averaging local illumination intensities along a trajectory, thus eliminating this systematic error.

[0016] The scattered field and the reference field detected by a bright-field microscope produce interference patterns that constitute a hologram of the nanoparticles. Many authors have created holograms of nanoparticles using a reference laser beam illuminating the camera to interfere with the scattered field. However, phase fluctuations between the scattered and reference fields limit the sensitivity of these devices. In contrast, as explained below, the invention uses an in-line setup where the paths followed by the two fields are identical to overcome this difficulty. It is then possible to digitally reconstruct the field scattered by the nanoparticles.

[0017] There is also a technique called Digital in-line Holography Microscopy (DIHM) which benefits from the interferometric stability of an "in-line" setup where the paths followed by the two fields are identical but which does not have a microscope objective to collect the scattered light and does not allow the detection of particles smaller than 100 nm as indicated in the document "Advancing the science of dynamic airborne nanosized particles using Nano-DIHM" Pal et al., Communications Chemistry vol. 4,p170 (2021), doi:10.1038 / s42004-021-00609-9.

[0018] One motivation underlying the invention is to provide an optical device for analyzing a sample comprising nanoparticles suspended in a solution, enabling the determination of the three-dimensional position of the suspended nanoparticles and providing accurate localization and hydrodynamic diameter measurements for biological nanoparticles with sizes ranging from 20 to 300 nm. A second motivation is to accurately determine the scattered intensity in order to deduce the mass or refractive index of the nanoparticle.

[0019] According to one embodiment, the invention provides an optical analysis device comprising: - a light source emitting illumination light adapted to illuminate a sample placed on a support, said sample comprising nanoparticles suspended in a solution, - an optical detector adapted to produce digital images comprising a pixel matrix in an image acquisition plane, - an optical device comprising a microscope objective, the optical device being arranged to collect outgoing light and direct the outgoing light from the sample to the optical detector to form an image of the sample on the optical detector, the outgoing light comprising scattered light corresponding to a portion of the illumination light scattered by the nanoparticles of the sample and unscattered light corresponding to a non-scattered portion of the illumination light.scattered and unscattered light following the same optical path, and interfering together to form a hologram of the scattering nanoparticles; - a computer image acquisition and processing device configured to: acquire a first time series of images produced by the optical detector, the first time series comprising a plurality of images of the sample acquired at successive times over time, each pixel of said image of the first time series having a light intensity measured by the optical detector at one of the successive times; determine a representative image of the intensity of the unscattered light over a given period of time, each pixel of said representative image having a light intensity equal to a value of a statistical indicator of the light intensities of the corresponding pixel of the images of the first time series.subtract said representative image from at least one or each image of the first time series to obtain a differential image or a second series of differential images and from at least one said or each or some differential image(s) and the representative image of the intensity of the unscattered light obtain at least one normalized differential image or a time series of normalized differential images, said or each normalized differential image being representative of the real part of the field scattered by the nanoparticles at one of the successive instants, from at least one said or each or some normalized differential image(s), determine by an electromagnetic propagation calculation a three-dimensional distribution of scattered field comprising images of propagated scattered field,The images of the propagated scattered field being representative of the amplitude and phase of the field scattered by nanoparticles in planes parallel to the image acquisition plane and located at a plurality of depths in a depth direction perpendicular to the image acquisition plane, in said three-dimensional distribution of scattered field, identify nanoparticles as sources of the scattered field and determine their three-dimensional positions in the sample.

[0020] Thanks to the device described above, it is possible to identify the nanoparticles present in a three-dimensional sample and to determine their positions in space with satisfactory accuracy.

[0021] In summary, the optical analysis device employs a digital holographic microscopy technique to detect and individually characterize nanoparticles, including biologically relevant nanoparticles with masses between 1 MDa and 100 MDa, in a droplet of solution deposited on a glass slide. Biologically relevant nanoparticles with masses in this range correspond to nanospheres with diameters generally between 20 and 300 nm.

[0022] In practice, the microscope objective of the optical device is arranged to collect outgoing light and direct the outgoing light from the sample to the optical detector.

[0023] In addition, it is possible to determine other characteristics of the sample, as described below.

[0024] The optical analysis device provides the position (x, y, z) of each nanoparticle at a given time, as well as the intensity of light scattered by that nanoparticle. A series of measurements at different times allows for the creation of a video and the reconstruction of the nanoparticle's trajectory due to Brownian motion. This makes it possible to deduce the nanoparticle's hydrodynamic diameter.

[0025] This optical analysis device and the corresponding analysis process make it possible to meet several needs for the characterization of biological nanoparticles such as viruses, phages, vesicles or lipid nanoparticles: - measurement of the concentration of nanoparticles by counting in a given volume, - measurement of the state of aggregation of nanoparticles, - measurement of the hydrodynamic diameter of nanoparticles - measurement of the cargo of a nanoparticle used as a vector for biological applications from the measurement of the diffusion intensity.

[0026] A particularly advantageous application of the invention concerns the ability to determine whether nanoparticles used as drug carriers are full or not, that is, whether they contain the molecules they are to transport to a biological target. This determination is also called "cargo measurement." For this purpose, the invention provides a highly sensitive optical analysis device with a high mass / scattering cross-section measurement resolution, which does not exist in the prior art.

[0027] The interference between scattered and unscattered light produced for the analysis device allows the formation of a hologram of the scattering nanoparticles, which allows the nanoparticles to be focused in order to measure their intensity / mass with precision.

[0028] Another property of the optical analysis device is its ability to measure the intensity of freely scattering nanoparticles using a holographic setup immune to phase fluctuations, thus maintaining high sensitivity. The optical analysis device allows the intensity scattered by the same nanoparticle to be measured many times along a long trajectory thanks to holographic reconstruction in an extended volume. This averaging reduces the effect of illumination inhomogeneities and increases the accuracy of the measurements.

[0029] Sensitivity is defined as the smallest detectable scattering cross-section of an optical device. This can be expressed as the smallest detectable mass or diameter for a given material.

[0030] The resolution is given by DX / <x>where DX is the variance of the Gaussian distribution of measurements of a quantity X, which can be mass or diameter and <x>is the average value of the quantity. This characterizes the ability of the optical analysis device to distinguish between different particles.

[0031] Silver nanoparticles of 7 nm are detectable.

[0032] The optical analysis device described above has a sensitivity that allows the detection of biological nanoparticles in water with sizes between 20 and 60 nm through the use of a bright-field setup.

[0033] In practice, the device is programmed to deduce from said three-dimensional distribution of scattered field a three-dimensional intensity distribution, and, in said three-dimensional intensity distribution, identify the nanoparticles and determine their three-dimensional positions in the sample.

[0034] According to one embodiment, the normalized differential image is obtained by performing a step of dividing the light intensity of each pixel of the differential image by the square root of the light intensity of a corresponding pixel of the image representing the intensity of the unscattered light.

[0035] According to one embodiment, the computer image acquisition and processing device is configured to filter said or each differential image through a high-pass filter to produce a filtered differential image.

[0036] According to one embodiment, the computer image acquisition and processing device is configured to obtain the normalized differential image by performing a step of dividing the light intensity of each pixel of the filtered differential image by the square root of the light intensity of a corresponding pixel of the representative image.

[0037] According to one embodiment, the electromagnetic propagation calculation performed to determine the scattered field distribution corresponds to propagation in the depth direction.

[0038] According to one embodiment, the electromagnetic propagation calculation includes the following steps: calculate a Fourier transform of the scattered field image, multiply the Fourier transform by a propagation term, and calculate an inverse Fourier transform of the Fourier transform multiplied by the propagation term.

[0039] Such calculations can be performed efficiently using known numerical methods.

[0040] According to one embodiment, the computer image acquisition and processing device is configured so that each image of the propagated scattered field represents the amplitude and phase of the field scattered by the nanoparticles in said planes parallel to the image acquisition plane at a plurality of depths summed with the image of the conjugate field propagated in these parallel planes.

[0041] According to one embodiment, the computer image acquisition and processing device is configured to estimate, from a normalized differential image of the time series of normalized images, an image of twice the real part of the field scattered by the nanoparticles at a said instant.

[0042] According to one embodiment, the step of identifying nanoparticles as sources of the scattered field involves searching for areas of the scattered field distribution exhibiting a light intensity greater than a predetermined intensity threshold or searching for areas of the scattered field distribution exhibiting a local maximum in light intensity. The center of this area gives the position of the nanoparticle.

[0043] According to one embodiment, the computer image acquisition and processing device is further configured to count the identified nanoparticles and determine a concentration of nanoparticles in the sample.

[0044] According to one embodiment, the computer image acquisition and processing device is further configured to determine a scattering cross section for at least one or each nanoparticle.

[0045] According to one embodiment, the computer image acquisition and processing device is further configured to determine a trajectory for said nanoparticle or each nanoparticle during said successive instants.

[0046] According to one embodiment, the computer image acquisition and processing device is programmed to iterate at several successive times the steps of: determining the three-dimensional distribution of scattered field, identifying nanoparticles in the three-dimensional distribution of scattered field and determining their positions in three dimensions, and allocating successive positions to said nanoparticle.

[0047] According to one embodiment, the computer image acquisition and processing device is further configured to determine a hydrodynamic diameter for each nanoparticle from the nanoparticle trajectory.

[0048] According to one embodiment, the computer image acquisition and processing device is further configured to count nanoparticles whose hydrodynamic diameter is within a range of diameter values ​​and to determine a concentration in the sample of nanoparticles whose hydrodynamic diameter is within said range.

[0049] According to one embodiment, the computer image acquisition and processing device is further configured to count nanoparticles whose mass or scattering cross section or index is within a range of diameter values ​​and to determine a concentration in the sample of nanoparticles whose hydrodynamic diameter or scattering cross section or index is within said range.

[0050] According to one embodiment, the computer image acquisition and processing device is further configured to determine an index of said nanoparticle or each nanoparticle.

[0051] According to one embodiment, the computer image acquisition and processing device is configured to determine a scattering cross section and / or a mass and / or a refractive index of said nanoparticle or each nanoparticle from a value statistically representative of the values ​​of a quantity derived from the scattered intensity measured for the nanoparticle along its trajectory.

[0052] The quantity derived from the scattered intensity can be the measured scattered intensity itself, the scattering cross section measured from the scattered intensity, or another quantity calculated from the measured scattered intensity.

[0053] According to one embodiment, the statistically representative value is a median value.

[0054] According to one embodiment, the computer image acquisition and processing device is further configured to count the identified nanoparticles whose hydrodynamic diameter and scattering cross section or mass or refractive index is within certain intervals, and to determine a sample concentration within said intervals.

[0055] According to one embodiment, the light source illuminates the sample with an angle of incidence of less than 15 degrees.

[0056] According to one embodiment, the nanoparticles are in solution in water.

[0057] According to one embodiment, the nanoparticles have dimensions between 20 and 300 nanometers.

[0058] According to one embodiment, the undiffused light is reflected off the sample support before reaching the sample.

[0059] According to one embodiment, unscattered light is transmitted through the sample without being scattered by the nanoparticles.

[0060] According to another embodiment, the invention provides a method for analyzing a sample comprising: - illuminating the sample with a light source emitting illumination light, the sample being placed on a support, said sample comprising nanoparticles suspended in a solution placed on a support; - collecting outgoing light and directing the outgoing light from the sample to an optical detector with an optical device comprising a microscope objective to form an image of the sample on the optical detector, the outgoing light comprising scattered light corresponding to a portion of the illumination light scattered by the nanoparticles of the sample and unscattered light corresponding to an unscattered portion of the illumination light, the scattered and unscattered light following the same optical path and interfering with each other; producing digital images with the optical detector.digital images comprising a pixel matrix in an image acquisition plane, with a computer image acquisition and processing device, perform the steps of: acquiring a first time series of images produced by the optical detector, the first time series comprising a plurality of images of the sample acquired at successive times over time, each pixel of said image of the first time series having a light intensity measured by the optical detector at one of the successive times, determining a representative image of the intensity of the unscattered light over a given period of time, each pixel of said representative image having a light intensity equal to a value of a statistical indicator of the light intensities of the corresponding pixel of the images of the first time series,subtract said representative image from at least one or each or some image(s) of the first time series to obtain a differential image or a second series of differential images and from at least one said or each or some differential image(s) and the representative image of the intensity of the unscattered light, obtain a normalized differential image or a time series of normalized differential images, the normalized differential image being representative of the real part of the field scattered by the nanoparticles at one of the successive instants, from at least one said or each or some normalized differential image(s), determine by an electromagnetic propagation calculation a three-dimensional distribution of scattered field comprising images of propagated scattered field,Since the images of the propagated scattered field are representative of the amplitude and phase of the field scattered by nanoparticles in planes parallel to the image acquisition plane and located at a plurality of depths in a depth direction perpendicular to the image acquisition plane, in said three-dimensional distribution of the scattered field, nanoparticles can be identified as sources of the scattered field and their three-dimensional positions in the sample determined. In practice, the microscope objective is arranged to collect the outgoing light and direct the outgoing light from the sample to the optical detector.

[0061] According to one embodiment, the normalized differential image is obtained by performing a step of dividing the light intensity of each pixel of the unmodified differential image by the square root of the light intensity of a corresponding pixel of the representative image.

[0062] According to one embodiment, said or each differential image is filtered by a high-pass filter to produce a filtered differential image.

[0063] According to one embodiment, the normalized differential image is obtained by performing a step of dividing the light intensity of each pixel of the filtered differential image by the square root of the light intensity of a corresponding pixel of the representative image. Brief description of the figures

[0064] The invention will be better understood, and other objects, details, features and advantages thereof will become more apparent from the following description of several particular embodiments of the invention, given solely by way of illustration and not limitation, with reference to the accompanying drawings.

[0065] Lare represents schematically an optical device for analyzing a sample comprising nanoparticles in suspension according to a first embodiment operating by reflection.

[0066] Lare represents schematically an optical device for analyzing a sample comprising nanoparticles in suspension according to a second embodiment operating in transmission.

[0067] The diagram schematically represents a block diagram of the implementation steps of a method for analyzing a sample containing suspended nanoparticles.

[0068] This schematically represents a series of raw images captured by the image capture device of the optical analysis system, with the pixel light intensity I in arbitrary units (au) represented as a grayscale value in the (x, y) plane of the image, and the z-coordinate of the acquired image (Im Nb). The x and y coordinates of the image plane are graduated in micrometers (microns). The arbitrary unit used corresponds to a digital unit of the camera. Here, the camera's digital unit is a 12-bit value ranging from 1 to 4096.

[0069] This is a graph showing the effect of normalizing the power fluctuations of the illumination laser beam: the variations of the spatial mean intensity (SMI) in arbitrary units (AU) (on the ordinate) as a function of the number of the captured image (Im Nb) on the abscissa, are represented with (dashed lines, curve C2) and without (solid lines, curve C1) intensity normalization.

[0070] The diagram schematically represents the series of images after subtracting an average image. The light intensity I, in arbitrary units, of the differential images in this series is represented in grayscale in the x, y plane, and the image number Im Nb is noted along the third axis. The x and y axes are graduated in microns.

[0071] Figure 1 schematically represents the series of images after normalization. The light intensity I, in arbitrary units, of the normalized differential images of this series is represented in grayscale in the x, y plane, and the image number Im nb is noted along the third axis. The x and y axes are graduated in microns.

[0072] This is a schematic representation of a three-dimensional scattered field distribution determined from an image in the series of images. The images are intensity images, which therefore represent the square of the scattered field magnitude. The light intensity I in arbitrary units of the propagated field images in this series is represented in grayscale in the x, y plane, and the order number of the scattered field (Ch Nb) in the distribution, corresponding to a depth in the sample, is denoted along the third axis z prop The x and y axes are graduated in micrometers (microns).

[0073] This is a schematic representation of propagation phase masks. The order number of the mask in the distribution, corresponding to a depth in the sample, is noted along the z-axis. prop The gray levels represent the phase (Ph) in radians of the phase mask. The K axes x and K y correspond to wave vectors in micrometers -1 .

[0074] This is a schematic representation of the three-dimensional distribution of the scattered field after finding the local maxima of light intensity at each depth represented on the z-axis. prop The detected local intensity maxima are shown in white on a black background. The x and y axes are graduated in microns. The white spots represent areas identified on each propagated field image as containing a nanoparticle.

[0075] This represents the trajectory of a nanoparticle within the sample. The disk diameter is proportional to the light intensity scattered by a nanoparticle. Variations in intensity are due to various measurement noises, including photon noise, and local illumination fluctuations (speckle), which can be corrected. The x, y, and z axes represent the sample volume and are graduated in micrometers.

[0076] This is a schematic representation of a histogram of the number of occurrences Oc Nb of the signal value S measured for a set of nanoparticles from a sample along a 10-point trajectory. The histogram is fitted by a Gaussian curve, giving a mean signal of 3.71 x 10 -3 and a standard deviation equal to 0.46x10 -3 .

[0077] This is a schematic representation of a histogram of the number of occurrences Oc Nb of the signal value S measured for a set of nanoparticles from a sample along a trajectory of 50 points. The histogram is fitted by a Gaussian curve, giving a mean signal of 3.75 x 10 -3 and a standard deviation equal to 0.22x10 -3 .

[0078] These two figures illustrate that longer trajectories allow for a longer average of the measurement of the intensity scattered by a nanoparticle, and for obtaining better accuracy in determining this intensity and therefore the cross section of the nanoparticle, as shown by the lower standard deviation of the Gaussian fitted with the longer trajectory.

[0079] This is a schematic representation of a nanoparticle mass histogram from a sample, allowing comparison of the mass distribution of nanoparticles in a biological sample containing viruses (CCMV) and in a sample containing silica nanoparticles (S i O2). The ordinate gives the number of nanoparticles (Part Nb) identified and the abscissa the mass of the nanoparticle in MDa. Silica nanoparticles are centered around 12 MDa while viruses are centered around 5 MDa.

[0080] This is a schematic representation of the results of three successive measurements on three different types of polystyrene nanospheres with diameters of 30, 40, and 50 nm (poly 30, poly 40, and poly 50 in the figure). The figure shows, in the lower right corner, for each measurement and each nanoparticle, a measurement of its scattered light intensity signal S (ordinate) as a function of its hydrodynamic diameter (Dh) in nanometers (abscissa). The dashed line indicates the function x 3 The upper part of the figure shows a histogram of the percentage of nanoparticles identified for each type of nanosphere as a function of the hydrodynamic diameter Dh (x-axis). The left part of the figure shows a histogram of the percentage of nanoparticles identified for each type of nanosphere (horizontal axis) as a function of the signal intensity S (vertical axis).

[0081] We describe below embodiments of an optical device for analyzing a sample containing nanoparticles suspended in a solvent.

[0082] This optical analysis device enables high-performance detection of light scattered by very small nanoparticles, for example, those with characteristic dimensions between 20 and 300 nm, and precise characterization of these nanoparticles. Specifically, the optical analysis device allows for the determination of the hydrodynamic diameter and mass or refractive index of a plurality or of each detected nanoparticle and the construction of a size and refractive index distribution. The optical analysis device offers improved sensitivity and resolution compared to prior art devices.

[0083] The optical analysis device described below is a holographic microscope.

[0084] Figures 1 and 2 represent two embodiments of the optical analysis device. Lamontre shows a first embodiment in which the optical analysis device 100 includes a light source 10 illuminating the sample in reflection, while Lamontre shows a second embodiment in which the optical analysis device 200 includes a light source 10 illuminating the sample in transmission.

[0085] Figures 1 and 2 use identical references for identical or corresponding elements.

[0086] The optical analysis device 100, 200 comprises: - a support 20 adapted to receive the sample 1 comprising nanoparticles in suspension, - the light source 10 which emits an illumination light adapted to illuminate the sample placed on the support 20, - an optical detector 30 adapted to produce digital images comprising an array of optical sensors in an image acquisition plane, - an optical device comprising a microscope objective 40, the optical device being arranged to collect outgoing light and direct the outgoing light from the sample to the optical detector 30 to form an image of the sample on the optical detector 30.

[0087] The sample support 20 is a transparent support. For example, it is a glass slide. The support 20 has two parallel, opposite principal faces: one facing upwards into the Earth's gravitational field and the other facing downwards.

[0088] Alternatively, the support can consist of a glass slide with thin-film deposits that modify the reflectivity of the slide / water interface. This is notably the case in the 100 optical analysis device, which operates by reflection. This allows for lower reflectivity, the use of a more intense laser, and increased device sensitivity.

[0089] Sample 1 is a droplet of solution containing nanoparticles suspended in a solvent. Sample 1 is deposited onto the receiving face of support 20.

[0090] Suspended nanoparticles can include organic or inorganic nanoparticles. Examples of organic nanoparticles include organic molecules, biological objects such as viruses, vesicles, nanolipids, or phages. Inorganic nanoparticles include metallic nanoparticles.

[0091] The solution containing the nanoparticles is, for example, an aqueous solution. The solvent is, for example, water. Alternatively, the solution may contain a solvent other than water.

[0092] The light source 10 is a laser source. This light source illuminates sample 1 with an incident light beam 110, which is an incident laser beam. The laser source is arranged to emit an incident laser beam with a wavelength, for example, between 400 nanometers and 1300 nanometers. The incident light beam 110 can be a wide beam illuminating a large area of ​​sample 1. Preferably, the incident light beam 110 is a collimated beam.

[0093] In one embodiment, the incident light beam 110 is mobile and sweeps across the area of ​​interest of the sample 1.

[0094] The laser source has the advantage of providing the brightest illuminating light, which ensures optimal sensitivity.

[0095] Alternatively, the light source may include or be composed of a light source other than a laser. The light source may, in particular, include a light-emitting diode (LED) or a superluminescent diode. The illuminating light consists of this incident light beam 110.

[0096] The microscope objective 40 is an immersion objective onto which the support 20 is mounted. It presents a Fourier plane 41.

[0097] The light source 10 illuminates the sample support and therefore the sample 1 under normal or near-normal incidence.

[0098] In the embodiments represented and described here, the light source 10 illuminates the sample under normal incidence.

[0099] However, lighting with a normal angle of incidence can generate back reflections on the optical elements of the device which can be bothersome.

[0100] To avoid these back reflections, it is possible to design the light source to illuminate the sample holder, and therefore the sample, at a small, non-zero angle of incidence less than 10 degrees. It is also possible to design the light source to illuminate the sample holder at normal incidence, but with a slight lateral offset from the optical axis AO. In other words, the light source is focused at the center of the Fourier back plane of the objective lens and passes through it at a non-zero mean angle of incidence.

[0101] Two configurations can be considered, as shown in figures 1 and 2.

[0102] In the first embodiment, the light source 10 illuminates the sample 1 by reflection. The light source is placed on the side of the support 20 opposite to the receiving face of the support receiving the sample 1.

[0103] The light beam emitted by the light source 10 passes through a first lens 60 and is then reflected towards the optical device and the sample 1 by a polarization splitter 50 centered on the optical axis AO.

[0104] The first lens 60 is a converging lens. It allows for precise control of the field and a well-defined phase. The polarizing splitter 50 has a semi-reflective surface, positioned so that the angle of incidence of the beam emitted by the light source 10 on the polarizing splitter 50 is 45°.

[0105] The polarization splitter blade 50 reflects a linear polarization component of the light beam emitted by the light source 10 but allows another linear polarization component to pass through in transmission.

[0106] The light beam emitted by the light source 10 is reflected on the polarizing splitter 50 and then directed towards the microscope objective 40.

[0107] The optical device 100 further includes a delay plate, which, in one embodiment, is a quarter-wave plate 42. The delay plate introduces a phase difference between two polarization components of the beam transmitted by this delay plate. The microscope objective 40, the sample 1, and the quarter-wave plate 42 are aligned along the optical axis AO of the microscope objective 40 and centered on it. The microscope objective 40 is positioned between the support 20 and the quarter-wave plate 42.

[0108] The light beam emitted by the light source 10 passes through the quarter-wave plate 42 then the microscope objective 40, the support 20 and finally the sample 1. The incident light beam 110 arrives on the support 20 on the side opposite the sample, the transmitted part is the transmitted light beam 111 through the sample.

[0109] The microscope objective 40 is arranged to collect outgoing light and direct outgoing light from the sample to the optical detector 30.

[0110] The microscope objective 40 can be an immersion objective, comprising an immersion oil having a refractive index identical to a refractive index of glass.

[0111] The outgoing light corresponds to the light beam emitted by sample 1 after interaction between the incident light beam 110 and sample 1. The outgoing light comprises scattered light, corresponding to a portion of the illumination light scattered by the nanoparticles of sample 1, and unscattered light, corresponding to an unscattered portion of the illumination light. The scattered and unscattered light follow the same optical path and interfere with each other.

[0112] Here the undiffused light includes for example a transmitted light beam 111 by the sample 1 and a reflected light beam 113 by the support 20.

[0113] The light scattered by the nanoparticles of sample 1 is collected by the microscope objective 40, passes through the quarter-wave plate 42 and is transmitted by the beam splitter 50. The collected scattered light forms a scattered light beam 112. A second lens 70 conjugates the optical detector 30 with a plane placed a few micrometers above the glass-water interface, typically 4-10µm above.

[0114] In other embodiments, the optical detector can also be combined with the glass / water interface or combined with a plane located in the thickness of the glass plate forming the support.

[0115] The second lens 70 is a converging lens which also directs the reflected light beam 113 towards the optical detector 30. The scattered light beam 112 propagates over a wider range of angles than the reflected light beam 113.

[0116] The second lens 70 and the optical detector 30 are also aligned on the optical axis AO.

[0117] The second converging lens 70 can be a tube lens. The image acquisition plane of the optical detector 30 can be placed at a focal length of the second lens 70.

[0118] The first lens 60 and the microscope objective 40 allow control of the illuminated field.

[0119] The detection system comprising the second lens 70 and the optical detector 30 collects the reflected light beam 113 and the scattered light beam 112 and makes the image on the optical detector 30 of a plane placed a few micrometers above the glass-water interface.

[0120] The optical detector 30 includes, for example, an image capture device such as a camera or a photographic sensor, for example, of the CMOS or CCD type. Sensors limited to between 10,000 and 100,000 electrons per pixel can be used. A sensor with 100,000 electrons per pixel is preferred. The device preferably includes a sensor with the largest possible well capacity.

[0121] The optical detector 30 includes a memory to record several successive images. The optical detector 30 records the images in an image acquisition plane that corresponds to the plane in which the sensors of the optical detector 30 are arranged. The optical detector 30 comprises, for example, a sensor array where each sensor corresponds to a pixel of the image acquired by the optical detector 30.

[0122] The image acquisition plane is conjugate to a plane located approximately 10 µm above the lamella. This conjugate plane of the image acquisition plane is the plane imaged by the optical detector 30.

[0123] The scattered light beam 112 and the reflected light beam 113 interfere, thus producing a hologram.

[0124] The optical analysis device 100 is configured so that the scattered light beam 112 and the reflected light beam 113 propagate along the same optical path, thereby eliminating the influence of phase fluctuations along this path on the hologram.

[0125] In the second embodiment of the optical analysis device shown in the figure, the light source 10 of the optical analysis device 200 illuminates the sample 1 in transmission. The light source 10 is placed on the side of the support 20 opposite to the receiving face of the support receiving the sample 1. In addition, two lenses 61, 62, which form a telescope, are provided between the light source 10 and the sample.

[0126] The light beam emitted by the light source 10 directly illuminates the sample 1 through the first two lenses 61, 62. It arrives on the support 20 on the side of the sample 1.

[0127] It passes successively through sample 1, support 20, microscope objective 40, then the second lens 70. The second embodiment of the device does not include a quarter-wave plate.

[0128] As in the first embodiment, the outgoing light corresponds to the light beam emitted by sample 1 after interaction between the incident light beam 110 and sample 1. Here, the outgoing light comprises scattered light, corresponding to a portion of the illumination light scattered by the nanoparticles of sample 1, and unscattered light, corresponding to an unscattered portion of the illumination light. The scattered and unscattered light follow the same optical path and interfere with each other.

[0129] Here the undiffused light includes the transmitted light beam 111 by sample 1 without being scattered.

[0130] As in the first embodiment, the light scattered by the nanoparticles of the sample 1 is collected by the microscope objective 40. The collected scattered light forms a scattered light beam 112. The second lens 70 combines the optical detector 30 with a plane placed a few micrometers above the interface between the support and the sample, i.e. here the glass-water interface.

[0131] The second lens 70 directs the transmitted light beam 111 to the optical detector 30.

[0132] The second lens 70 and the optical detector 30 are also centered on the optical axis AO.

[0133] The first lenses 60 and 61 allow control of the illuminated field. They form a telescope which allows for an illuminated field of the correct size as well as a very regular wavefront.

[0134] The detection system comprising the second lens 70 and the optical detector 30 collects the transmitted light beam 111 and the scattered light beam 112 and makes the image of them on the optical detector 30.

[0135] The optical detector 30 is identical to that of the first embodiment.

[0136] The scattered light beam 112 and the transmitted light beam 111 interfere, thus producing a hologram.

[0137] The optical analysis device 200 is configured so that the scattered light beam 112 and the transmitted light beam 111 propagate along the same optical path, thereby eliminating the influence of phase fluctuations along this path on the hologram.

[0138] The optical analysis device 100; 200 of the two embodiments is further configured to perform a digital processing described below to reconstruct in space the field scattered by the nanoparticles present in the sample and having scattered the incident light beam.

[0139] The camera of the optical detector 30 receives a final signal which is a superposition of the reflected light beam 113 or transmitted light beam 111 and the scattered light beam 112.

[0140] Capturing multiple images at successive times allows us to reconstruct the trajectories of the nanoparticles within the sample during the measurement period and to measure their hydrodynamic diameter. The movement of the nanoparticles in the solution is Brownian motion.

[0141] Furthermore, the light intensity scattered by a given nanoparticle is proportional to its scattering cross-section and the local illumination intensity. Using a laser light source reduces photon noise and allows for the detection of small nanoparticles thanks to its high illumination intensity. However, this results in spatial fluctuations in illumination (speckle).

[0142] As explained in more detail below, the optical analysis device 100;200 exploits the temporal stability of the setup and the time-dependent nature of the images related to the Brownian motion of the nanoparticles in sample 1 to determine the illumination field averaged over several acquired images and subtract it from each image in order to eliminate speckle noise.

[0143] Furthermore, the optical analysis device 100; 200 can be programmed to determine an average value of the light intensity scattered by one of the nanoparticles along its trajectory in the sample during the acquisition period. This makes it possible to improve the signal-to-noise ratio of the measurement and reduce the effect of spatial fluctuations in illumination intensity.

[0144] The 100;200 optical analysis device enables the detection of nanoparticles, including biological objects with masses greater than 1 MDa. It can detect nanoparticles (NPs) of biological interest with masses between 1 MDa and 100 MDa and diameters between 20 and 300 nm. In general, the 100;200 optical analysis device is suitable for detecting nanoparticles with sizes between 10 and 300 nm.

[0145] The optical analysis device 100; 200 includes for this purpose a computer image acquisition and processing device configured to acquire a time series S1 of images 2 produced by the optical detector 30.

[0146] Lamontre such a series S1 of images 2 captured by optical detector 30.

[0147] Nanoparticles can be seen positioned at the center of concentric rings. The image contrast is proportional to the mass of the nanoparticle.

[0148] Sample 1, for example, is an aqueous solution containing silica nanoparticles with a diameter of 50 nm and a concentration of 10 9 nanoparticles / ml.

[0149] In practice, the optical detector 30 is a camera. The optical detector 30 records a film comprising the images 2. The film constitutes the time series S1 containing a plurality of images 2 of the sample 1 acquired at successive times over time.

[0150] Each pixel of an image 2 of the time series S1 records a light intensity measured by one of the sensors of the optical detector 30 at one of the successive instants at a corresponding position of the image acquisition plane of the optical detector 30.

[0151] The computer system for image acquisition and processing is also programmed to determine a representative image 3 of the intensity of the outgoing unscattered light over a given period of time.

[0152] Each pixel of said representative image 3 has a light intensity equal to a value of a statistical indicator of the set of light intensities of the pixel corresponding to the same position on the image acquisition plane of the optical detector 30 on the images 2 of the time series S1 recorded by the optical detector 30.

[0153] The statistical indicator is, for example, an average or a median.

[0154] In the example described below, each pixel of image 2 is normalized by the average intensity value so that all images have the same average value, thus compensating for spatial and temporal fluctuations in laser illumination. This provides a series of normalized images 4 of the S2 series ().

[0155] The intensity measured by the camera is written , whereE s is the scattered field (s for scattered) by the nanoparticle, and E r the undiffused field, referred to hereafter as the reference field of the outgoing light.

[0156] E r summer s are complex amplitudes that are functions of the position on the image acquisition plane of the optical detector 30 and of time, and are formally written ) And ) with (x,y) the position on the image acquisition plane, but to simplify reading, this will be omitted thereafter.

[0157] This intensity can be written as: using the notation for the conjugate of , And the phase difference between the reference field and the scattered field.

[0158] Here, the representative image 3 of the intensity of the outgoing unscattered light over a given period of time is an average image.

[0159] The computer system for image acquisition and processing is programmed to calculate the average image, which is the time average of the time series S1.

[0160] The intensity of each pixel in the average image is written as:

[0161] The computer image acquisition and processing device is programmed to subtract said representative image 3 from at least one image 2 and preferably from each image 2 of the time series S1 to obtain at least one differential image 4 and, preferably, a differential image 4 for each image 2 of the time series S1.

[0162] The intensity of each pixel in the differential image 4 is written as:

[0163] The set of differential images obtained forms a series S2 of differential images 4. The series S2 of differential images 4 obtained is represented on the. We can begin to see in these images the signal of the nanoparticles contained in sample 1.

[0164] Optionally, the computer system for image acquisition and processing is programmed to perform an intermediate denoising step. This is particularly useful when low spatial frequency fluctuations in light intensity, originating from fluctuations in the laser light source, are visible. These fluctuations are preferably filtered with a filter that only allows high spatial frequencies to pass through. Each differential image 4 is filtered by a high-pass filter, producing a filtered differential image 5. Preferably, a series S3 of filtered differential images 5 is obtained when filtering is applied to several differential images 4.

[0165] For said differential image 4 or said filtered differential image 5 or for each differential image 4 of the series S2 of differential images 4 or each filtered differential image 5 of the series S3 of filtered differential images 5, the computer image acquisition and processing device is then programmed to estimate the field scattered by the nanoparticles of sample 1.

[0166] To do this, it is programmed to divide the light intensity of each pixel of said or each differential image 4, or of said or each filtered differential image 5 when filtering is carried out, by the square root of the light intensity of the pixel of the representative image 3 corresponding to the same position on the image acquisition plane of the optical detector 30, so as to obtain, for said or each differential image 4 or for said or each filtered differential image 5, a normalized differential image.

[0167] The square root of the average intensity corresponds to the amplitude of the reference field. We have: where φ(E r ) is the phase of the reference field.

[0168] The set of successive normalized differential images forms a series S4 of normalized differential images, represented for example on the when this step is applied to several differential images or several filtered differential images.

[0169] Each normalized differential image is representative of the field scattered by the nanoparticles in the image acquisition plane of the optical detector 30.

[0170] More precisely, each normalized differential image is here an image of twice the real part of the scattered field. Consequently, the direction of propagation of the field along the axis is not distinguished, and therefore positions above and below the plane imaged by the optical device are not differentiated.

[0171] The normalized differential image of twice the real part of the field scattered by the nanoparticles of sample 1 at a given time is referred to hereafter as the "scattered field image 6". A series S4 of scattered field images 6 is thus obtained when this step is applied to several normalized differential images.

[0172] From said image or each scattered field image 6, the computer image acquisition and processing system is programmed to determine, by an electromagnetic propagation calculation, a three-dimensional scattered field distribution D1, representing the amplitude and phase of the field scattered by the sample nanoparticles in planes parallel to the image acquisition plane and at a plurality of depths in a depth direction perpendicular to the image acquisition plane. The planes parallel to the image acquisition plane are hereafter referred to as field reconstruction planes.

[0173] In the figures and in the rest of the description, the coordinates of the points in space are identified by x and y along two perpendicular axes of this image acquisition plane and by z along the depth direction.

[0174] Each three-dimensional scatter distribution D1 thus comprises a set of propagated scatter image 7 to reconstruct the scattered field in three dimensions. The pixels of each propagated scatter image 7 have a complex amplitude corresponding to the complex amplitude of the light scattered by the nanoparticles of the sample, estimated in a field reconstruction plane located at a given depth in the sample, i.e., at a given distance from the image acquisition plane.

[0175] Each field reconstruction plane of the propagated scattered field images 7 is parallel to the image acquisition plane of the optical detector 30.

[0176] A series S5 of three-dimensional D1 scatter field distributions is thus obtained (represented schematically on the) when this step is applied to several scatter field images 6.

[0177] The electromagnetic propagation calculation performed to determine one of the D1 scattered field distributions corresponds to propagation in the depth direction, for one of the scattered field images 6.

[0178] The electromagnetic propagation calculation includes, for example, the following steps: - calculate a Fourier transform of the image of the scattered field 6, - multiply the Fourier transform of the image of the scattered field 6 by a propagation term, and - calculate an inverse Fourier transform of the Fourier transform of the image of the scattered field 6 multiplied by the propagation term.

[0179] The computer system for image acquisition and processing is programmed to perform these steps, then determine the square of the absolute value of the inverse Fourier transform of the Fourier transform of the scattered field image 6 multiplied by the propagation term.

[0180] In practice, the scattered field Es is estimated at the depth z=z prop . z prop is the z-position at which we want to estimate the field:

[0181] With the estimation of the E field s in z prop , TF and TF -1 the Fourier transform and inverse Fourier transform operators and k x the wave vector propagating along the x-axis, k y the wave vector propagating along the y and k axes z The wave vector propagates along the depth direction. The refractive index of the medium through which the light propagates is denoted n. Here, for example, this is the refractive index of the solvent in the solution, such as water. is the wave number in a vacuum, with , c being the speed of light.

[0182] This technique is called a spectrum propagation technique, or in English "Angular Spectrum Propagation" because it considers that the field in z prop is a sum of plane waves propagating in different directions, each term of the Fourier transform being the complex amplitude of the plane wave propagating in the direction (k x , k y The expression for this phase mask is For And For The phase mask allows the selection of propagating waves by eliminating evanescent waves. The phase for several values ​​of z is represented graphically in.

[0183] The images of the propagated scattered field 7 are thus obtained. A 2D image extending in the (x, y) image acquisition plane taken at a given time t gives access to a 3D volume in a coordinate system (x, y, z) after this processing.

[0184] The propagated scattered field images 7 of the D1 distribution correspond to complex amplitude images more or less defocused with respect to the microscope objective setting.

[0185] These steps are preferably repeated for each diffused field image 6 of the S4 series.

[0186] In a subsequent step, the computer image acquisition and processing system determines the square of the norm of the complex amplitude of the field for each image of the propagated scattered field 7, so as to obtain for each image of the propagated scattered field 7 an image of the propagated field intensity 7a. A distribution D1a of images of propagated field intensity 7a is obtained () and an example is shown on the.

[0187] A series S5a of D1a distributions is thus obtained (represented schematically on the) when this step is applied to several images of propagated scattered field 7.

[0188] The propagated scatter field intensity images 7a represent several sections parallel to the image capture plane, distributed along the depth direction z, of a dark-field intensity volume. The quantity represented by each pixel of each propagated scatter field image 7 is the square of the absolute value of the field.

[0189] The calculation method described here allows us to determine the images of the propagated field summed with the image of the propagated conjugate field.

[0190] In practice, this means that a nanoparticle identified using the described method can be above or below the plane imaged by the optical device. Indeed, for a nanoparticle above the imaged plane, propagation towards positive z-coordinates will focus the E field. s and thus detect the nanoparticles that are sources of scattered light located on the positive z-axis, and propagation towards the negative z-axis will allow focusing the conjugate of the E field s and therefore to detect the nanoparticles that are sources of scattered light located on the negative z side.

[0191] A correction can be made to compensate for the propagated conjugate field.

[0192] In practice, for a largely defocused nanoparticle, for example located a few microns from the plane of the imaged sample, when the Es field is digitally focused by the propagation step, the conjugate field Es* is defocused even further. Thus, when the computer system for image acquisition and processing determines the square of the norm to form a dark-field image and measures the signal from this nanoparticle, the conjugate field has a negligible influence on the measurement because the corresponding conjugate field is extremely defocused and corresponds to a signal much weaker than the signal from the field of this nanoparticle.

[0193] For a nanoparticle located in the imaged plane and therefore perfectly in focus, when the computer device for image acquisition and processing determines the square of the norm and measures the corresponding intensity, the device measures the signal of the field Es and the conjugate field Es*, that is to say a signal twice as large as that which would be detected by another method not taking into account the conjugate field.

[0194] For a slightly defocused nanoparticle, for example, one less than one micron from the image plane, when the image acquisition and processing system performs the field propagation step, the nanoparticle's field Es is in focus and its conjugate field Es* is defocused, but not sufficiently so for its influence on the scattering cross-section measurement to be negligible. Thus, in this case, the Es field and the conjugate field Es* interfere with each other when the square of the field magnitude is calculated. The image acquisition and processing system then estimates an intensity greater than when the nanoparticle is in focus, and less than when it is largely defocused.

[0195] The variation in the detected signal related to the nanoparticle's position depends only on the particle's position in the z-depth direction. This variation is systematic and deterministic. The image acquisition and processing computer system is preferably programmed to correct for this variation, for example, through calibration.

[0196] The light source 10 of the optical analysis device 100; 200 is carefully collimated. We can thus consider that the phase of the reference field E r is uniform.

[0197] The computer image acquisition and processing device is programmed to, in said three-dimensional distribution D1a of scattered field intensity, identify nanoparticles as sources of the scattered field and determine their three-dimensional positions in sample 1.

[0198] To identify nanoparticles as sources of the scattered field, the computer image acquisition and processing device is programmed to search for areas where the intensity (i.e., the squared magnitude of the scattered field) is high enough to consider that there is a light-scattering nanoparticle located in that area.

[0199] In practice, the computer image acquisition and processing device is programmed to search for areas, for example pixels, of the propagated scattered field intensity distribution D1a exhibiting a light intensity greater than a predetermined intensity threshold or to search for areas, for example pixels, of the propagated scattered field intensity distribution D1a exhibiting a local maximum of light intensity.

[0200] The computer system for image acquisition and processing is programmed to detect the nanoparticles of the sample in each of the propagated scattered field intensity distributions D1a.

[0201] A schematic representation of a D2 distribution of 8 images showing the identified areas is shown in Figure 8. In these 8 images, the pixels of the areas identified as corresponding to a nanoparticle are represented in white, and the rest of the image is black.

[0202] Therefore, the positions of the nanoparticles at time t are identified and the intensities scattered by the nanoparticles are determined. The scattered intensities can be calibrated with reference objects of known scattering cross section.

[0203] We obtain a series S6 of distribution D2 () when these steps are applied to each three-dimensional distribution D1.

[0204] For example, a difference of Gaussian (DoG) is used. This technique consists of convolving each propagated field image of the D1 distribution with an image that is a difference of Gaussians. This highlights the areas of interest, which can then be identified by thresholding. This technique is described, for example, in the document I. Smal, M. Loog, W. Niessen and E. Meijering, "Quantitative Comparison of Spot Detection Methods in Fluorescence Microscopy," in IEEE Transactions on Medical Imaging, vol. 29, no. 2, pp. 282-301, Feb. 2010, doi: 10.1109 / TMI.2009.2025127.

[0205] Alternatively, the computer system for image acquisition and processing is programmed to use a technique called "matched filtering," which involves convolving the field image propagated by the pattern being sought. This technique optimizes the signal-to-noise ratio if the noise is Poissonian. This technique is also described, for example, in the document I. Smal, M. Loog, W. Niessen and E. Meijering, "Quantitative Comparison of Spot Detection Methods in Fluorescence Microscopy," in IEEE Transactions on Medical Imaging, vol. 29, no. 2, pp. 282-301, Feb. 2010, doi: 10.1109 / TMI.2009.2025127.

[0206] Finally, the computer system for image acquisition and processing can also be programmed to use inverse problem solving, in which, through optimization, the positions of the sources that create the scattered field distribution are found. Such a technique is also described, for example, in the document Alexey Brodoline, Nitin Rawat, Daniel Alexandre, Nicolas Cubedo, Michel Gross. 4D compressive sensing holographic microscopy imaging of small moving objects. Optics Letters, 2019, 44 (11), pp. 2827, 10.1364 / OL.44.002827, hal-02189448.

[0207] In this technique, the position of a source is first determined as a region. The image acquisition and processing computer can then be configured to determine the centroid of the pixels within this region. The three-dimensional position of this centroid is assigned to a nanoparticle in the sample. More precisely, the image acquisition and processing computer calculates the position of the particle's center as the centroid of the pixels in the region (positive pixels in D2 images), weighted by the intensity of the scattered field.

[0208] The computer system for image acquisition and processing is therefore programmed to identify and determine the position of at least one and preferably every nanoparticle of the sample present in the portion of the sample imaged by the device (block B1 of the).

[0209] The computer system for image acquisition and processing is then configured, for example, to count the nanoparticles identified as sources of the scattered field and to determine a concentration of nanoparticles in sample 1 (block B2 of the).

[0210] After identifying the areas corresponding to the nanoparticles in sample 1 and determining the three-dimensional position of each nanoparticle, the computer-based image acquisition and processing system counts the nanoparticles. It further determines the sample volume represented by the three-dimensional scattering field distribution based on the dimensions of the optical detector and the maximum depth of the field reconstruction planes. It can be programmed to deduce an instantaneous concentration of the nanoparticles, that is, the number of nanoparticles per unit volume of the sample estimated from a single scattering field image 6.

[0211] The size of the optical detector in the object plane of the sample imaged by the analysis device is, for example, approximately 20 micrometers long by 20 micrometers wide. The depth of the sample explored in the depth direction is, for example, approximately 4 micrometers.

[0212] The three-dimensional distribution D1 of the scattered field includes, for example, 100 images of the propagated scattered field.

[0213] In addition or alternatively, after identifying the areas corresponding to the nanoparticles of sample 1 and determining the three-dimensional position of each nanoparticle, the computer image acquisition and processing device can also be programmed to determine a scattering cross section for each identified nanoparticle (block B3 of the).

[0214] The computer system for image acquisition and processing is programmed to estimate the diffusion cross section of each nanoparticle identified using the fact that the intensity The center of its 3D diffraction spot is proportional to its scattering cross-section and the intensity .of the beam that illuminated it in the object plane, i.e.

[0215] either .

[0216] The diffraction spot corresponds to the area determined previously.

[0217] With a proportionality term α that depends on microscope parameters, such as its numerical aperture and where is the intensity that illuminates the nanoparticle in the object plane imaged by the optical detector 30. These quantities are known because the light source 10 is known, as well as all the parameters of the optical analysis device 100 or 200.

[0218] It should be noted is a function of x, y, but also z: indeed, the laser beam from the light source 10 is a Gaussian beam that varies relatively quickly in x and y, and necessarily varies slightly in z. In practice, we approximate that the intensity is equal to the average intensity of the average image 3 that was estimated previously from which all the highest spatial frequencies are removed.

[0219] For example, by approximation, we consider that the quantity is a function of x and y only. However, a correction taking into account the variations of three-dimensional approaches could be considered.

[0220] In practical terms, the computer system for image acquisition and processing is, for example, programmed to calculate the filtered intensity with * the convolution operator and N(d) a Gaussian of width d chosen smaller than the beam waist of the beam.

[0221] In addition or alternatively, after identification of the areas corresponding to the nanoparticles of sample 1 and determination of the three-dimensional position of each nanoparticle, the computer image acquisition and processing device can also be programmed to determine a trajectory for at least one and preferably for each nanoparticle during said successive instants (block B4 of the).

[0222] To this end, the computer system for image acquisition and processing connects the identified nanoparticles on the three-dimensional D1 scattering field distributions corresponding to successive volumes acquired at successive times, so as to track the same nanoparticle from one time t to another. The computer system for image acquisition and processing uses, for example, a nearest neighbor algorithm or Hungarian algorithm to connect the identified nanoparticles.

[0223] This allows for the identification of nanoparticles at each image acquisition time t, the determination of each nanoparticle's position along the film, and, most importantly, the acquisition of numerous measurements of the intensity scattered by each nanoparticle over time. Consequently, this enables the deduction of many measured values ​​of the scattering cross-section of that nanoparticle, allowing for an average that improves accuracy.

[0224] Lamontre shows the successive x, y, z positions of a nanoparticle tracked over time, as well as the light intensity scattered by the nanoparticle measured at each instant, represented by circles of varying sizes depending on the measured intensity.

[0225] From this data, the computer system for image acquisition and processing is programmed to determine an average concentration of nanoparticles in sample 1 using all the determined trajectories of the identified nanoparticles (block B5 of the diagram). The average concentration is equal to the number of nanoparticles detected on average over time in the digitally constructed volume, divided by this volume.

[0226] This is a schematic representation of a histogram of the number of occurrences Oc Nb of the signal value S measured for a set of nanoparticles from a sample along a 10-point trajectory. The signal S is represented on the x-axis. It is the intensity measured in arbitrary units. The histogram is fitted by a Gaussian curve, giving a mean signal of 3.71 x 10⁻³ -3 and a standard deviation equal to 0.46x10 -3 .

[0227] This is a schematic representation of a histogram of the number of occurrences Oc Nb of the signal value S measured for a set of nanoparticles from a sample along a 50-point trajectory. The signal S is represented on the x-axis. It is the intensity measured in arbitrary units. The histogram is fitted with a Gaussian curve, giving a mean signal of 3.75 x 10⁻¹⁰ -3 and a standard deviation equal to 0.22x10 -3 .

[0228] These measurements demonstrate the ability to improve measurement resolution by increasing the length of the trajectories. They also show that long trajectories allow for spatial and temporal averaging of illumination fluctuations.

[0229] Furthermore, the computer system for image acquisition and processing is programmed to determine the mass of one or more nanoparticles (block B6 in Figure 3). The scattered light intensity is proportional to the square of the nanoparticle's volume and therefore to the square of its mass. As shown by the formula either As previously stated, the proportionality term contains the illumination intensity. In practice, the mass of each nanoparticle is preferably determined from the median value of the diffusion cross-section (Is / I). PO ) measured for this nanoparticle along its trajectory.

[0230] Thus, in practice, we use the median value of the diffusion cross section (Is / (α.I)). PO )).

[0231] The computer system for image acquisition and processing is programmed to preferably determine the mass of each identified nanoparticle. It then preferably deduces a mass distribution of the nanoparticles in sample 1.

[0232] An example of such a mass distribution is shown on the.

[0233] The computer system for image acquisition and processing is preferably programmed to determine the mass of each identified nanoparticle from a statistically representative value of the nanoparticle's scattering cross sections measured along its trajectory. This statistically representative value is, for example, a mean or median value.

[0234] The mass distribution is obtained by calculating for each nanoparticle the square root of the median scattering cross section measured for the nanoparticle along its trajectory and multiplying it by a proportionality coefficient obtained experimentally or theoretically.

[0235] From a set of images, it is possible to establish a histogram of nanoparticle masses. The image represents a distribution of CCMV viruses and a distribution of silica nanoparticles.

[0236] Viruses of known mass or silica nanoparticles of calibrated mass can be used to calibrate the device and experimentally obtain the aforementioned proportionality coefficient. Optionally, it is possible to correct for a factor that accounts for a difference in density and a difference in refractive index.

[0237] The 100;200 optical analysis device can thus differentiate nanoparticles of varying masses. This can be used in biology for detecting nanoparticles corresponding to biological objects. When the nanoparticle is a virus, a vesicle, or more generally a vector that may or may not contain an object of biological interest, the 100;200 optical analysis device can determine the fraction of vectors containing the biological object of interest and the empty fraction, since these two vector populations have different masses. Furthermore, according to the same principle, when the sample contains nanoparticles that can aggregate together, the optical analysis device can quantify the state of aggregation of the nanoparticles, that is, determine how many nanoparticles form the aggregates and what the relative proportions of the aggregates present are.

[0238] The computer image acquisition and processing device can also be configured to determine a hydrodynamic diameter for each nanoparticle from the nanoparticle trajectory (block B7 of the).

[0239] Estimating the hydrodynamic diameter of a particle from its trajectory is done in two steps.

[0240] First, the trajectory is used to estimate a diffusion coefficient D of the nanoparticle. The estimation of the diffusion coefficient D can, for example, be carried out according to the method described in the following document: Michaelt X., “Mean square displacement analysis of single-particle trajectories with localization error: Brownian motion in an isotropic medium”, Phys Rev E Stat Nonlin Soft Matter Phys, 2010; 82(4 Pt 1): 041914, doi:10.1103 / PhysRevE.82.04191.

[0241] Then we use the Stokes-Einstein equation, which relates the hydrodynamic diameter Dh to the diffusion coefficient D according to the formula: Dh = k B .T / (3 π µ D) with- k B The Boltzmann constant, the temperature, and the viscosity of the solution containing the nanoparticle (in this case, water) are all factors considered. The computer system for image acquisition and processing is further configured to count nanoparticles whose hydrodynamic diameter falls within a specified range of diameter values ​​and to determine the concentration of nanoparticles with this hydrodynamic diameter within the sample.

[0242] Lamontre presents the results of three successive measurements on polystyrene nanospheres with diameters of 30, 40, and 50 nm. The figure shows, for each measurement and for each nanoparticle, a measurement of its hydrodynamic diameter as well as the light intensity scattered by that nanoparticle. The dotted line indicates the function x 3 whose adjustment to the data allows the optical index to be measured .

[0243] The computer-based image acquisition and processing system can be configured to determine the nanoparticle scattering cross-section from a statistically representative value of the nanoparticle scattering cross-section measurements taken along its trajectory. Further details on calculating the cross-section of particles small compared to the wavelength can be found in "Absorption and Scattering of Light by Small Particles" by Bohren and Huffman (Wiley, 1981), particularly its dependence on the particle refractive index, the solvent refractive index, the diameter, and the wavelength.

[0244] The computer image acquisition and processing device can also be configured to determine a real refractive index of the nanoparticle (B8 block of the) from a statistically representative value of the intensities scattered by the nanoparticle along its trajectory and knowledge of the solvent index, wavelength and hydrodynamic diameter measurement.

[0245] In practice, the optical index of the medium containing the particles is that of the solution, here water with an index n=1.33. The statistically representative value of the scattered light intensities is for example a time average value or a median value.

[0246] The computer system for image acquisition and processing is further configured to determine the refractive index contrast of one or each nanoparticle relative to the refractive index of the solvent in which the nanoparticle is suspended. The refractive index contrast is, for example, equal to the difference between the refractive index of the nanoparticle and the refractive index of the solvent in the solution. Therefore, in this example, it is the difference between the refractive index of the nanoparticle and the refractive index of water.

[0247] The computer system for image acquisition and processing can also be programmed to place the particles in a diagram with the hydrodynamic diameter on the x-axis and the index contrast on the y-axis.

[0248] This pattern is interesting because the particle does not scatter when the index contrast is zero and has a scattering coefficient that increases with the index contrast.

[0249] Thus, the described optical analysis device allows for the implementation of an analysis process including the following steps:

[0250] illuminate the sample with a light source emitting illuminating light, the sample comprising nanoparticles suspended in a solution arranged on a support,

[0251] collect outgoing light and direct the outgoing light from the sample to an optical detector with an optical device comprising a microscope objective to form an image of the sample on the optical detector, the outgoing light comprising scattered light corresponding to a portion of the illumination light scattered by the nanoparticles of the sample and unscattered light corresponding to a non-scattered portion of the illumination light, the scattered and unscattered light following the same optical path and interfering with each other,

[0252] to produce digital images with the optical detector, the digital images comprising a pixel matrix in an image acquisition plane,

[0253] Using a computer-based image acquisition and processing device, perform the following steps:

[0254] to acquire a time series of images produced by the optical detector, the time series comprising a plurality of images of the sample acquired at successive times over time, each pixel of an image in the time series having a light intensity measured by the optical detector at one of the successive times,

[0255] determine a representative image of the intensity of unscattered light over a given period of time, each pixel of said representative image having a light intensity equal to a value of a statistical indicator of the light intensities of the corresponding pixel of the images in the time series, and subtract said representative image from each image in the time series to obtain a time series of differential images, then normalize each pixel of this image by the square root of the statistical indicator of the intensity of the same pixel,

[0256] From a normalized differential image of the time series of normalized images, estimate an image twice the size of the real part of the field scattered by the nanoparticles at a given instant.

[0257] From the scattered field image, determine by an electromagnetic propagation calculation a three-dimensional scattered field distribution, representing an amplitude and a phase of the field scattered by the nanoparticles in planes parallel to the image acquisition plane and at a plurality of depths in a depth direction perpendicular to the image acquisition plane,

[0258] in said three-dimensional distribution of scattered field, identify nanoparticles as sources of the scattered field and determine their three-dimensional positions in the sample.

[0259] This provides three pieces of information about the sample: the concentration of nanoparticles, the distribution of the hydrodynamic diameters of the nanoparticles, and the distribution of the diffusion cross section, which can be related to the mass distribution or the index of the nanoparticles.

[0260] The use of a microscope and a laser light source maximizes the produced and collected signal. Using a setup in which the scattered light beam and the transmitted or reflected light beam interfering with the scattered beam travel the same optical path reduces phase fluctuations. Speckle noise determination and subtraction are made possible by the time averaging of the acquired images and the stability of the setup.

[0261] The use of the verb "comporter", "comprendre" or "include" and its conjugated forms does not exclude the presence of other elements or steps than those stated in a claim.

[0262] In claims, any reference sign in parentheses shall not be interpreted as a limitation of the claim.< / x> < / x>

Claims

Optical analysis device (100; 200) comprising: - a light source (10) emitting illumination light adapted to illuminate a sample (1) placed on a support (20), said sample (1) comprising nanoparticles suspended in a solution, - an optical detector (30) adapted to produce digital images comprising a pixel matrix in an image acquisition plane, - an optical device comprising a microscope objective (40), the microscope objective (40) being arranged to collect outgoing light and direct the outgoing light from the sample (1) to the optical detector (30) to form an image (2) of the sample on the optical detector (30), the outgoing light comprising scattered light corresponding to a portion of the illumination light scattered by the nanoparticles of the sample and unscattered light corresponding to an unscattered portion of the illumination light,scattered light and unscattered light following the same optical path and interfering with each other, - a computer image acquisition and processing device configured to: acquire a first time series (S1) of images produced by the optical detector (30), the first time series (S1) comprising a plurality of images (2) of the sample (1) acquired at successive times over time, each pixel of one of the images (2) of the first time series (S1) having a light intensity measured by the optical detector (30) at one of the successive times, determine a representative image (3) of the intensity of the unscattered light over a given time period, each pixel of said representative image (3) having a light intensity equal to a value of a statistical indicator of the light intensities of the corresponding pixel of the images of the first time series (S1),subtract said representative image (3) from at least one image (2) of the first time series (S1) to obtain a differential image (4), from the differential image, obtain a normalized differential image (6) taking into account the representative image of the intensity of the unscattered light, said normalized differential image being representative of the real part of the field scattered by the nanoparticles at one of the successive instants, from said normalized differential image (6), determine by an electromagnetic propagation calculation a three-dimensional distribution (D1) of scattered field, comprising images of propagated scattered field (7), the images of propagated scattered field being representative of the amplitude and phase of the field scattered by the nanoparticles in planes parallel to the image acquisition plane and located at a plurality of depths in a depth direction perpendicular to the image acquisition plane,in said three-dimensional distribution (D1) of scattered field, identify nanoparticles and determine their three-dimensional positions in the sample (1). Optical analysis device (100; 200) according to claim 1, wherein the normalized differential image is obtained by performing a step of dividing the light intensity of each pixel of the differential image by the square root of the light intensity of a corresponding pixel of the representative image (3) of the intensity of the unscattered light. Optical analysis device (100; 200) according to any one of claims 1 and 2, wherein the electromagnetic propagation calculation comprises the following steps: - calculate a Fourier transform of the scattered field image, - multiply the Fourier transform by a propagation term, and - calculate an inverse Fourier transform of the Fourier transform multiplied by the propagation term. Optical analysis device (100; 200) according to any one of claims 1 to 3, wherein identifying nanoparticles as sources of the scattered field involves searching for areas of the scattered field distribution exhibiting a light intensity greater than a predetermined intensity threshold or searching for areas of the scattered field distribution exhibiting a local maximum of light intensity. Optical analysis device (100; 200) according to any one of claims 1 to 4, wherein the computer image acquisition and processing device is further configured to count the identified nanoparticles and determine a concentration of the identified nanoparticles in the sample (1). Optical analysis device (100; 200) according to any one of claims 1 to 5, wherein the computer image acquisition and processing device is further configured to determine a scattering cross section for at least one of the identified nanoparticles or each identified nanoparticle. Optical analysis device (100; 200) according to any one of claims 1 to 6, wherein the computer image acquisition and processing device is further configured to determine a trajectory and hydrodynamic diameter for at least one of the identified nanoparticles or each identified nanoparticle during said successive instants. Optical analysis device (100; 200) according to claim 7, wherein the computer image acquisition and processing device is further configured to determine the index of one or each of the identified nanoparticles. Optical analysis device (100; 200) according to any one of claims 7 and 8, wherein the computer image acquisition and processing device is further configured to determine an index contrast of said or each identified nanoparticle with respect to a refractive index of the solution in which the identified nanoparticle is suspended. Optical analysis device (100; 200) according to claims 7 to 9, wherein the computer image acquisition and processing device is configured to determine a scattering cross section and / or a mass and / or a refractive index of said or each identified nanoparticle from a statistically representative value of the values ​​of a quantity derived from the scattered intensity measured along its trajectory. Optical analysis device (100; 200) according to claim 10, wherein the statistically representative value of the values ​​of the quantity derived from the intensity scattered by the identified nanoparticle measured along its trajectory is a median value. Optical analysis device (100; 200) according to claim 7 to 11 wherein the computer image acquisition and processing device is further configured to count the identified nanoparticles whose hydrodynamic diameter and scattering cross section or mass or refractive index is within certain intervals, and to determine a sample concentration within said intervals. Optical analysis device (100; 200) according to claim 7 to 12 wherein the light source illuminates the sample with an angle of incidence less than 15 degrees. A method for analyzing a sample (1) comprising: - illuminating the sample (1) with a light source (10) emitting illumination light, the sample (1) being placed on a support (20) and comprising nanoparticles suspended in a solution, - collecting outgoing light and directing the outgoing light from the sample (1) to an optical detector (30) with an optical device comprising a microscope objective (40) to form an image (2) of the sample (1) on the optical detector (30), the microscope objective (40) being arranged to collect the outgoing light and direct the outgoing light from the sample (1) to the optical detector (30), the outgoing light comprising scattered light corresponding to a portion of the illumination light scattered by the nanoparticles of the sample (1) and unscattered light corresponding to an unscattered portion of the illumination light,the scattered light and the unscattered light following the same optical path and interfering with each other, - produce digital images (2) of the sample with the optical detector, the digital images comprising a matrix of pixels in an image acquisition plane, - with a computer image acquisition and processing device, perform the steps of: - acquire a first time series (S1) of images produced by the optical detector (30), the first time series (S1) comprising a plurality of images (2) of the sample (1) acquired at successive times over time, each pixel of said image (2) of the first time series (S1) presenting a light intensity measured by the optical detector (30) at one of the successive times, determine a representative image (3) of the intensity of the unscattered light over a given period of time,each pixel of said representative image (3) having a light intensity equal to a value of a statistical indicator of the light intensities of the corresponding pixel of the images of the first time series (S1), subtract said representative image (3) from at least one image (2) of the first time series (S1) to obtain a differential image (4) and, from the differential image, obtain a normalized differential image (6) taking into account the representative image of the intensity of the unscattered light, the normalized differential image (6) being representative of the real part of the field scattered by the nanoparticles at one of the successive instants, from said normalized differential image (6), determine by an electromagnetic propagation calculation a three-dimensional distribution (D1) of scattered field, comprising images of propagated scattered field,Since the images of the propagated scattered field are representative of the amplitude and phase of the field scattered by the nanoparticles in planes parallel to the image acquisition plane and located at a plurality of depths in a depth direction perpendicular to the image acquisition plane, from said three-dimensional distribution (D1) of scattered field, identify nanoparticles as sources of the scattered intensity and determine their three-dimensional positions in the sample (1).

Images