DEVICE FOR IMAGING THE ELECTRICAL ACTIVITY OF A SAMPLE, IMAGING SYSTEM, METHOD FOR ANALYZING A SAMPLE AND ASSOCIATED MANUFACTURING METHOD

Abstract

DEVICE FOR IMAGING THE ELECTRICAL ACTIVITY OF A SAMPLE, IMAGING SYSTEM, METHOD FOR ANALYZING A SAMPLE AND ASSOCIATED MANUFACTURING METHOD One aspect of the invention relates to a device (1) for imaging the electrical activity of a sample (50), comprising: a polymer film (10) in which liquid crystal droplets are dispersed, called PDLC film (10), a transparent fluidic component (20) in which at least one cavity (210) is arranged, adapted to contain the sample (50), a transparent reference electrode (30), the PDLC film (10) being arranged between said at least one cavity (210) and the reference electrode (30) and configured to convert an electric field (E1) created in a region (R1) of the PDLC film (10) in response to an electric potential (Ve) generated by the sample (50) in a variation of transparency of said region (R1) of the PDLC film (10). Figure to be published with the abstract: Figure 4

Description

Title of the invention: DEVICE FOR IMAGING THE ELECTRICAL ACTIVITY OF A SAMPLE, IMAGING SYSTEM, METHOD FOR ANALYZING A SAMPLE AND ASSOCIATED MANUFACTURING METHOD TECHNICAL FIELD OF THE INVENTION

[0001] The technical field of the invention is linked to the analysis of a sample, for example a biological sample, by an imaging system.

[0002] The invention relates to an imaging device for analyzing a sample, an imaging system, a method for analyzing a sample and a method for manufacturing the imaging device.

[0003] The invention finds application in the fields of biology and health, in particular in the field of organs on chips, or "organ-on-chip" in English. TECHNOLOGICAL BACKGROUND OF THE INVENTION

[0004] In the field of neuroscience and medicine, the ability to detect, visualize and record the electrical activity of neurons is key to understanding the functioning and dysfunction of the brain, and to directly see the effects of drugs or other stimulations on the functioning of a population of neurons.

[0005] A particularly important application concerns the understanding of neurodegenerative diseases, such as Parkinson's disease or Alzheimer's disease, as well as the development of drugs against these diseases.

[0006] Electrophysiological imaging techniques generally rely on an electronic detection / transduction approach.

[0007] There are thus commercially available microelectrode arrays, also called MEAs (acronym for Micro-Electrode Arrays https: / / en.wikipedia.org / wiki / Microelectrode_array). Each microelectrode is electrically connected by an electrical connection (via, wire, etc.) to conditioning and addressing electronics.

[0008] There are also transistor matrices, called MTA (acronym for "multi-transistor-array" in English). The transistors are then used in a configuration called EOS (acronym for "Electrolyte-oxide-silicon" in English). The document "Electrical imaging of neuronal activity by multi-transistor-array (MTA) recording at 7.8 pm resolution" by Lambacher et al., Applied Physics A., 2004 describes a matrix of 16384 field effect transistors manufactured with CMOS technology (acronym for "complementary metal oxide Silicon" in English) and covered of a thin insulating layer of titanium dioxide (TiO2).

[0009] These imaging devices based on an electronic approach have been optimized to achieve satisfactory spatial resolutions (less than 10 pm). However, their field of analysis is limited to 1 mm2 or a few mm2, for example 3.5 mm2, due to the space taken up by the electrical connections and electronic circuits.

[0010] This limitation prevents imaging large populations of neurons, or testing, on the same device and the same sample, several experimental conditions (for example several drugs) in parallel.

[0011] Furthermore, these electronic devices are complex and expensive to manufacture.

[0012] There is therefore a need for electrophysiological imaging devices that are simple to manufacture and capable of analyzing large samples.

[0013] This need exists for the study of neuron populations, and more broadly for the study of samples comprising at least one source capable of generating an electrical signal. Summary of the invention

[0014] The invention offers a solution to the problem mentioned above by proposing to use the electro-optical transduction capabilities of a PD LC film (acronym for “polymer-dispersed liquid crystal” in English).

[0015] A first aspect of the invention relates to a device for imaging the electrical activity of a sample, comprising: • a polymer film in which liquid crystal droplets are dispersed, called a PDLC film, • a transparent fluidic component in which at least one cavity is fitted, suitable for containing the sample, • a transparent reference electrode, the PDLC film being arranged between said at least one cavity and the reference electrode and configured to convert an electric field created in a region of the PDLC film in response to an electric potential generated by the sample into a variation in transparency of said region of the PDLC film.

[0016] The term “transparent” designates a material or element having an optical transmission coefficient greater than 85% for at least one wavelength of the 400-800 nm spectral band.

[0017] According to the invention, three elements are sufficient (a fluidic component with at least one cavity, a PDLC film and a reference electrode) to obtain a device for spatio-temporal visualization of the electrical activity of a sample.

[0018] The cavity allows the sample to be positioned opposite the PDLC film and the reference electrode. It is well suited to “in-vitro” applications.

[0019] The reference electrode is used to detect electrical potentials generated in a plurality of regions of the sample.

[0020] The PDLC film is used as a transducer, to convert these detected electrical potentials into a variation in the transparency state of regions of the PDLC film corresponding to (i.e., facing) the plurality of regions of the sample. This variation in transparency is measurable by optical imaging.

[0021] The PDLC film allows direct viewing, without the use of a polarizer and analyzer arranged on either side of the film. This absence of polarizing elements, in addition to avoiding potential attenuation of the optical signal, contributes to the simplicity of the imaging device.

[0022] The PDLC film also has adaptable mechanical properties (strength, flexibility), which facilitate its integration into the imaging device. The imaging device, already simple in its structure, is thus easy to manufacture.

[0023] The PDLC film can furthermore be easily manufactured in dimensions as large as desired, for example 4 cm x 4 cm. It also has passive transduction capabilities, i.e. without the need for detection electrodes, electrical connections or electronic addressing circuits which usually limit the number of measurement channels, or the surface area of ​​the sensitive surface.

[0024] These advantages specific to the PDLC film give the imaging device according to the invention, in addition to great simplicity, an intrinsic field of analysis clearly superior to the state of the art. The imaging device is thus intrinsically suitable for imaging large samples, extending over areas greater than 1 cm2.

[0025] The device according to the first aspect of the invention may also have one or more of the characteristics below, considered individually or in all technically possible combinations.

[0026] The PDLC film is further configured to vary in transparency at electrical potentials in the range 100 pV - 100 mV.

[0027] Thus, the imaging device is configured to detect and convert into the optical domain electrical signals whose amplitude is typical of the extracellular potentials generated by excited cells, such as neurons.

[0028] The liquid crystal droplets of the PDLC film have on average a diameter of between 1 pm and 10 pm, preferably between 1 pm and 5 pm.

[0029] The droplet diameter determines the spatial resolution of the imaging device. The droplet diameter thus specified is less than, or within the range of, the size of neuronal cells. Thus, the imaging device is suitable for imaging the electrical activity of an individual neuron.

[0030] The PDLC film has a liquid crystal loading rate in the polymer of between 10% and 60% in bulk.

[0031] Thus, the variation in transparency is obtained in response to electric fields in the range of 0.1 V.pm to 5 V.pm.

[0032] The liquid crystal droplets are dispersed in the PDLC film such that there are at least five liquid crystal droplets in the PDLC film in a volume of 10 pm x 10 pm x 10 pm.

[0033] Preferably, the liquid crystal droplets are homogeneously dispersed in the polymer film. The term "homogeneous" herein means that the number of liquid crystal droplets 110 per unit volume exhibits a variation of less than 5% within the PDLC film. Thus, the transduction capabilities of the PDLC film are substantially the same over the entire surface area of ​​the PDLC film.

[0034] Furthermore, the quantity of liquid crystal droplets per unit volume is sufficient for a variation in transparency of at least 30% to be obtained in a region of the PDLC film corresponding to an electrically active region of surface area 10 μm x 10 μm.

[0035] Since the neurons extend over an area of ​​10 pm x 10 pm, this configuration of the PDLC film ensures that a neuron, regardless of its position with respect to the PDLC film (and the reference electrode), is associated with a transductive zone of the PDLC film (i.e., an area whose transparency varies in response to an electric field).

[0036] Unlike state-of-the-art devices based on matrix arrangements (of detection electrodes, transistors, etc.), the imaging device is devoid of a non-sensitive zone, i.e., opposite which the electrical activity of the sample cannot be detected. The imaging device thus has better detection efficiency.

[0037] The liquid crystal droplets preferably comprise doping particles chosen from the following particles: dye nanoparticles, gold nanoparticles, silver nanoparticles, zinc oxide nanoparticles, gold nanowires, silver nanowires.

[0038] Thus, the PDLC film is more sensitive and reacts to electric fields in the range of 2 V and 50 V.

[0039] The fluidic component further comprises polarization means interacting with the interior volume of said at least one cavity, to polarize the sample.

[0040] Thus, the device is well suited to the analysis of a sample immersed in a solution such as a culture solution, for example a neuronal media.

[0041] The polarization means comprise a transparent electrode, called a counter-electrode.

[0042] When the PDLC film reacts to an electric field less than a bias of 0.1 V and the fluidic component is insulating, the polarization means and the reference electrode can be electrically connected to ground

[0043] Alternatively, the polarization means and the reference electrode are electrically connected to the terminals of a voltage generator to create an electric field in the PDLC film equal to or greater than a threshold electric field of the PDLC film from which the variation in transparency of the PDLC film is obtained.

[0044] Thus, the electric field generated by the sample is added to the threshold electric field generated by the voltage generator. This configuration makes it possible to take into account the fact that the PDLC film varies in transparency from a threshold electric field.

[0045] Said at least one cavity extends to the PDLC film, the polymer of the PDLC film being a material suitable for receiving the sample, such as polydimethylsiloxane (PDMS).

[0046] Using PDMS as the polymer of the PDLC film gives the PDLC film sufficient rigidity so that the reference electrode and the fluidic component can be assembled from the PDLC film. Thus, the fabrication of the device is simplified.

[0047] Said at least one cavity extends to a transparent and insulating layer disposed between the PDLC film and the cavity.

[0048] A second aspect of the invention relates to a system for imaging the electrical activity of a sample, comprising: • An imaging device according to the first aspect of the invention, • A light source capable of emitting a light wave, • An image sensor adapted to form an image of the light transmitted by the imaging device under the effect of the electrical activity of the sample, the imaging device being arranged between the light source and the image sensor, the light source being opposite one of the elements chosen from the transparent electrode and the cavity of the imaging device, and the image sensor being arranged opposite the other of said chosen elements.

[0049] A third aspect of the invention relates to a method of analyzing a sample using the imaging device according to the first aspect of the invention, comprising the steps of: • Arranging the sample in the cavity of the fluidic component of the imaging device, said imaging device being arranged between a light source and an image sensor, • Illumination of the imaging device and the sample by a light wave produced by the light source, called the incident wave, the incident wave propagating through the imaging device to the image sensor, • Optical detection of the electrical activity of the sample including: • a step of acquisition, by the image sensor, of an image representative of the light wave having been propagated through the imaging device, called the transmitted wave, • a step of detecting, by a processor communicating with the image sensor, a modification of the transmission properties of the imaging device under the effect of the electrical activity of the sample, from the previously acquired image.

[0050] The step of detecting the modification of the transmission properties of the imaging device may comprise the following steps: • Determination of characteristics of the acquired image, • Correlation of these determined characteristics with the electric field generated by the sample, from calibration characteristics established by carrying out the steps of sample arrangement, illumination and optical detection using a standard sample.

[0051] A fourth aspect of the invention relates to a method of manufacturing a device for imaging the electrical activity of a sample, comprising the following steps: • Provision of a sacrificial substrate, • Manufacturing a polymer film in which liquid crystal droplets are dispersed, called a PDLC film, the PDLC film being configured to convert an electric field created in a region of the PDLC film in response to an electric potential generated by the sample into a variation in transparency of said region of the PDLC film, • Fabrication, on a first face of the PDLC film opposite the sacrificial substrate, of a transparent reference electrode, • Removal of the sacrificial substrate from the PDLC film, so as to leave free a second face of the PDLC film opposite the first face, • Supply of a transparent fluidic component in which at least one cavity is fitted, suitable for containing the sample, • Assembling the fluidic component on the second face of the PDLC film, such that the PDLC film is arranged between said at least one cavity of the fluidic component and the reference electrode.

[0052] A fifth aspect of the invention relates to a method of manufacturing a device for imaging the electrical activity of a sample, comprising the following steps: • Provision of a transparent substrate, • Fabrication of a transparent reference electrode on the substrate transparent, • Fabrication, on the reference electrode, of a polymer film in which liquid crystal droplets are dispersed, called a PDLC film, the PDLC film being configured to convert an electric field created in a region of the PDLC film in response to an electric potential generated by the sample into a variation in transparency of said region of the PDLC film, • Supply of a transparent fluidic component in which at least one cavity is fitted, suitable for containing the sample, • Assembling the fluidic component on the face of the PDLC film opposite the reference electrode, such that the PDLC film is arranged between said at least one cavity of the fluidic component and the reference electrode.

[0053] The invention and its various applications will be better understood upon reading the following description and examining the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0054] The figures are presented for information purposes only and in no way limit the invention. • [Fig.l] schematically represents in cross-sectional view a first embodiment of a device for imaging the electrical activity of a sample, • [Fig.2] schematically represents in cross-sectional view a second embodiment of the imaging device, • [Fig.3] schematically represents in cross-sectional view a third embodiment of the imaging device, • [Fig.4] illustrates the imaging device of [Fig.3] in use with a biological sample, • [Fig.5] is a photograph of an example of a fluidic component used in the imaging devices of Figures 1 to 3, • [Fig.6] schematically represents in cross-sectional view an imaging system using the imaging device of [Fig.3]. • [Fig.7] is a block diagram illustrating the sequence of the main steps of a process for analyzing the electrical activity of a sample, • Figures 8A to 8H schematically represent in sectional view steps or sub-steps of a method of manufacturing the imaging device of Figures 1 to 3, and • Figures 9A to 9F schematically represent in sectional view steps or sub-steps of an alternative manufacturing method to the manufacturing method of Figures 8A to 8H.

[0055] Unless otherwise specified, the same element appearing in different figures presents a unique reference. DETAILED DESCRIPTION

[0056] As indicated previously, the present invention relates in particular to an imaging device for detecting and visualizing the electrical activity of a sample, for example of a biological sample such as neuronal tissue, or neuronal cells in culture in a culture medium.

[0057] The imaging device advantageously has a large analysis field, greater than 1 cm2, continuity of detection over this entire analysis field, and a micrometric spatial resolution, between 1 pm and 20 pm, corresponding to the scale of an individual neuron.

[0058] A first embodiment and a second embodiment of the imaging device are shown in [Fig.l] and [Fig.2] respectively.

[0059] A third embodiment, compatible with the first or second embodiment, is shown in [Fig.3].

[0060] This third embodiment is also shown in [Fig.4] in use with a sample 50 of neuronal cells 520, 521 immersed in a liquid culture medium 510, such as a neuronal media. An example of a neuronal media is a phosphate buffered saline solution or DBPS (acronym for “Dubelcco's Phosphate Buffered Saline” in English).

[0061] Common to the three embodiments, the device 1 comprises: • a film 10 of polymer 120 in which liquid crystal droplets 110 are dispersed, called PDLC film 10, • a transparent fluidic component 20 in which one or more cavities 210 are arranged, adapted to contain the sample, and • a transparent reference electrode 30.

[0062] Also in common to the three embodiments, the PDLC film 10 is arranged between the cavity(ies) 210 and the reference electrode 30. As illustrated in [Fig.4], the PDLC film 10 is configured to convert an electric field El created in a region RI of the PDLC film 10 in response to an electric potential Ve generated by the sample 50 into a variation in transparency of said region RI of the PDLC film 10.

[0063] The variation in transparency is obtained thanks to the intrinsic electro-optical properties of the PDLC film 10. These are described below.

[0064] Preferably, the reference electrode 30 is in direct contact with the PDLC film 10. It can be formed by depositing a layer of a conductive and transparent material on the PDLC film 10.

[0065] The transparent and insulating material is then chosen according to the deposition method. to be chemically compatible with PDLC film 10.

[0066] In another configuration, not shown in [Fig.l], the imaging device 1 further comprises a transparent substrate on which the reference electrode 30 has been formed. The reference electrode 30 is then arranged between said transparent substrate and the PDLC film 10.

[0067] The transparent substrate may be made of glass or a transparent polymer material chosen from the following polymers: polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polycarbonate (PC).

[0068] When the transparent substrate is made of a polymer, the reference electrode 30 is advantageously formed by a conductive and transparent material chosen from the following materials: transparent conductive oxides or TCO (acronym for “Transparent Conductive Oxides” in English), including aluminum-doped zinc oxide (AZO), zinc oxide (ZnO), or by a transparent conductive ink, comprising for example nanowires of silver (Ag), gold (Au) or poly(3,4-ethylenedioxythiophene) (PEDOT).

[0069] When the transparent substrate is made of glass, the reference electrode 30 comprises a layer of a conductive and transparent material chosen from the following materials: indium tin oxide (ITO), zinc oxide (ZNO), aluminum-doped zinc oxide (AZO), tin oxide (SnO2), silver (Ag), aluminum (Al), titanium (Ti), copper (Cu), or a stack of layers formed by one or more of these materials.

[0070] The thickness e30 of the reference electrode 30 is preferably between 10 μm and 2 mm.

[0071] Laterally, the reference electrode 30 preferably extends over the entire surface of the PDLC film 10.

[0072] The fluidic component 20 is formed from a transparent material. This material is preferably polymethylsiloxane or PDMS. This material has the advantage of being biocompatible, therefore suitable for the sample 50, and transparent.

[0073] The fluidic component 20 may be formed from a material that is further electrically insulating, such as PDMS, or from an electrically conductive material.

[0074] With reference to [Fig.l], the fluidic component 20 comprises a cavity 210. As mentioned previously, and with reference to [Fig.4], this cavity 210 is adapted to contain the sample 50.

[0075] The dimensions of the cavity 210 are preferably between 10 mm2 and 100 mm2.

[0076] The fluidic component 20 may further comprise at least one inlet (not shown in [Fig.4]) connected to the cavity 210 to convey the sample 50 into the cavity 210. This inlet is for example equipped with a valve, making it possible to ensure the fluidic seal between the cavity 210 and the exterior of the fluidic component 20. The valve is preferably formed from a transparent material, for example PDMS.

[0077] With reference to [Fig.5], the fluidic component 20 can also comprise several cavities, for example six cavities 211, 212, 213, 214, 215 and 216, connected to each other by conduits 217. In the case of a biological sample, these conduits 217 are adapted to the culture solutions 50.

[0078] These cavities preferably have dimensions between 1x1 mm and 10x10 mm.

[0079] Each cavity 211-216 is then opposite the PDLC film 10 and the reference electrode 30.

[0080] The PDLC film 10 is a film of a composite material comprising a polymer material and a liquid crystal. This composite material is formed by polymerization of a mixture of a solution of the polymer and a solution of a liquid crystal material (or, more simply put, a liquid crystal). Under the effect of the polymerization, and with reference to [Fig.l], the liquid crystal is in the form of droplets 110 dispersed discretely (separated from each other) in the network of the polymer 120.

[0081] The PDLC film 10 preferably has a thickness ei0 which depends on the size of the droplets 110. The minimum thickness may be 5 μm.

[0082] Laterally, the PDLC film 10 can extend over dimensions as large as desired. For example, it extends over dimensions greater than 1 mm x 1 mm, preferably greater than 1 cm x 1 cm, for example 4 cm x 4 cm.

[0083] The PDLC film can operate in a so-called “normal” mode, that is to say that the PDLC film 10 has the property of varying between an opaque or semi-transparent state (off state) and a transparent state (on state) after application of an electric field perpendicular to the plane of the PDLC film 10. However, other modes of use (inverse, etc.) can be envisaged.

[0084] In the first embodiment represented by [Fig.l], the cavity 210 extends to the PDLC film 10. In other words, it opens onto the PDLC film 10. The polymer of the PDLC film 10 is then a material suitable for receiving the sample, such as polydimethylsiloxane (PDMS). The PDMS is advantageously biocompatible, that is to say suitable for receiving biological samples.

[0085] In the second embodiment shown in [Fig. 2], the cavity 210 does not extend to the PDLC film 10 but to a transparent and insulating layer 220, arranged between the PDLC film 10 and the cavity 210. This transparent and insulating layer 220 is preferably part of the fluidic component 20. The advantage of using such an intermediate layer is to improve the mechanical strength of the imaging device 1. This embodiment is preferred when the film has insufficient rigidity to directly support (i.e. via direct contact) the component fluidics 20.

[0086] In the third embodiment represented by [Fig.3], the imaging device 1 further comprises a counter-electrode 230 used as polarization means, this transparent counter-electrode 230 being arranged at least partly in the cavity 210 to polarize the sample 50. The counter-electrode 230 is preferably formed from a conductive and transparent material.

[0087] For example, the counter-electrode 230 has the shape of a rod which passes through a wall of the fluidic component 20 to open inside the cavity 210.

[0088] Alternatively, the counter-electrode 230 may have the form of a conductive layer arranged on an interior surface of the fluidic component 20 (delimiting the cavity 210), other than the bottom 210a (cf. [Fig.3]) of the cavity 210, for example on one of the side walls of the cavity 210 or on the surface 10a of the PDLC 10.

[0089] Another possibility is that the counter electrode 230 can be formed of a conductive and transparent layer disposed on the bottom 210a of the cavity 210. The fact that it is transparent allows light to pass through the imaging device 1, for optical imaging.

[0090] It will be noted that when the fluidic component 20 is formed from a conductive material, the walls and the bottom 210a of the cavity 210 are also conductive. In this case, the cavity 210 intrinsically forms the counter-electrode 230.

[0091] When the fluidic component is formed from an insulating material and the counter-electrode 230 is not formed from a conductive and transparent layer arranged on the bottom 210a of the cavity 210, the means for polarizing the sample (comprising the counter-electrode 230) and the reference electrode 30 can be: • either electrically connected to ground (configuration not shown in [Fig.3]) when the PDLC film reacts to an electric field lower than a bias of 0.1 V, • either electrically connected to the terminals of a voltage generator 40 to create an electric field E in the PDLC film equal to or greater than a threshold electric field Es of the PDLC film from which the variation in transparency of the PDLC film is obtained. This threshold electric field Es typically corresponds to a polarization between the reference electrode 30 and the counter-electrode 230 of between 3 V and 5 V.

[0092] When the fluidic component is formed from a conductive material or the counter-electrode 230 is formed from a conductive and transparent layer arranged on the bottom 210a of the cavity 210, the polarization means are electrically connected to the terminals of the voltage generator 40.

[0093] Thus, the polarization means 230 make it possible to configure the initial electric field Es (i.e. in the absence of electrical activity of the sample) in the film PDLC 10.

[0094] The operating principle of the imaging device 1 is described below in relation to [Fig.4].

[0095] The reference electrode 30 acts as a means for generating an electric field El in a region RI of the PDLC film 10 in response to the electrical activity Ve of a neuron 520.

[0096] When the sample 50 is not electrically active, an electric field Es pre-exists in the PDLC film 10, created under the effect of the voltage generated between the reference electrode 30 and the counter-electrode 230 by the voltage generator 40.

[0097] When a region 520 of the sample 50 is electrically active, charges accumulate at the level of this active region 520, generating an electric potential Ve. This electric potential Ve leads to a field El which is added locally (i.e. in the zone RI of the active region 520) to the electric field Es created in the PDLC film 10 by the voltage generator 40. The resulting electric field E is therefore greater than the threshold electric field Es.

[0098] When there is no voltage generator 40 and the reference electrode 30 is grounded, the operating principle is identical, except that the pre-existing electric field in the PDLC film 10 is zero.

[0099] It should be noted that the imaging device operates in the same way when no polarization means is used, as in the first and second embodiments. Simply, the reference electrode 30 is brought to ground, and the pre-existing electric field depends on the polarization of the sample.

[0100] The PDLC film serves as an electro-optical transducer to convert the electrical potentials into a variation in the transparency of the PDLC film in the area RI of the active region 520. In the other areas R0 of the PDLC film 10, since the voltage between the reference electrode 30 and the sample 50 is less than or equal to the threshold voltage Vs, the transparency has not been modified compared to the off state (the film is opaque or semi-transparent in these areas R0).

[0101] The variation in transparency is linked to the birefringence properties of the liquid crystal droplets 110.

[0102] Thus, with reference to [Fig.4], when no electric field is applied, or when it is lower than the threshold electric field Es of the PDLC film, the liquid crystal molecules in the droplets 110a are randomly oriented relative to each other. There are therefore differences in refractive indices between each droplet 110a and the polymer 120, which causes diffusion: the R0 region of the PDLC film 10 appears opaque.

[0103] On the other hand, when the applied electric field El is greater than the threshold field of the PDLC film 10, the director axis of each droplet 110b aligns in the same direction. Thus, the refractive index between each droplet 110b is the same and the RI region of the PDLC film 10 appears transparent (disappearance of diffusion).

[0104] The transduction capabilities of the PDLC film 10 are thus passive: there is no need for electronic circuits to condition the electrical potentials generated by the sample or the output signals of the transducer.

[0105] The fact that the PDLC film 10 reacts to electric fields and not to electric currents avoids the use of a counter-electrode which would be arranged on the other side of the PDLC film 10 relative to the reference electrode 30, and which would have the function of bringing the electric potentials of the sample to the PDLC film 10.

[0106] The PDLC film advantageously has a homogeneous distribution of the liquid crystal droplets 110. The term “homogeneous” here means that the number of liquid crystal droplets 110 per unit volume has a variation of less than 5% within the PDLC film. Thus, the transduction capacities of the PDLC film are substantially the same over the entire surface area of ​​the PDLC film. In other words, the transduction capacities of the film are spatially regular and continuous.

[0107] Preferably, the distribution of the droplets is such that there are at least five droplets of liquid crystal 110 in a volume of 10 pm x 10 pm x 10 pm of the PDLC film 10.

[0108] Since the neurons extend over an area of ​​10 pm x 10 pm, this configuration of the PDLC film ensures that a neuron, regardless of its position with respect to the PDLC film (and the reference electrode), is associated with a transductive zone of the PDLC film (i.e., an area whose transparency varies in response to an electric field). With reference to [Fig. 4], an active neuron 520 is associated with a transduction region RI which has a diameter drl equal to the diameter of the active neuron 520.

[0109] More generally, the spatial resolution is no longer limited, as is the case in state-of-the-art devices based on matrix transducers, by the pitch of the matrix.

[0110] The liquid crystal droplets 110 advantageously have an average diameter dn0 of between 1 pm and 10 pm, preferably of between 1 pm and 5 pm.

[0111] The spatial resolution is thus adapted to the scale of a neuron, the latter having a size of 15 pm.

[0112] The polymer of the PDLC film 10 is for example a MOA-65 Norland polymer having a refractive index equal to 1.524.

[0113] The liquid crystal is a nematic liquid crystal. For example, it is the MERCK MDA003969 liquid crystal having an ordinary refractive index of nol.498, an extraordinary refractive index of ne 1.719, and an isotropic temperature of 106°C. It has an anisotropy of index A e equal to 2.7 at a frequency greater than or equal to 50 kHz.

[0114] Furthermore, the liquid crystal loading rate in the polymer can be between 10% and 60%.

[0115] Thus, the variation in transparency is obtained in response to electric fields in the range of 30 mV.pm to 100 mV.pm, which corresponds to the range of electric fields generated by a biological sample such as sample 50.

[0116] The liquid crystal droplets may comprise doping particles chosen from the following particles: dye nanoparticles, gold nanoparticles, silver nanoparticles, ZnO nanoparticles, gold nanowires, silver nanowires. A method for encapsulating dopants in PDLC films is for example described in the document “Preparation and electrooptic study of reverse mode polymer dispersed liquid crystal: performance augmentation with the doping of nanoparticles and dichroic dye”, by Vandna Sharma et al., Journal of Applied Polymer Science, 48745, 2020.

[0117] These doping particles have the effect of increasing the sensitivity of the PDLC film 10. In other words, the PDLC film can vary in transparency in response to electric fields with an amplitude of between 10 mV.pm and 1 V.pm, thus covering the range of electric fields generated by neurons. Since, moreover, the surface area of ​​the PDLC film 10 is centimetric, an imaging device I is available which combines a large field of analysis and a micrometric spatial resolution.

[0118] [Fig.6] represents an imaging system 6 of the electrical activity of a sample using the imaging device 1 described previously.

[0119] With reference to [Fig.6], the imaging system 6 comprises: • The imaging device 1 according to any one of the embodiments, • A light source 62 arranged opposite the reference electrode 30 and the cavity 210 of the imaging device 1, adapted to emit light 621 towards the imaging device 1, • An image sensor 61 arranged opposite the cavity 210, and adapted to form an image of the light transmitted 622 by the imaging device 1 under the effect of the electrical activity of the sample.

[0120] The imaging device 1 is thus arranged between the light source 62 and the image sensor 61.

[0121] The light source 62 is for example a white light source. It may comprise optical elements (polarizer, lenses, optical fibers, etc.) for conveying and / or shaping the emitted light 621.

[0122] The image sensor 61 is for example a CCD sensor (acronym for “Charged Coupled Device” in English) integrated into a microscope.

[0123] The light source 62 and the image sensor 61 can be interchanged with respect to the imaging device 1.

[0124] This imaging system 6 makes it possible to image the electrical activity of the sample in a simple and direct manner.

[0125] A method of analyzing 100 the sample 50 using the imaging device 1 is described below in relation to [Fig.7].

[0126] The analysis method 100 comprises the following main steps: • Arrangement S101 of the sample 50 in the cavity 210 of the fluidic component 20 of the imaging device 1, said imaging device 1 being arranged between a light source and an image sensor such as the light source 62 and the image sensor 4 of the imaging system 6 described previously (see [Fig. 6]). The sample is preferably introduced into the cavity once the latter has been assembled on the assembly formed by the PDLC film and the electrode 30. • Illumination S102 of the imaging device 1 and the sample 50 by a light wave 622 produced by the light source 62, called incident wave 622, incident wave 621 propagating through the imaging device 1 to the image sensor 61, • Optical detection S103 of the electrical activity of sample 50 comprising: • an acquisition step S1031, by the image sensor 61, of a representative image of the light wave 622 having been propagated through the imaging device 1, called transmitted wave 621, • a detection step S1302, by a processor communicating with the image sensor, of a modification of the transmission properties of the imaging device 1 under the effect of the electrical activity of the neurons 520,521 of the sample, from the previously acquired image.

[0127] The step S1032 of detecting the modification of the transmission properties of the imaging device 1 may comprise the following steps: • Determination S 1032a of characteristics of the acquired image, such as regions of interest, average value of the pixels in this region of interest, etc. • Correlation S 1032b of these determined characteristics with the location of the electrically active region of the sample 520 and the electrical potential generated by this region 520, from calibration characteristics established by carrying out steps S101, S102 and S103 of arrangement of the sample, illumination and optical detection using a standard sample.

[0128] A method of manufacturing the imaging device 1 is described below. Generally, it comprises the following steps: • Production of PDLC 10 film, • Supply of fluidic component 20, • Manufacture of the reference electrode 30, • Assembly of the PDLC film 10, the reference electrode 30, and the fluidic component 20 such that the PDLC film 10 is arranged between the cavity(ies) 210 of the fluidic component 20 and the reference electrode (30).

[0129] A first embodiment of this manufacturing method is illustrated in FIGS. 8A to 8H.

[0130] According to this first embodiment, the PDLC film 10 is first manufactured and serves as a support for the manufacture of the reference electrode 30.

[0131] According to this first mode of implementation, the method 7 begins with step S701, illustrated in [Fig.8A], of providing a sacrificial substrate 710.

[0132] Step S701 continues with a step S702 of manufacturing the PDLC film 10 on the sacrificial substrate 710.

[0133] The manufacturing comprises a first sub-step S702A, illustrated in [Fig.8B] of spin-coating T of the mixture of the polymer solution and the liquid crystal solution, and a second sub-step S702B, illustrated in [Fig.8C], of polymerization, for example by exposure to ultraviolet UV, of the mixture deposited in step S702A.

[0134] Step S702 is followed by a step S703, illustrated in [Fig.8D], of forming the reference electrode 30 on a first face 10b of the PDLC film opposite the sacrificial substrate 710.

[0135] Step S703 is extended by a step S704, illustrated in [Fig.8E], of removing the sacrificial substrate 710 from the PDLC film 10.

[0136] With reference to [Fig.8F], at the end of this step S704, a second face 10c of the PDLC film 10 opposite the first face 10b is left free, and the PDLC film 10 and the reference electrode 30 have been assembled to form a PDLC film-reference electrode assembly 1030.

[0137] Steps S705 and S706 are illustrated in [Fig.8G]. Step S705 is a step of providing the fluidic component 20.

[0138] Step S706 consists of assembling the fluidic component 20 on the second face 10c of the PDLC film 10 so that said cavity 210 faces the reference electrode 30, the PDLC film 10 then being arranged between the cavity 210 and the reference electrode 30.

[0139] Two additional steps S707 and S708, illustrated in [Fig.8H], are then successively carried out to, respectively, produce the polarization means 230 of the fluidic component 20, and electrically connect these polarization means. Step S706 thus consists of inserting the counter-electrode 230 into the cavity 210 through the wall of the fluidic component 20. Step S707 consists of providing the ge voltage generator 40 and electrically connecting its terminals on the one hand to the reference electrode 30 and on the other hand to the counter-electrode 230.

[0140] At the end of step S707, the PDLC film-reference electrode assembly 1030 and the fluidic component 20 have been assembled, and the imaging device 1 is ready to be used.

[0141] A second mode of implementation of the manufacturing method is illustrated in Figures 9A to 9F.

[0142] The method 8 according to this second embodiment differs from the first embodiment in that the reference electrode 30 is formed on a transparent substrate and serves as a support for manufacturing the PDLC film 10. Such an embodiment will be preferred when the rigidity of the PDLC film 10 is insufficient to serve as a support for the reference electrode 30.

[0143] The method 8 thus begins with a step S801, illustrated in [Fig.9A], of providing the transparent substrate 810.

[0144] The method 8 is extended by a step S802, illustrated in [Fig.9B], of manufacturing, on the transparent substrate 810, the reference electrode 30.

[0145] Method 8 continues with step S803 consisting of manufacturing the PDLC10 film on the reference electrode 30. The reference electrode is thus arranged on the first face 10b of the PDLC film 10.

[0146] This step S803 comprises the sub-steps S803A and S803B, illustrated respectively in [Fig.9C] and in [Fig.9D]. These sub-steps are identical to the sub-steps S702A and S702B illustrated in FIGS. 8B and 8C.

[0147] Step S803 is followed by steps S804 and S805, illustrated in [Fig.9E], and steps S806 and S807, illustrated in [Fig.9F]. These steps S804, S805, S806 and S807 are identical, respectively, to steps S705, S706, S707 and S708 illustrated in FIGS. 8G and 8H.

Claims

Claims

1. Imaging device (1) of the electrical activity of a sample (50), characterized in that it comprises: - a film (10) of polymer (120) in which liquid crystal droplets (110) are dispersed, called PDLC film (10), - a transparent fluidic component (20) in which at least one cavity (210) is arranged, adapted to contain the sample (50), - a transparent reference electrode (30), the PDLC film (10) being arranged between said at least one cavity (210) and the reference electrode (30) and configured to convert an electric field (El) created in a region (RI) of the PDLC film (10) in response to an electric potential (Ve) generated by the sample (50) into a variation in transparency of said region (RI) of the PDLC film (10).

2. The imaging device (1) of claim 1, wherein the PDLC film (10) is further configured to vary in transparency at electrical potentials (Ve) in the range 100 pV - 100 mV.

3. Imaging device (1) according to one of claims 1 to 2, wherein the liquid crystal droplets (110) of the PDLC film (10) have on average a diameter (dn0) of between 1 pm and 10 pm, preferably of between 1 pm and 5 pm.

4. Imaging device (1) according to one of claims 1 to 3, wherein the PDLC film (10) has a liquid crystal (110) loading rate in the polymer (120) of between 10% and 60% by mass.

5. The imaging device (1) according to one of claims 1 to 4, wherein the liquid crystal droplets (110) are homogeneously dispersed in the PDLC film (10) such that there are at least five liquid crystal droplets (110) in the PDLC film (10) in a volume of 10 pm x 10 pm x 10 pm.

6. Imaging device (1) according to one of claims 1 to 5, wherein the liquid crystal droplets (110) comprise doping particles selected from the following particles: dye nanoparticles, gold nanoparticles, silver nanoparticles, ZnO nanoparticles, gold nanowires, silver nanowires.

7. Imaging device (1) according to one of claims 1 to 6, in which the fluidic component (20) further comprises polarization means (230) interacting with the interior volume of said at least one cavity (210), to polarize the sample (50).

8. Imaging device (1) according to claim 7, wherein the polarization means (230) comprise a transparent electrode (230), called counter-electrode (230).

9. Imaging device (1) according to one of claims 7 to 8, wherein the polarization means (230) and the reference electrode (30) are electrically connected to ground, the fluidic component (20) being formed from an electrically insulating material.

10. Imaging device (1) according to one of claims 7 to 8, wherein the polarization means (230) and the reference electrode (30) are electrically connected to the terminals of a voltage generator (40) to create an electric field in the PDLC film equal to or greater than a threshold electric field (Es) of the PDLC film (10) from which the variation in transparency of the PDLC film (10) is obtained.

11. Imaging device (1) according to one of claims 1 to 10, wherein said at least one cavity (210) extends to the PDLC film (10), the polymer (120) of the PDLC film (10) being a material suitable for receiving the sample (50), such as PDMS.

12. An imaging device (1) according to one of claims 1 to 10, wherein said at least one cavity (210) extends to a transparent and insulating layer (220) disposed between the PDLC film (10) and the cavity (210).

13. Imaging system (6) of the electrical activity of a sample (50), comprising: - An imaging device (1) according to claims 1 to 12, - A light source (62) capable of emitting a light wave (621), - An image sensor (621) adapted to form an image of the light transmitted (622) by the imaging device (1) under the effect of the electrical activity of the sample (50), the imaging device (1) being arranged between the light source (62) and the image sensor (61), the light source (61) being opposite one of the elements chosen from the reference electrode (30) and the cavity (210) of the imaging device (1), and the image sensor (61) being in

14.

15. regard to the other of the said chosen elements. Method for analyzing (100) a sample (50) using an imaging device (1) according to one of claims 1 to 12, comprising the steps of: - Arranging (S 101) the sample (50) in the cavity (210) of the fluidic component (20) of the imaging device (1), said imaging device (1) being arranged between a light source (62) and an image sensor (61), - Illumination (S102) of the imaging device (1) and the sample (50) by a light wave (622) produced by the light source (62), called incident wave (622), the incident wave (622) propagating through the imaging device (1) to the image sensor (61), - Optical detection (S 103) of the electrical activity of the sample (50) comprising: • a step of acquisition (S1031), by the image sensor (61), of an image representative of the light wave having been propagated through the imaging device, called transmitted wave (621), • a step of detecting (S 1302), by a processor communicating with the image sensor (61), a modification of the transmission properties of the imaging device (1) under the effect of the electrical activity of the sample (50), from the previously acquired image. The analysis method (100) of claim 14, wherein the step of detecting (S 1032) the change in the transmission properties of the imaging device (1) comprises the following steps: - Determination (S 1032a) of characteristics of the acquired image, - Correlation (S 1032b) of said determined characteristics with the electric field (Ve) generated by the sample (50), from calibration characteristics established by carrying out the steps of arrangement (S101) of the sample, illumination (S 102) and optical detection (S 103) using a standard sample.

16. Method (7) of manufacturing an imaging device (1) of the electrical activity of a sample (50), comprising the following steps: - Provision (S701) of a sacrificial substrate (710), - Manufacturing (S702A, S702B) a film (10) of polymer (120) in which liquid crystal droplets (110) are dispersed, called PDLC film (10), the PDLC film (10) being configured to convert an electric field (El) created in a region of the PDLC film (10) in response to an electric potential (Ve) generated by the sample (50,520) into a variation in transparency of said region (RI) of the PDLC film (10), - Fabrication (S703), on a first face (10b) of the PDLC film (10) opposite the sacrificial substrate (710), of a transparent reference electrode (30), - Removal (S704) of the sacrificial substrate (710) from the PDLC film (10), so as to leave free a second face (10c) of the PDLC film (10) opposite the first face (10b), - Supply (S705) of a transparent fluidic component (20) in which at least one cavity (210) is arranged, suitable for containing the sample (50), - Assembling (S706, S707, S708) the fluidic component (20) on the second face (10c) of the PDLC film (10), such that the PDLC film (10) is arranged between said at least one cavity (210) of the fluidic component (20) and the reference electrode (30).

17. Method (8) of manufacturing an imaging device (1) of the electrical activity of a sample (50), comprising the following steps: - Providing (S801) a transparent substrate (810), - Manufacturing (S802) a transparent reference electrode (30) on the transparent substrate (810), - Manufacturing (S803A, S803B), on the reference electrode (30), a film (10) of polymer (120) in which liquid crystal droplets (110) are dispersed, called PDLC film (10), the PDLC film (10) being configured to convert a electric field (El) created in a region of the PDLC film (10) in response to an electric potential (Ve) generated by the sample (50, 520) in a variation of transparency of said region (RI) of the PDLC film (10), Supply (S804) of a transparent fluidic component (20) in which at least one cavity (210) is arranged, adapted to contain the sample (50), Assembly (S805, S806, S807) of the fluidic component (20) on the face (10c) of the PDLC film (10) opposite the reference electrode (30), so that the PDLC film (10) is arranged between said at least one cavity (210) of the fluidic component (20) and the reference electrode (30).

Images