Optical system for monitoring a sample by fluorescence

Abstract

The invention relates to an optical system for monitoring a sample by fluorescence, comprising an excitation light source (2) generating an excitation light beam (F1) and a microfluidic card (1), said sample being placed in a microfluidic circuit integrated in said microfluidic card, said system comprising a device (3) for detecting the fluorescence signals (F2) generated by said sample after excitation, characterized in that: The system comprises optical polarization means positioned on the path of said light beam (F1), The optical polarization means being composed of at least a first optical polarizer (P1) and a second optical polarizer (P2),The optical polarization means being configured to select and illuminate a viewing area by acting on the orientation of the polarization direction of the first optical polarizer (P1) and on the polarization direction of the second optical polarizer (P2). Figure to be published with the abstract: Figure 2,

Description

Title of the invention: Optical system for monitoring a sample by fluorescence Technical field of the invention

[0001] The present invention relates to an optical system for monitoring a sample by fluorescence during a reaction in a microfluidic card. State of the art

[0002] Microfluidics is a scientific and technical field whose objective is in particular the manipulation of small volumes of fluid within a microfluidic network. Its applications are diverse, such as medical diagnosis, the environment, agri-food or veterinary.

[0003] Microfluidic technologies are capable of integrating complex protocols autonomously. In the context of medical diagnosis, for example, a microfluidic cartridge performs all operations from the deposit of the liquid sample (blood, urine, saliva), its manipulation (transfer, contact with biological reagents) to the reading of the analysis result and its interpretation (computer-assisted).

[0004] The analyses can for example be implemented using nucleic acid amplification protocols, for example of the PCR ("Polymerase Chain Reaction") or LAMP ("Loop Mediated Isothermal Amplification") type. Such protocols are used to detect pathogens in various matrices (blood, food, saliva, etc.) and are based on the principle of exponential amplification of nucleic acids. From a DNA sequence, suitably designed biochemical reagents allow the DNA sequence to be replicated exponentially by creating millions of copies, thanks to heating of the solution. One way to visualize the multiplication of DNA sequences in real time is to add a fluorescent probe to the solution. During amplification, the fluorescence released is proportional to the number of DNA copies created.Thus, by following the evolution of fluorescence over time, it is possible to plot the associated amplification curves and thus go back to the initial concentration of DNA before amplification, and sometimes to the initial concentration of the pathogen sought (qPCR).

[0005] Today, nucleic acid amplification protocols are routinely performed on commercial instruments, carrying out the steps of heating the solution and recording fluorescence data, up to data processing with the display of amplification curves and associated analyte concentrations.

[0006] The implementation of nucleic acid amplification protocols in microfluidic cards is the subject of much research and has many advantages (speed of heat transfer, reduction of reagent volumes, reduced analysis times). The solution containing the DNA and the reaction mix for amplification is located in dedicated microfluidic chambers, and a fluorescent reagent is used in the amplification solution. The greater the quantity of DNA, the greater the fluorescence. In order to follow the evolution of the fluorescence as a function of time, the solution in the microfluidic chambers is excited by lighting (LED + filter for example) centered on the absorption spectrum of the fluorophore. After excitation, fluorescence signals are emitted by the fluorophores present in the solution and recorded by a camera equipped with a filter at the chosen wavelength.Processing this data (fluorescence curves as a function of time) allows us to know the DNA concentration in the sample.

[0007] As described in patent application CN109187367 and in the referenced publication "High-sensitivity, disposable lab-on-a-chip with thin-film organic electronics for fluorescence detection" - Andrea Pais, Ansuman Banerjee, David Klotzkin, Ian Papautsky - Department of Electrical and Computer Engineering, University of Cincinnati, 814 Rhodes Hall, ML030, Cincinnati, OH, USA - Publication DOI:10.1039 / b715143h, the optical setup used is arranged in transmission, with polarized excitation light and an analyzer at the output of the fluidic card to cut the excitation wavelength. The excitation source is placed on one side of the card and illuminates the microfluidic chambers, and the camera is positioned on the other side of the card to capture the images.This architecture does not work for some systems in which the microfluidic card is manufactured using birefringent materials or when the microfluidic card itself is composed of a material that is non-transparent at the wavelengths considered (such as silicon in the visible).

[0008] The aim of the invention is to propose a system that can measure fluorescence signals in a microfluidic chamber after excitation, without having the limitations of the transmission principles of the state of the art. The solution of the invention can also be adapted to visualize distinct parts of the microfluidic circuit of a card, even those which are in lower layers of the card. Statement of the invention

[0009] This aim is achieved by an optical system for monitoring a sample by fluorescence, comprising a controlled excitation light source for generating a light beam and a microfluidic card, said sample being placed in a microfluidic circuit integrated into said microfluidic card, said system comprising a device for detecting fluorescence signals generated by said sample after excitation by said light beam, said system being characterized in that: - The excitation light source and the detection device are arranged relative to the microfluidic circuit according to a reflection assembly, - The system comprises optical polarization means positioned on the path of said light beam, - The optical polarization means being composed of at least a first optical polarizer and a second optical polarizer, - The optical polarization means being configured to select and illuminate a viewing area by acting on the orientation of the polarization direction of the first optical polarizer and on the polarization direction of the second optical polarizer.

[0010] According to one feature, the viewing area includes at least a portion of the microfluidic circuit.

[0011] According to another feature, the viewing area is an area not polarized by the second optical polarizer or an area polarized with a third optical polarizer having a polarization direction distinct from that of the second optical polarizer.

[0012] According to a particular embodiment, the optical polarization means are passive so that the polarization direction of the first optical polarizer and the polarization direction of the second optical polarizer have a given and fixed orientation.

[0013] According to another particular embodiment, the optical polarization means are active, so that the system comprises means for adjusting the orientation of the polarization direction of the first optical polarizer and / or the orientation of the polarization direction of the second optical polarizer.

[0014] According to a particular embodiment, the active polarization means comprise mechanical means configured to rotate one or more of said optical polarizers in order to orient their polarization direction according to a given orientation depending on the viewing area to be illuminated.

[0015] According to a particular embodiment, the microfluidic card is produced by assembling one or more superimposed layers, one or more layers integrating said microfluidic circuit of the microfluidic card.

[0016] According to another particular embodiment, the optical polarization means comprise at least a fourth optical polarizer inserted between at least two layers of the microfluidic card.

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] According to a particular feature, the fourth optical polarizer is realized in the form of a layer of the microfluidic card. According to another feature, the fourth optical polarizer comprises at least one opening positioned opposite at least a second part of the microfluidic circuit. According to another particular embodiment, the optical polarization means comprise a fifth optical polarizer placed in said opening. According to a particularity, each layer of the card located above an optical polarizer is made of a non-birefringent material. According to a particular embodiment, the active optical polarization means comprise at least one liquid crystal part positioned between the first optical polarizer and the second optical polarizer and voltage-controlled to transmit or block the light beam between the excitation light source and the microfluidic card and to enable one or more areas to be illuminated on the microfluidic card to be selected. According to a particularity, the liquid crystal part is single-pixel or pixelated. According to another feature, the detection device includes a camera. The invention also relates to a method for monitoring a liquid sample by fluorescence, implemented using an optical monitoring system as defined above, the method consisting of: - Activate the excitation light source to direct the light beam towards the microfluidic card, - Position the detection device relative to the microfluidic circuit of the card according to a reflection assembly, - Capture the fluorescence signals emitted by the liquid sample present in the microfluidic circuit, after excitation, - The first optical polarizer and the second optical polarizer being oriented with a polarization direction adapted so that the viewing area comprises at least part of the microfluidic circuit. Brief description of the figures Other features and advantages will appear in the detailed description which follows, given with reference to the attached drawings in which: - [Fig.l] shows an exemplary embodiment of a microfluidic card that can be used in the system of the invention; - [Fig.2] schematically shows the architecture of the system of the invention; - [Fig.3] shows, in an exploded view, a first example of the production of the microfluidic card used in the system of the invention; - [Fig.4] shows an embodiment of the system of the invention integrating the microfluidic card of [Fig.3]; - Figures 5A to 5C illustrate the operating principle of the system with the microfluidic card of [Fig.3]; - Figures 6A and 6B show a top view of another example of embodiment of the microfluidic card of the invention and illustrate its operating principle; - [Fig.7] shows, in an exploded view, another example of the embodiment of the microfluidic card used in the system of the invention; - Figures 8A to 8C illustrate, by top views of the microfluidic card, different modes of operation of the system of the invention with a microfluidic card of [Fig.7]; - Figures 9A and 9B show a first example of the operating principle of a liquid crystal device; - Figures 10A to 10F schematically show several other alternative embodiments of the system of the invention;

[0026] Detailed description of at least one embodiment

[0027] For the remainder of the description, we define an orthonormal reference frame X, Y, Z to represent the three dimensions.

[0028] The terms "upper", "lower", "above", "below" and equivalents are to be considered along the Z axis of the reference frame.

[0029] The invention applies to a system for monitoring a sample by fluorescence and mainly comprises a microfluidic card 1, an excitation light source 2 and a detection device 3. Microfluidic card

[0030] [Fig.l]

[0031] A microfluidic card 1 is notably used in the medical field to analyze a fluid, such as a liquid sample (blood for example) by connecting to an analysis machine. The microfluidic card 1 can thus integrate an entire microfluidic network composed of microfluidic elements such as valves, chambers and microfluidic channels.

[0032] A microfluidic card can be made in a single layer or by assembling several layers 10 together. Its layers 10 are for example assembled together by thermal sealing. Each layer 10 can be machined so as to create at least a part of the microfluidic network of the card 1, the assembly of the layers 10 by stacking making it possible to form the entire microfluidic network of the microfluidic card 1.

[0033] Depending on the materials used, the application of mechanical constraints during the production of microfluidic cards can generate photoelasticity and make the card material birefringent even if it is isotropic in nature.

[0034] The microfluidic card 1 comprises two opposite faces, each extending in the two dimensions X, Y and having, for example, a small thickness (a few mm) along Z with respect to the other two dimensions.

[0035] The microfluidic card 1 is advantageously made of a transparent material of the COP (Cyclo-Olefin Polymer), COC (Cyclo-Olefin Copolymer), PMMA (Polymethyl Acrylic Methacrylate), PDMS (Polydimethyl Siloxane), Silicon type.... But we will see that, in certain embodiments, the material of the card will have to meet certain requirements (non-birefringence) so that the principle of the invention works best. As a reminder, a material has the physical property of birefringence when light propagates through this material in an anisotropic manner.

[0036] In the context of the invention, we are interested in a microfluidic network comprising at least one microfluidic chamber 11, used to implement optical monitoring by fluorescence of a liquid sample placed in said microfluidic chamber 11. At a minimum, the microfluidic chamber 11 has on the top a wall transparent to the light beam F1 emitted by the excitation light source 2 and to the fluorescence signals F2 emitted after excitation. Excitation light source

[0037] [Fig.2]

[0038] This is a light source 2, which can be associated with a filter (not shown), to emit a light beam Fl over a range of wavelengths centered on the absorption spectrum of the fluorophore compounds present in the sample to be monitored.

[0039] The light source 2 is placed above the microfluidic card 1 to illuminate a zone ZI of the microfluidic card 1 including the microfluidic chamber 11.

[0040] In a non-limiting manner, the light source 2 may be composed of one or more light-emitting diodes, for example of the OLED type (for "Organic Light Emitting Diode"). The source may also be a laser, which makes it possible - for certain types of lasers - to dispense with the spectral filter and the first optical polarizer PI (see below). Detection device

[0041] [Fig.2]

[0042] The detection device 3 may comprise a camera for reading and recording the fluorescence signals emitted by the fluorophore compounds present in the sample. and excited by the light beam Fl emitted by the excitation light source 2. The camera may in particular include a CMOS (Complementary Metal-Oxide-Semiconductor) or CCD (Charge Coupled Device) type sensor. Associated with the camera, the detection device 3 may integrate a tube lens, a spectral filter and an objective (not shown) to obtain a clear image on the camera sensor. Optical assembly - principle

[0043] The excitation light source 2 and the detection device 3 are arranged relative to the microfluidic card 1 and the microfluidic chamber 11 according to a reflection assembly. The excitation and the detection are therefore placed on the same side relative to the microfluidic card 1, for example above the microfluidic card 1. The light beam F1 is emitted by the light source towards the card, on the side of its upper face, and the fluorescence signals F2 generated are emitted towards the detection device 3, also located above the card, on the side of its upper face.

[0044] The light source 2 is oriented so as to illuminate a zone ZI of the card including the microfluidic chamber 11, and the detection device 3 is positioned to acquire images of this illuminated zone ZI or of at least a part of it (including for example at least a part of the microfluidic circuit of the card), in order to be able to visualize the fluorescence signals F2 emitted by the sample, for example when it is placed in the microfluidic chamber 11 in the presence of an excitation (as in [Fig.2]).

[0045] According to the invention, the optical assembly of the system also incorporates optical polarization means. The optical polarization means are used to highlight and select certain areas of the microfluidic card 1, ensuring that the light beam F1 emitted by the light source 2 does not pass through the entire microfluidic card 1, but that it penetrates only into the chosen area, for example the microfluidic chamber 11 containing the fluorophores (area Z3 for example in [Fig. 2] described below).

[0046] The polarization means can be chosen passive, that is to say that they always keep the same configuration during the experiment or chosen active, that is to say that they can be activated to select a particular zone of the microfluidic card 1.

[0047] For the remainder of the description, the so-called crossed configuration is defined when two polarizers have their polarization directions perpendicular and the so-called aligned configuration when two polarizers have their polarization directions parallel. The polarization direction is defined by the parallel orientation of the polarization lines of the polarizer considered. Passive optical polarization means

[0048] [Fig.2]

[0049] In this configuration, the system comprises at least two optical polarizers PI, P2, called first optical polarizer PI and second optical polarizer P2.

[0050] The two optical polarizers PI, P2 are for example linear polarizers, that is to say that they each have a given and fixed direction of polarization.

[0051] The optical assembly as presented in [Fig.2] aims to ensure that the light beam Fl emitted by the light source 2 does not pass through the entire microfluidic card 1, but that it only penetrates into the microfluidic chamber 11 containing the fluorophores (zone Z3 for example in [Fig.2]). First optical polarizer

[0052] [Fig.2]

[0053] In the context of the configuration presented in [Fig.2], the first optical polarizer PI is positioned in front of the excitation light source 2 so as to polarize the light beam Fl emitted by the light source 2. It is therefore placed between the excitation light source 2 and the microfluidic card 1.

[0054] Its direction of polarization is for example oriented along a first direction (for example along X). Second optical polarizer

[0055] [Fig.2]

[0056] [Fig.3]

[0057] The second optical polarizer P2 is arranged between the first optical polarizer PI and the microfluidic card 1. Its polarization direction is oriented along a second direction distinct from the first direction, so as to form crossed lines with those of the first optical polarizer PL. Its polarization direction is advantageously oriented perpendicular to that of the first optical polarizer PI (along Y for the previous example).

[0058] According to the invention, this second optical polarizer P2 is configured to form a second zone Z2 where the light is polarized according to the direction imposed by the second optical polarizer P2 and a third zone Z3 comprising or not comprising a polarizer, and where the light is de facto polarized differently from that present at the zone Z2 (see [Fig.6A]). In other words, this second optical polarizer P2 does not extend over the entire surface of the illuminated zone Z1. The third zone Z3 advantageously includes the microfluidic chamber 11 containing the sample to be monitored by fluorescence.

[0059] The third zone Z3 is for example formed by the creation of an opening O opposite the microfluidic chamber 11.

[0060] In this way, the light beam Fl emitted by the light source 2 does not pass through the microfluidic card in the zone Z2 and only passes through the zone Z3 to illuminate the microfluidic chamber 11 of the microfluidic card. The detection device 3 can thus easily distinguish the relevant reaction zone (the one which contains the microfluidic analysis chamber) of the card from the other non-relevant zones, and thus capture the fluorescence signals F2 emitted by the fluorophores present in the sample when they are excited. First embodiment

[0061] [Fig.2]

[0062] [Fig.3]

[0063] [Fig.4]

[0064] [Fig.5A]

[0065] [Fig.5B]

[0066] [Fig.5C]

[0067] Advantageously, in connection with Figures 2 and 3, the second optical polarizer P2 is in the form of a film applied to the upper face of the microfluidic card 1, this film having the opening O made around the microfluidic chamber 11, this part, for example cut and removed from the film forming the zone Z3 defined above. The film can be applied to the microfluidic card 1 by gluing, using screws, magnets or simply deposited by electrostatic effect. This polarizer P2 can also be produced by a thin layer deposition with masking of the zone Z3 during the deposition, making this zone non-polarized.

[0068] With reference to [Fig.4], the excitation light source 2 can be positioned to emit its beam Fl in a plane parallel to the X, Y directions and an optical device 4 (cube + dichroic mirror) can be positioned above the card to orient the light beam Fl generated by the excitation light source 2 towards the microfluidic card 1, along the Z axis. The detection device 3 can be positioned above the microfluidic card 1, the surface of its sensor perpendicular to the Z axis. The optical device 4 is chosen to allow the fluorescence signals F2 generated by the excitation to pass through.

[0069] Figures 5A to 5C illustrate the operation of the invention with the microfluidic card of [Fig.3].

[0070] [Fig.5A]: Excitation light source 2 off.

[0071] [Fig.5B]: The first optical polarizer PI and the second optical polarizer P2 are in crossed configuration, allowing the light beam Fl to only pass through the opening O, thus making it possible to select the zone Z3, in which the microfluidic chamber 11 is located.

[0072] [Fig.5C]: The first optical polarizer PI and the second optical polarizer P2 are in aligned configuration, allowing the light beam Fl to cross the entire map and therefore to visualize zones Z2 and Z3 and to follow the fluid movements throughout the map. Other embodiments

[0073] [Fig.6A]

[0074] [Fig.6B]

[0075] [Fig.7]

[0076] [Fig. 8 A]

[0077] [Fig.8B]

[0078] [Fig.8C]

[0079] According to another embodiment variant illustrated by Figures 6A and 6B, it is also possible to add an optical polarizer P20 within each opening O produced through the second optical polarizer P2, this polarizer P20 having a polarization direction oriented crosswise with respect to that of the optical polarizer P2. Thanks to this configuration, the zone Z3 is also polarized, which makes it possible, depending on the orientation of the polarization applied to the light source 2 via the first optical polarizer PI, to select the zone to be "turned on" or "turned off" between the zone Z2 and the zone Z3.

[0080] [Fig.6A] thus shows that when the light source 2 is polarized by the first optical polarizer PI, in a manner aligned with the optical polarizer P20, it "lights up" the microfluidic chamber 11. In the case where the polarization of PI is in an intermediate orientation, for example at 45° between aligned and crossed, the entire card is illuminated with, however, reduced beam transmission over the different polarized zones.

[0081] [Fig.6B] shows another configuration in which the first optical polarizer P1 and the second optical polarizer P2 are in an aligned configuration, and the polarizer P20 is in a crossed configuration relative to them. In this configuration, it is possible to observe the zone Z2 by masking the zone Z3, which makes it possible to follow the fluid circulation in the card without exciting the fluorophores contained in the microfluidic chamber 11 and thus to avoid photobleaching.

[0082] According to another advantageous embodiment illustrated by [Fig.7], it is possible to integrate optical polarizers (for example two optical polarizers P2, P3) at several levels in the layers of the microfluidic card 1, in order to highlight different layers of the microfluidic network of the microfluidic card 1 and several zones Z3, Z4 of the card. By playing on the orientation of the polarization direction of each optical polarizer P2, P3 integrated into the microfluidic card 1, it is possible to visualize the different layers 10 of the microfluidic card 1 and to select zones of interest.

[0083] By way of example, a layer of the microfluidic card may comprise at least one microfluidic chamber 11 and another layer of the microfluidic card may comprise at least one other microfluidic chamber 12.

[0084] The optical polarizer P2 may comprise an opening 01 made around the microfluidic chamber 11 and the optical polarizer P3 may comprise another opening 02 made around the microfluidic chamber 12. Each opening may advantageously integrate an optical polarizer P20, P30 oriented differently (in crossed configuration for example), as described above according to the configuration of [Fig.7].

[0085] In the various configurations proposed, it is however necessary to ensure that the upper layer or layers of the microfluidic card 1 located above each optical polarizer P2, P3 are made of a non-birefringent material. On the other hand, the lower layer or layers located under the lowest optical polarizer (P3 in this configuration) can be made of a birefringent material. As a reminder, a non-birefringent material is a so-called isotropic material, which does not depend on the direction of polarization of the light wave. It can for example be glass.

[0086] Figures 8A to 8C thus show different operating configurations depending on the state of the light source 2 and its type of polarization, for a microfluidic card 1 having two microfluidic chambers 11 arranged in a layer of the microfluidic card located between the optical polarizer P2 and the third optical polarizer P3, and two other microfluidic chambers 12 arranged in a lower layer of the microfluidic card located under the third optical polarizer P3. The second optical polarizer P2 is for example a film having for example a polarization direction oriented along Y and the third optical polarizer P3 is a film having a cross-polarization direction, for example oriented along X. The first optical polarizer P1 is placed in front of the excitation light source 2.We also add the polarizer P20 in each opening of the polarizer P2, in crossed configuration with respect to the polarizer P2, and the polarizer P30, in crossed configuration with respect to the polarizer P3.

[0087] [Fig.8A]: Excitation light source 2 off.

[0088] [Fig.8B]: Excitation light source 2 switched on and first PI polarizer in confi guration aligned with the polarizer P20 and crossed with the second polarizer P2. The camera observes the fluorescence emitted at the level of each zone Z3 occupied by the two microfluidic chambers 11 of the upper layer.

[0089] [Fig.8C]: Excitation light source 2 switched on and first polarizer PI in configuration aligned with polarizer P2 and with polarizer P30, and crossed with polarizer P20 and with polarizer P3. The camera observes the fluorescence emitted at level of each zone Z4 occupied by the two microfluidic chambers 12 of the lower layer.

[0090] Mechanical rotation system of polarizers for selection of viewing areas

[0091] Advantageously, it is possible to provide active technical means for mechanically rotating the first optical polarizer PI and / or possibly each other optical polarizer, and thus adjusting the orientation of their polarization directions and adjusting the polarization level, either between the two extreme polarization situations, i.e. aligned polarization and 90° crossed polarization, or in an intermediate position (typically at 45°). In the case of the first optical polarizer PI, mechanical means can for example be added to the system to control its orientation.

[0092] By the term “active”, we mean that it is possible to play at any time on the direction of polarization applied to the light beam Fl.

[0093] This mechanical rotation advantageously makes it possible to select the viewing areas according to the need and in particular to be able to find the same configurations as those described above: - [Fig.5B]: illumination of zone Z3 only if the polarization directions of polarizers PI and P2 are crossed - [Fig.5C]: illumination of zones Z2+Z3 if the polarization directions of polarizers PI and P2 are aligned. - [Fig.ôA]: illumination of zone Z3 if the polarization directions of polarizers PI and P20 are aligned and that of polarizer P2 is crossed. - [Fig.ôB]: illumination of zone Z2 alone if the polarization directions of polarizers PI and P2 are aligned and that of polarizer P20 is crossed.

[0094] An intermediate configuration makes it possible to illuminate the zones Z2+Z3 if the polarization direction of PI is in an intermediate position (typically 45°) relative to the polarization directions of P2 and P20 (which are in a crossed configuration).

[0095] It should be noted that the illumination of zone Z2 only can be advantageous when one wants to follow the fluid circulation in the card without exciting the fluorophores contained in the fluid chamber (11) - delimited by zone Z3 and thus avoid photobleaching. Active polarization means

[0096] [Fig.9A]

[0097] [Fig.9B]

[0098] [Fig.10A]

[0099] [Fig.lOB]

[0100] [Fig.10C]

[0101] [Fig.10D]

[0102] [Fig.10E]

[0103] [Fig. 10F]

[0104] In particularly advantageous embodiments, it is also possible to use active polarization means, so as to be able to control the polarization directions without any mechanical movement of the polarizer. By the term "active" is meant that it is possible to act at any time on the direction of polarization applied to the light beam Fl.

[0105] For this, we can also use the principle of liquid crystals.

[0106] In a known manner, a liquid crystal device makes it possible to control the polarization of light by means of an electric field applied to the crystals. These rotators are based on a variation of the birefringence properties of the liquid crystals under the effect of an electric field. Different types of liquid crystal polarization rotator architectures are possible.

[0107] A particular configuration that can be used within the framework of the invention is described in a simplified manner in Figures 9A and 9B: The liquid crystal part comprises two glass substrates (not shown), between which the liquid crystal 52 is placed. On either side of these substrates, the device comprises an analyzer 50 and a polarizer 51.

[0108] The analyzer and the polarizer have polarization directions in crossed configuration. The two glass substrates are covered with a transparent electrode (typically ITO). When an electric field V is applied between the two electrodes, the molecules of the liquid crystal 52 gradually align themselves along the direction of the field generated between the two electrodes ([Fig.9B]). The light is no longer deflected by the molecules, it is therefore stopped by the analyzer 50 because it is in crossed configuration with respect to the polarizer 51. The device is switched off. If the electric field is deactivated, the molecules of the liquid crystal 52 form a helical structure, and the device switches back on ([Fig.9A]).

[0109] This type of component therefore allows, when a voltage is applied to it, to modify the direction of polarization in the desired manner (depending on the chosen polarization rotator architecture, it is possible to obtain aligned polarization, or crossed at 90°, or according to another orientation). This solution based on liquid crystals has the advantage of being able to illuminate or not rooms without mechanical movement of the different polarizers.

[0110] A distinction is made between the so-called single-pixel liquid crystal device, i.e. one which can be switched on or off over its entire surface, and the pixelated liquid crystal device, i.e. one having several individually voltage-controllable pixels.

[0111] Figures 10A to 10F show several possible configurations - based on the architecture of figures 9A and 9B of helical nematic cells, to be considered in a non-limiting manner.

[0112] [Fig.lOA]:

[0113] The excitation light source 2 is unpolarized. A single-pixel liquid crystal device 5 is positioned on the card with an opening O forming the zone Z3 at the level of the microfluidic chamber 11. It can be deposited directly on the upper face of the microfluidic card or positioned just above the card 1, close to the upper face thereof.

[0114] The device 5 thus comprises its three superimposed “layers”, analyzer 50, liquid crystal part (helical nematic cells) 52 and polarizer 51. In operation, and depending on the voltage applied to the device 5, it is thus possible to illuminate only the zone Z3 or the entire microfluidic card 1 (zones Z2+Z3).

[0115] [Fig.lOB]: This is a configuration similar to that of [Fig.lOA], except that the excitation light source 2 is polarized with the first polarizer PI placed directly in front of the light source 2, this polarizer PI replacing that of the liquid crystal device 5.

[0116] [Fig. 10C]: This is a configuration identical to that of [Fig. 10B], except that the liquid crystal part 52 is offset in front of the excitation light source 2, in front of the polarizer PL. Only the analyzer 50 is maintained on the microfluidic card 1 (in this case the analyzer can be in the form of a film stuck on the fluidic card) or close to it. The operation is identical to that of the configuration of [Fig.lOB].

[0117] [Fig. 10D]: In this configuration, the liquid crystal device 5 is pixelated, that is to say that it is possible to turn on or off each pixel of the device 5 individually, to let the light pass or not. The arrangement is identical to that of [Fig.10A] described above, except that it is not necessary to create the opening and the zone Z3 to visualize the microfluidic chamber.

[0118] The advantage of this configuration is that it can be adapted to the microfluidic network of the microfluidic card 1.

[0119] [Fig.lOE]: This is a similar configuration to that of [Fig.lOB], except that a pixelated liquid crystal device 5 is used. The polarizer PI is positioned in front of the excitation light source 2 and it is not necessary to create an opening opposite the microfluidic chamber, since the device 5 is pixelated. Each pixel of the device 5 can be activated / deactivated individually to allow or not allow light to pass into the microfluidic card 1.

[0120] [Fig. 10F]: A first liquid crystal device 5a is placed on a first microfluidic card 1a, then a second microfluidic card 1b on this first liquid crystal device 5a, then a second liquid crystal device 5b on the second microfluidic card 1b. The second microfluidic card 1b must be made of a non-birefringent material for the principle to work.

[0121] In this configuration, each pixel can be activated / deactivated individually to allow light to pass through or not into each of the microfluidic cards 1a, 1b.

[0122] The orientation of the polarizer of the liquid crystal device 5a must necessarily be the same as that of the analyzer of the liquid crystal device 5b.

[0123] As long as the upper microfluidic card(s) are made of non-birefringent materials and the analyzers and polarizers of the consecutive liquid crystal devices are aligned, it is possible to add additional layers. The polarizer of the second device 5b can be offset in front of the excitation light source 2.

[0124] Advantageously, the liquid crystal device 5 is for example connected to a control unit responsible for generating the voltages adapted to display the polarization pattern, according to the desired configuration.

[0125] It should be noted that the use of a liquid crystal solution offers many possibilities and that many other variant embodiments could be imagined, possibly combining the different configurations presented above.

[0126] The invention thus presents numerous advantages listed below.

[0127] The solution of the invention makes it possible to obtain several optical visualization modes on microfluidic cards. The use of a polarizing mask makes it possible to choose certain areas that one wants to excite and / or visualize, and therefore opens up interesting perspectives for the use of such optical systems in combination with other parts of microfluidic cards where fluorescence is not excited.

[0128] The solution of the invention - where only the area of ​​interest is illuminated - makes it possible to overcome the problems of parasitic noise present in the microfluidic card technologies used (for example autofluorescence or Raman), and inherent in the materials used. The resolution of the noise problem makes it possible to envisage improvements in various biological protocols. As an example, we can cite: • Nucleic acid amplification protocols (PCR, LAMP). Fluorescence data as a function of time gives a "Ct" ("cycle threshold") time, corresponding to the threshold where the fluorescence level increases significantly, and allows the skilled person to calculate the initial concentration of DNA template. The reduction of parasitic noise on the microfluidic card should allow better distinction of the signal from the background noise, and therefore faster detection. Detection protocols using linear amplification of the fluorescent signal, for example ELIS A type. Contrast enhancement and noise reduction allow the linear range of such tests to be extended.

Claims

Claims

1. Optical system for monitoring a sample by fluorescence, comprising a controlled excitation light source (2) for generating a light beam (Fl) and a microfluidic card (1), said sample being placed in a microfluidic circuit integrated in said microfluidic card, said system comprising a device for detecting (3) the fluorescence signals (F2) generated by said sample after excitation by said light beam (Fl), characterized in that: - The excitation light source (2) and the detection device (3) are arranged relative to the microfluidic circuit in a reflection arrangement, - The system comprises optical polarization means positioned on the path of said light beam (Fl), - The optical polarization means being composed of at least a first optical polarizer (PI) and a second optical polarizer (P2),- The optical polarization means being configured to select and illuminate a viewing area by acting on the orientation of the polarization direction of the first optical polarizer (PI) and on the polarization direction of the second optical polarizer (P2).,

2. System according to claim 1, characterized in that the viewing area includes at least part of the microfluidic circuit.

3. System according to claim 1 or 2, characterized in that the viewing area is an area not polarized by the second optical polarizer (P2) or an area polarized with a third optical polarizer (P20) having a polarization direction distinct from that of the second optical polarizer (P2).

4. System according to one of claims 1 to 3, characterized in that the optical polarization means are passive so that the polarization direction of the first optical polarizer (PI) and the polarization direction of the second optical polarizer (P2) have a given and fixed orientation.

5. System according to one of claims 1 to 3, characterized in that the optical polarization means are active, so that the system comprises means for adjusting the orientation of the polarization direction of the first optical polarizer (PI) and / or the orientation of the polarization direction of the second optical polarizer (P2).

6. System according to claim 5, characterized in that the active polarization means comprise mechanical means configured to rotate one or more of said optical polarizers in order to orient their polarization direction according to a given orientation depending on the viewing area to be illuminated.

7. System according to one of claims 1 to 6, characterized in that the microfluidic card (1) is produced by assembling several superimposed layers (10), one or more layers integrating said microfluidic circuit of the microfluidic card (1).

8. System according to claim 7, characterized in that the optical polarization means comprise at least a fourth optical polarizer (P3) inserted between at least two layers of the microfluidic card.

9. System according to claim 8, characterized in that the fourth optical polarizer (P3) is produced in the form of a layer of the microfluidic card.

10. System according to claim 9, characterized in that the fourth optical polarizer (P3) comprises at least one opening (02) positioned opposite at least a second part of the microfluidic circuit.

11. System according to claim 10, characterized in that the optical polarization means comprise a fifth optical polarizer (P30) placed in said opening (02).

12. System according to one of claims 7 to 11, characterized in that each layer of the card located above an optical polarizer is made of a non-birefringent material.

13. System according to claim 5, characterized in that the active optical polarization means comprise at least one liquid crystal part (52) positioned between the first optical polarizer (PI) and the second optical polarizer (P2) and voltage-controlled to transmit or block the light beam (Fl) between the excitation light source (2) and the microfluidic card (1) and to allow one or more areas to be illuminated on the microfluidic card to be selected.

14. System according to claim 13, characterized in that the liquid crystal part (52) is single-pixel or pixelated.

15. System according to one of claims 1 to 14, characterized in that the detection device (3) comprises a camera.

16. Method for monitoring a liquid sample by fluorescence, implemented using an optical monitoring system as defined in one of claims 1 to 15, characterized in that it consists of: - Activate the excitation light source (2) to direct the light beam towards the microfluidic card (1), - Position the detection device (3) relative to the microfluidic circuit of the card according to a reflection assembly, - Capture the fluorescence signals (F2) emitted by the liquid sample present in the microfluidic circuit, after excitation, - The first optical polarizer (PI) and the second optical polarizer (P2) being oriented with a polarization direction adapted so that the viewing area comprises at least part of the microfluidic circuit.

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