Diffractive optical element including a metasurface for TIRF microscopy

Abstract

Diffractive optical element (10) comprising: - a substrate (SB) having a first surface (Sm) and a second surface opposite to the first surface (Se), transparent to light in at least one spectral range and having, in said spectral range, a refractive index greater than that of water;- at least one metasurface adapted to diffract light radiation (Lin) of wavelength within said spectral range, incident at an angle of incidence, into diffracted radiation (Ldiff), such that the diffracted radiation propagates in the substrate and reaches said second substrate surface (Se) at a diffracted angle greater than or equal to a limiting angle ( ) of total internal reflection between said substrate and water, said metasurface being configured to exhibit, for said angle of incidence, a transmission of the diffraction order 0 of less than 5% and a transmission of the diffracted radiation (Ldiff) corresponding to a diffraction order -1 or +1 greater than 50%. Figure 2;

Description

Title of the invention: Diffractive optical element comprising a me- tasurface for TIRF microscopy Technical field

[0001] The present invention relates to the field of TIRF microscopy (or total internal reflection fluorescence microscopy). Previous technique

[0002] Hereafter, a metasurface will be called a component comprising a periodic or quasi-periodic repetition of an elementary pattern, of dimension and period less than a wavelength of incident radiation and diffusing said incident radiation by modifying its phase and amplitude.

[0003] Total Internal Reflection Fluorescence (TIRF) microscopy is a fluorescence microscopy technique in which the excitation of fluorescent molecules in the observed sample is confined to a nanometer-thick region located in the immediate vicinity of the sample holder. In particular, it allows for the selective observation of structures and processes located on a cell membrane, with spatial resolution in an axial direction far exceeding the diffraction limit. Furthermore, compared to more conventional epifluorescence techniques, it provides better contrast in the fluorescence image and reduces the effects of photobleaching and irradiation damage to cells.

[0004] The principle underlying evanescent wave fluorescence microscopy is illustrated in Figure IA. Consider the case of a substrate SB with refractive index n2, having a surface Se in contact with an ambient medium MA with refractive index ni <n2. Par exemple, le substrat SB peut être constitué par une lamelle porte-échantillon, ou par un élément en verre sur lequel est posée une telle lamelle, tandis que le milieu ambiant MA peut être une solution aqueuse contenant, en suspension, des cellules marquées par des fluorophores. Un faisceau lumineux FLI, provenant du substrat SB, est incident sur la surface Se ; sa direction de propagation forme avec la normale z s à la surface un angle 0 supérieur à une valeur critique 0C (angle limite) _ arcsin^ (1)-

[0005] Consequently, the FLI beam undergoes total internal reflection (the reference FLR designates the reflected beam) and an evanescent wave OE appears in the surrounding medium MA. This evanescent wave has an intensity that decreases exponentially with the distance z from the surface S: j , , ^ / 3, where the length of pe Netration ô is given by g — (n2sin20d - nj) (Ibis), X being the length of the light radiation wave. The evanescent wave excites the fluorophores contained in the surrounding medium, but only over a thickness on the order of λ, because beyond this point its intensity quickly becomes negligible. For example, for λ = 488 nm, n2 = 1.514 (BK7 glass), n1 = 1.33 (water) and θ = 67°C > 0°C^61.45°, we find λ^93 nm, which means that only the fluorophores located in a layer approximately 100 nm thick are excited and contribute to the formation of a fluorescence image.

[0006] Figure IB illustrates the most commonly used configuration in TIRF microscopy.

[0007] In the case of Figure IB, the same microscope objective OBJ, located on the substrate side opposite the medium MA, is used both to generate the evanescent waves by total internal reflection and to collect the fluorescence radiation. The objective is generally of the oil immersion type and can have a large numerical aperture (NA), for example, on the order of 1.45, which in turn allows for a high lateral spatial resolution (perpendicular to the z-direction) since the lateral spatial resolution is given by d = X / 2NA. Furthermore, an objective OBJ with a high numerical aperture NA is essential because it allows the incident light beam FLI to be displaced relative to the optical axis and thus obtain a strong deflection of the incident beam FLI by the objective. The FLI beam can therefore propagate at a high angle of incidence θ, sufficient to obtain total internal reflection.However, such a lens is very expensive and introduces significant aberrations. Furthermore, aligning such a setup is complex.

[0008] The invention aims to overcome the aforementioned drawbacks of the prior art. More particularly, it aims to provide a diffractive optical element comprising a metasurface, enabling, for example, TIRF microscopy and eliminating the need for high numerical aperture objectives of the prior art. Summary of the invention

[0009] For this purpose, an object of the invention is a diffractive optical element comprising: - a substrate having a first surface and a second surface opposite to the first surface, transparent to light in at least one spectral range and having, in said spectral range, a refractive index greater than that of water; - at least one metasurface made of a dielectric material and disposed on said first surface of the substrate, the metasurface being formed by the repetition of a periodic pattern along two directions x and y perpendicular to each other and parallel to a surface of the substrate, said pattern comprising a sub-pattern comprising a first portion of a straight band along y; said metasurface being adapted to diffract a light radiation of wavelength / 1 included in said spectral range, incident at an angle of incidence, according to diffracted radiation, such that the diffracted radiation propagates in the substrate and reaches said second surface of the substrate at a diffracted angle greater than or equal to a limiting angle 0c of total internal reflection between said substrate and water, the metasurface being configured to exhibit, for said angle of incidence, a transmission of the diffraction order 0 less than 5% and a transmission of the diffracted radiation corresponding to a diffraction order -1 or +1 greater than 50%.

[0010] According to particular modes of the invention: - the metasurface has a first band of dotted lines along the y direction, formed from said sub-motif and the periodic pattern further includes a second portion of a straight band along the y direction, so that the metasurface has a second continuous band along a y direction; - within the pattern, the area of ​​the first band is less than that of the second band and in which a width of the first band is between 30nm and 500nm, a width of the second band is between 100nm and 700nm and a length of the dotted line is between 60nm and 800nm; - the sub-motif includes a disc, said disc being placed between two successive dotted lines of said first band; - the sub-motif comprises a semi-disc attached to a first side of said first straight portion, and in which said first portions form a first continuous band; - the first straight portion also includes an indentation on a second side of said first straight portion, opposite the first side, and located opposite the semi-disc; - the dimension of the pattern along the x direction is such that Px < A / neau, with n«au the refractive index of water; - the total dimension of the metasurface along the x direction, xMS is such that xMS < with e a thickness of the substrate; - the dimension of the pattern along the y direction is between 300nm and 1000nm; - the refractive index of the metasurface material is greater than the refractive index of a substrate material; - the diffractive optical element comprises a plurality of metasurfaces arranged on said first surface of the substrate, with a pattern dimension along said x direction different for each metasurface, so that each metasurface is adapted to diffract light radiation of wavelength within said range and different from that of the other metasurfaces.

[0011] Another object of the invention is a device for imaging a sample by total internal reflection fluorescence microscopy comprising an optical element including: - at least one light source suitable for emitting said light radiation in said spectral range, incident on said diffractive optical element at said angle of incidence, said light radiation diffracted by said diffractive optical element producing evanescent waves in at least one region of the second surface of the substrate; - a sample placed in correspondence with at least one region of the second surface where said evanescent waves are produced and generating fluorescence radiation excited by said evanescent waves; - a detector suitable for detecting said fluorescence radiation emitted by said sample.

[0012] Yet another object of the invention is a device for detecting a biological sample by surface plasmon resonance comprising a diffractive optical element, said diffractive optical element comprising: - a plurality of metasurfaces with a different pattern dimension along said x direction for each metasurface, such that the diffracted radiation reaches said second substrate surface at a different diffracted angle for each metasurface, said device including further; - at least one suitable light source to emit said light radiation in said spectral range, incident on said diffractive optical element with said angle of incidence; - a sample comprising at least a second substrate, a metallic layer disposed above the second substrate and a layer to be analyzed disposed above the metallic layer, said sample being disposed above the first substrate in such a way that said diffracted radiation is reflected at least partially by the metallic layer of the sample; said diffractive optical element further comprising an optical coupling element adapted to couple said light radiation reflected by the sample to free space; - a detector adapted to detect the intensity of said light radiation reflected by the sample and coupled to free space by said coupling element; at least one metasurface being adapted so that said diffracted angle associated with the metasurface produces, upon reflection on the metallic surface, a resonant excitation of surface plasmons and at least partial absorption of said diffracted radiation.

[0013] According to a particular embodiment of the invention, the optical coupling element comprises at least one output metasurface, formed by the repetition of a periodic pattern along two directions x and y perpendicular to each other and parallel to a surface of the substrate, said pattern comprising a sub-pattern comprising a first portion of a straight band along y, said output metasurface being disposed on said first surface of the substrate. Brief description of the drawings

[0014] Other features, details and advantages of the invention will become apparent from the description given with reference to the accompanying drawings provided by way of example, which represent, respectively:

[0015] [fig.lA] and

[0016] [fig.lB], the evanescent wave microscopy technique known from the prior art;

[0017] [fig.2], a schematic view of a diffractive optical element according to the invention;

[0018] [fig.3A] and

[0019] [fig.3B], respectively a schematic view and a graph showing an effect diffraction efficiency of a diffractive optical element according to a first embodiment of the invention;

[0020] [fig.4A] and

[0021] [fig.4B], respectively a schematic view and a graph showing an effect diffraction efficiency of a diffractive optical element according to a variant of the first embodiment of the invention;

[0022] [fig.5A] and

[0023] [fig.5B], respectively a schematic view and a graph showing an effect diffraction efficiency of a diffractive optical element according to a second embodiment of the invention;

[0024] [fig.6], a schematic view of a device for imaging a sample by mid total internal reflection fluorescence cross-scopy comprising a diffractive optical element according to the invention;

[0025] [fig.7], a schematic view of a biological sample detection device by resonance of surface plasmons comprising a diffractive optical element according to the invention;

[0026] In the figures, unless otherwise indicated, the elements are not to scale. Description of the implementation methods

[0027] Figure 2 shows a schematic view of a diffractive optical element according to the invention. This diffractive optical element 10 comprises a substrate SB of refractive index n2, having a first surface Sm and a second surface opposite the first surface Se, transparent to light in at least one spectral range and having, in said spectral range, n2 being greater than that of water. The surface Se of the substrate is in contact with an ambient medium MA of index ni <n2.

[0028] Furthermore, the diffractive optical element of the invention comprises at least one metasurface MS made of a dielectric material, disposed on the first surface of the substrate Sm. According to a preferred embodiment of the invention, this metasurface is made of titanium dioxide. Alternatively, according to another embodiment of the invention, this metasurface is made of silicon.

[0029] According to the invention, the metasurface is adapted to diffract a light ray Lin of wavelength 2 within said spectral range, incident at an angle of incidence along a diffracted ray Ldiff, such that the diffracted ray propagates through the substrate and reaches said second substrate surface Se at a diffracted angle greater than or equal to a limiting angle 0c of total internal reflection between said substrate and water. By way of non-limiting example, the wavelength 2 of the light ray Lin is between 400 nm and 800 nm.

[0030] Furthermore, the structure of the metasurface is configured so that the metasurface exhibits, for said angle of incidence 9^, a transmission of the diffraction order 0 less than 5% and a transmission of the diffracted radiation Ldiff corresponding to a diffraction order -1 or +1 greater than 50%.

[0031] In the following, in all figures, the diffracted radiation Ldiff is considered to represent the diffraction order towards which the incident radiation is predominantly diffracted (or redirected).

[0032] In the following, the angle Ga formed by the diffracted radiation Ldiff with the normal zs to the second surface of the substrate will be referred to as the "diffraction angle" or "diffracted angle." According to a preferred embodiment of the invention, the two opposing surfaces Se and Sm of the substrate are parallel, and the diffraction angle is equal to that formed by a normal to the first surface Sm and the diffracted radiation. In the remainder of this description of the invention, and not by way of limitation, the two surfaces Se and Sm of the substrate are parallel.

[0033] According to one embodiment of the invention, the substrate is made of optical glass transparent over the spectral range 400nm-800nm ​​such as borosilicate glasses or BK-7. In this embodiment, as mentioned above, the limiting angle 0c is 0c ~ 61°, so the diffraction angle is greater than 61°.

[0034] Alternatively, according to another embodiment, the substrate is made of quartz, or of fused silica.

[0035] The diffractive optical element 10 of the invention allows for very low transmission of the diffraction order 0 and makes it possible to redirect an intensity of the incident radiation predominantly towards a diffraction order -1 relative to the order +1 (or vice versa), like a lattice, with a sufficiently high angle to allow a Total internal reflection (TIR) ​​of diffracted radiation occurs when the surrounding medium (MA, above the substrate) is aqueous. Total internal reflection of the diffracted radiation within the substrate allows the excitation of evanescent waves in the surrounding medium above the region where the diffracted radiation undergoes total internal reflection.

[0036] Also, the optical element of the invention is particularly suited to TIRF microscopy applications that require the excitation of evanescent waves at the interface between a substrate (typically a glass slide) and a sample stored in an aqueous medium. Indeed, in TIRF microscopy, it is desirable to have the lowest possible diffraction order 0 transmission in order to reduce the background noise from the epifluorescence radiation emitted by a sample containing fluorophores during the transmission of the incident radiation.

[0037] To achieve the aforementioned effects, using numerical simulations, the inventors have developed a particular type of metasurface which is presented in a first and a second embodiment of the invention, detailed in Figures 3A and 5A respectively, and a variant of the first embodiment, illustrated in Figure 4A. In all embodiments of the invention, the metasurface is formed of a periodic pattern along two directions x and y perpendicular to each other and parallel to a surface of the substrate, the dimension of the pattern Px being less than the wavelength of the incident radiation h. The pattern includes a sub-pattern E2 comprising a first portion P of a straight band along y.

[0038] The metasurfaces of the invention are produced on the substrate by methods known to those skilled in the art. By way of non-limiting example, they can be produced by electron beam lithography or by nanoimprint lithography and then structured by plasma etching to form the patterns and, if necessary, the sub-patterns. These methods are fast, repeatable, and relatively inexpensive.

[0039] In a known manner, the dimension of the pattern Px along the direction x is related to the diffraction order m, to the wavelength of the incident radiation z and to the diffraction angle dj by the following equation: p — mÀ (2). A rc2sin (0J

[0040] Here, the dimension of the pattern Px along the x direction can be understood as the repetition period of the pattern of the metasurface.

[0041] Based on equation (2) and equation (1), in all embodiments of the invention, the dimension Px of the pattern along the x direction is such that: PX < 2 / n}.

[0042] Thus, the diffraction angle of the diffracted beam will be greater than the critical angle at the interface between the surface Se of the substrate and the ambient medium MA with refractive index nh. When the ambient medium MA is water, the dimension Px of the pattern along the x direction is such that P x < 2 / 1.33.

[0043] As shown in equation (2), the smaller the dimension of the pattern along the x-direction, the larger the diffraction angle. Furthermore, as shown in equation (1bis), the larger the diffraction angle, the shorter the penetration length of the evanescent waves. Thus, for application to TIRF microscopy, the smaller the dimension of the metasurface pattern, the more localized the excitation of the fluorophores in a sample will be on a thin layer, thereby improving the axial resolution of the device.

[0044] In order to maximize the efficiency of the -1 or +1 diffraction order and to limit the transmission of the 0th order, in all embodiments, the refractive index of the metasurface material nmat is greater than the refractive index of the substrate material n2. This condition arises from the relationship between the diffraction efficiency of the different diffraction orders and the coupling between the Bloch modes excited in the metasurface along the z-direction by incident radiation Lin. The excited propagating Bloch modes have coupling coefficients for the transmission of the 0, +1, and -1 diffraction orders with substantially the same amplitude but different phase profiles, and are thus constructively added for the -1 or +1 diffraction order and destructively added for the 0th order. Thus, with the metasurface according to the invention, the incident radiation is predominantly directed towards the +1 or -1 diffraction order.

[0045] Figure 3A illustrates a schematic view of a diffractive optical element 10 according to a first embodiment of the invention. This first embodiment has a first band M1 of dotted lines P, i.e., a discontinuous band, along the y-direction, formed from said sub-pattern E2. The dotted line P corresponds to the first portion of a straight band that repeats along the y-axis. Furthermore, the periodic pattern E1 also includes a second portion of a straight band, called the second portion SP, along the y-direction extending over the entire y-dimensional dimension of the pattern E1, so that the metasurface has a second continuous band M2 along a y-direction.

[0046] In this embodiment, the metasurface therefore comprises a network along x of continuous straight bands M2 along y with, between each continuous band, a dashed band M1 also along y.

[0047] In order to generate an effective refractive index ramp within a metasurface pattern sufficient to establish the effective media regime, and thus deflect or diffract the incident radiation, it is necessary that, within a pattern El, the area of ​​the first band (i.e., of a dashed line P) be smaller than the area of ​​the second portion SP. These areas are determined by numerical simulations so as to generate a sufficient variation in the effective refractive index between the region formed by the dashed lines and the region formed by the continuous band ML.

[0048] Similarly, the spacing between the dotted lines P influences the effective index of the region formed by the dotted lines and is determined by numerical simulation.

[0049] The relative position of the first dashed band M1 with respect to the second continuous band M2, Ay, results in a symmetry breaking along the x-direction, which allows the metasurface to redirect the intensity of the incident radiation primarily towards one diffraction order rather than another. This relative position of the first band with respect to the second band depends on the dimensions of all the elements of the metasurface and is optimized by RCWA (rigorous coupled wave analysis) numerical simulation.

[0050] By way of non-limiting example, in this first embodiment, the width of the first band 11 is between 30 nm and 500 nm, the width of the second band 12 is between 100 nm and 700 nm, and the length of the dotted line P is between 60 nm and 800 nm. The dimension Px of the pattern along the x direction is between 300 and 1000 nm, and the dimension Py along the y direction of the sub-pattern is between 300 and 1000 nm. The depth of the bands (dimension of the metasurface along the z direction) is between 100 and 500 nm. Typically, the wavelength 2 is between 400 nm and 800 nm.

[0051] Figure 3B illustrates the diffraction efficiency of the diffractive optical element according to the first embodiment of the invention, for an incidence angle range between -5° and 5°. In this non-limiting example, the width of the first band is 160 nm, the width of the second band is 61 nm, and the length of the dotted line is 322 nm. The relative position of the first band with respect to the second band is 186 nm. The dimension Px of the pattern along the x-direction is 465 nm, and the dimension Py of the sub-pattern along the y-direction is 465 nm. These transmissions are obtained for 640 nm.

[0052] The efficiency of the -1 diffraction order is greater than that of the +1 diffraction order and significantly greater than that of the 0 diffraction order. For an angle of incidence of 0°, the efficiency of the -1 diffraction order is 55%, that of the +1 order is 15%, and that of the 0 diffraction order is approximately 3%. In this example, the diffraction angle is 65°.

[0053] The structure of the metasurface in Figure 3A therefore allows the incident radiation to be redirected mainly towards the diffraction order -1 while having a very low transmission of the 0th order.

[0054] Figure 4A illustrates a schematic view of a diffractive optical element according to a variant of the first embodiment of the invention. In this variant, each sub-pattern E2 of the metasurface of the diffractive optical element comprises a disk C arranged between two successive dotted lines P of the first band ML. The first band ML is therefore formed by an alternation of dotted lines and disks C along the direction y-

[0055] In this variant of the first embodiment, the diameter of the disk C and its position relative to the dashed line P along the y-direction in a sub-pattern E2 are optimized by RCWA simulation so as to generate an effective index adapted in the sub-pattern E2 with respect to the effective index of the pattern E1

[0056] By way of non-limiting example, in this variant of the first embodiment, the diameter of the disks is between 30 and 500 nm and its spacing along the y direction with the dotted line of the sub-pattern is between 20 and 100 nm.

[0057] Figure 4B illustrates the diffraction efficiency of the diffractive optical element according to the variant of the first embodiment of the invention, for an incidence angle range between -5° and 5°. In this non-limiting example, the width of the first band M1 is 170 nm, the width of the second band M2 is 31 nm, and the length of the dashed line P is 246 nm. The relative position of the first band M1 with respect to the second band M2 is 123 nm. The diameter of the disks is 112 nm, and their spacing along the y-direction with the dashed line of the sub-pattern is 53 nm. The dimension Px of the pattern along the x-direction is 465 nm, and the dimension Py along the y-direction of the sub-pattern is 465 nm. These transmissions are obtained for 640 nm.

[0058] In this variant, the metasurface therefore comprises a network along x of continuous rectilinear bands M2 along y with, between each continuous band, a dashed band M1 also along y, the dashed band comprising disks C between each dash.

[0059] The efficiency of diffraction orders -1 and +1 is significantly greater than that of diffraction order 0. Thus, for an angle of incidence of -5°, the efficiency of diffraction order -1 is 51%, that of order +1 is 20%, and that of diffraction order 0 is less than 1%. The efficiency of diffraction order +1 is comparable to that of diffraction order -1. In this example, the diffraction angle Oj is 65°.

[0060] The structure of the metasurface in Figure 4A therefore allows the incident radiation to be redirected mainly towards the diffraction order -1 while having an extremely low transmission of the order 0.

[0061] Alternatively, according to a second embodiment of a diffractive optical element, illustrated in Figure 5A, the first straight portions P are contiguous between two adjacent sub-patterns, forming a first continuous band BC. Furthermore, the sub-pattern E2 comprises a semi-disk DC attached to a first side of said first straight portion P. According to one variant, the straight portion P further comprises an indentation I on a second side of the first straight portion P, opposite the first side, located opposite the half-disk. The indentation is optimized by numerical simulation and allows maximizing the diffraction efficiency of a previously selected order preferentially compared to another diffraction order.

[0062] The diameter of the semicircle of each sub-motif is optimized by RCWA simulation so as to generate an effective index ramp between the region formed by the first straight portion P and the region formed by the semicircle. Within a sub-motif E2, the area of ​​the semicircle DC is less than the area of ​​the straight portion P to which the semicircle DC is attached.

[0063] In this variant, the metasurface therefore comprises a network along x of continuous bands BC along y comprising a straight part P and additional motifs having a half-disk shape DC, attached to this straight part, and distributed periodically along y.

[0064] By way of non-limiting example, in this second embodiment, the width of the straight portion P is between 30 nm and 500 nm, and the diameter of each half-disk is between 100 nm and 500 nm. The dimension Px of the pattern is between 300 and 1000 nm, and the dimension Py of the sub-pattern is between 300 and 1000 nm. The dimension along the x-direction of the indentation is between 15 and 100 nm. In order to provide a sufficient effective refractive index ramp to deflect the incident beam, the width of the straight portion P is less than 2 / 3 of the dimension Px of the pattern along the x-direction. The depth of the bands (dimension of the metasurface along the z-direction) is between 100 and 500 nm.

[0065] Figure 5B illustrates the diffraction efficiency of the diffractive optical element of the second embodiment of the invention, for an incidence angle range of -5° to 5°. In this example, given by way of non-limiting example, the width of the rectilinear portion P is 149 nm, and the diameter of each half-disk is 293 nm. The dimension Px of the pattern along the x-direction is 467 nm, and the dimension Py of the sub-pattern along the y-direction is 467 nm. In this example, the diffraction angle is 65°. These transmissions are obtained for 640 nm.

[0066] The efficiency of the diffraction order -1 is greater than that of the diffraction order +1 and much greater than that of the diffraction order 0. For an angle of incidence of 0°, the efficiency of the diffraction order -1 is about 57%, that of the order +1 is 10% and that of the diffraction order 0 is about 3%.

[0067] The structure of the metasurface in Figure 5A therefore allows the incident radiation to be redirected mainly towards the diffraction order -1 while having a very low transmission of the 0th order.

[0068] Another object of the invention is a DI imaging device for a sample by total internal reflection fluorescence microscopy comprising a diffractive optical element 10 as described above. A schematic view of the DI device is illustrated by figure 6.

[0069] The DI device comprises at least one light source SL for emitting light radiation Lin in the spectral range in which the substrate is transparent. The diffractive optical element is adapted so that light radiation emitted by the light source SL at wavelength 2 is incident on the metasurface MS with the angle of incidence such that the light radiation diffracted Ldiff by said diffractive optical element is reflected by total internal reflection by the second face of the substrate and produces evanescent waves OE in at least one region of the second surface Se of the substrate.

[0070] The DI device further comprises a sample Ech placed in correspondence with at least one region of the second surface where said evanescent waves are produced and generating fluorescence radiation Fl excited by said evanescent waves.

[0071] In one embodiment, the sample Ech is a cell culture sample, the cells being cultured on a slide in a material identical to that of the substrate SB and stored in an aqueous medium. Since the diffraction angle dj of the diffracted radiation Ldiff is greater than the critical angle of total internal reflection between said substrate and water, the total internal reflection of the diffracted radiation at the interface between the second surface Se and the aqueous medium generates evanescent waves OE. These waves excite fluorophores of the sample located in a thin layer (approximately 100 nm) which contribute to the formation of a fluorescence image

[0072] The DI device further includes an objective Obj adapted to collect the fluorescence radiation Fl and to direct it to a detector Det adapted to detect said fluorescence radiation emitted by the sample.

[0073] Since the angle of propagation of the diffracted radiation remains constant within the substrate, the diffracted radiation propagates along the entire length of the substrate and generates evanescent waves in each region of reflection on a surface of the substrate. This means that when the dimension along the x-direction of the substrate allows it, there are a plurality of reflection regions on a surface of the substrate where evanescent waves are produced, and therefore a plurality of regions where fluorescence radiation within a sample can be excited.

[0074] The transverse dimension (along the x and y directions) of the regions where the evanescent waves are produced is fixed by the smaller of the two dimensions: the transverse dimension of the metasurface MS and the diameter of the incident radiation beam Lin

[0075] The total lateral dimension along the x-direction of the metasurface MS is determined by the diffraction angle and the thickness e of the substrate. Indeed, the radiation diffracted and reflected by the second surface Se of the substrate (which is called reflected radiation Lr) ) must not be extracted from the substrate by the metasurface MS. The lateral dimension of the metasurface along the x direction, xMS is therefore such that xMS < 2esinOd.

[0076] The DI device enables total internal reflection fluorescence microscopy (TIRF microscopy) of a sample Ech using a diffractive optical element 10 according to the invention. This diffractive optical element 10 generates sufficient deflection of the incident radiation so that it propagates within the substrate of the diffractive optical element in a direction suitable for generating evanescent waves at the interface between the substrate of the diffractive optical element and the sample.

[0077] Thus, the DI device allows for the selective observation of structures and processes located on a cell membrane, with a spatial resolution in an axial direction much better than the diffraction limit.

[0078] Furthermore, the DI device allows TIRF microscopy to be performed without using a high numerical aperture ON objective, greatly reducing the cost of the DI device compared to a prior art TIRF microscopy device.

[0079] Furthermore, since the diffraction order 0 transmission of the diffractive optical element of the DI device is very low, the intensity of the epifluorescence radiation emitted by the sample during direct transmission of the incident radiation is greatly reduced. The background noise produced by this radiation is decreased, and the contrast of the fluorescence image of the thin sample layer is improved.

[0080] According to one embodiment of the invention, the path of the incident light radiation between the light source SL and the diffractive optical element is traced in free space. This experimental arrangement is simple and allows for easy control of the excitation region of the sample. In this embodiment, the light source can be a laser source or an LED source placed in the immediate vicinity of the metasurface MS. When the light source is an LED source placed in the immediate vicinity of the metasurface MS, it is advantageous to place the sample Ech above a region of the second surface Se of the substrate where evanescent waves are produced, which is transversely distant along the x-direction from the metasurface MS. In other words, it is advantageous to place the sample above a region corresponding to a plurality of reflections on the surfaces of the substrate.Indeed, the intensity of the stray radiation emitted by the LED, scattered by the metasurface and illuminating the sample, will be greatly reduced. Thus, the background noise produced by this stray radiation is decreased and the contrast is improved.

[0081] Alternatively, according to another embodiment, the path of the incident light radiation between the light source SL and the diffractive optical element is fiber-linked, and the device DI includes an optical fiber coupler (not shown in Figure 6) for directing the light radiation Lin towards the metasurface MS of the diffractive optical element. The advantage of this embodiment is that it does not require No further alignment is required after proper placement of the fiber optic coupler. In this embodiment, the light source SL is a laser source.

[0082] Alternatively, according to another embodiment, the objective Obj is further adapted to collimate the incident radiation Lin onto the metasurface MS. In this embodiment, the device includes another lens (not shown) focusing the incident radiation Lin emitted by the light source SL onto the object focal plane of the objective Obj, the image of the radiation then being projected to infinity in the image plane of the objective Obj. In this embodiment, the angle of incidence θ of the incident radiation is adapted so that the reflected radiation Lr is not collected by the objective.

[0083] According to one embodiment of the invention, the diffractive optical element comprises a plurality of metasurfaces MSb, MS2, MSn, arranged on said first surface of the substrate Sm, with a different dimension Px of the pattern along said x-direction for each metasurface. Thus, each metasurface is adapted to diffract a light ray Lin with a wavelength different from that of the other metasurfaces. In this embodiment of the invention, the DI imaging device of Figure 6 has the advantage of being wavelength-tunable.

[0084] Another object of the invention is a device for detecting a biological sample by surface plasmon resonance D2 comprising a diffractive optical element according to the invention. A schematic view of the device D2 is illustrated in Figure 7.

[0085] The diffractive optical element of the device in Figure 7 comprises a plurality of n metasurfaces MSb MS;, ..., MSn, with a dimension of said pattern Px along said direction x different for each metasurface, so that the diffracted radiation Ldiff reaches said second surface of the substrate Se at an angle i associated with the i-th metasurface MS;.

[0086] The device D2 of Fig. 7 further comprises at least one light source SL adapted to emit the light radiation Lin in the spectral range in which the substrate is transparent. The source is adapted so that the light radiation is incident on the metasurfaces with the angle of incidence 6,

[0087] The device D2 of figure 7 further includes a sample Ech comprising at least a second substrate S2, a metallic layer Met disposed above the second substrate and a layer to be analyzed An disposed above the metallic layer.

[0088] According to one embodiment of the invention, the layer to be analyzed is an aqueous layer.

[0089] The sample Ech is arranged above the first substrate SB so that said diffracted radiation is reflected at least partially by the metallic layer of the sample, forming a reflected radiation Lr.

[0090] The diffractive optical element of the device D2 further includes an optical coupling element MSout placed on the first surface of the substrate Sm and adapted to couple said light radiation reflected by the sample Lr to free space.

[0091] According to one embodiment of the invention, the optical coupling element MSout comprises at least one output metasurface formed by the repetition of a periodic pattern Ei along two directions x and y perpendicular to each other and parallel to a surface of the substrate, said pattern comprising a sub-pattern E2 comprising a first portion of a straight band along y P, said output metasurface disposed on said first surface of the substrate Sm the metasurface.

[0092] According to a preferred embodiment of the invention, the optical coupling element MSout is the "mirror" of the diffractive optical element MSin. That is to say, the optical element MSout is the axial reflection across an axis parallel to the z-direction of the diffractive optical element MSin.

[0093] The device D2 of figure 7 includes a detector Det adapted to detect the intensity of said light radiation reflected by the sample and coupled to free space by said coupling element.

[0094] According to one embodiment, the device D2 further includes an objective Obj adapted to collect the reflected radiation Lr coupled to free space by said coupling element, and to direct it towards the detector Det.

[0095] The diffractive optical element comprises at least one metasurface MS; adapted such that the diffraction angle θi associated with the metasurface produces, upon reflection from the metallic surface, a resonant excitation of surface plasmons and at least partial absorption of said diffracted radiation Ldiff. When the incident radiation is deflected by a metasurface MS; with an associated diffraction angle θd, which allows the excitation of the resonance of surface plasmons upon reflection from the metallic layer Met, the diffracted radiation is partially absorbed and the intensity of the reflected radiation Lr decreases.

[0096] According to one embodiment, above the metallic layer Met of the sample is disposed a chemical functionalization layer adapted to allow the adsorption of specific biological molecules that one wishes to detect in the layer to be analyzed An.

[0097] In order to detect the presence of molecules, it is therefore necessary to illuminate each MS metasurface successively with the light source SL and to detect the intensity of the reflected radiation Lr with the detector Det. Let 0SPR 0 be the excitation angle of the surface plasmon resonance of the Met layer, without the presence of molecules to be detected in the layer to be analyzed An. The surface plasmon resonance is then excited by the k-th metasurface MSk to which is associated a diffraction angle k = 0spR 0. For this single metasurface MSk, the intensity of the reflected radiation Lr is decreased compared to the intensity of the reflected radiation Lr obtained with the other metasurfaces.

[0098] When the molecules to be detected are present within the layer to be analyzed, they will adsorb to the metallic layer Met, thereby changing the angle at which the surface plasmon resonance is excited. Let &SPR j be the angle at which the surface plasmon resonance is excited.

[0099] The detection of molecules adsorbing to the metasurface is therefore carried out when a change in the metasurface MSm is observed, which is associated with a diffraction angle m = @spr 1 9ui. Allows the excitation of the resonance of surface plasmons and for which the intensity of the reflected radiation Lr decreases.

[0100] The device D2 in Figure 7 enables biological detection by surface plasmon resonance with a significantly simplified setup compared to prior art devices. More specifically, this device enables surface plasmon resonance detection without requiring variation of the wavelength or angle of incidence 9 of the incident radiation Lin. Furthermore, this device is easy to align, unlike most prior art devices which use coupling prisms to excite surface plasmon resonance, as these prisms are bulky and complex to align.

Claims

Claims

1. Diffractive optical element (10) comprising: - a substrate (SB) having a first surface (Sm) and a second surface opposite the first surface (Se), transparent to light in at least one spectral range and having, in said spectral range, a refractive index greater than that of water; - at least one metasurface (MS) made of a dielectric material and arranged on said first surface of the substrate (Sm), the metasurface being formed by the repetition of a periodic pattern (Ei) along two directions xety perpendicular to each other and parallel to a surface of the substrate, said pattern comprising a sub-pattern (E2) comprising a first portion (P) of rectilinear strip along y;said metasurface being adapted to diffract light radiation (Lin) of wavelength 2 included in said spectral range, incident with an angle of incidence, according to a diffracted radiation (Ldiff), so that the diffracted radiation propagates in the substrate and reaches said second surface of the substrate (Se) according to a diffracted angle 0d greater than or equal to a limit angle 0c of total internal reflection between said substrate and water, said metasurface being configured to have, for said angle of incidence, a transmission of the order of diffraction 0 less than 5% and a transmission of the diffracted radiation (Ldiff) corresponding to a diffraction order -1 or +1 greater than 50%.;

2. Diffractive optical element according to the preceding claim, wherein: - the metasurface has a first band (Ml) of dotted lines (P) in the y direction, formed from said sub-pattern; said periodic pattern further comprising a second portion of rectilinear band in the y direction, so that the metasurface has a second continuous band (M2) in a y direction.

3. Diffractive optical element according to the preceding claim, wherein, within the pattern, the area of the first band is less than the area of the second band and wherein a width of the first band (11) is between 30nm and 500nm, a width of the second band (12) is between 100nm and 700nm and a length of the dotted line (P) is between 60nm and 800nm.

4. Diffractive optical element according to one of claims 2 or 3, in which the sub-pattern comprises a disc (C), said disc being arranged between two successive dotted lines (P) of said first band.

5. Diffractive optical element according to claim 1, in which said sub-pattern comprises a half-disc (DC) attached to a first side of said first rectilinear portion, and in which said first portions form a first continuous band (BC).

6. Diffractive optical element according to the preceding claim, in which said first rectilinear portion further comprises an indentation (I) on a second side of said first rectilinear portion, opposite the first side, and located opposite the half-disc.

7. Diffractive optical element according to any one of the preceding claims, in which the dimension of the pattern (Px) in the x direction is such that Px <2! neait, with a refractive index of water.

8. Diffractive optical element according to any one of the preceding claims, in which the total dimension of the metasurface in the x direction, xMS is such that xMS < 2.e.sinOd, with e a thickness of the substrate.

9. Diffractive optical element according to any one of the preceding claims, in which the dimension of the pattern (Py) in the y direction is between 300nm and 1000nm.

10. A diffractive optical element according to any preceding claim, wherein the refractive index of the metasurface material is greater than the refractive index of a substrate material.

11. Diffractive optical element according to any one of the preceding claims, comprising a plurality of metasurfaces (MSb MS2, MSn) arranged on said first surface of the substrate (Sm), with a dimension of the pattern in said direction x (Px) different for each metasurface, so that each metasurface is adapted to diffract light radiation (Lin) of wavelength included in said range and different from that of the other metasurfaces.

12. Imaging device (Dl) of a sample by total internal reflection fluorescence microscopy comprising a diffractive optical element (10) according to any one of the preceding claims and comprising: - at least one light source (SL) adapted to emit said light radiation (Lin) in said spectral range, incident on said diffractive optical element with said angle of incidence, said light radiation diffracted by said diffractive optical element producing evanescent waves (EW) in at least one region of the second surface (Se) of the substrate; - a sample (Ech) placed in correspondence with at least one region of the second surface where said evanescent waves are produced and generating fluorescence radiation (Fl) excited by said evanescent waves; - a detector (Det) adapted to detect said fluorescence radiation (Fl) emitted by said sample.

13. Device for detecting (D2) a biological sample by surface plasmon resonance comprising a diffractive optical element (10) according to any one of claims 1 to 10, said diffractive optical element comprising: - a plurality of metasurfaces (MSb MS2, MSn,) with a dimension of the pattern according to said direction x (Px) different for each metasurface, so that the diffracted radiation (Ldiff) reaches said second surface of the substrate (Se) according to a diffracted angle ^d different for each metasurface, said device further comprising; - at least one light source (SL) adapted to emit said light radiation (Lin) in said spectral range, incident on said diffractive optical element with said angle of incidence; - a sample (Ech) comprising at least a second substrate (S2), a metal layer (Met) arranged above the second substrate and a layer to be analyzed (An) arranged above the metal layer, said sample being arranged above the first substrate (SB) so that said diffracted radiation is reflected (Lr) at least partially by the metal layer of the sample; said diffractive optical element further comprising an optical coupling element (MSout) adapted to couple said light radiation reflected by the sample towards free space; - a detector (Det) adapted to detect the intensity of said light radiation reflected by the sample and coupled to free space by said coupling element; at least one metasurface being adapted so that said diffracted angle associated with the metasurface produces, upon reflection on the metal surface, a resonant excitation of surface plasmons and at least partial absorption of said diffracted radiation (Ldiff).

14. Device according to the preceding claim, wherein said element optical coupling comprises at least one output metasurface, formed by the repetition of a periodic pattern (EJ) along two directions x and y perpendicular to each other and parallel to a surface of the substrate, said pattern comprising a sub-pattern (E2) comprising a first portion of rectilinear strip along y (P), said output metasurface arranged on said first surface of the substrate (Sm).

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