Optical slide for a total internal reflection optical device, method for manufacturing same, and optical device comprising a slide of this kind

Abstract

The invention relates to an optical slide (LO) for receiving a biological sample (E) in a total internal reflection optical device, the optical slide comprising: - a so-called substrate (SB) layer, which is transparent in a predetermined spectral range; - a so-called optimised stack arranged above the substrate (SB) and comprising a number M > 7 of successive and alternating thin dielectric layers of a first dielectric material and a second dielectric material, each of the layers respectively having a so-called optimised thickness, the optimised thicknesses and the number M of thin dielectric layers being such that the optical slide has at least: o a first resonant absorption at a so-called illumination wavelength λin and at a first angle of incidence θ 1 in total internal reflection; and - a second resonant absorption at the illumination wavelength λin and at a second angle of incidence θ 2 in total internal reflection, where Δθ = |θ 1 - θ 2| ∈ [0.4°; 3°] and θ 2 < θ in < θ 1 or θ 1 < θ in <sb / >< θ 2.

Description

DESCRIPTION Title of the invention: Optical plate for an optical device by total internal reflection, its manufacturing method and optical device comprising such a plate Technical field:

[0001] The invention relates to the field of optical devices. More particularly, the invention relates to a new concept of optical plate based on a multilayer stack as a support for enhancing the electromagnetic field suitable for optical devices by total internal reflection, such as for example a total internal reflection fluorescence microscope.

[0002] The invention applies in particular, but not exclusively, to the field of imaging of biological samples by total internal reflection fluorescence microscopy or TIRF microscopy (acronym in English for “Total Internal Reflection Fluorescence”). Such an imaging technique is particularly well suited to the visualization, analysis and quantification of molecular events occurring in particular at the plasma membrane of biological cells. Previous technique:

[0003] Evanescent wave fluorescence microscopy is a fluorescence microscopy technique in which the excitation of fluorescent molecules contained in the observed sample is confined to a nanometer-thick region, located in the immediate vicinity of the sample slide. In particular, it allows selective observation of structures and processes located on a cell membrane, with an axial resolution much better than the diffraction limit. In addition, compared to more conventional epi-fluorescence techniques, it allows for better contrast of the fluorescence image and reduces the effects of photobleaching and irradiation damage to cells.

[0004] It is known from the scientific publication "Directing Fluorescence with Plasmonic and Photonic Structures", Sharmistha Dutta Choudhhury et al, which describes among other things the use of dielectric materials called, "1 dimensional photonic crystal" to direct an excitation beam and control fluorescence emission from molecules placed on a surface. This publication does not, however, mention the rapid decrease, or even loss, of fluorescence emission enhancement when the excitation beam is divergent, which is minimally the case in optical devices such as TIRF microscopes.

[0005] The principle underlying evanescent wave fluorescence microscopy is illustrated in Figure 1A. We consider the case of a substrate SB with refractive index n2, having a surface Se in contact with an ambient medium MA with index m <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 at the surface an angle 0 greater than a critical value Oc (limit angle) (1).

[0006] Therefore, the FLI beam undergoes a total internal reflection (TIR) ​​forming a reflected beam FLR and producing an evanescent wave OE in the ambient medium MA. This evanescent wave has an intensity which decreases exponentially with the distance z from the surface S: where the s? 4 penetration length ô is given by ô = (1 bis), X being the wavelength of the light radiation. The evanescent wave excites the fluorophores contained in the ambient medium, but only over a thickness of the order of δ, because beyond this its intensity quickly becomes negligible. For example, for 7=488 nm, n2=1.514 (BK7 glass), m=1.33 (water) and 0 = 67° > 0 C ~ 61.45°, we find ô «93 nm, which means that only the fluorophores located in a layer of approximately 100 nm thickness are excited and contribute to the creation of a fluorescence image.

[0007] Figure 1 B illustrates the most commonly used configuration in TIRF microscopy. In the case of Figure 1 B, the same microscope objective OBJ, located on the side of the substrate opposite to the MA medium, is used both to generate the evanescent waves by RTI and to collect the fluorescence radiation.

[0008] Thus, using this known technique, the images obtained have multiple qualities: first of all, they benefit from low background noise (because the fluorophores located in the deep layers of the sample (outside the evanescent field) are only very weakly excited) and relatively high axial resolution.

[0009] Microscope slides with complex structures, such as those based on surface metallization, have also been designed to locally enhance the evanescent electromagnetic field. Such optical slides, which are based on the principle of surface plasmon resonance, can improve the sensitivity of microscopy imaging. However, this known solution remains limited in terms of field enhancement value and in the choice of materials, i.e. noble metals, which limit the usable illumination conditions as well as biocompatibility, which is not optimal.

[0010] Another known technique, described in patent document US 2016 / 0238830, is based on a multilayer waveguide whose layer thicknesses and refractive indices are chosen to support a guided leakage mode. However, microscopy imaging experiments carried out with this technique remain limited in terms of sensitivity and resolution in particular.

[0011] Furthermore, it is known from application FR2108879 to produce microscopy slides covered with stacks of nanometric layers of dielectric materials designed to enhance the evanescent electromagnetic field at the interface between the slide and the biological objects deposited on it. This type of structure makes it possible to improve the sensitivity of total internal reflection microscopes which use a microscope objective to reach the critical angle of total reflection as illustrated in Figure 1 B.

[0012] The use of these dielectric stacks makes it possible to generate this enhancement at all wavelengths of the visible / near infrared spectrum and at all illumination angles beyond the critical angle of RTI. However, this method is optimized for illuminations with plane waves, which is not the case in a microscope illuminating the object through the objective.

[0013] Indeed, one of the main limitations of resonant structures in RTI is their low angular tolerance, which is all the lower the more resonant the structure is. However, unlike sensor applications which seek the most intense and finest spectrally and angularly enhanced evanescent field, in microscopy, the desired resonance is associated with a weaker enhanced (from ten to a few hundred) with an angular tolerance at least of the order of magnitude of the divergence of the illumination by the microscope objective.

[0014] Consequently, there is a need for an optical slide intended for a total internal reflection optical device, in particular a fluorescence microscope, making it possible to enhance the evanescent field at the interface between the slide and the biological objects deposited on it with increased angular tolerance compared to the slides of the prior art. Summary of the invention:

[0015] For this purpose, an object of the invention is a method of manufacturing an optical slide intended to receive a biological sample in an optical device by total internal reflection, said method comprising a step of designing said optical slide and a step of material manufacturing of said optical slide thus designed, said method being characterized in that the design phase comprises the following steps: A. the selection of a first dielectric material having a refractive index n a and a second dielectric material having a refractive index n b such that n a > n b B. the selection of a so-called illumination wavelength in and an angle of incidence called optimal 6 in depending on said optical device by total internal reflection for which said optical blade is intended C. the design of a first structure comprising a first stack of thin dielectric layers arranged above a so-called substrate (SB) layer transparent in a predetermined spectral region comprising said illumination wavelength in , said first stack comprising N > 7 of successive and alternating thin layers of the first dielectric material and of the second dielectric material, a first respective thickness of each dielectric thin layer and said number N of dielectric thin layers being adapted so that the first structure exhibits resonant absorption at the illumination wavelength in and at the optimal angle of incidence 0 in , in total internal reflection mode D. determining a first function F representative of a value of a transmission of the first structure in the predetermined spectral region as a function of a number of dielectric thin layers and a thickness of each dielectric thin layer, and determining a second function F2 representative of an intensity of an evanescent electric field generated by the optical plate in the total internal reflection regime at the illumination wavelength in and at the optimal angle of incidence 6 in depending on a number of dielectric thin layers and said first thickness of each dielectric thin layer E. the design of said optical blade which comprises a second stack of dielectric thin layers called optimized stack arranged above the substrate (SB), the optimized stack comprising a number M < N, M > 7 of successive and alternating dielectric thin layers of the first dielectric material and of the second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number M of dielectric thin layers being determined by an optimization of a merit function F based on the first function F and the second function F2 so that said optical blade has a first resonant absorption at the illumination wavelength in and at a first angle of incidence 0 in total internal reflection regime and has a second resonant absorption at the illumination wavelength i nand at a second angle of incidence 02 in the total internal reflection regime, with

[0016] Preferably, the merit function F is such that F = aF^ + (1 - a)F2, with 0 < a < 1 a parameter making it possible to weight a respective weight of the first function F and the second function F2 in said optimization.

[0017] Preferably, a can be understood as 0.3 and 0.7.

[0018] Preferably, the optimization can be such that A0 = \0 - 02| G [0.4°; 1.5°].

[0019] According to one embodiment, the optimization may consist of finding the optimized thicknesses and the number M of dielectric thin layers for which said transmission value of the first structure in the predetermined spectral region is maximum and for which said intensity of the evanescent electric field is maximum.

[0020] According to one embodiment, in the first stack, an upper layer of the first stack may be in the second dielectric material and have a first thickness of between 1.1 in / 4 and 1.8 in / 4 and in which the N - 1 lower layers of the first stack each have a first thickness of between 0.9 in / 4 and l.l in / 4.

[0021] According to one embodiment, the first function F1 may be representative of a transmission of fluorescence radiation induced indirectly by an absorption of said illumination wavelength in in total internal reflection in said first stack illuminated with said optimal angle of incidence 0 in, the fluorescence radiation having a spectrum included in said spectral region, the transmission being integrated over an angular range of incidence of the fluorescence radiation on said optical plate between 0 and 80°.

[0022] Another object of the invention is an optical slide intended to receive a biological sample in an optical device by total internal reflection, the optical slide comprising: - a so-called substrate layer transparent in a predetermined spectral region - a so-called optimized stack arranged above the substrate and comprising a number M > 7 of successive and alternating thin dielectric layers of a first dielectric material and a second dielectric material, each layers having a respective so-called optimized thickness, the optimized thicknesses and the number M of dielectric thin layers being adapted so that said optical plate has at least: o a first resonant absorption at a so-called illumination wavelength in and at a first angle of incidence 6 ± in total internal reflection regime, and o a second resonant absorption at the illumination wavelength in and at a second angle of incidence 02 in total internal reflection regime,

[0023] According to an advantageous embodiment of the optical blade according to the invention: - the first dielectric material may have a refractive index n a between 1.8 and 3.5; - the second dielectric material may have a refractive index n b between 1.2 and 1.7.

[0024] According to one embodiment of the optical blade according to the invention, the first material may be based on Nb2O5, and the second material may be based on SiO2.

[0025] According to one embodiment of the optical blade according to the invention, the number M of thin dielectric layers may be less than 30, preferably less than 20.

[0026] According to one embodiment of the optical blade according to the invention, the optimized thicknesses and the number M of thin dielectric layers may be such that said optical blade has a total thickness of less than 5 μm and preferably between 0.5 μm and 2 μm.

[0027] According to one embodiment, the optimized thicknesses may be greater than or equal to 10 nm.

[0028] Another object of the invention is an optical device by total internal reflection comprising: - an optical blade according to the invention as defined above, - a light source adapted to emit a light beam having the illumination wavelength in - an optical device adapted to shape the light beam so that it illuminates said optical blade with said optimized angle of incidence d in - a detector adapted to detect fluorescence radiation emitted when a sample is deposited in correspondence with a region of a surface of said optical plate where evanescent waves are produced by total internal reflection in said optical plate of the shaped light beam, said fluorescence radiation being excited by said evanescent waves.

[0029] Preferably, the total internal reflection optical device according to the invention may consist of a total internal reflection fluorescence microscope.

[0030] Preferably, the optical device may be a microscope objective. Brief description of the figures:

[0031] Other characteristics, details and advantages of the invention will emerge from reading the description given with reference to the appended drawings given by way of example and which represent, respectively:

[0032] [Fig.1 A] a schematic diagram of the operation of a prior art total internal reflection fluorescence microscope,

[0033] [Fig.1 B] a schematic view of a prior art total internal reflection microscope;

[0034] [Fig.2], a schematic view of a total internal reflection microscopy device according to the invention;

[0035] [Fig.3], a schematic view of an optical slide according to the invention, intended to receive a biological sample in a total internal reflection microscopy device;

[0036] [Fig.4A], the absorption of an optical plate according to a preferred embodiment of the invention in the total internal reflection regime, as a function of the angle of incidence and the wavelength of the incident beam;

[0037] [Fig.4B], the enhancement factor of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in the total internal reflection regime, as a function of the angle of incidence of the incident beam;

[0038] [Fig.5A], a first graph representing the evolution of the enhancement factor of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam, for a first divergence different from the incident beam,

[0039] [Fig.5B], a second graph representing the evolution of the enhancement factor of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam, for a second different divergence of the incident beam;

[0040] [Fig.6], the normalized enhancement factor relative to a glass plate of the evanescent electric field generated on the surface of an optical plate according to a preferred embodiment of the invention in total internal reflection mode, as a function of the angle of incidence of the incident beam,

[0041] [Fig.7], a schematic representation of the steps of the design phase of the manufacturing process according to the invention.

[0042] In the figures, unless otherwise indicated, elements are not to scale and identical references designate identical elements or identical steps. Detailed description:

[0043] Figure 2 illustrates in a simplified manner a device D for total internal reflection microscopy according to the invention. The device D comprises an optical slide LO according to the invention, a light source SL, an optical device Obj and a fluorescence detector Del

[0044] We will first describe the operation of the device D before detailing the structure of the optical blade LO. Finally, we will describe a manufacturing method according to the invention for obtaining the optical blade LO.

[0045] The optical slide LO is a slide, preferably biocompatible, intended to receive a biological sample E for microscopy imaging purposes according to an RTL configuration. An example of an optical slide structure in accordance with the invention is described further in relation to figure 3 which schematically illustrates a section along the xz plane of the optical slide LO according to the invention.

[0046] The SL light source is adapted to emit a Lin light beam having a so-called illumination wavelength in predetermined. The SL source is a laser source configured to emit at a wavelength typically between 350 and 1300 nm so as to excite the fluorophores contained in the sample E and thus produce the emission of fluorescence radiation FL while being at least partially transmitted by the optical plate LO.

[0047] The optical device Obj is adapted to shape the light beam Un so that it illuminates the optical blade with a so-called optimized angle of incidence 6 in greater than the critical angle.

[0048] Preferably, as illustrated in Figure 2, the optical device Obj is a microscope objective. The microscope objective can have a variable focal length and a variable numerical aperture (greater than 1.45). It comprises an optic or a more or less complex assembly of optical lenses capable of allowing the formation of the light beam Lin by focusing it in the direction of the optical plate. It is also capable of collecting the reflected and / or backscattered beam from the optical plate in an angular cone from 0 to a maximum angle given by the numerical aperture of the objective. The objective is generally of the immersion type in oil and can have a large numerical aperture (NA), for example of the order of 1.45, which makes it possible to obtain a high lateral spatial resolution (perpendicular to the z direction) because the lateral spatial resolution d is given by d = Additionally, an objective Obj with a high numerical aperture ON is advantageous because it allows the incident light beam L to be moved in relative to the optical axis OA and thus obtain a strong deviation of the incident beam L in through the lens, the beam L in thus being able to propagate with an angle of incidence of 0 in high and sufficient to obtain an RTI.

[0049] Alternatively, the optical device Obj comprises one or more lenses and / or mirrors to focus the light beam Lin and possibly a prism or metasurface so that the light beam Lin illuminates the optical plate with the optimized angle of incidence 0in .

[0050] This optimized incidence angle 0 in is greater than the critical angle 0 c at the interface between a surface SE of the blade intended to receive the sample E and the ambient medium (typically liquid) comprising the sample. The optimized angle of incidence d in is typically between 62 and 80 degrees for a biological environment with a refractive index between 1.33 and 1.35. Thus, the RTI of the Lin light beam on said interface produces evanescent waves OE. The sample E being placed in correspondence with a region of the SE surface where the evanescent waves are produced, the fluorophores of the sample will generate FL fluorescence radiation excited by the evanescent waves OE.

[0051] The emitted fluorescence radiation FL is then collected by the objective Obj and is then directed towards the detector Det in order to be detected. This detector Det is typically a CCD or CMOS camera whose spectral band is adapted to the detection of the re-emitted fluorescence radiation FL coming from the sample E. It converts the received light intensity into an electrical signal intended for a processing unit (not shown in the figures). The processing unit is electrically connected to the light source SL, the detector Det and the microscope objective Obj so as to be able to control these elements for the purpose of acquiring images of the sample E in RTI mode.

[0052] As a non-limiting example, the device D illustrated in Figure 2 is based on the principle of epifluorescence, the observation of the fluorescence of which is carried out in a configuration by reflection using a slide or a dichroic mirror 50 for example. This particular configuration makes it possible to dissociate the optical path taken by the excitation light L in , of the optical path taken by the FL fluorescence radiation.

[0053] Preferably, the optimized angle of incidence 0 inis between 62 and 80 degrees. The lower limit of the above-mentioned range (62 degrees) is given by the refractive index value of the sample studied. As for the upper limit of the above-mentioned range (80 degrees), it is defined according to the value of the numerical aperture used for microscopy observation. The typical numerical aperture of the microscope objective Obj of the device D is greater than or equal to 1.45. The microscope objective Obj is preferably of numerical aperture and variable focal length.

[0054] The structure of the optical blade according to the invention, as shown in Figure 3, will be described in more detail below.

[0055] The optical slide LO has the first face SE, and a second face SI, opposite to the first, and defines a stacking axis extending between these two opposite faces in the z direction. The first face SE is intended to receive the biological sample E to be observed and the second face SI is the face illuminated by the light beam L in The first face SE constitutes the interface where an exaltation of the evanescent electromagnetic field OE is produced by the optical blade 10.

[0056] According to the embodiment illustrated in Figure 2, the optical slide LO and the microscope objective Obj are arranged so that the stacking axis of the slide coincides with the optical axis OA of the objective. In other words, the optical slide LO and the microscope objective Obj are oriented relative to each other so that the optical interface formed between the optical slide LO and the sample E is perpendicular to the optical axis OA, along the x direction.

[0057] According to the invention, the optical blade LO comprises an optically transparent substrate SB in a spectral region which comprises the illumination wavelength in and which includes the spectral range of radiation of FL fluorescence. For example, the SB substrate is a microscope slide made of soda-lime glass with an index of 1.5 (or any other optically transparent support calibrated in thickness).

[0058] In addition, the optical blade LO comprises a stack of dielectric layers (hereinafter referred to as the optimized stack EO) arranged directly above the substrate SB. This optimized stack EO is particularly suitable for enabling an enhancement of the evanescent electric field OE produced at the surface SE in an RTI configuration of the beam L in . Thus, when the device of Figure 2 is in operation, the biological sample E which is placed on the upper layer is illuminated by the beam Lin at the illumination wavelength i n after passing through the optical plate. More precisely, the beam L in passes through the SB substrate, then the EO dielectric stack until reaching the interface between the upper layer and the sample E at the optimized angle of incidence 6 in The evanescent wave OE created by the RTI sees its intensity amplified thanks to the resonant structure of the EO stack, which makes it possible to significantly increase the imaging performance of TIRF microscopy, in particular in terms of sensitivity and axial resolution.

[0059] As illustrated in Figure 3, this EO stack is formed from a succession of M > 7 alternating thin layers of a first dielectric material with refractive index n a (thin layers referenced C) and a second dielectric material with refractive index n b (thin layers referenced C2), with n b < na ■

[0060] The thin layer intended to be in contact with the sample E (called the upper layer) is made of one of the two dielectric materials. Preferably, this layer is made of a biocompatible dielectric material, for example SiO2, TiO2, Nb2O5 ... . The upper face of this upper layer corresponds to the face SE previously introduced. As for the face SI illuminated by the beam L in , it corresponds to the lower face of the SB substrate.

[0061] In the optical plate LO of the invention, each of the dielectric layers has a respective so-called optimized thickness chosen in particular as a function of the illumination wavelength  £n , of the optimized incidence angle 0 in , of the incident polarization and refractive indices n1 and n2. The manufacturing method and the design face of the LO optical plate of the invention will be detailed later in the description of figure 7. We will briefly limit ourselves to specifying that the optimized thicknesses and the number M of thin dielectric layers are adapted so that the LO optical plate has at least one double resonance at the illumination wavelength in in RTI for two distinct angles of incidence. More precisely, the optimized thicknesses and the number M of thin dielectric layers are such that the optical blade has: - a first resonant absorption at the illumination wavelength in and at a first angle of incidence 6 ± in RTI, and - a second resonant absorption at the illumination wavelength in and at a second angle of incidence 02 in RTI.

[0062] Angles of incidence 6± and 02 are chosen so that A0 = \6 - 02\ is between 0.4° and 3°, preferably between 0.4° and 1.5°. More precisely, we want to have A0 of the order of magnitude of the divergence of the microscope, therefore according to the systems

[0063] It is understood that the optical blade LO of the invention can comprise more than two resonances as long as the aforementioned conditions remain verified.

[0064] The EO stack of dielectric multilayers coupled to the SB substrate provides significant enhancement of the evanescent electromagnetic field OE at the interface between the optical plate and the sample with a much higher angular tolerance than prior art optical plates. This enhancement increases the intensity of the FL fluorescence radiation, which improves the sensitivity of microscopic imaging as well as the axial resolution of the device D.

[0065] Indeed, the resonant optical plates of the prior art are optimized to exhibit a narrow resonance spectrally and angularly, for a predetermined optimal angle of incidence and for a theoretically planar illumination wave. They thus exhibit a very high "theoretical" enhancement factor of the evanescent electromagnetic field. However, according to the laws of geometric optics, the beam L in has a significant natural angular divergence after passing through the lens (typically between 0.5° and 1°). Also, the beam incident on the optical plate LO actually has a plurality of angles of incidence centered around the optimal angle of incidence. This implies that the evanescent wave enhancement factor OE induced by the optical plate for rays at angles of incidence other than the optimal angle of incidence is in practice significantly lower than the "theoretical" enhancement factor.

[0066] In the invention, the presence of two (or more) angularly close resonances allows a partial overlap between the resonance bands. This overlap makes it possible to widen the angular tolerance of the optical plate LO so that it has a "real" enhancement factor six to ten times greater than the optical plates of the prior art (see figure 6) (or more in the case of multi-resonances). The angular tolerance of the plate of the invention is specifically chosen to be greater than or equal to the divergence of the beam L in after passing through the microscope objective Obj which is typically between 0.5° and 1°.

[0067] Through numerous simulations and experiments, the inventors observed that this multiple resonance was only possible with an EO stack comprising a number M > 7 of thin dielectric layers.

[0068] As mentioned previously, the optimized thickness of each layer of the stack is chosen in particular according to the illumination wavelength A in , of the optimized incidence angle 0 in , polarization and refractive indices n and n2. The optimized angle of incidence 0 in corresponds here to the angle of incidence at which the beam L in must illuminate the optical slide at illumination wavelength i n to obtain an evanescent wave enhancement factor OE with an optimized angular tolerance. It is between and 02 (or between 02 > 02) to benefit from the overlap of the band of the first resonant absorption and the band of the second resonant absorption. Preferably, the optimized thicknesses and the number M are such that 0 in = (0 X + 02) / 2.

[0069] The optimized thicknesses are generally between 5 and 500 nanometers. Preferably, the optimized thicknesses are greater than or equal to 10 nm in order to facilitate the manufacture of the LO optical blade. Indeed, the Precise control of the thickness of layers less than 10 nm thick is technically complex to achieve.

[0070] Preferably, the first dielectric material has a refractive index n a between 1.8 and 3.5 and the second dielectric material has a refractive index n b between 1.2 and 1.7. For example, the first material is based on Nb2O5, the second material is based on SiO2.

[0071] Preferably, the number M of thin dielectric layers is less than 30, preferably less than 20. Indeed, the inventors have observed that, compared to an optical plate LO with a number of layers less than 20, a number of dielectric layers greater than or equal to 20 increases the complexity of the manufacturing process but only allows a minimal gain (or even zero) in the evanescent wave enhancement factor.

[0072] Preferably, in order to limit the optical aberrations induced by the use of the optical plate in the device D, the optimized thicknesses and the number M of thin dielectric layers are such that said optical plate has a thickness of less than 5 μm and preferably between 0.5 μm and 2 μm. Favorite example:

[0073] According to a preferred embodiment of the invention, the optimized stack EO of the optical blade LO comprises 14 alternating dielectric layers of SiO2 and Nb2O5. The upper layer of the stack (the one intended to be in contact with the sample) is made of SiO2. The optimized thicknesses of the dielectric thin layers are, from the upper layer to the lower layer in contact with the substrate, respectively: 20 nm, 26.5 nm, 91.6 nm, 101.8 nm, 242.7 nm, 60.25 nm, 30.2 nm, 20 nm, 273.4 nm, 29.1 nm, 83.1 nm, 20 nm, 530.6 nm, 97.6 nm.

[0074] This structure is optimized for an illumination wavelength in = 561 nm and for an optimized incidence angle 6 in = 68°. These optimized thicknesses are determined by the method of Figure 7 described later. The indices of the layers are respectively n2(SiO2) = 1.486 and ni(Nb2O5) = 2.292 at 561 nm.

[0075] The average transmission of this structure, angularly integrated between 0 and 80 degrees, and spectrally between 561 and 700 nm is 53%.

[0076] Figure 4A is a graph showing the optical absorption of the EO stack according to the preferred embodiment of the invention, with a liquid medium above the upper layer, as a function of the angle of incidence and the illumination wavelength. Four resonance bands are observed, including the band of the first resonant absorption (referenced B1 in Figure 4A) and the band of the second resonant absorption (referenced B2 in Figure 4A). At the illumination wavelength  £n, the first resonant absorption is centered on the angle 01= 68.4° and the second resonant absorption is centered on the angle 02= 67.65°. It is recalled here that the plot in Figure 4A represents an absorption curve and, consequently, is only an indirect plot of the enhancement of the evanescent field generated in RTI on the SE surface. Indeed, the dependence between enhancement and absorption is not linear, and the angular tolerance of the resonant enhancement cannot be directly determined from Figure 4A. The exact calculation shows that it is nevertheless about 1°. As mentioned previously, this angular tolerance value corresponds to the upper limit of the typical divergence of the beam L in after passing through the microscope objective Obj.

[0077] Figure 4B is a curve which represents the value of the enhancement factor of the evanescent field generated in RTI by the optical blade according to the preferred embodiment of the invention as a function of the angle of incidence of the beam L in , at the illumination wavelength in = 561nm. The curve in Figure 4B corresponds to a horizontal section of the previous figure for the illumination wavelength i n = 561nm, but for the evanescent field intensity and not the absorption. As expected, the curve in Figure 4B includes the two exaltation peaks associated with the first and second resonant absorption fî1,fî2. It is observed that these resonant absorptions make it possible to obtain a respective exaltation factor of the evanescent field intensity of the order of 75. However, this value of the exaltation factor is a "theoretical" value obtained for illumination with a plane wave. In practice, the divergence of the beam L must be taken into account in to estimate the real value of the exaltation factor.

[0078] For this, figures 5A and 5B represent a curve of the value of the factor of the exaltation of the intensity of the evanescent field generated in RTI as a function of the angle of incidence of the beam L in , at the illumination wavelength in = 561nm and for two different divergences of the beam L in The curve in Figure 5A is obtained for a divergence of 0.6° while the curve in Figure 5B is obtained for a divergence of 0.9°.

[0079] It is observed that taking into account the beam divergence produces an angular integration of the first and second resonant absorption which makes this double B resonance disappear 1( B2 for the benefit of a single “averaged” resonance centered on the optimized angle of incidence 6 in= 68°. This “averaged” resonance has an exaltation factor of a lower value than the values ​​associated with simple B resonances 1( B2 (i.e. the “theoretical” value of Figure 4B) but higher than what can be obtained with a single optimized resonance.

[0080] Thus, for a beam L in at the illumination wavelength in = 561nm and for the optimized incidence angle 6 in = 68°, the optical plate LO according to the preferred embodiment of the invention makes it possible to obtain an enhancement factor of approximately 35 for a divergence of 0.6° and approximately 30 for a divergence of 0.9°. Logically, a greater divergence implies a greater angular averaging of the first and second resonant absorption B 1( B2 and therefore a greater reduction in the excitement factor.

[0081] Figure 6 is a curve which shows the value of the enhancement factor of the fluorescence radiation produced by the optical blade LO according to the preferred embodiment of the invention as a function of the divergence of the incident beam, for the optimized angle of incidence 0 in = 68°. The value of the enhancement factor is calculated with an integration over an angular cone ranging from 0° to 80° of the FL fluorescence radiation, taking into account the average transmission of the LO optical slide, then is normalized with respect to the value of the enhancement factor obtained by a glass slide.

[0082] Additionally, Figure 6 includes a shaded area that corresponds to the divergence range of a laser beam focused by a commercial microscope.

[0083] Figure 6 shows that the LO optical slide according to the preferred embodiment of the invention makes it possible to obtain high enhancement factors for a wide divergence range which covers most existing commercial microscopes. More precisely, the LO optical slide makes it possible to obtain a normalized fluorescence enhancement factor of the order of 7.

[0084] Although the curves of Figures 4A to 6 have been described in relation to the optical plate LO according to the preferred embodiment of the invention, those skilled in the art will be able to reproduce equivalent results without departing from the scope of the invention with different dielectric materials, and / or different optimized thicknesses and / or a different number of layers M by adapting them to a wavelength in and an optimized angle of incidence 6 in predetermined.

[0085] We now describe the manufacturing process of the optical blade LO according to the invention. This manufacturing process comprises a design phase of the optical blade and then a physical manufacturing phase of the designed optical blade. The different steps A to E of the design phase are illustrated in Figure 7.

[0086] In a first step A, the first dielectric material of index r^ and the second dielectric material of index n2 are selected such that n > n2. The material of index n2 is preferably silica for biological compatibility and the material of index m can be for example Nb2O5, TiO2, HfO2 or Ta2O5.

[0087] In a second step B, we select the illumination wavelength i n and the optimal angle of incidence 6 independing on the total internal reflection microscopy device for which the optical slide is intended. This typically involves choosing an illumination wavelength in equal to the wavelength of the light source of the microscopy device. The optimal angle of incidence 0j n is chosen to be greater than the critical angle of RTI for an interface formed between a liquid medium and a layer in the second dielectric material and depending on the value of the numerical aperture used for microscopy observation.

[0088] After step B, the design phase comprises a step C consisting of designing a first structure comprising a first stack of dielectric thin layers disposed above a substrate SB. The substrate SB is transparent in a predetermined spectral region comprising the illumination wavelength inand the FL fluorescence radiation generated indirectly by the illumination wavelength in .

[0089] The first stack comprises N > 7 successive and alternating thin layers of the first dielectric material and the second dielectric material, each dielectric thin layer having a respective first thickness. The first thicknesses and the number N of dielectric thin layers are adapted so that the first structure has a single resonant absorption at the illumination wavelength in and at the optimal angle of incidence 6 in in a regime of total internal reflection.

[0090] This step C is known in itself and is carried out using optical thin layer simulation and optimization software such as Optilayer or by direct calculation.

[0091] According to one embodiment, in the first stack, the upper layer of the first stack is in the second dielectric material and has a first thickness of between 1.1 in / 4 and 1.8 in / 4 . The exact value of the thickness of the top layer depends on the indices of the dielectric materials, the desired resonance wavelength and the desired resonance angle. In addition, the lower N - 1 layers of the first stack (i.e. those below the top layer) each have a first thickness between 0.9 in / 4 and l.l in / 4. Preferably, these are layers of thickness  in / 4.

[0092] For example, in order to design the optical plate of the preferred embodiment of the invention previously described, the first stack of step B comprises N=15 alternating dielectric layers of SiO2 and Nb2O5. The layer upper is in SiO2 and has a first thickness equal to 1.44 in / 4. The other layers have a first thickness equal to  in / 4.

[0093] After step C, the design phase includes a step D consisting of determining two functions F and F2, each representative of the two critical parameters of the optical blade for the intended application: the transmission of the fluorescence radiation and the enhancement of the evanescent field generated in RTI.

[0094] More precisely, the function F is representative of the value of the transmission of the FL fluorescence radiation as a function of the number of dielectric thin layers and the thickness of each dielectric thin layer. To clarify, this is the FL fluorescence radiation induced indirectly by absorption in the first stack of the illumination wavelength in in RTI with the optimal angle of incidence 6 inIn order to take into account the Lambertian emission of fluorescence radiation, the transmission is integrated over a wide angular range of incidence angle of fluorescence radiation on the optical plate, for example for an incidence angle between 0° and 80° as well as over the spectral range associated with fluorescence emission.

[0095] The function F2 is representative of the intensity of the evanescent electric field generated by the first stack in RTI at the illumination wavelength in and at the optimal angle of incidence 6 in depending on the number of dielectric thin layers and the thickness of each dielectric thin layer

[0096] In fact, the actual enhancement factor of the optical slide, that is to say that of the fluorescence radiation collected by the microscope objective, is multifactorial and depends on: - the transmission of the structure for fluorescence radiation. This must be maximized in order to increase the collected fluorescence radiation. This amounts, for example, to maximizing the transmission of the blade for an angle of incidence between 0 and 80 degrees, for illumination with a wavelength between 400 nm and 800 nm, - the exaltation factor of the evanescent field generated in RTI, which must also be maximized - the angular tolerance of this exaltation factor, as explained previously.

[0097] Simultaneous optimization of these three parameters is only really possible by increasing the number of degrees of freedom. This is why it is necessary to construct the functions F and F2 which will be used for the next step E.

[0098] For example, according to the preferred embodiment of the invention, the functions F and F2 have the following form for the design of the optical blade LO: with T the transmission of the structure for a plane wave illuminated under incidenceO at wavelength A, with E the amplitude of the electric field at the upper interface of the structure, AO the angular interval chosen for field optimization, typically between 0.2 and 1.5 degrees.

[0099] Finally, after the determination of the functions F and F2, the design phase includes a last step E of optimization of the first stack so as to obtain the optimized stack EO of the optical blade LO. The optimized thicknesses and the number M < N, M > 7 of thin dielectric layers of the optimized stack EO are determined by the optimization of a merit function F based on the first function F and the second function F2 so that the optical blade has: - the first resonant absorption at the illumination wavelength in and at the angle of incidence 6 ±in RTI, and the second resonant absorption at the illumination wavelength in and at the second angle of incidence 02 in RTI.

[0100] As mentioned above, the optimization is such that A0 = - 021 e [0.4°; 3°] and 02< Vin < 01 OR i < 0 in < 02.

[0101] This optimization is carried out using optical thin layer simulation and optimization software such as Optilayer.

[0102] Preferably, the merit function F to be maximized is such that F = aF + (1 - a)F2, with 0 < a < 1 a parameter allowing to weight a respective weight of the first function F and the second function F2 in the optimization. The alpha parameter influences the respective weights of the transmission of fluorescence by the blade with respect to the exaltation of the evanescent field and is preferably between 0.3 and 0.7 because the two parameters being important, it is advisable to consider them both in a non-negligible manner.

[0103] This form of merit function has a sufficient number of degrees of freedom to simultaneously optimize the three parameters influencing the actual enhancement factor of the optical plate.

[0104] The optimization typically consists of finding the optimized thicknesses and the number M of dielectric thin layers for which the value of the transmission of the fluorescence radiation is maximum and for which the intensity of the evanescent electric field is maximum.

[0105] According to a preferred embodiment, the optimization is such that A0 = \0 - 02| G [0.4°; 1.5°]. This makes it possible to obtain a double resonance angularly separated by a value corresponding to the typical divergence of a laser beam focused by a commercial microscope objective. This maximizes the value of the real enhancement factor of the optical plate LO of the invention.

[0106] Concerning the material manufacturing phase, the deposition of each thin layer is carried out using any technique known to those skilled in the art, for example by one of the following techniques: vacuum evaporation, vacuum spraying, sol-gel process, spin coating, chemical vapor deposition, plasma deposition.

[0107] The invention thus offers the possibility of producing optical blades with electromagnetic field enhancement whose characteristics can be easily adapted according to the imaging parameters required by the microscopy system.

[0108] As indicated above, the thickness, number and type of material are characteristics of the stack according to the invention which can be adapted on a case-by-case basis, depending in particular on the imaging parameters of the system and the desired or imposed lighting conditions. Optically transparent materials in the spectral band used to carry out the study will be preferred, the dispersion values ​​of which for the refractive index and the absorption coefficient are known and controlled. Such characteristics must allow, at a predetermined optimized angle of incidence and illumination wavelength of the optical plate in total reflection regime, optical absorption with improved angular tolerance in the upper layer of the optimized stack, thus enhancing the evanescent electromagnetic field at the interface of said stack.

Claims

Claims 1. Method for manufacturing an optical slide (LO) intended to receive a biological sample (E) in an optical device by total internal reflection, said method comprising a step of designing said optical slide (LO) and a step of materially manufacturing said optical slide (LO) thus designed, said method being characterized in that the design phase comprises the following steps: A. the selection of a first dielectric material having a refractive index n a and a second dielectric material having a refractive index n b such that n a > n b B. the selection of a so-called X illumination wavelength in and an angle of incidence called optimal 0 in depending on said optical device by total internal reflection for which said optical blade is intended C. the design of a first structure comprising a first stack of dielectric thin layers arranged above a so-called substrate (SB) layer transparent in a predetermined spectral region comprising said illumination wavelength X in , said first stack comprising N > 7 of successive and alternating thin layers of the first dielectric material and of the second dielectric material, a first respective thickness of each dielectric thin layer and said number N of dielectric thin layers being adapted so that the first structure exhibits resonant absorption at the illumination wavelength X in and at the optimal angle of incidence 0 in , in total internal reflection mode D. the determination of a first function F rrepresentative of a value of a transmission of the first structure in the predetermined spectral region as a function of a number of dielectric thin layers and a thickness of each dielectric thin layer, and the determination of a second function F2 representative of an intensity of an evanescent electric field generated by the optical plate in the total internal reflection regime at the illumination wavelength X in and at the angle of incidence CORRECTED SHEET (RULE 91) ISA / EP optimal 6 in depending on a number of dielectric thin layers and said first thickness of each dielectric thin layer E. the design of said optical blade which comprises a second stack of dielectric thin layers called optimized stack arranged above the substrate (SB), the optimized stack comprising a number M < N, M > 7 of successive and alternating dielectric thin layers of the first dielectric material and the second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number M of dielectric thin layers being determined by an optimization of a merit function F based on the first function F and the second function F2 so that said optical blade has a first resonant absorption at the illumination wavelength X in and at a first angle of incidence in the total internal reflection regime and exhibits a second resonant absorption at the illumination wavelength X inand at a second angle of incidence 02 in the total internal reflection regime, with A0 = |0 X - 02| e [o.4°; 3°] and e2< e in < e1 or 0i < e in < e2.

2. Manufacturing method according to the preceding claim, in which said merit function F is such that F = aF r + (1 - a)F2, with 0 < a < 1 a parameter allowing to weight a respective weight of the first function F t and the second function F2 in said optimization.

3. Manufacturing method according to the preceding claim, in which a is between 0.3 and 0.

7.

4. Manufacturing method according to any one of the preceding claims, wherein said optimization is such that A0 = |0 X - 02| e [0.4°; 1.5°].

5. Manufacturing method according to any one of the preceding claims, wherein said optimization consists of finding the optimized thicknesses and the number M of dielectric thin layers for which said value of the transmission of the first structure in the predetermined spectral region is maximum and for which said intensity of the evanescent electric field is maximum. CORRECTED SHEET (RULE 91) ISA / EP 6. A manufacturing method according to any one of the preceding claims, wherein, in the first stack, an upper layer of the first stack is in the second dielectric material and has a first thickness of between 1.1A in / 4 and 1.8A in / 4 and in which the N - 1 lower layers of the first stack each have a first thickness of between 0.9A in / 4 and l.lA in / 4.

7. Manufacturing method according to any one of the preceding claims, in which the first function F1 is representative of a transmission of fluorescence radiation induced indirectly by an absorption of said illumination wavelength A. in in total internal reflection in said first stack illuminated with said optimal angle of incidence 0 in , the fluorescence radiation having a spectrum included in said spectral region, the transmission being integrated over an angular range of incidence of the fluorescence radiation on said optical plate between 0 and 80°.

8. Optical slide (LO) intended to receive a biological sample (E) in an optical device (D) by total internal reflection, the optical slide comprising: - a so-called substrate (SB) layer transparent in a predetermined spectral region - a so-called optimized stack arranged (EO) above the substrate (SB) and comprising a number M > 7 of successive and alternating thin dielectric layers (C 1; C2) of a first dielectric material and a second dielectric material, each of the layers having a respective so-called optimized thickness, the optimized thicknesses and the number M of thin dielectric layers being adapted so that said optical plate has at least: o a first resonant absorption at a so-called illumination wavelength A in and at a first angle total internal reflection regime, and o a second resonant absorption at the illumination wavelength A in and at a second angle of incidence 02 in the total internal reflection regime, with A0 = |0 X - 02| e [0.4°; 3°].

9. Optical blade according to the preceding claim, in which: CORRECTED SHEET (RULE 91) ISA / EP - the first dielectric material has a refractive index n a between 1.8 and 3.5; - the second dielectric material has a refractive index n b between 1.2 and 1.

7.

10. Optical blade according to the preceding claim, in which the first material is based on Nb20s, the second material is based on SiC>2.

11. Optical blade according to any one of claims 8 to 10, in which the number M of thin dielectric layers is less than 30, preferably less than 20.

12. Optical blade according to any one of claims 8 to 11, in which the optimized thicknesses and the number M of thin dielectric layers are such that said optical blade has a thickness of less than 5 μm and preferably between 0.5 μm and 2 μm.

13. Optical blade according to any one of claims 8 to 12, in which the optimized thicknesses are greater than or equal to 10 nm.

14. Optical device (D) by total internal reflection comprising: - an optical blade (LO) according to any one of claims 8 to 13, - a light source (SL) adapted to emit a light beam (Lin) having the illumination wavelength A in - an optical device (Obj) adapted to shape the light beam (Ln) so that it illuminates said optical blade with said optimized angle of incidence 0 in - a detector (Det) adapted to detect fluorescence radiation (FL) emitted when a sample (E) is deposited in correspondence with a region of a surface (SE) of said optical plate where evanescent waves (OE) are produced by total internal reflection in said optical plate of the light beam (Ln ) shaped, said fluorescence radiation (Fl) being excited by said evanescent waves (OE).

15. Device according to the preceding claim 14, consisting of a total internal reflection fluorescence microscope. CORRECTED SHEET (RULE 91) ISA / EP 16. Device according to claim 15, in which the optical device (Obj) is a microscope objective. CORRECTED SHEET (RULE 91) ISA / EP

Images